anticancer compounds from medicinal plants biologica nyssana 4 (1-2)  december 2013: 1-7 stešević, d. caković, d.  contribution to the alien flora of montenegro … 97 short communication first record of erpobdella concolor (annandale, 1913) (hirudinida: erpobdellidae) from greece clemens grosser 1 and vladimir pešić 2 1 am wasserturm 20, 04523 elstertrebnitz, germany; 2 department of biology, university of montenegro, cetinjski put bb, 81000 podgorica, montenegro * e-mail: vladopesic@gmail.com abstract: grosser, c., pešić, v.: first record of erpobdella concolor (annandale, 1913) (hirudinida: erpobdellidae) from greece. biologica nyssana, 4 (1-2), december 2013: 97-98. erpobdella concolor (annandale, 1913) was recorded for the first time from a spring in the peloponnese, greece. this locality represents the westernmost spot of the species range known so far. key words: hirudinida, erpobdellidae, erpobdella concolor, greece, first record introduction erpobdella concolor (a n n a n d a l e , 1913) is presumably considered as the east-mediterranean species (n e s e m a n n & n e u b e r t 1999), but the current knowledge of the geographical distribution of this species is still far from complete. recently, g r o s s e r & p e š i ć (2006) revealed possibly the eastern border of range of this species in the kerman province in iran. however, it has not been clear how far the range extends to the west in the mediterranean region. this research deals with the finding of erpobdella concolor in the peloponnese, greece, and establishes the westernmost locality of the species known so far. material and methods on 4 th july, 2017, one leech specimen was collected by hand under stones within 10-50 cm off the shore of the small spring (sentenikos spring, 37°11'31.06"ν, 22°21'53.61"ε, close to zoros spring, region of sparti town) in the central peloponnese. the specimen was collected by junior author and preserved in 96% ethanol. the specimen is deposited in the collection of senior author. results and discussion the specimen was identified as erpobdella concolor (a n n a n d a l e , 1913). the anterior part of the body is cylindrical; posteriorly, there are two blunt keels. the body is unicoloured without paramedian stripes. the male genital pore is situated in furrow b2/a2, the female in furrow b5/c11. the genital pores are separated by two annuli. the size of the leech is 13 mm in the length and 3 mm in the width. the single specimen of erpobdella concolor (a n n a n d a l e , 1913) from the peloponnese agree well with the description of n e s e m a n n & n e u b e r t (1999). this species was a long time treated as subspecies of dina lineata (m ü l l e r , 1774). however, recent phylogenetic studies (t r o n t e l j & s k e t 2000; s i d d a l l 2002) based on morphology and dna sequence data showed that a revision of the family was necessary because the morphological characters (first the annulation) used to distinguish former erpobdellid 4 (1-2) • december 2013: 97-98 biologica nyssana 4 (1-2)  december 2013: 97-98 grosser, c. pešić, d.  first record of erpobdella concolor … 98 genera are not informative. for this reason, s i d d a l l (2002) after morphogenetic analysis synonymized all the genera of erpobdellidae with erpobdella. furthermore, s i d d a l l (2002) elevated erpobdella concolor to full species status. according to the most comprehensive revision of the european leeches by nesemann & n e u b e r t (1999), erpobdella concolor is known from cyprus, southern turkey, syria, iraq, israel, jordan and the northern part of saudi arabia. the stream in village sirch in the kerman province (se iran) represent the easternmost locality of this species (g r o s s e r & p e š i ć 2006). on other side, our finding in the peloponnese represent the westernmost spot of the species range known so far. no informations on the ecology of this species are available. we found this species in the peleponnese under stones of a spring with a stream outflow (fig. 1). the accompaining fauna includes: planorbis atticus b o u r g u i g n a t , 1852 (gastropoda), eylais mutila k o e n i k e , 1897 (hydrachnidia) and scarodytes roberti f e r y , 2011 (coleoptera). in greece, two erpobdellid species have been found (see: n e s e m a n n & n e u b e r t 1999): erpobdella testacea (s a v i g n y , 1822) and e. latestriata (n e s e m a n n & n e u b e r t , 1995). additional field work is highly needed for appropriate evaluation of leech biodiversity in greece as well the extant distribution of erpobdella concolor. references grosser, c. & pešić, v. (2006) on the diversity of iranian leeches (annelida: hirudinea). archives of biological sciences, 58 (1): 21-24. nesemann, h. & e. neubert (1999): annelida, clitellata: branchiobdellida, acanthobdellea, hirudinea. – in: schwoerbel, j., & p. zwick (eds): süßwasserfauna von mitteleuropa. begründet von a. brauer 6/2, 178 pp., (spektrum) heidelberg siddall, m. e. (2002). phylogeny of the leech family erpobdellidae (hirudinea: oligochaeta). invertebrate systematics, 16: 1–6. trontelj, p. & sket. b. (2000) molecular reassessment of some phylogenetic, taxonomic and biogeographic relationships between the leech genera dina and trocheta (hirudinea: erpobdellidae). hydrobiologia, 438: 227-235. fig. 1. the westernmost locality of erpobdella concolor (annandale, 1913): spring in region of sparti town, peloponnese, greece anticancer compounds from medicinal plants biologica nyssana 4 (1-2)  december 2013: 71-74 sućur, j.,et al.  oxidativ e stress in soy bean seedings treated with… 71 original article oxidative stress in soybean seedlings treated with thymus serpyllum aqueous extract jovana šućur, milan popović, dejan prvulović, đorđe malenčić, biljana kiprovski faculty of agriculture, university of novi sad, trg dositeja obradovića 8, 21000 novi sad, serbia * e-mail: bkiprovski@polj.uns.ac.rs abstract: šućur, j., popović, m., prvulović, d., malenčić, đ., kiprovski, b.: oxidative stress in soybean seedlings treated with thymus serpyllum aqueous extract . biologica nyssana, 4 (1-2), december 2013: 71-74. the effect of different concentrations (0.05, 0.1 and 0.2%) of thymus serpyllum l. aqueous extracts on lipid peroxidation process (lp), as well as reduced glutathione content (gsh) in leaves and roots of soybean seedlings were examined 24 and 72 h after the treatment. our results showed that only highest concentration of the extract used (0.2%) enhanced process of lipid peroxidation, while concentration of 0.1% stimulated gsh accumulation in soybean seedlings. key words : allelopathy, antioxidants, biopesticides, glycine max l., thymus serpyllum l. introduction the genus thymus l. (lamiaceae) consists of about 215 species of herbaceous perennial and subshrubs. thymus species are considered as medicinal plants due to their pharmacological and biological properties (h u s s a i n et al., 2013). it is commonly used as tonic, herbal tea and flavouring agent. thymus oils and extracts are widely used in pharmaceutical, cosmetic and perfume industry also for flavouring and bio-preservation of several food products (v e r m a et al., 2011). in the mediterranean region, which can be described as the centre of this genus, annual aromatic herb, thymus serpyllum l. inhabits cultivated and uncultivated lands including wastelands (v e r m a et al., 2011). research on allelopathy of t. serpyllum on weeds shown that water extract of t. serpyllum promoted test plants' growth at low concentration but inhibited them at high, and inhibition became stronger as concentration increased (z h a n g , 2009). the use of allelochemicals as weed control agents, becomes widely investigated, however the impact of these bioherbicide on cultivated plants is less known. since ros (reactive oxygen species)generation resulting in oxidative damage has been proposed as one of the modes of action of allelochemicals, the aim of this study was to examine the impact of thymus serpyllum l. aqueous extract on soybean antioxidant properties so as to assess its possible side effects when applied as bioherbicide in soybean organic production. material and methods the wild, aromatic plant, thymus serpyllum l., was collected in northern serbia (vojvodina province) in may of 2010. voucher specimens of collected plant was confirmed and deposited at the herbarium of the department of biology, faculty of natural sciences, university of novi sad. seeds of soybean (glycine max l.) cv. sava were obtained 11 th sfses • 13-16 june 2013, vlasina lake 4 (1-2) • december 2013: 71-74 biologica nyssana 4 (1-2)  december 2013: 71-74 sućur, j.,et al.  oxidativ e stress in soy bean seedings treated with… 72 from the institute of field and vegetable crops, novi sad, serbia. the aqueous extract of t. serpyllum was prepared with air-dried plant material (10 g) in boiling distilled water (100 ml). after 24 h, the extract was filtered through filter paper and kept at 4 °c until application. the experiment was performed at the laboratory of biochemistry, faculty of agriculture, novi sad and conducted under controlled conditions (temperature 25 °c, 60-70% relative humidity, 16 h photoperiod). soybean seedlings were grown for 14 days in plastic pots (500 ml) containing sterile sand, after which they were transferred on plastic pots (700 ml) containing the hoagland’s solution and 3.5, 7 and 14 ml of t. serpyllum aqueous extract, while 3.5, 7 and 14 ml of the hoagland’s solution were added in pots of control. seedlings were harvested for determining the investigated biochemical parameters 24 and 48 h after the treatments. biochemical analyses were carried out spectrophotometrically using an uv/vis spectrophotometer model 6105 (jenway, uk). lipid peroxidation (lp) was measured at 532 nm using the thiobarbituric acid (tba) test. the total amount of tbars (tba-reactive substances) is given as nmol malondialdehyde (mda) equivalents mg -1 proteins (m a n d a l et al., 2008). reduced glutathione (gsh) was determined according to s e d l a k a n d l i n d s a y (1968) and expressed as µmol gsh mg -1 proteins. values of the biochemical parameters were expressed as means  standard error of determinations made in triplicates and tested by anova followed by comparison of the means by duncan’s multiple range test (p<0.05). data were analysed using statistica for windows version 11.0. results and discussion the toxicity of many allelochemicals can largely be attributed to the formation of semiquinone radicals that donate electrons to molecular oxygen, forming reactive oxygen species (ros), such as superoxide anions (o2 .), and more reactive hydroxyl ( . oh) or hydroperoxyl (ho2 ) radicals (h a m m o n d k o s a k & j o n e s , 1996). this plant-plant allelopathic interaction generates a cascade of signaling events-including ca 2+ influx and proton efflux that actives the nadph-oxidase complex (generating . o2 ) and ph-sensible cell wall peroxidases (producing h2o2), which produces an oxidative burst (d o s s a n t o s et al., 2008). enhancement of ros production during the oxidative burst is one of the earliest reactions elicited in response to various abiotic and biotic stimuli. some allelochemicals rapidly depolarize the cell membrane, increasing membrane permeability, inducing lipid peroxidation and causing a generalized cellular disruption that ultimately leads to cell death (d e v i & p r a s a d , 1996; z e n g et al., 2001; y u et al., 2003). the peroxidation of unsaturated lipids of biological membranes is the most prominent symptom of oxidative stress in animals and plants (m a n d a l et al., 2008). malondialdehyde (mda) content, an end-product of lipid peroxidation process, is used as oxidant biomarker. it is suggested that different concentrations of allelochemicals induce production of mda, however due to their antagonistic and synergistic effect mda content is not always in reciprocity with these chemicals (d i n g et al., 2007; h a d d a d c h i & g e r i v a n i , 2009). our results showed that 24 h after the treatment with t. serpyllum aqueous extracts no significant difference in the lp intensity in soybean leaves and roots between plants form control and treatments were recorded. the significant increase was recorded 72 h after the treatment only in leaves of soybean plants treated with the highest concentration of t. serpyllum aqueous extract (fig. 1). allelochemicals might directly inhibit oxidizing enzymes in some way, leaving the plant vulnerable to oxidative damage, similar to the reports of q i a n et al. (2009). it has been documented that gsh can remove ros and also protect proteins from the oxidation of protein thiol groups into denaturation (n o c t o r , 2002). t. serpyllum aqueous extract with concentration of 0.1% induced production of gsh in leaves of soybean plants 24 h, and in leaves and roots 72 h after treatment (fig. 2). also, 24 h after the treatment with t. serpyllum aqueous extract with concentration of 0.2%, roots of soybean had highest level of gsh content (fig. 2). conclusion in conclusion, our results showed that low concentration of thymus serpyllum l. aqueous extract did not induce lipid peroxidation in soybean seedlings, while the highest concentration used (0.2%) enhanced lipid peroxidation process 72 h after the treatment. furthermore, concentration of 0.1% of t. serpyllum aqueous extract stimulated gsh accumulation in soybean seedlings which propose its possible stimulative effect on antioxidant system. biologica nyssana 4 (1-2)  december 2013: 1-7 balian, d., mujagić-pašić, a..  morphological v ariability of the f ruit… 73 fig. 1. lipid peroxidation level in leaves and roots of soybean seedlings 24 and 72 h after treatment with different concentrations (%) of t. serpyllum aqueous extracts (v/v) and in control (c). values marked with different letter differ significantly at p<0.05 (duncan’s test). fig. 2. reduced glutathione content in leaves and roots of soybean seedlings 12 and 48 h after treatment with different concentrations (%) of t. serpyllum aqueous extracts (v/v) and in control (c). values marked with different letter differ significantly at p<0.05 (duncan’s test). acknowledgements . this study was carried out within a project of the ministry of education, science and technological development, republic of serbia, grant n o tr-31022. references devi, s.r., prasad, m.n.v. 1996: ferulic acid mediated changes in oxidative enzymes of maize seedlings: implications in growth. biology of plants, 38 (3):387-395. ding, j., sun, y., xiao, c. l., shi, k., zhou, y. h., yu, j. q. 2007: physiological basis of different allelopathic reactions of cucumber and figleaf gourd plants to cinnamic acid. journal of experimental botany, 58 (13): 3765-3773. dos santos, w.d., ferrarese, m.m.l., ferraresefilho, o. 2008: ferulic acid: an allelochemical troublemaker. functional plant science and biotechnology, 2 (1): 47-55. haddadchi, g.r., gerivani, z. 2009: effects of phenolic extracts of canola (brassica napuse l.) biologica nyssana 4 (1-2)  december 2013: 71-74 sućur, j.,et al.  oxidativ e stress in soy bean seedings treated with… 74 on germination and physiological responses of soybean (glycine max l.) seedlings. international journal of plant production, 3 (1): 63-74. hammondkosak, k.e., jones, j.d.g. 1996: resistance gene-dependent plant defense responses. plant cell, 8 (1):1773-1791. hussain a.i., anwar f., chatha s.a.s., latif s., sherazi s.t.h., ahmad a., worthington j., sarker s.d. 2013: chemical composition and bioactivity studies of the essential oils from two thymus species from the pakistani flora. food science and technology, 50: 185-192. mandal, s., mitra, a., mallick, n. 2008: biochemical characterization of oxidative burst during interaction between solanum lycopersicum and fusarium oxysporum f. sp. lycopersici. physiological and molecular plant pathology, 72 (1): 56-61. noctor, g., gomez, l., vanacker, h., foyer, c.h. 2002: interactions between biosynthesis, compartmentation and transport in the control of glutathione homeostasis and signalling. jornal of experimental botany, 53 (372): 1283–304. qian, h., xu, x., chen, w., jiang, h., jin, y., liu, w., fu, z. 2009: allelochemical stress causes oxidative damage and inhibition of photosynthesis in chlorella vulgaris. chemosphere, 75 (3): 368-375. sedlak, j., lindsay, h. 1968: estimation of total protein bound and non protein sulphydryl groups in tissue with ellman’s reagent. analytical biochemistry, 25: 192-205. verma r.s., verma r.k.,chauhan a., yadav a.k. 2011: seasonal variation in essential oil content and composition of thyme, thymus serpyllum l. cultivated in uttarakhand hills. indian journal of pharmaceutical sciences, 73 (2): 233-235. yu, j.q., ye, s.f., zhang, m.f., hu, w.h. 2003: effects of root exudates and aqueous root extracts of cucumber (cucumis sativus), and allelochemicals on photosynthesis and antioxidant enzymes in cucumber. biochemical systematics and ecology, 31 (2):129-139. zeng, r.s., luo, s.m., shi, y.h., shi, m.b., tu, c.y. 2001: physiological and biochemical mechanism of allelopathy of secalonic acid f on higher plants. agronomy journal, 93 (1):72-79. zhang y., m.sc., may 2009: studies of thymus serpyllum l. var. asiaticus kitag. allelopathy on weeds. http://www.globethesis.com/?t=2143360245465855 novica ranđelović: tireless botanist explores on the verge of his ninth decade 1 novica ranđelović: tireless botanist explores on the verge of his ninth decade fig. 1. novica ranđelović and his crocuses: a crocus rujanensis n. randj. et d.a. hill; b c. jablanicensis n. randj. et v. randj.; c – c. danubensis h. kernd., e. pasche, n. randj. & v. randj. exactly ten years ago, 9th symposium on the flora of southeastern serbia and neighboring regions has been organized in honor of 70th birthday of novica randjelović. he was a founder of non-formal south serbian botanical school, symposium on the flora of southeastern serbia and neighboring regions, the first professor and founder of the department for biology and ecology at the university of niš (s t a m e n k o v i ć , 2007). even though he was in his eight decade, professor randjelović tirelessly continues his research activities. he published many scientific papers. especially important are those describing a new species from his favorite genus crocus c. jablanicensis (r a n đ e l o v i ć et al., 2011) i c. danubensis (h a r p k e et al., 2014). two species belonging to this genus were named in his honor – crocus novicii (m i l j k o v i ć et al., 2016) and c. randjeloviciorum (h a r p k e et al., 2017). he published 13 papers (6 in international journals) in the period from 2007 to 2017. besides that, he is still a regular participant of all botanical scientific symposiums held on the territory of entire balkan peninsula. 8 (1) • september 2017: 01-03 doi: 10.5281/zenodo.962581 biologica nyssana 8 (1)  september 2017: 01-03  novica ranđelović: tireless botanist explores on the verge... 2 bibliography of n. ranđelović in period from 2007 to 2017 ranđelović, n., sekovski, ž., dimeska, g. 2007: systematic, chorological and genetic research into the genus crocus l. macedonia collection of papers devoted to academician kiril micevski, 97-131. ranđelović, v., zlatković, b., milosavljević, v., ranđelović, n. 2008: the endemic flora of bosilegrad surroundings (krajište region) in southeastern serbia. phytologia balcanica, 14 (3): 367-375. milosavljević, v., ranđelović, v., zlatković, b., ranđelović, n. 2008: phytocenologic diversity of krajište in southeastern serbia. natura montenegrina, 7 (3): 193-204. stanisavljević, d. m., đorđević, s. m., ristić, m. s., veličković, d.t., ranđelović, n.v. 2010: effects of different drying methods on the yield and the composition of essential oil from herb mentha longifolia (l.) huds. biologica nyssana, 1 (1-2): 89-93. zlatković, b., nikolić, lj., ranđelović, v., ranđelović, n., stevanović, v. 2011: comparative analyses of the vascular flora of the pcinja river gorges in serbia and macedonia. archives of biological sciences, 63 (4): 11571166. ranđelović, n., ranđelović, v., hristovski, n. 2012: crocus jablanicensis (iridaceae), a new species from the republic of macedonia, balkan peninsula. annales botanici fennici, 49 (1-2): 99102. velickovic, d.t., ristic, m.s., bjelakovic, l.l., karabegovic, i.t., stojicevic, s.s., lazic, m.l., randjelovic, n.v. 2012: chemical composition of dictamnus albus l. essential oil from serbia. agro food industry hi tech, 23, 3. miljković, m., ranđelović, n., ranđelović, v. 2012: phytogeographical analysis of the flora of miljkovačka gorge in eastern serbia. biologica nyssana 3 (2): 77-90. harpke, d., peruzzi, l., kerndorff, h., karamplianis, th., constantinidis, th., ranđelović, v., ranđelović, n., jušković, m., pasche, e., blattner, f.r., 2014: phylogeny, geographic distribution and new taxonomic circumscription of the crocus reticulatus species group (iridaceae). turkish journal of botany, 38 (6): 1182-1198. stanisavljevic, d., dordevic, s., milenkovic, m., lazic, m., velickovic, d., randelovic, n., zlatkovic, b. 2014: antimicrobial and antioxidant activity of the essential oils obtained from mentha longifolia (l.) hudson, dried by three different techniques. records of natural products, 8 (1), 61. papović, o., miljković, m., ranđelović, n., ranđelović, v., 2014: analysis of the flora of rogozna mountain in southwestern serbia. biologica nyssana, 5 (1): 17-30. papović, o., miljković, m., ranđelović, n., ranđelović, v. 2014: phytogeographical characteristics and endemism of the flora of rogozna mt. (sw serbia). biologica nyssana, 5 (2): 103-112. fig. 2. novica ranđelović with associates from serbia and germany on the field biologica nyssana 8 (1)  september 2017: 01-03  novica ranđelović: tireless botanist explores on the verge... 3 harpke, d., carta, a., tomović, g., ranđelović, v., ranđelović, n., blattner, f. r., peruzzi, l. 2015: phylogeny, karyotype evolution and taxonomy of crocus series verni (iridaceae). plant systematics and evolution, 301(1): 309-325. references harpke, d., peruzzi, l., kerndorff, h., karamplianis, th., constantinidis, th., ranđelović, v., ranđelović, n., jušković, m., pasche, e., blattner, f.r. 2014: phylogeny, geographic distribution and new taxonomic circumscription of the crocus reticulatus species group (iridaceae). turkish journal of botany, 38(6): 1182-1198. harpke, d., kerndorff, h., raca, i., pasche, e. 2017: a new serbian endemic species of the genus crocus (iridaceae). biologica nyssana, 8 (1): 0713. miljković, m., ranđelović, v., harpke, d. 2016: a new species of crocus (iridaceae) from southern albania (sw balkan peninsula). phytotaxa, 265 (1), 39-49. ranđelović, n., ranđelović, v., hristovski, n. 2012: crocus jablanicensis (iridaceae), a new species from the republic of macedonia, balkan peninsula. annales botanici fennici, 49 (1-2): 99102. stamenković, v. 2007: 70 godina života prof. dr novice ranđelovića i 45 godina obrazovnovaspitnog i naučno-istraživačkog rada. proceedings of 9th symposium on flora of southeastern serbia and neighboring regions, niš, 1-7. lakušić, d., rat, m., anačkov, g., jovanović, s.: datura inoxia mill. (solanaceae), a new alien species in serbia. biologica nyssana, 8 (1), september 2017 biologica nyssana 8 (1)  september 2017: 47-51 lakušić, d. et al.  datura inoxia mill. (solanaceae), a new alien species … 47 original article received: 25 july 2017 revised: 12 august 2017 accepted: 30 august 2017 datura inoxia mill. (solanaceae), a new alien species in serbia dmitar lakušić1, milica rat2, goran anačkov2, slobodan jovanović1 1university of belgrade, faculty of biology, institute of botany and botanical garden “jevremovac”, takovska 43, 11 000 belgrade, serbia 2university of novi sad, faculty of science, department of biology and ecology, trg dositeja obradovića 2, 21 000 novi sad, serbia * e-mail: dlakusic@bio.bg.ac.rs abstract: lakušić, d., rat, m., anačkov, g., jovanović, s.: datura inoxia mill. (solanaceae), a new alien species in serbia. biologica nyssana, 8 (1), september 2017: 47-51. as ornamental plant originated from central america, datura inoxia mill. has been introduced to many areas beyond its natural range. however, in many places, mainly in the mediterranean region d. inoxia has escaped from cultivation and is being established as an alien. in relevant botanical literature its occurrence on the balkan peninsula has not been reported for albania, bosnia & herzegovina, macedonia (fyrom) and serbia. based on our field and herbarium studies, we have found that d. inoxia is also present in serbia, both as cultivated ornamental plant, but as well as an escape, with established populations. we have registered this species in 23 localities in srem, bačka, banat, šumadija and ne serbia regions, which are situated in 16 utm 10 km x 10 km squares. most of the findings (16 localities) refer to a small group of individuals which are cultivated, while the remaining seven relate to plants that have escaped from cultivation and have established small wild populations in the surrounding ruderal habitats, edges of natural or planted forest, waste and arable fields. although we have registered only seven small groups of individuals that have escaped from cultivation, due to its capacity to invade natural habitats d. inoxia can be considered as potential threat for natural biodiversity in serbia. key words: datura inoxia, alien species, distribution, cultivation, invasion apstrakt: lakušić, d., rat, m., anačkov, g., jovanović, s.: datura inoxia mill. (solanaceae), nova strana vrsta u srbiji. biologica nyssana, 8 (1), septembar 2017: 47-51. kao ukrasna biljka koja potiče iz centralne amerike, datura inoxia mill. je uvedena u mnoge oblasti izvan njenog prirodnog areala. međutim, na mnogim mestima, uglavnom u mediteranu, ona je pobegla iz kultivacije i postala strana vrsta. u relevantnoj botaničkoj literaturi njeno prirustvo na balkanskom poluostrvu nije registrovano za albaniju, bosnu i hercegovinu, makedoniju i srbiju. na osnovu našeg istraživanja utvrđeno je da je d. inoxia prisutan i u srbiji, i to kao kultivisana ornamentalna biljka, ali i kao strana vrsta sa uspostavljenim populacijama i na staništima izvan kultivacije. ova vrsta je registrovana na 23 lokaliteta u regionima srem, bačka, banat, šumadija i ne srbija. registrovani lokaliteti se nalaze u 16 utm kvadrata 10 x 10 km. većina nalaza (16 lokaliteta) odnosi se na male grupe gajenih individual koje, dok se preostalih sedam nalaza odnosi na biljke koje su uspostavile male divlje populacije u okolnim ruderalnim staništima, ivicama prirodnih ili zasađenih šuma, deponijama ili obradivim površinama. iako smo registrovali samo sedam malih 8 (1) • september 2017: 47-51 doi: 10.5281/zenodo.963583 biologica nyssana 8 (1)  september 2017: 47-51 lakušić, d. et al.  datura inoxia mill. (solanaceae), a new alien species … 48 grupa pojedinaca koji su pobegli iz kultivacije, zbog svoje sposobnosti za invaziju prirodnih staništa d. inoxia se može smatrati potencijalnom pretnjom za autohtoni biodiverzitet u srbiji. ključne reči: datura inoxia, strane vrste, rasprostranjenje, gajenje, invazija introduction datura inoxia mill. is an annual, ornamental but extremely toxic plant. it originates from central america. due to its attractive flowers and increased growing in the gardens for decorative purposes, it has been introduced to many areas beyond its natural range (africa, asia, australia and europe). however, in many places, mainly in the mediterranean region (spain, portugal, france, italy, turkey), d. inoxia has escaped from cultivation and is being established as an alien species while in other european countries its alien status is unknown (daisie, 2017). there is the evidence that in vojvodina province this species is cultivated as medicinal plant and that it occasionally escapes from cultivation (o b r a d o v i ć & j a n j a t o v i ć , 1982). in relevant botanical literature its occurrence on the balkan peninsula has not been reported for albania, bosnia & herzegovina, macedonia (fyrom) and serbia (daisie, 2017, c a k o v i ć et al., 2014). according to g r e u t e r and r a u s (2005) datura inoxia has been known from bulgaria (sofia district) since 1933. in romania it is a casual alien, distributed along black sea coastal area, mainly in habitats close to harbors (a n a s t a s i u et al., 2011). the first data for greece refer to plants collected by a. yannitsaros in october 1966, on a rubbish-tip on waste ground on the south peloponnesian island of kithira (y a n n i t s a r o s , 1998). later, the plant was recorded in several localities in the coastal and mainland greece (c a k o v i ć et al., 2014). in croatia the species was reported approximately 30 years ago (h e ć i m o v i ć , 1981). like in greece, in croatia datura inoxia has spread along roads, in courtyards, gardens and in ruderal vegetation, not only to a number of locations in the littoral region, but also within the continental parts (h e ć i m o v i ć , 1981, 1982, f r a n j i ć , 1993, p a n d ž a & s t a n č i ć , 1990, t r i n a j s t i ć et al., 1993, f r a n j i ć et al., 1998, p a n d ž a et al., 2001). recently, in 2012, species was reported for the first time in montenegro, where it was recorded on waste ground at long ulcinj beach (c a k o v i ć et al., 2014). material and methods the study was carried out on recent field studies, analysis of herbarium material deposited at beou and buns (herbarium acronyms according to t h i e r s , 2017+), as well as literature data. the locations for occurrences of the species in the field were recorded with gps (garmin etrex legend hcx and garmin etrex vista c). all other data on the distribution were georeferenced in oziexplorer 3.95 4s program. the chorological data are presented according to the grid map with squares of c. 10 km × 10 km, based on the universal transverse mercator (utm) projection (l a m p i n e n , 2001), greed zone 34t. latitudes and longitudes are given according to the world geodetic system 84 (wgs84). results and discussion based on our field and herbarium studies, we have found that d. inoxia is also present in serbia, both as cultivated ornamental plant, but as well as an escape, with established populations. we have registered this species in 23 localities in srem, bačka, banat, šumadija and ne serbia regions, which are situated in 16 utm 10 km x 10 km squares (fig. 1). most of the findings (16 localities) refer to a small group of individuals which are cultivated, while the remaining seven relate to plants that have escaped from cultivation and have established small wild populations in the surrounding ruderal habitats, edges of natural or planted forest, waste and arable fields (fig. 2). monitoring of few populations (stepojevac, ritopek and golubac ponikva) from 2013 to 2016 did not show any significant spreading into the surrounding hinterland. chorological data bačka: cr45  apatin, in surroundings, cultivated (perić, r. summer 1998, buns) cr75  rumenka, cemetery (ujhelji, s. 23.10.2003, buns) cs90  subotička peščara kelebija-radanovac, planted pinus-robinia forest, 46.134546 n, 19.642232 e, 124 m (radak, b., bokić, b., rat, m. field obs., 12.08.2011) banat: dr57  kikinda 45.83 n, 20.45 e, 80 m, cultivated (fodulović, g. field obs., 09. 2013) srem: dq25  kupinovo, forest edge, waste and the arable fields in vicinity of the settlement, 44.711295 biologica nyssana 8 (1)  september 2017: 47-51 lakušić, d. et al.  datura inoxia mill. (solanaceae), a new alien species … 49 n, 20.045730 e, 74m (knežević, j., kovački, m., rat, m. field obs., 14.10.2016) šumadija: dq42 stepojevac, 44.511594 n, 20.294598 e, 130 m, escaped from cultivation (lakušić, d. field obs., 10.09.2015, ibid 09.10.2016) dq55 beograd braće jerković, 44.772 n, 20.490 e, 150 m, cultivated (fodulović, g. field obs., 09. 2013) dq55 beograd braće jerković, 44.769621 n, 20.498191 e, 175 m, cultivated (fodulović, g. field obs., 08.09. 2016) dq55 beograd železnik, 44.738512 n, 20.386443 e, 90 m, cultivated (lakušić, d. field obs., 10.09.2015) dq56 beograd dorćol, visokog stevana 21, 44.825409 n, 20.458966 e, 80 m, cultivated (dnevne novine blic 12.7.2013) dq56 beograd zvezdara, marka oreškovića, 44.803009 n, 20.487444 e, 120 m, cultivated (lakušić, d. field obs., 10.2015) dq56 beograd zvezdara, vojvode brane, 44.804743 n, 20.483909 e, 120 m, escaped from cultivation (lakušić, d. field obs., 10.09.2015) dq56 beograd zvezdara, vojvode savatija, 44.804695 n, 20.483720 e, 125 m, cultivated (lakušić, d. field obs., 09. 2013) dq65 beograd ritopek, 44.764100 n, 20.564839 e, 200 m, escaped from cultivation (lakušić, d. field obs. 12.02.2016, ibid. 09.10.2016) dq65 beograd selo rakovica ispod avale, 44.720943 n, 20.499923 e, 200 m, cultivated (lakušić, d. field obs., 10.09.2015) dq66 beograd karaburma, marijane gregoran, 44.813257 n, 20.508549 e, 115 m, cultivated (lakušić, d. field obs., 09. 2013) dq72 mladenovac, 44.468310 n, 20.666330 e, 150 m, cultivated (lakušić, d. field obs. 11.08.2015) dq74 grocka 44.682760 n, 20.706863 e, 110 m, cultivated (lakušić, d. field obs., 06.09. 2016) ne serbia: eq24 veliko gradište, sirakovo 44.679089 n, 21.338209 e, 160 m, escaped from cultivation (lakušić, d. field obs., 25.09. 2013) eq35 veliko gradište, kumane 44.730360 n, 21.466415 e, 75 m, cultivated (lakušić, d. field obs., 25.09. 2013) eq35 veliko gradište, topolovnik 44.720534 n, 21.443139 e, 75 m, cultivated (lakušić, d. field obs., 25.09. 2013) eq44 golubac, ponikva 44.689409 n, 21.570992 e, 120 m, escaped from cultivation (lakušić, d. 37683, 25.09. 2013, beou) eq92 donji milanovac, centar 44.465961 n, 22.153158 e, 70 m, cultivated (lakušić, d. field obs., 25.09. 2013) conclusion in conclusion, although we have registered only seven small groups of individuals that have escaped from cultivation, due to its capacity to invade natural – fig. 1. distribution of datura innoxia mill. in serbia. (utm grid zone 34t; basic square 10 x 10 km, according to l a m p i n e n , 2001). locality symbols:  cultivated plants  established populations biologica nyssana 8 (1)  september 2017: 47-51 lakušić, d. et al.  datura inoxia mill. (solanaceae), a new alien species … 50 habitats d. inoxia can be considered as potential threat for natural biodiversity in serbia. according to field records, its status in serbia is established alien species, with low invasiveness impact, i.e. it is casual species. nevertheless, on a local level it can be considered an important invader. the most vulnerable habitats are sandy areas along danube and continental sands, that are close to human settlements. in the same time, the wide range of adaptations on different artificial habitats and our records altogether with already published data show that datura inoxia is spreading in the continental and pannonian part of the se europe and thus can be expected in albania, bosnia & herzegovina and macedonia (fyrom) as well. acknowledgements. research was supported by the ministry of education and science, the republic of serbia, grant 173030, and financial mechanism of the european economic area 2009-2014, programme bg03 biodiversity and ecosystem services д-33-51/30.06.2015 (esenias-tools). references anastasiu, p., negrean, g., samoila, c., memedemin, d., cogalniceanu, d. 2011: a comparative analysis of alien plant species along the romanian black sea coastal area. the role of harbours. journal of coastal conservation, 15: 595-606. caković, d., stešević, d., vuksanović, s., tan, k. 2014: colchicum cupanii guss. subsp. glossophyllum (heldr.) rouy, datura innoxia mill. and eclipta prostrata (l.) l., new floristic records in montenegro and western balkan. acta botanica croatica, 73(1): 255–265. daisie european invasive alien species gateway, 2017. datura innoxia. available from: http://www.europealiens.org/speciesfactsheet.do?speciesid=20353# [accessed 30th june 2017]. franjić, j., trinajstić, i., škvorc, ž. 1998: a contribution to the knowledge of the spreading of some neophytes in croatia. fragmenta phytomedica et herbologica, 26: 5–17. fig. 2. naturalized plants of datura innoxia mill. golubac, ponikva – eq44, 44.689409 n, 21.570992 e, 120 m (lakušić, d. field obs., 25.09. 2013, photo d. lakušić). biologica nyssana 8 (1)  september 2017: 47-51 lakušić, d. et al.  datura inoxia mill. (solanaceae), a new alien species … 51 franjić, j. 1993: new findings of datura innoxia miller (solanaceae) in croatia. acta botanica croatica, 52: 97–100. greuter, w., raus, t. 2005: med-checklist notulae, 23. willdenowia, 35: 55–64. hećimović, m. 1981: report and the analysis of the island of šipan flora. acta botanica croatica, 40: 205–227. hećimović, s. 1982: flora on the islands of lokrum, bobara and mrkan. acta botanica croatica, 41: 155–170. lampinen, r. 2001: universal transverse mercator (utm) and military grid reference system (mgrs). http://www.fmnh.helsinki.fi/english /botany/afe/map/utm.htm obradović, m., janjatović, v. 1982: florogeneza najznačajnijih lekovitih semenica vojvodine. matica srpska. zbornik za prirodne nauke, 63: 6181. pandža, m., franjić, j.,trinajstić, i., škvorc, z., stančić, z. 2001: the most recent state of affairs in the distribution of some neophytes in croatia. natura croatica, 10: 259–275. pandža, m., stančić, z. 1990: new localities of the species datura innoxia miller and solanum elaeagnifolium cav. (solanaceae) in croatia. natura croatica, 8: 117–124. thiers, b. 2013 (continuously updated). index herbariorum: a global directory of public herbaria and associated staff. new york botanical garden's virtual herbarium. available from: http://sweetgum.nybg.org/ih/ (accessed 2017). trinajstić, i., pavletić, z., franić, j., liber, z. 1993: contribution to the knowledge of the neophytic flora of makarska littoral (dalmacija, hrvatska) . fragmenta phytomedica et herbologica, 21: 57– 62. yannitsaros, a. 1998: additions to the flora of kithira (greece) i. willdenowia, 28: 77–94. novica ranđelović: diary of a collaboration. biologica nyssana, 8 (1) 5 essay received: 17 january 2017 novica ranđelović: diary of a collaboration david a. hill budapest, hungary * e-mail: futured@hu.inter.net in summer 1980 i was appointed as the british council lektor at the primary teacher training college (viša pedagoška škola) in prizren, kosovo. i had specifically chosen that particular lektor post out of various that were offered me (priština, niš, skopje, belgrade) because of what i already knew about the natural history of the area and the serbian monasteries. i had started studying botany at the age of 12, written my first article and given my first public lecture aged 16, and had articles published in watsonia and the alpine garden society journal by the age of 27. and as well as 25 years in the uk, i has also spent 2 years in northern italy, honing my alpine and mediterranean botanical skills. thus when i arrived in prizren i was ready for a new experience with the balkan flora. of course, i was fairly badly equipped for this in terms of botanical field guides. i had polunin’s flowers of europe, polunin & everard’s trees & bushes of europe, and various uk, northern european and mediterranean guides, though this was fortunately soon augmented by polunin’s flowers of greece & the balkans, which came out in 1980, and i bought when home in the uk in summer 1981. so i did the best i could. i should say that as well as flowers, i was also studying any other wildlife that came my way, especially birds and butterflies, for which i had adequate books for the region. my first finds of local crocuses came on 17.03.81, when i discovered a lilac-flowered species with a netted corm, which i misidentified as crocus reticulatus. polunin (1980) told me that crocus dalmaticus, which it later turned out to be, was in the velebit mountains, so i didn’t consider it. this turned out to be an important find, extending the known range of the species considerably. i also found it at nearby landovica. and so i trawled the prizren area, kosovo, the mountains around finding many, many new and interesting flowers, besides the first crocus. i also found crocus chrysanthus on dulje between prizren and priština in spring 1982. but then i left prizren and moved to niš university to be british council lektor there, starting in september 1982. i was by this time living with a serbian lektor in english, and it was she who found novica ranđelovic for me. she was travelling on a bus between priština and niš in february 1983, when she overheard two gentlemen talking about flowers. she interrupted them and explained my situation – british amateur botanist, no contacts with local botanists, could they help? and novica – one of the gentlemen! – did. and so on 25.02.83, i drove down to his home in doljevac to meet novica, show him some of my botanical work, and discuss flowers with him, not least crocuses, in which we soon found we were both very interested. he told me about the jelašnica and sičevo gorges west of niš, and i visited both on the next two days, finding crocus adami in flower as he’d suggested. then on 11.03.83, novica and i went out into the field for the first time together. he took me to seličevica to see crocus alexandrii, amongst other things. it was the start of a long, happy and fruitful period of collaborative botanical work. i was very pleased when novica spoke to my partner 8 (1) • september 2017: 05-06 doi: 10.5281/zenodo.962791 biologica nyssana 8 (1)  september 2017: 05-06 hill, d.a.  novica ranđelović: diary of a collaboration 6 after that first meeting and said ‘i thought you said he was an amateur – he really knows a lot!’ and so the idea gradually took shape that we would work to revise the information on the genus crocus in serbia, but also do lots of other botany together outside the spring and autumn crocusflowering seasons. this list will give you an idea of the intensity of our field work: 1983: 11.03: seličevica; 13.03: rača; 24.04: rujan planina / pohor pčinjski; 15.05 stolovi; 29.05: ostrozub; 05.06 suva planina; 15.10: rujan planina; 1984: 03.03: vučje; 18.03: bujanovacpreševo; 24.03: trgovište; 01.04: bujanovacpreševo; 15.04: vlasinsko jezero; 03.06: preševo; 10.06: basara; 04.07: vlasinsko jezero; 20.10: trgovište / radovnica; 1985: 17.03: velika kopašnica / velika grabovnica / vučje; 24.03: prosek / sićevo / staničenje; 03.04: rujan planina; 06.04: trnava; 27.04: subotinac; 03.05: radovnica / novo selo; 19.05: vidlič / basara; 08.06: svrljig; 14.06: subotinac; 22.09: subotinac; 1986: 08.03: konjuh; 15.03: preševo / rujan planina / bujanovac / kopašnica; 22.03: preševo / katlanovo / prilep / pletvar / belavodna / prisad; 30.03: skopje: kitka / skopska crna gora; 02.04: staničenje; 05.04: vrska čuka; 09.04: subotinac; 12.04: vlasotinsko jezero; 23.04: šar planina (stojkova kuća / prevalac) / prizren / landovica / beli drim gorge / pojata / štimlje. my involvement in the fieldwork stopped then, because in august 1986 my six year limit of time in yugoslavia was up, and i moved to milan, italy to work at the british council there. i returned to serbia each holiday up until the wars began in 1990, and so met novica from time to time. the last time we met face-to-face was on 26.08.89. however, we kept in close touch, because from december 1988 we were working on our book the genus crocus l. in serbia which was published by sanu, belgrade which i finally got a copy of in october 1990. the fieldwork listed above was often done in conjunction with other botanists, most frequently spas sotirov and vlastimir stamenković. we drove everywhere either in novica’s yellow citroen dyane, or my red renault 4tl; in fact, we often used my car because in those days petrol was issued using coupons, and locals could only have so many per month, while foreigners could buy as many as they wanted (at a higher rate, of course!)… so i always had plenty. many of the places listed above were visited primarily to look for crocuses, and repeat visits were to check on species we knew about. others were general botanizing expeditions aimed at deepening knowledge about the flora of southern parts of serbia. the various visits to subotinac were for a project on the flora of oil shale, which i presented at a symposium in belgrade in june 1986. a number of articles were published in different places describing our finds. of course, i have not listed the numerous meetings and phone calls that novica and i had to talk over our finds, examine material, check the books, especially with reference to the new species we found for science, crocus rujanensis randjelović et hill, first seen on 18.03.84. so who was this novica randjelović that i shared so much time, so many car journeys, so much foot-slogging over rough terrain, so many cups of coffee, and so much discussion with over a period of three years? first of all, he was a kind and generous man, someone who was able to accept this younger, enthusiastic foreigner into his home and make him feel welcome, and that in a time when foreigners were still widely treated with suspicion. secondly, he was someone who wore his knowledge lightly and with humility – he never wanted to push what he knew at you to show it off; rather, it was there as a constant resource. thirdly, he was endlessly curious and enthusiastic, always wanting to do new projects, discover new plants in different areas. i appreciated all of these characteristics greatly. we had a lot of fun on all those trips as well as doing a lot of serious work, too. i regret that things ended as they did. the wars came, and i never got back to niš. however, the bond i feel to novica has remained a deep and important part of me. i salute him and thank him on his 80th birthday. may there be many more. anticancer compounds from medicinal plants biologica nyssana 2 (2)  december 2011: 00-00 ljupković r.b. et al..  removal cu(ii) ions from water... 17 original article a new species of bythiospeum bourguignat, 1882 (hydrobiidae, gastropoda) from montenegro vladimir pešić* 1 , peter glöer 2 1 department of biology, faculty of sciences, university of montenegro, cetinjski put b.b., 81000 podgorica, montenegro 2 biodiversity research laboratory, schulstraße 3, d-25491 hetlingen, germany * e-mail: vladopesic@gmail.com abstract: pešić, v., glöer, p.: a new species of bythiospeum bourguignat, 1882 (hydrobiidae, gastropoda) from montenegro. biologica nyssana, 3 (1), september 2012: 17-20. a new minute hydrobid species, bythiospeum bogici sp. nov. from one small spring near podgorica, montenegro is described. the list of subterranean species of the family hydrobiidae from montenegro is given. key words: bythiospeum, new species, subterranean, montenegro. introduction the rissooidean family hydrobiidae troschel, 1857 is one of the largest gastropod families with more than 400 recent genera assigned. the gastropods of the family hydrobiidae of montenegro have been studied by several authors during the 20th and 21th century (e.g., s c h ü t t 1960, b o l e 1961, r a d o m a n 1973, 1983, r e i s c h ü t z & r e i s c h ü t z 2008, g l ö e r & p e š i ć 2010, f a l n i o w s k i et al. 2012a, b). however, our knowledge still remains incomplete. to date, 42 hydrobioid species and subspecies belonging to 15 genera have been recorded in montenegro. it is notable that 62% of these species and subspecies are endemic for montenegro. the iucn red list of threatened species includes 18 hydrobiid species from montenegro. five of them are classified as critically endangered, six as endangered, two as data deficient and the rest as least concern (c u t t e l o d et al. 2011). some of hydrobiid gastropods are stygobiote species, i.e. living in underground waters. however, most of these species rarely been sampled in subterranean habitats and only being recorded in springs which flows directly out of the ground (see table 1). many of these springs are intermittent, and this may explain the limited recording of hydrobioid species, especially during the summer months when the freshwater springs dry up. because acess to, and sampling in hypogean habitats are difficult, subterranean hydrobiid gastropods have been described mainly from very few living animals or from empty shells only, and often collected outside the subterranean networks. consequently, ecological status (strict vs. occasional subterranean species) for most of these species are still unclear and subject to various opinions. recently, first author collected the spring in central montenegro, empty shells of one hydrobiid gastropod species which did not correspond to any described species. description of this species is given in this paper. 3 (1) • september 2012: 17-20 biologica nyssana 3 (1)  september 2012: 17-20 pešić, v., glöer, p.  a new species of bythiospeum… 18 figure 1. photo of the locus typicus (spring taban near spuž, podgorica). photo. v. pešić. materials and methods the empty shells of one minute hydrobiid gastropod from a spring taban (42º31.653 n, 19º13.145 e, 105 m asl. – fig. 1) near spuž, podgorica, were collected by hand and fixed with 80% ethanol. the spring water and sediment had a smell of hydrogen sulphide. since population abundance of this species seems to be low in the spring where it was found, only 10 specimens were collected. shell morphometric variables (namely shell height and width, aperture height and width) were measured using a stereo microscope (zeiss). shells were photographed with a leica digital camera system. the type material is stored in the zoological museum of hamburg (zmh). table 1. list of subterranean species of the family hydrobiidae from montenegro taxa sampling localities references red list category & criteria: bracenica spiridoni radoman, 1973 spring „spirov izvor“ (skadar lake area) radoman (1973) endangered b1ab(iii)+2ab(iii) ver 3.1 saxurinator orthodoxus schütt, 1960 spring of river zeta near straganik and tunjevo schütt (1960) critically endangered b1ab(iii)+2ab(iii) ver 3.1 paladilhiopsis tarae bole & velkovrh, 1987 groundwater system underlying the tara valley bole & velkovrh (1987) data deficient ver 3.1 plagigeyeria montenigrina bole, 1961 spring in obodska pećina cave near rijeka crnojevića bole (1961) critically endangered b1ab(iii)+2ab(iii) ver 3.1 plagigeyeria zetaprotogona zetaprotogona schütt, 1960 spring of river zeta near tunjevo village schütt (1960) endangered b2ab(ii,iii) ver 3.1 p.zetaprotogona pageti schütt, 1960 spring of river zeta near tunjevo village schütt (1960) endangered b2ab(ii,iii) ver 3.1 p. zetaprotogona zetadidyma schütt, 1960 spring of river zeta near tunjevo village schütt (1960) endangered b2ab(ii,iii) ver 3.1 p. zetaprotogona vitoja reischütz & reischütz, 2008 spring vitoja (skadar lake area) reischütz & reischütz, (2008) endangered b2ab(ii,iii) ver 3.1 vinodolia matjasici (bole, 1961) small spring in lipovik village (skadar lake area) bole (1961) critically endangered b1ab(iii)+2ab(iii) ver 3.1 vinodolia gluhodolica (radoman, 1973) spring at velje oko below the gluhi do village ( skadar lake area) radoman (1973) endangered b1ab(iii)+2ab(iii) ver 3.1 biologica nyssana 3 (1)  september 2012: 17-20 pešić, v., glöer, p.  a new species of bythiospeum… 19 results and disscusion systematics hydrobiidae genus bythiospeum bourguignat, 1882 bythiospeum bogici sp. nov. (fig. 2) material examined: 10 ex. from the type locality, 05.06.2012, leg. pešić & gligorović. holotype: shell height 2.3 mm, shell width 1.2 mm, zmh 79649. paratypes: 4 ex. zmh 79650; 5 ex. in coll. glöer type locality: montenegro, pogorica, spring taban, 42º31.653 n, 19º13.145 e, 105 m asl. description: the conically cylindrical shell consists of 5.5 convex whorls with a deep suture and a blunt apex. the shell is whitish and translucent. the umbilicus is slit-like. the surface is smooth and silky. the periostome in adults is bent to the outside from the lateral view. the basis of the aperture is pulled forward. shell height 1.85 – 2.3 mm, width 0.9 – 1.0 mm. etymology: named after phd student bogić gligorović who lead the first author to the spring and helped in the collecting. diferential diagnosis: due to the shape of the periostome which is bent to the outside in the adults, we ascertained the new species to the genus bythiospeum. the new species resembles bythiospeum montenegrina (schütt, 1959) (= paladilhiopsis montenegrina schütt, 1959) a species described from čeplica spring near bileća, bosnia and hercegovina (schütt, 1959). from the latter species bythiospeum bogici sp. nov. can easily be distinguished by the lower number of whorls and the aperture which is not oblique. remark: this taxon is a typical stygobiont. its very likely that bythiospeum represents a complex of closely related genera, probably with a limited distributions (e.g. balkanospeum georgiev, 2012 from bulgaria see georgiev 2012). further researches are certainly needed to collect living specimens for providing a proper anatomical description. distribution: known only from the type locality. figure 2. bythiospeum bogici sp. nov.: shell (holotype). acknowledgements. this study was supported by the research project cbfecomtg from the ministry of science, montenegro. references bole, j. 1961: nove hidrobide (gastropoda) iz podzemeljskih voda zahodnega balkana. (neue hydrobiiden (gastropoda) aus den unterirdischen gewässern westbalkans). biološki vestnik, 9: 59-69. bole, j. & velkovrh, f. 1987: nove vrste podzemeljskih polzev. jugoslavije. (new species of the subterranean snails of yugoslavia). sazu, ljubljana, 28 (3): 69-83. cuttelod a., seddon m. & neubert e. 2011: european red list of non-marine molluscs. iucn global species programme, iucn regional office for europe, iucn species survival commission, 98 pp. falniowski, a., szarowska, m., glöer, p. & pešić, v. 2012a: molecules vs morphology in the taxonomy of the radomaniola/grossuana group of balkan rissoiidea (mollusca: caenogastropoda). journal of conchology, 41 (1): 19-36 falniowski, a., szarowska, m., glöer, p., pešić, v., georgiev, d., horsák, m. & sirbu, i. 2012b: radiation in bythinella moquin-tandon, 1856 (mollusca: gastropoda: rissooidea) in the balkans. folia malacologica, 20 (1): 1-10 georgiev, d. 2012: new taxa of hydrobiidae (gastropoda: risooidea) from bulgarian cave h=2,3mm biologica nyssana 3 (1)  september 2012: 17-20 pešić, v., glöer, p.  a new species of bythiospeum… 20 and spring waters. acta zoologica bulgarica, 64 (2): 113-121 glöer, p. & pešić, v. 2010: the freshwater snails of the genus bythinella moquin-tandon (gastropoda: rissooidea: hydrobiidae) from montenegro. archives of biological sciences, 62: 441-447. radoman, p. 1983: hydrobioidea a superfamily of prosobranchia (gastropoda). i. systematics. monographs 547, serbian academy of sciences and arts, (department of science), 256 p. radoman, p. 1973: anagasta un nouveau genre prosobranchia et sa spéciation dans la bassin du lac skadar. zoologischer anzeiger, 190: 421429. reischütz, a. & reischütz, p.l. 2008: neue hydrobiiden (gastropoda, prosobranchia, hydrobiidae) aus dem becken des skutari-see (montenegro/albanien). basteria, 72: 143-145. schütt, h. 1960: neue höhlenschnecken aus montenegro. archiv für molluskenkunde, 89, 46: 11. schütt, h. 1959: zur höhlenschneckenfauna montenegros. archiv für molluskenkunde, 88 (4/6): 185-190. anticancer compounds from medicinal plants biologica nyssana 4 (1-2)  december 2013: 49-55 mitic, v. et al.  chemometric analysis of chlorophyll… … 49 original article chemometric analysis of chlorophyll a, b and carotenoid content in green leafy vegetables violeta mitić, vesna stankov jovanović, marija dimitrijević, jelena cvetković, , goran petrović, gordana stojanović university of nis, faculty of science and mathematics, department of chemistry, nis, serbia e-mail: violetamitic@yahoo.com abstract: mitić, v., stankov jovanović, v., dimitrijević, m., cvetković, j., petrović, g., stojanović, g.: chemometric analysis of chlorophyll a, b and carotenoid content in green leafy vegetables. biologica nyssana, 4 (1-2), december 2013: 49-55. effects of cooking on chlorophyll a and b and total carotenoid content in seven leafy green vegetables (brussels sprout, white cabbage, kale, chard, garden patience, broccoli and spinach) were evaluated. pigment content varied between species. chlorophyll a content was higher in all analyzed vegetables, compared to chlorophyll b and carotenoid content. boiling caused significant loss of chlorophyll a, chlorophyll b and carotenoids in kale (90.30%, 96.32% and 98.09%, respectively). cluster analysis was applied on pigment content in fresh and boiled vegetables and two statistically significant clusters were obtained, with difference in spinach position. key words: carotenoid content, chlorophyll content, chemometrics, cluster analysis, leafy green vegetables introduction leafy green vegetables are widely consumed in serbia. most of the vegetables are cooked by boiling in water or consumed fresh. fresh leafy vegetables contain valuable food components, such as chlorophylls, carotenoids, vitamins and phenolic compounds. these compounds are associated with dietary activities (k i m u r a & r o d r i g u e z a m a y a , 2003; g u t i e r r e z e t a l ., 2008). leafy green vegetables are well known for their characteristic flavor, color, and therapeutic value (f a l l e r & f i a l h o , 2009). these vegetables also contain chlorophylls and carotenoids (k i m u r a & r o d r i g u e z a m a y a , 2002), which are responsible for visual quality attributes (x u e & y a n g , 2009). chlorophylls are the earth’s most important organic molecules as they are necessary for photosynthesis (m a r k o v i ć e t a l ., 2012). chlorophyll is green pigment, which contain magnesium ion at the center of the porphyrin ring. there are various types of chlorophyll structure (chlorophyll a, chlorophyll b, chlorophyll c1, chlorophyll c2, chlorophyll d), but plants contain chlorophyll a and b. chlorophyll b differs from chlorophyll a only in aldehyde group bonded to the porphyrin, compared to the methyl group for chlorophyll a. chlorophyll a is photosynthetic pigment and absorbs blue, red and violet wavelengths in the visible spectrum, while chlorophyll b absorbs blue light and is used to complete the absorption spectrum of chlorophyll a. carotenoids are an important group of pigments in bacteria, algae and higher plants, where they function as accessory photosynthetic pigments covering regions of the visible spectrum not utilized by chlorophylls. carotenoids belong to tetraterpenoids, and they are split into two classes: 11 th sfses • 13-16 june 2013, vlasina lake 4 (1-2) • december 2013: 49-55 http://en.wikipedia.org/wiki/magnesium http://en.wikipedia.org/wiki/ion biologica nyssana 4 (1-2)  december 2013: 49-55 mitic, v. et al.  chemometric analysis of chlorophyll… … 50 xanthophylls (which contain oxygen) and carotenes (hydrocarbons, contain no oxygen). chlorophylls and carotenoids have an important role in the prevention of various diseases such as cancer, cardiovascular diseases and other chronic diseases (s a n g e e t h a & b a s k a r a n , 2010). cooking process can cause changes in physical characteristics and chemical composition of vegetables (z h a n g & h a m a u z u , 2004). chlorophylls are known to be easily degraded by acids, heat and light (t o n u c c i & v o n e l b e , 1992). food color plays important role in product acceptability, so it is important to prevent chlorophyll loss. the reason for the green color loss during processing is attributed to conversion of chlorophylls to pheophytins. this study was undertaken to evaluate pigment content in fresh and boiled broccoli (brassica oleracea var. silvestris), brussels sprouts (brassica oleracea var. gemmifera), white cabbage (brassica oleracea var. capitata), kale (brassica oleracea var. sabauda), chard (beta vulgaris), spinach (spinacia oleracea) and garden patience (rumex patientia). cluster analysis was applied to group vegetables based on their pigment content. material and methods fresh broccoli, brussels sprouts, white cabbage, kale, chard, spinach and garden patience were purchased from local market in niš – serbia. the contents of chlorophyll and carotenoids were determined spectrophotometrically. pigment content was determined in fresh and boiled vegetables. vegetable (10 g) was added to 50 ml of water and cooked for 5 minutes. samples were drained off and cooled. vegetables were cut into small pieces and weighed to 0.5000 g. measured vegetable material was mixed with acetone and homogenised in a mortar. mgco3 was added before vegetable homogenization to prevent chlorophyll pheophytinization. mixtures were filtered, mortar and pestle were washed several times with acetone, and the content was quantitatively transferred to the filter. the filter was washed with acetone so that the rest of the filter was completely white. filtrate was diluted with acetone to a total volume of 25 ml. apsorbance of prepared mixtures was recorded at 662, 644 and 440 nm using acetone as blank and pigment content was calculated using the formula of holm and wetsttein: chlorophyll а = 9.784·a662 – 0.990·a644 chlorophyll b = 21.426·a644 – 4.650·a662 chlorophyll a + b = 5.134·a662 + 20.436·a644 carotenoids = 4.695·a440 – 0.268·(a + b); а = absorbency at corresponding wave length, values 9.784, 0.990, 21.426, 4.650 and 0.288 is the molar absorptivity coefficient according to holm (1954) and wetsttein (1957) for acetone (absorption of 1 cm). after calculating the concentrations, the amounts of pigment per g of fresh matter were calculated applying the formula: c = content of pigment (mg/g) of fresh matter; c1 = the concentration of pigment calculated by the previous formula (mg/l); v = the starting volume of extract (ml); r = dilution; m = the weighed fresh plant (g). statistical analysis all results are expressed as mean of three determinations. arithmetic mean, the standard deviation and the coefficient of variation and correlations were calculated applying software statistica 7 (statsoft, inc., tulsa, ok, usa). cluster analysis (ca) is a multivariate technique, with the purpose of classifying the objects of the system into categories or clusters based on their similarities (richard & dean, 2002). ca is reported in several publications for the analysis of foodstuff (cam et al., 2009, tomsone et al., 2012). results obtained by cluster analysis are typically illustrated by a dendrogram. cluster analysis was done by means of ward’s method using euclidean distances as a measure of similarity. ward’s method attempts to minimize the sum of squares of any two (hypothetical) clusters that can be formed at each step. euclidean distance is most common way to measure distance between objects. the distances can be affected by differences in scale among the dimensions from which the distances are computed, so the variables must be standardized. the linkage distance is reported as dlink/dmax, which represents the quotient between the linkage distances for a particular case divided by the maximal linkage distance. the data derived from this study were originally centered by logarithmic transformation with all variables standardized. statistica 7 software was used to compile the dendrogram. biologica nyssana 4 (1-2)  december 2013: 49-55 mitic, v. et al.  chemometric analysis of chlorophyll… … 51 results and discussion analyses performed on selected vegetables showed a large variety of chlorophyll a, b and carotenoid content. chlorophyll and carotenoid content in green leafy vegetables was measured in fresh and boiled vegetables and the results were presented in figure1, 2 and 3. chlorophyll a content varied between species, from 0.047 mg/g fresh vegetables (f.v.) in brussels sprout, to 0.837 mg/g f.v. in garden patience (figure 1). amount of chlorophyll a was higher than chlorophyll b and carotenoids in all selected vegetables, which is in agreement with literature (lopez-ayerra et al., 1998). in fresh vegetables chlorophyll b ranking was: spinach kale garden patience chard broccoli cabbage brussels sprout (figure 2). these results showed that the chlorophyll concentration in the leafy green vegetables is dependent on species. the highest carotenoid content (figure 3) in spinach (0.191 mg/g f.v.) was seven times higher than than the lowest content in brussels sprout (0.024 mg/g f.v.). fig. 1. chlorophyll a content in fresh and boiled vegetables fig. 2 chlorophyll b content in fresh and boiled vegetables biologica nyssana 4 (1-2)  december 2013: 49-55 mitic, v. et al.  chemometric analysis of chlorophyll… … 52 fig. 3. carotenoid content in fresh and boiled vegetables most vegetables are cooked by boiling before consumed. cooking process has influence on chemical composition of vegetables (ismail et al., 2004; z h a n g and h a m a u z u ; 2004). s a h l i n et al. (2004) concluded that boiling, baking and frying had effect on the ascorbic acid, total phenolic, lycopene and antioxidant activity of the tomatoes. few data on the chlorophyll and carotenoid content of these vegetables are found in the literature, and disagreement with literature data may be caused by differences in varieties used and analytical methods applied. t u r k m e n et al. (2006) reported that chlorophyll a+b content in boiled spinach and broccoli was lower than in fresh vegetables, which is in agreement with our study. boiling caused significant loss of chlorophyll a, chlorophyll b and carotenoids in kale (90.30%, 96.32% and 98.09%, respectively). reduction of chlorophyll a and b content is attributed to the degradation of chlorophylls in their main derivatives pheophytin a and b, respectively (b o e k e l , 1999). pheophytin formation is result of mg 2+ eliminiation of chlorophyll. pheophytin can be produced by an acidic cooking environment or by prolonged cooking. in boiled vegetables, chlorophyll a content was lower than in fresh ones, except in brussels sprout. boiled brussels sprout contained 0,534 mg/g f.v. chlorophyll a, which is almost twice higher than chlorophyll a content in fresh vegetables. chlorophyll b content was lower in boiled than in fresh vegetables. chlorophyll a and b content decreased the most in kale (96.32% and 90.30%, respectively). carotenoid content was higher in boiled brussels sprout and chard than in fresh ones. in other vegetables tested, carotenoid content decreased, ranged from 28.07% in broccoli to 98.09% in kale. chemometrics is the branch of chemistry dealing with the analysis of chemical data (extracting information from data) and ensuring that experimental data contain maximum information (the design of experiments) (wold, 1995). chemometrics is used to classify of food products based on their main compounds (woodcock et al., 2007). in cluster analysis (ca), samples are grouped based on similarities. in this study ca was performed to the standardised data, on chlorophyll a, chlorophyll b and carotenoid content in fresh and boiled vegetables. two clusters were formed after applying cluster analysis to pigment content in fresh vegetables. brussels sprout, cabbage, broccoili and kale formed one cluster, while spinach, garden patience and chard grouped in second cluster. brussels sprout and cabbage showed lowest pigment content, so it was expected that they are „nearest neighbors“. euclidean distance between this two species was lowest (0.09). largest distance was observed between brussels sprout and garden patience (0.84), and accordingly differences between their pigment content was highest. biologica nyssana 4 (1-2)  december 2013: 49-55 mitic, v. et al.  chemometric analysis of chlorophyll… … 53 t ree diagram for 7 cases ward`s met hod euclidean dist ances 0 20 40 60 80 100 120 (dlink/dmax)*100 chard garden p atience sp inach kale broccoli cabbage brussels sprouts fig. 4. dendrogram obtained by cluster analysis using means of pigment content in fresh vegetables table 1. euclidean distances among analyzed fresh vegetables euclidean distances (fresh vegetables) brussels sprouts broccoli cabbage kale spinach garden patience chard brussels sprouts 0.00 0.24 0.09 0.36 0.80 0.84 0.58 broccoli 0.24 0.00 0.18 0.17 0.57 0.63 0.37 cabbage 0.09 0.18 0.00 0.27 0.75 0.80 0.55 kale 0.36 0.17 0.27 0.00 0.57 0.65 0.42 spinach 0.80 0.57 0.75 0.57 0.00 0.11 0.23 garden patience 0.84 0.63 0.80 0.65 0.11 0.00 0.28 chard 0.58 0.37 0.55 0.42 0.23 0.28 0.00 ca was performed on pigment content in boiled vegetables (figure 5). according to ca, boiled leafy green vegetables can be grouped as follows: brussels sprout, cabbage, kale, broccoli and spinach (cluster 1) and garden patience and chard (cluster 2). clustering according boiled vegetables pigment content shows different grouping of vegetables. compared to dendrogram compiled using fresh vegetables pigment content, this one differs in spinach position. boiled spinach have similar pigment content as brussels sprout, cabbage, kale and broccoli, while fresh spinach is more similar to garden patience and chard. nearest neighbors are boiled cabbage and kale, with euclidean distance of 0.01. biologica nyssana 4 (1-2)  december 2013: 49-55 mitic, v. et al.  chemometric analysis of chlorophyll… … 54 tree diagram for 7 cases ward`s method euclidean distances 0 20 40 60 80 100 120 (dlink/dmax)*100 chard garden patience spinach broccoli kale cabbage brussels sprouts fig. 5. dendrogram obtained by cluster analysis using means of pigment content in boiled vegetables table 2. euclidean distances among analyzed boiled vegetables euclidean distances (fresh vegetables) brussels sprouts broccoli cabbage kale spinach garden patience chard brussels sprouts 0.00 0.10 0.08 0.08 0.18 0.55 0.43 broccoli 0.10 0.00 0.18 0.18 0.08 0.46 0.33 cabbage 0.08 0.18 0.00 0.01 0.26 0.63 0.51 kale 0.08 0.18 0.01 0.00 0.26 0.62 0.51 spinach 0.18 0.08 0.26 0.26 0.00 0.39 0.26 garden patience 0.55 0.46 0.63 0.62 0.39 0.00 0.27 chard 0.43 0.33 0.51 0.51 0.26 0.27 0.00 conclusion chlorophyll a, b and carotenoid content varied between species. chlorophylls and carotenoids have significant role in human diet, so it is important to determine their loss during cooking process. largest decrease of all analysed pigments was recorded in kale. cluster analysis can be used to classify vegetables according to certain properties. according to pigment content in fresh vegetables, two statistical significant clusters were obtained. pigment content in boiled vegetables also yelds dendrogram with two clusterts, but with different grouping. biologica nyssana 4 (1-2)  december 2013: 49-55 mitic, v. et al.  chemometric analysis of chlorophyll… … 55 acknowledgements: the research was supported by ministry of education, science and technological development of the republic of serbia [oi 172047; oi 172051]. references boekel, m.a.j.s. 1999: testing of kinetic models: usefulness of the multiresponse approach as applied to chlorophyll degradation in foods. food research international, 32 (4): 261–269. cam m., hısıl y., durmaz g. 2009: classification of eight pomegranate juices based on antioxidant capacity measured by four methods, food chemistry 112 (): 721– 726 faller, a.l.k., fialho, e. 2009: the antioxidant capacity and polyphenol content of organic and conventional retail vegetables after domestic cooking. food research international, 42 (1): 210-215. gutierrez, j., barry-ryan, c., bourke, p. 2008: the antimicrobial efficacy of plant essential oil combinations and interactions with food ingredients. international journal of food microbiology, 124 (1): 91-97. ismail, a., marjan, z.m., foong, c.w. 2004: total antioxidant activity and phenolic content in selected vegetables. food chemistry, 87 (4): 581–586. kimura, m., rodriguez-amaya, d.b. 2002: a scheme for obtaining standards and hplc quantification of leafy vegetable carotenoid. food chemistry, 78 (3): 389–398. kimura, m., rodriguez-amaya, d.b. 2003: carotenoid composition of hydroponic leafy vegetables. journal of agricultural and food chemistry, 51 (9): 2603–2607. lopez-ayerra. b., murcia. m.a., garciacarmona. f. 1998: lipid peroxidation and chlorophyll levels in spinach during refrigerated storage and after industrial processing. food chemistry, 61 (1-2): 113– 118. marković m., pavlović d., tošić s., stankovjovanović v., krstić n., stamenković s., mitrović t., marković v. 2012: chloroplast pigments in post-fire-grown cryptophytes on vidlič mountain (southeastern serbia), archives of biological sciences, 64 (2): 531538. richard, a. johnson, & dean, w. wichern (2002). applied multivariate statistical analysis. london: prentice-hall. sahlin, e., savage, g.p., lister, c.e. 2004: investigation of the antioxidant properties of tomatoes after processing. journal of food composition and analysis, 17 (5): 635–647. sangeetha, r.k., baskaran, v. 2010: carotenoid composition and retinol equivalent in plants of nutritional and medicinal importance. efficacy of b-carotene from chenopodium album in retinoldeficient rats. food chemistry, 119 (4): 1584–1590. tomsone, l., kruma, z., alsina, i. 2012: the application of hierarchical cluster analysis for clasifying horseradish genotypes (armoracia rusticana l.) roots. cheminė technologija, 4 (62): 52-56. tonucci, l.h., von elbe j.h. 1992: kinetics of the formation of zinc complexes of chlorophyll derivatives. journal of agricultural and food chemistry, 40 (12): 2341-2344. turkmen, n., poyrazoglu, e.s., sari, f., sedat velioglu, y. 2006: effects of cooking methods on chlorophylls, pheophytins and colour of selected green vegetables. international journal of food science and technology, 41 (3): 281–288. wold, s. 1995: chemometrics; what do we mean with it, and what do we want from it? chemometrics and intelligent laboratory systems, 30 (1): 109-115 woodcock, t., downey, g., kelly, j.d., o’donnell, c. 2007: geographical classification of honey samples by nearinfrared spectroscopy: a feasibility study. journal of agricultural and food chemistry, 55 (22): 9128. xue, l., yang, l. 2009: deriving leaf chlorophyll content of green-leafy vegetables from hyperspectral reflectance. isprs journal of photogrammetry and remote sensing, 64 (1): 97–106. zhang, d., hamauzu, y. 2004: phenolics, ascorbic acid, carotenoids and antioxidant activity of broccoli and their changes during conventional and microwave cooking. food chemistry, 88 (4): 503–509. new locality of centaurea pichleri (asteraceae) in bulgaria biologica nyssana 7 (2)  december 2016: 87-90 bancheva, s., delcheva, m.  new locality of centaurea pichleri… 87 original article received: 01 september 2016 revised: 10 november 2016 accepted: 24 november 2016 new locality of centaurea pichleri (asteraceae) in bulgaria svetlana bancheva, malina delcheva institute of biodiversity and ecosystem research, bulgarian academy of sciences, acad. g. bonchev str., bl. 23, 1113-sofia, bulgaria * e-mail: sbancheva@yahoo.com abstract: bancheva, s., delcheva, m.: new locality of centaurea pichleri (asteraceae) in bulgaria. biologica nyssana, 7 (2), december 2016: 87-90. centaurea pichleri boiss. belongs to subgenus cyanus of asteraceae and has a very complex taxonomy. it is distributed in turkey (mainly in central anatolia), greece, bulgaria and lebanon. in the bulgarian flora the species has a conservation value being protected by the national biodiversity act and listed in the red list of vascular plants in bulgaria. during field investigations in the spring of 2016 a new locality of the species in toundzha hilly country (se bulgaria) was found. the new population of c. pichleri is in a good state and numbers over then 1000 individuals. from the spatial location of individuals it can be assumed that the reproduction of the species in this locality combined vegetative and seed model. in the new locality, no threats to the species' population were registrated. key words: asteraceae, centaurea, bulgarian flora, rare plant species apstrakt: bancheva, s., delcheva, m.: novi lokalitet vrste centaurea pichleri (asteraceae) u bulgarskoj. biologica nyssana, 7 (2), decembar 2016: 31-34. centaurea pichleri boiss. pripada podrodu cyanus porodice asteraceae i ima veoma kompleksnu taksonomiju. rasporostranjena je u turskoj (uglavnom u centralnoj anatoliji), grčkoj, bugarskoj i libanu. ova vrsta ima konzervacionu vrednost u bugarskoj flori, jer je zaštićena nacionalnim zakonom o biodiverzitetu i nalazi se na crvenoj listi vaskularnih biljaka bugarske. tokom terenskih istraživanja vršenih u proleće 2016. godine, nov lokalitet ove vrste je nađen u tundža oblasti (bugarska). nova populacija c. pichleri je u dobrom stanju i broji preko 1000 jedinki. iz prostornog rasporeda jedinki može se pretopstaviti da je reprodukcija vrste na ovom lokalitetu kombinacija vegetativnog i generativnog načina razmnožavanja. na novom lokalitetu nisu uočene pretnje populaciji ove vrste. ključne reči: asteraceae, centaurea, flora bugarske, retka biljna vrsta 7 (2) • december 2016: 87-90 12th sfses • 16-19 june 2016, kopaonik mt doi: 10.5281/zenodo.200404 biologica nyssana 7 (2)  december 2016: 87-90 bancheva, s., delcheva, m.  new locality of centaurea pichleri… 88 introduction during field investigations in the spring of 2016 a new locality of centaurea pichleri boiss. in toundzha hilly country (se bulgaria), was found. previously, the species was known from only three floristic regions of the country up to 800 m alt. (b a n c h e v a & d e n c h e v , 2000). a s s y o v & p e t r o v a (2012) erroneously indicate it also from thracian valley. centaurea pichleri belongs to subgenus cyanus of asteraceae and has a very complex taxonomy. cyanus was first mentioned by m i l l e r (1754) as a genus. de candolle (1838) was the first to considered it a section within the genus centaurea. in light of the molecular evidence, the latest compilations of the cardueae (s u s a n n a & g a r c i a -j a c a s 2007, 2009) suggest that cyanus should be left within centaurea. according to b o r š i ć et al. (2011) c. pichleri is a species of controversial delineation. the populations of this taxon appear to be diversely associated with other eastern relatives and collectively do not form a supported clade. bulgarian populations of the species seem to have less variability and they are more clearly identifiable. the species is distributed in turkey (mainly in central anatolia), greece, bulgaria and lebanon (w a g e n i t z , 1975; b o r š i ć et al., 2011). in bulgaria the species is rare and has conservation value – it is protected by the national biodiversity act and included in the red list of vascular plants in bulgaria as “vulnerable” (b a n c h e v a , 2009). material and methods this investigation was based on material collected by the authors in toundzha hilly country floristic region (se bulgaria) and on specimens deposited in the following herbaria: bgbm, iste, ma, p, prm, som, w. results and discussion new record centaurea pichleri boiss.: toundzha hilly country (se bulgaria), near to kostur village, svilengrad municipality, haskovo district, on the periphery of dry, stony meadows in termophilous oak forest, n 41,977788, e 26,270822, 572 m alt., 29 april 2016, coll. s. bancheva & m. delcheva, som – 172833. distribution and ecology the species is distributed in four (out of 20) floristic regions in bulgaria – strandzha mt, black sea coast, the rhodopes (east) and toundzha hilly country up to 800 m alt. the new population of c. pichleri inhabits the periphery of dry, stony meadows in termophilous oak forest and brushwood in the natural landmark "habitat of wild peony" fig. 1. habit of c. pichleri fig. 2. c. pichleri in its natural habitat biologica nyssana 7 (2)  december 2016: 87-90 bancheva, s., delcheva, m.  new locality of centaurea pichleri… 89 proclaimed in 1976 (figs 2, 3). the species grows together with: quercus frainetto ten., q. pubescens willd., fraxinus ornus l., carpinus orientalis mill, acer monspessulanum l., anthemis arvensis l., linaria genistifolia (l.) mill., paeonia peregrina mill., muscari botryoides (l.) mill., saxifraga rotundifolia l., veronica austriaca l. subsp. jacquinii (baumg.) eb. fisch., geranium sanguineum l., verbascum phoeniceum l., dactylis glomerata l., ranunculus illyricus l., lamium purpureum l., cornus mas l., potentilla neglecta baumg., centaurea thirkei sch. bip., achillea millefolium l., lychnis coronaria (l.) desr., plantago lanceolata l., hypericum perforatum l., ajuga laxmanii (l.) benth., lathyrus aphaca l., ranunculus neapolitanus ten., silene conica l. etc. this territory is a part of a site of community importance bg0000212 “sakar” from the ecological network natura 2000 in bulgaria. this habitat type named “pannonian-balkanic turkey oak – sessile oak forests – 91m0” is included in annex i of the “habitat” directive 92/43/eec. another specimen of centaurea pichleri from toundzha hilly country, south of the village of iglika, bolyarovo municipality (som 162724), is stored in som, which was collected about 50 km east of the new population. this is also unpublished record of the species. population observations the new population of c. pichleri is in a good state and numbers over then 1000 individuals. although finding a new population that is in good condition we consider regrettable that the conservation status of the species should not be changed. from the spatial location of individuals it can be assumed that the reproduction of the species in this locality combined vegetative and seed model. in the new locality no threats to the species' population were noticed. conclusion newly established population represents the northern boundary of the distribution of the species. acknowledgements. this research received support from the synthesys project http://www.synthesys.info/ which is financed by european community research infrastructure action under the fp7 integrating activities programme (es-taf-3669, fr-taf-3913, de-taf4725, cz-taf-4824). the field studies were supported by the financial mechanism of the european economic area 2009-2014, contract № д-33-88/28.08.2015 “mapping and assessment of sparsely vegetated land ecosystem services in bulgaria (spa-ecoservices)”. the authors thank vladimir trifonov from the regional inspectorate of fig. 3. map of distribution of c. pichleri biologica nyssana 7 (2)  december 2016: 87-90 bancheva, s., delcheva, m.  new locality of centaurea pichleri… 90 environment and water, haskovo for providing information about the newly established population. references assyov, b. & petrova, a. (eds). 2012. conspectus of the bulgarian vascular flora. bulgarian biodiversity foundation sofia. 452 p. bancheva, s. 2009. cyanus pichleri. in: petrova, a. & vladimirov, v. (eds). 2009. red list of bulgarian vascular plants. phytologia balcanica, 15 (1): 83. bancheva, s. & denchev, c. 2000. occurrence and taxonomic investigation of centaurea pichleri boiss. (asteraceae) in bulgaria. phytologia balcanica, 6 (2-3): 167-175. boršić, i., susanna, a., bancheva, s., garcia-jacas n. 2011. centaurea sect. cyanus: nuclear phylogeny, biogeography and life-form evolution. international journal of plant sciences, 172 (2): 238-249. de candolle, a.p. 1838: prodromus systematis naturalis regni vegetabilis. vol 6. treuttel & würtz, paris. miller, p. 1754: the gardeners dictionary. abridged ed. 4. privately published, london. wagenitz, g., 1975: genus centaurea l. pp. 465585. in: davis, p. h. (ed.): flora of turkey and the east aegean islands, 5. edinburgh university press, edinburgh. silene noctiflora l., present in the flora of kosovo and metohija (serbia) biologica nyssana 7 (2)  december 2016: 83-86 prodanović, d. et al.  silene noctiflora l., present in the flora of… 83 original article received: 15 october 2016 revised: 21 november 2016 accepted: 29 november 2016 silene noctiflora l., present in the flora of kosovo and metohija (serbia) danijela prodanović1, miloš stanojević2 , zoran krivošej2 1university of priština, faculty of agriculture lešak, lešak, serbia. 2university of priština, faculty of natural science, kosovska mitrovica, serbia *e-mail: danijela.prodanovic@gmail.com abstract: prodanović, d., stanojević, m., krivošej, z.: silene noctiflora l., present in the flora of kosovo and metohija (serbia). biologica nyssana, 7 (2), december 2016: 83-86. the genus silene (family caryophyllaceae) comprises more than 700 species and it is one of the larger genera of the world’s flora. studying the flora of the plain part of kosovo and metohija, in the village of gračanica, on the eastern rim of the kosovo basin (approximately 10 km to the south of priština), near the gračanka stream, we identified a weed and ruderal species, silene noctiflora. the part of the stream bank is urbanised and turned into a quay. the quay is regularly maintained implying that the banks are regularly weeded and the riverbeds are cleaned as the typha latifolia species rapidly grows; it may be the reason why this species was not immediately detected and appropriately identified. except for the afore mentioned locality, the species was also reported on the neglected arable fields and tilths, around the unkempt orchards, along the new tarmac road between gračanica and laplje selo, on the locality called labura. silene noctiflora is not mentioned for the region of kosovo and metohija in the new and revised edition of the book flora of serbia 2. key words: silene noctiflora, kosovo and metohija, serbia, new chorological data apstrakt: prodanović, d., stanojević, m., krivošej, z.: silene noctiflora l., prisutna u flori kosova i metohije (srbija). biologica nyssana, 7 (2), decembar 2016: 83-86. rod silene (familija caryophyllaceae) obuhvata više od 700 vrsta i jedan je od najvećih rodova u svetskoj flori. proučavajući floru nizijskog (ravničarskog) dela kosova i metohije, u selu gračanica, na istočnom obodu kosovske kotline (oko 10 km južno od prištine), pored rečice gračanke, konstatovali smo korovsko-ruderalnu vrstu, silene noctiflora. deo obale oko rečice urbanizovan je i pretvoren u kej, koji se redovno održava, što podrazumeva i stalno košenje korova na obalama ili čišćenje korita kada se prenamnoži typha latifolia; možda je to razlog zašto ova vrsta nije ranije uočena i pravilno determinisana. vrsta je sem na navedenom lokalitetu konstatovana i na zapuštenim oranicama i njivama, oko neodržavanih voćnjaka, duž novog asfaltnog puta između gračanice i lapljeg sela, na lokalitetu koji meštani gračanice zovu labura. u novom, prerađenom i dopunjenom izdanju knjige „flora srbije 2“, silene noctiflora se ne navodi za region kosova i metohije. ključne reči: silene noctiflora, kosovo i metohija, srbija, novi horološki podatak 7 (2) • december 2016: 83-86 12th sfses • 16-19 june 2016, kopaonik mt doi: 10.5281/zenodo.200403 biologica nyssana 7 (2)  december 2016: 83-86 prodanović, d. et al.  silene noctiflora l., present in the flora of… 84 introduction silene l. is one of the larger genera of the world’s flora. in the wide sense adopted here is comprised c. 700 species, about half of which occur in the mediterranean area (g r e u t e r , 1995). there are two major centers of diversity in silene: one in the mediterranean/middle east and one in central asia. a few taxa have been introduced to other continents (m a m a d a l i e v a et al., 2014). on the balkan peninsula as many as 64 species (41%) and 98 taxa (47%) are endemic. the high level of endemism has been recorded in the flora of turkey (48%) and iran (32%) (s t e v a n o v i ć , ed., 2012). genus silene has been placed in the tribe sileneae and the subfamily caryophylloideae. the tribe sileneae has a long history of controversial taxonomy at the genus level. in recent molecular phylogenetic studies by o x e l m a n et al. (2001) the genus silene along with lychis are distinct from viscaria, ixoca (=heliosperma), and atocion which form a well supported monophyletic group. the genus silene clusters in two major clades of approximately equal size, which are tentatively classified as silene subgenus silene and silene subgenus behen (moench) bunge (m a m a d a l i e v a et al., 2014). the genus consists mainly of herbaceous plants (annuals, biennials, and perennials), and, more rarely, of small shrubs or sub shrubs. silene also includes a number of cultivated species and widespread weeds. the flowers have free petals, with each petal consisting of a usually visible limb that can be divided or entire, and a claw that is included within the synsepalous calyx (e g g e n s et al., 2007). at the representatives of the caryophyllaceae family, particularly in the genus silene, the appearance of gynodioecy, dioecy and poliploidy are frequent so they present important model for genetic studies. silene noctiflora is gynomonoecious plant, namely individuals produce two types of flowers: hermaphrodite, which often occurs and functionally female flowers, which are less frequent (p e t r o v i ć , 2015). material and methods the results are based on field work in the plain part of central kosovo, during the year 2015, as well in year 2016. besides the field survey, checking of herbarium material and relevant literature sources were used to present an overall distribution for studied taxa in serbia. identification of the collected plant material is made according to flora of serbia 2, (s t e v a n o v i ć , ed., 2012) and flora europea 1 (t u t i n et al., eds., 1993); the nomenclature was adjusted to euro+med plantbase (2006). on the basis of relevant distribution data investigated species is mapped on 10x10 sq km at utm grid system (utm zone 34t) (l a m p i n e n , 2001). results and discussion fam. caryophyllaceae silene noctiflora l. floristic element: bor-submerid; col-plan life form: a mes-meg t scap the genus silene within the flora of serbia includes 44 species. silene noctiflora species appears as a weed or ruderal species in fallow fields, by the roads, on plowed, mostly shady places. its distribution in the flora of serbia is mostly related to lower areas/altitudes of the territory of srem and banat in vojvodina province, western, central, eastern, southeastern and southern parts of serbia, as well in šumadija and pomoravlje. vascular plants adapted to arable habitats are acknowledged to be among the most vulnerable groups in national floras to the land-use change, particularly in western european states. v r b n i č a n i n et al. (2009) consider that insufficient crop management in the mountain area has also led to the appearance of some rare weed species and similar result was obtained for some endangered 'red list' species, such as silene noctiflora. in a short list of rare or threatened european arable plants species silene noctiflora is present in 26 countries, and in 13 countries the species is rare, threatened or recently extinct (s t o r k e y et al., 2012). general distribution: europe, to ireland to the west, to the peninsula coolley to the north, to central apenines and northern greece to the south; small asia and caucasus, iran, south siberia and mountains of central asia. the species has been introduced to the most of continents. distribution in serbia: vojvodina: srem: fruška gora: čortanovci, karlovčić; banat pančevo: vojlovica, pančevački rit; deliblatska peščara; western serbia: mt jelica; mt zlatibor: gmizova ćuprija; šumadija: beograd; kosmaj; pomoravlje: jagodina; northeastern serbia: đerdap: kazan (pecka bara), sip, veliki štrbac; zlot; eastern serbia: niš: gorica, seličevica, niška banja: koritnik, pirot: basara, krupac, sukovo; babušnica; south eastern biologica nyssana 7 (2)  december 2016: 83-86 prodanović, d. et al.  silene noctiflora l., present in the flora of… 85 serbia: vlasotince, kruševica; vlasina; čemernik; southern serbia vranje (s t e v a n o v i ć , ed., 2012). new chorological data in serbia (fig. 1): village gračanicagračanka river banks (near priština town): 420 35' 99,9“ n, 210 11' 66“ e (559 m a.s.l) utm 34 ten12 (leg./det. krivošej, z., prodanović, d., stanojević, m., 29-may-2015; 9-june-2016); locality labura between gračanica and laplje selo village 420 35' 581“ n, 210 10' 075“ e 580 m a.s.l (30may-2015; 9-june-2016). fig. 1. distribution of the species silene noctiflora l. in serbia according to stevanović, ed., 2012 (black squares). new record marked with red circle. according to the insight into the available references of the botanist from kosovo and metohija and the latest edition of the flora of serbia 2 (s t e v a n o v i ć , ed., 2012), silene noctiflora has not been so far recorded on the territory of kosovo and metohija. during the field work in 2015, on the territory of central part of kosovo, this species was found in two relatively close localities, near gračanica village. in the first locality the species was detected on the banks of gračanka stream about 10 kilometers south from city of priština. the part of the stream bank is urbanised and turned into a quay. the quay is regularly maintained implying that the banks are regularly weeded and the riverbeds are cleaned as the typha latifolia species rapidly grows; it may be the reason why this species was not immediately detected and appropriately identified. the floristic structure in the gračanka river locality can be seen from next phytocenological record: typha latifolia l. 4.4., polygonum lapathifolium l. +.1, arenaria serpyllifolia l. 1.1., mentha aquatica l. +, polygonum aviculare l. +, convolvulus arvensis l. +, stellaria media l. (vill). +. floristic deficiency in this locality is due to the fact that the river banks (or riverbed) are paved by stony blocks so that only a few number of species have managed to inhabit them (fig. 2). fig. 2. habitat of species silene noctiflora l., on the locality gračanka stream later the species was also reported on the neglected arable fields and tilths, around the unkempt orchards, along the new tarmac road between gračanica and laplje selo, on the locality called labura. conclusion silene noctiflora l. is rather sporadically present in serbia, and the data of its presence in gračanica village and surrounding are the only available for kosovo and metohija flora and present a new locality for the serbian flora. new reports about the distribution of silene noctiflora l. species have complemented the picture of distribution and will certainly contribute to better understanding the chorology of this relatively rare species in serbia. biologica nyssana 7 (2)  december 2016: 83-86 prodanović, d. et al.  silene noctiflora l., present in the flora of… 86 references chater, a.o., walters, s.m., akeroyd, j.r. 1993: silene l. in: tutin, t.g., burges, n.a., chater, a.o., edmondson, j.r., heywood, v.h., moore, d.m., valentine, d.h., walters, s.m., webb, d.a. (eds.): flora europaea 1. second edition, university press, cambridge. pp. 191-218. eggens, f., popp, m., nepokroeff, m., wagner, w.l., oxelman, b., 2007: the origin and number of introductions of the hawaiian endemic silene species (caryophylaceae). american journal of botany, 94: 210-218. greuter, w., 1995: silene (caryophyllaceae) in greece: a subgeneric and sectional classification. taxon, 44 (4): 543-581. lampinen, r. 2001: universal transverse mercator (utm) and military grid reference system (mgrs). http://www.luomus.fi/english/botany/ afe/map/utm.htm mamadalieva, n., lafont, r., wink, m., 2014: diversity of secondary metabolites in the genus silene l. (caryophyllaceae) structures, distribution and biological properties. diversity, 6: 415-499. niketić, m., stevanović, v., 2012: silene l. in: stevanović, v. (ed.): flora srbije 2: 372-469. srpska akademija nauka i umetnosti. beograd. oxelman, b., lidén, m., rabeler, r.k., popp, m. 2001: a revised generic classification of the tribe sileneae (caryophyllaceae). nordic journal of botany, 20: 743-748. petrović, d. 2015: efekat gustine jedinki na pojavu funkcionalno ženskih i hermafroditnih cvetova kod vrste silene noctiflora l. master rad., prirodno-matematički fakultet, departman za biologiju i ekologiju, univerzitet u nišu. niš (manuscr.) 38 pp. storkey, j., meyer, s., still, k.s., leuschner, c., 2012: the impact of agricultural intensification and land-use change on the european arable flora. proceedings of the royal society b, 279: 14211429. the euro+med plantbase (2006-)the information resource for euro-mediterranean plant diversity. available at: http://ww2. bgbm.org/europlusmed vrbničanin, s., dajić-stevanović, z., jovanovićradovanov, k., uludaǧ, k., 2009: weed vegetation of small grain crops in serbia: environmental and human impacts. turkish journal of agriculture and forestry, 33: 325-337. a note on scavenging behaviour of adult hermann’s tortoise biologica nyssana 7 (1)  september 2016: 53-55 nikolić, m. et al.  a note on scavenging behaviour of adult hermann's tortoise... 53 short communication received: 26 december 2015 revised: 15 february 2016 accepted: 01 june 2016 a note on scavenging behaviour of adult hermann’s tortoise (testudo hermanni) marko nikolić1,2*, dimitrija savić1,2, maja ilić1,2, dragana stojadinović1, jelka crnobrnjaisailović1,3 1faculty of sciences and mathematics, university of niš, višegradska 33, 18000 niš, serbia 2biological society “dr. sava petrović”, visegradska 33, 18000 niš, serbia 3institute for biological research “siniša stanković”, university of belgrade, despota stefana 142, 11000 belgrade, serbia * e-mail: zerocool.axl@gmail.com abstract: nikolić, m., savić, d., ilić, m., stojadinović, d., crnobrnja-isailović, j.: a note on scavenging behaviour of adult hermann’s tortoise (testudo hermanni). biologica nyssana, 7 (1), september 2016: 53-55. report of the first observation of scavenging behaviour in the population of testudo hermanni boettgeri that has been monitored for six years in the village kunovica near the city of niš in serbia. on 31 may 2015 at 10:18 a.m., the adult tortoise was observed while eating a dead european green lizard (lacerta viridis). key words: testudo hermanni, diet, scavenging behavior, serbia apstrakt: nikolić, m., savić, d., ilić, m., stojadinović, d., crnobrnja-isailović, j.: beleška o strvinarskom ponašanju kod adulta šumske kornjače (testudo hermanni). biologica nyssana, 7 (1), septembar 2016: 5355. beleška o prvom zapažanju strvinarskog ponašanja u populaciji šumske kornjače testudo hermanni boettgeri, čiji monitoring se sprovodi već šest godina u selu kunovica, u blizini grada niša u srbiji. adultna kornjača je uočena prilikom hranjenja lešinom zelembaća (lacerta viridis) 31. maja 2015. u 10:18. key words: testudo hermanni, ishrana, strvinarsko ponašanje, srbija introduction testudinidae is the family of terrestrial chelonians (i.e. tortoises) which are, apart from a few species of lizards, the only terrestrial ectothermic vertebrates with generalized herbivorous or omnivorous feeding habits (d e l v e c c h i o et al., 2011). l u i s e l l i (2006) reviewed general dietary habits of 50 species from the family testudinidae and 15 species from the families geoemydidae and emydidae. of those, about 66% of terrestrial chelonians were exclusively herbivorous, 33% were omnivorous, and only one species (terrapene carolina) was predominantly carnivorous. herbivory in tortoises is not obligatory, as many species also feed on different food that 7 (1) • september 2016: 53-55 doi: 10.5281/zenodo.159104 biologica nyssana 7 (1)  september 2016: 53-55 nikolić, m. et al.  a note on scavenging behaviour of adult hermann's tortoise... 54 includes mushrooms, soil, sand, pebbles, and animal matter (m o s k o v i t s & b j o r n d a l , 1990; c e l s e et al., 2014). testudo hermanni is a southern european species (b e r t o l e r o et al., 2011). several studies about its diet, done in croatia, italy, france and spain, revealed the total of 134 plant species (46 families) on the menu, where the most frequently used plants were from the families asteraceae and fabaceae, and a bit less used species were from the families ranunculaceae and poaceae (for more detailed list see in v e t t e r , 2006). according to data from corsica, the hermann's tortoise is almost exclusively vegetarian (97.4% plants in 997 feedings observed in the wild), and its highly diverse food spectrum includes at least 250 species (c e l s e et al., 2014). although hermann’s tortoises avoid ligneous, aromatic, resinous, milky or hairy plant species, it has been shown that they consume some plants that are toxic to other animal species, such as tammus communis, arum sp. and ranunculus sp. (v e t t e r , 2006; b e r t o l e r o et al., 2011), perhaps to neutralize intestinal parasites (l o n g e p i e r r e & g r e n o t , 1999). in addition to various vascular plants, hermann’s tortoises occasionally feed on mushrooms, colonies of cyanobacteria (nostoc sp.), different species of invertebrates, and feces from various mammal species (human, dog, rabbit, goat, and pig) which seem to be appreciated for the hair and bone fragments or moisture that they contain, as well as on carrion (v e t t e r , 2006). heterospecific coprophagy was also reported elsewhere (v e t t e r , 2006). the ingestion of soil and stones can be a common albeit rarely observed behavior of desert tortoises (i.e. gopherus sp.). the deficiency of a mineral other than calcium or a ratio of minerals may be responsible for the ingestion of bones, stones, and soils by tortoises (e s q u e & p e t e r s , 1994). also, it is known that hermann’s tortoises occasionally ingest soil (geophagy) to acquire minerals (đ o r đ e v i ć & g o l u b o v i ć , 2014). here we report on the first observation of scavenging behaviour in the population of testudo hermanni boettgeri that has been monitored for six years in the village kunovica near the city of niš, serbia (43° 18’ 00’’ n; 22° 05’ 29’’ e). feeding behavior was previously observed in 27 of 1398 records collected during the fieldwork from 2010 to 2015. in all those 27 cases tortoises were feeding on plants. however, on 31 may 2015, at 10:18 a.m., the adult tortoise was observed while eating the remains of a dead european green lizard (lacerta viridis) (fig. 1). kunovica is a typical highland village area, situated at 621 m a.s.l., with the surface of 13.41 km2 and agricultural part covering about 4.44 km2. it is characterized by diverse vegetation and habitat composition, as about 58% of the land is covered with forests, 19% with fields and gardens, pastures constitute 6%, and there are also abandoned and active vineyards, and orchards (t u r n š e k , 2006). all these facts suggest optimal plant resources for local hermann’s tortoises. fig. 1. the adult hermann’s tortoise (testudo hermanni boettgeri) eating the remains of dead european green lizard (lacerta viridis) biologica nyssana 7 (1)  september 2016: 53-55 nikolić, m. et al.  a note on scavenging behaviour of adult hermann's tortoise... 55 b u d ó et al. (2009) recorded scavenging behaviour in t. h. hermanni in albera, (catalonia, spain) (one tortoise was observed while eating the remains of a dead bird). to our knowledge, this kind of feeding behaviour was reported in c e l s e et al. (2014) as sporadically detected in tortoise populations in serbia, but no details were provided. on the basis of relations between habitat preferences and dietary habits in tortoises, their sporadical carnivorous diet is considered as relic (n a t c h e v et al., 2015, but see j a c k s o n et al., 1999). therefore, further investigation about variation in its feeding habits is necessary for building more comprehensive knowledge on hermann’s tortoise ecological requirements. acknowledgements. this study was supported by grant no173025 ministry of education, science and technological development of republic of serbia. references bertolero, a., cheylan, m., hailey, a., livoreil, b., & willemsen, r.e. 2011: testudo hermanni (gmelin 1789): hermann's tortoise. in: rhodin, a.g.j., pritchard, p.c.h., van dijk, p.p., saumure, r.a., buhlmann, k.a., iverson, j.b., mittermeier, r.a. (eds.), conservation biology of freshwater turtles and tortoises: a compilation project of the iucn/ssc tortoise and freshwater turtle specialist group. chelonian research monographs, 5: 059.1–059.20. budó, j., capalleras, x., fèlix, j., font, j. 2009: aportacions sobrel’estudi de l’alimentació de la tortuga mediterrània (testudo hermanni hermanni) (gmelin, 1789) a la serra de l’albera (catalunya). butlletí de la societat catalana d’herpetologia, 18: 109–115. celse, j., catard, a., caron, s., ballouard, j.-m., gagno, s., jardé, n., cheylan, m., astruc, g., croquet, v., bosc, m., petenian, f. 2014: management guide of populations and habitats of the hermann's tortoise. life, 8. del vecchio, s., burke, r.l., rugiero, l., capula, m., luiselli, l. 2011: seasonal changes in the diet of testudo hermanni hermanni in central italy. herpetologica, 67: 236–249. đorđević, s., golubović, a. 2013: geophagy in the hermann’s tortoise, testudo hermanni. photo note hyla herpetological bulletin, 2013 (1): 4647. esque, t. c., peters, e. l. 1994: ingestion of bones, stones, and soil by desert tortoises. fish and wildlife research, 13: 73-84. jackson, d. r., ostertag, t. e. 1999: gopherus polyphemus (gopher tortoise). scavenging. herpetological review, 30 (1): 40. longepierre, s., grenot, c. 1999: some effects of intestinal nematodes on plant foraging behaviour of testudo hermanni hermanni in the south of france. in: miaud, c., guyetant, g. (eds.), current studies in herpetology, 277-284, le bourget du lac (seh). luiselli, l. 2006: resource partitioning in the communities of terrestrial turtles: a review of the evidences. revue d ecologie la terre et la vie, 61: 353–365. moskovits, d.k., bjorndal, k.a. 1990: diet and food preferences of the tortoises geochelone carbonaria and g. denticulata in northwestern brazil. herpetologica, 46(2): 207–218. natchev, n., tzankov, n., werneburg, i., heiss, e. 2015: feeding behavior in a “basal” tortoise provides insights on the transitional feeding mode at the dawn of modern land turtle evolution. peerj, 3: e1172. turnšek, b.a. 2006: the village of kunovica in the sustainable development context. facta universitatis – series: architecture and civil engineering, 4 (1): 25–39. vetter, h. 2006: chelonian library 2. hermann's tortoise. testudo hermanni, t. boettgeri and t. hercegovinensis. – frankfurt/main (edition chimaira), 325 pp. aćimović et al., 2019, biologica nyssana 10(1) 10 (1) september 2019: 23-28 doi: 10.5281/zenodo.3463994 the chemical composition of the essential oil of dracocephalum moldavica l. from vojvodina province (serbia) original article milica aćimović institute of field and vegetable crops novi sad, maksima gorkog 30, 21000 novi sad, serbia acimovicbabicmilica@gmail.com (corresponding author) jovana stanković institute of chemistry, technology and metallurgy, njegoševa 12, 11000 belgrade, serbia jovana_stankovic@hotmail.com mirjana cvetković institute of chemistry, technology and metallurgy, njegoševa 12, 11000 belgrade, serbia miracvet13@gmail.com marina todosijević university of belgrade, faculty of chemistry, studentski trg 12-16, 11000 belgrade, serbia marinab@chem.bg.ac.rs milica rat university of novi sad, faculty of science, trg dositeja obradovića 3, 21000 novi sad, serbia milica.rat@dbe.uns.ac.rs received: mart 7, 2019 revised: may 21, 2019 accepted: august 26, 2019 abstract: dracocephalum moldavica l., also called moldavian balm or moldavian dragonhead, is native to temperate climate of asia, but it was naturalized in eastern and central europe, north africa, china and north-eastern united states. this is an annual plant, with numerous stems (up to 6), 22-45 cm high, and blue flowers arranged in pseudo-whorls growing in leaf axils. essential oil accumulates in exogenous oil-containing cells at the dorsal sides of the leaves, and in the inflorescence. because of this, the entire plant has a citrus-like flavor, resembling that of lemon balm and catnip. this plant is extensively used as a spice and for composition of tea blends, in food aromatization (canned fish, jams, candies, syrups), perfumery, alcohol industry, soaps and detergents. dracocephalum moldavica from vojvodina province, serbia contains geranial (29.6%), geranyl acetate (27.2%) and neral (19.4%) as the most abundant compounds. further investigations will be focused on the influence of weather conditions on essential oil composition, as well as on bioactive potential of this essential oil. key words: moldavian balm, moldavian dragonhead, gc-ms analysis, geranial, geranyl acetate, neral apstract: hemijski sastav etarskog ulja dragocephalum moldavica l. iz vojvodine (srbija) biljka dracocephalum moldavica l., poznata je kao moldavska melisa ili moldavska zmajeglavka. poreklom je iz umerenog klimata azije, ali je naturalizovana u istočnoj i centralnoj evropi, severnoj africi, kini i severo-istočnom delu sad. iz korena ove jednogodišnje biljke izbija veći broj stabljika (do 6), koje su 22-45 cm visine, sa plavim cvetovima raspoređenim u lažnim pršljenastim cvastima u pazusima listova. etarsko ulje se akumulira u egzogenim uljanim ćelijama sa donje strane listova i u cvetovima. zbog etarskog ulja koje ima citrusnu notu ova biljka podseća na matičnjak i macinu travu. biljka se intenzivno koristi kao začin i kao dodatak čajnim mešavinama, za aromatizaciju hrane (konzervirana riba, džemovi, slatkiši, sirupi), parfimeriji, alkoholnoj industriji, proizvodnji sapuna i deterdženata. etarsko ulje d. moldavica iz ap vojvodine, srbije sadrži geranial (29,6%), geranil-acetat (27,2%) i neral (19,4%) kao najzastupljenije komponente. dalja istraživanja biće usmerena na uticaj vremenskih uslova na sastav etarskog ulja, kao i na njegov biološki potencijal. ključne reči: moldavska melisa, moldavska zmajeglavka, gc-ms analiza, geranial, geranil acetat, neral introduction dracocephalum moldavica l., also called moldavian balm or moldavian dragonhead, is native to temperate climate of asia, but it was naturalized in eastern and central europe, north africa, china and north-eastern united states. this species belongs to the subtribe nepetinae, tribe menthae of lamiaceae family. the genus contains 71 species, widespread across northern hemisphere regions (naderifar et al., 2015; amirnia et al., 2017). this annual plant is very beautiful, with numerous stems (up to 6), 22-45 cm high, and blue flowers arranged in pseudo-whorls growing in leaf axils. © 2019 aćimović et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 23 essential oil accumulates in exogenous oil-containing cells at the dorsal sides of the leaves, and in the inflorescence. because of this, the entire plant has a citrus-like flavor, resembling that of lemon balm (melissa officinalis l.) and catnip (nepeta cataria l.). apart from essential oil, the plant produces nectar, due to which it is grown as a honey-bearing plant and cultivated in gardens and parks as an ornamental plant (aćimović et al., 2019). this plant with its citrus like flavor is extensively used as a spice and for composition of tea blends, because of neral and geranial as major constituents of essential oil. dracocephalum moldavica is used in food aromatization (canned fish, jams, candies, syrups), perfumery, alcohol industry, soaps and detergents. apart from this, dried leaves have the potential of being used as a functional additive for extruded crisps with high nutritional value, especially because of the dietary fiber and rosmarinic acid content, strong antioxidant potential and acceptable sensory properties (wojtowicz et al., 2017). seed is a good source of fatty oil with spicy taste and aromatic odor, rich in unsaturated fatty acids, principally the linolenic and linoleic acids. this categorizes d. moldavica seed into the group of raw materials suitable for nutraceuticals, food supplements, and functional food applications (aćimović et al., 2019). moreover, the application of d. moldavica residues as bagasse waste (oilcake) collected after pressing and added to corn crisps could be an effective way of limiting the oil waste after pressing and increasing the sustainability of waste management. a new range of nutritionally valuable snacks could be introduced to the market (oniszczuk et al., 2017). furthermore, numerous investigations show that this plant possesses good antioxidative (aprotosoaie et al., 2016; aslanipour et al., 2017; weremczukjeżynaet al., 2017; ehsani et al., 2017; fallah et al., 2018), antimicrobial (pak et al., 2016; ehsani et al., 2017; keikhaie et al., 2018) and insecticidal activity (chu et al., 2011; ding et al., 2015). it is also used as antinociceptive (maham et al., 2013), sedative (martínez-vázquez et al., 2012), neuroprotective (sun et al., 2014), as well as cardiotonic agent (najafi et al., 2009; zeng et al., 2018), and for treating chronic mountain sickness (maimaitiyiming et al., 2014). this species is not well known in serbia. it was introduced in serbia in the collection garden of institute of field and vegetable crops, from romania (kišgeci et al., 1982). the aim of this paper is to analyze the essential oil obtained from d. moldavica grown in agroecological conditions of vojvodina province, serbia. materials and methods plant material the plant (fig. 1) grown at institute of field and vegetable crops novi sad, during 2018, were confirmed by m. rat and deposited at the herbarium of biology and ecology (buns herbarium), faculty of natural sciences, university of novi sad, as d. moldavica, voucher specimens 2-1468. during the flowering stage (june), the aboveground parts were cut, dried and used for essential oil extraction. essential oil extraction the dried aboveground parts of d. moldavica were subjected to hydro-distillation using an all glass clevenger-type apparatus to extract essential oils. the samples were ground, homogenized and made into a fine powder. in order to extract the essential oils, 100 g of the powder was placed in 1 l conical flask and connected to the clevenger apparatus. 500 ml of distilled water was added to the flask and heated to the boiling point. the steam in combination with the essential oils was distilled into a graduated cylinder for 4 h and then separated from aqueous layer. the yield essential oil was very low, so it is extracted with n-hexane, dried over anhydrous sodium sulfate and evaporated. the obtained oil was kept refrigerated at +4 °c until required for further analysis. 24 biologica nyssana ● 10 (1) september 2019: 23-28 aćimović et al. ● the chemical composition of the essential oil of dracocephalum moldavica l. from vojvodina province (serbia) fig. 1. dracocephalum moldavica gc/ms analysis gas chromatographic-mass spectrometric analysis was performed using an agilent 6890 gas chromatograph coupled with an agilent 5973 network mass selective detector (msd) (both agilent, santa clara, usa), in positive ion electron impact (ei) mode. the separation was effected using agilent 19091s-433 hp-5ms fused silica capillary column with 30 m × 0.25 mm i.d., 0.25 μm film thickness. the gc oven temperature was programmed from 60 °c to 285 °c at a rate of 3 °c/min. helium was used as carrier gas; inlet pressure was 20.3 kpa; linear velocity was 1 ml/ min at 210 °c. injector temperature: 250 °c; injection mode: splitless. ms scan conditions: ms source temperature, 230 °c; ms quad temperature, 150 °c; energy, 70 ev; mass scan range, 40–550 amu. 25 identification of volatile compounds the identification of components was carried out on the basis of kovats retention index and by comparison with reference mass spectra (wiley and nist databases). results and discussion in d. moldavica essential oil 88 compounds were detected, among which geranial (29.6%), geranyl acetate (27.2%) and neral (19.4%) were the most abundant, comprising 76.2%. other significant compounds were: geraniol (5.4%), neryl acetate (3.0%), piperitone (1.8%) and vulgarone b (1.6%). other compounds were present in the amount less than 1.0%, among which 31 unidentified compounds table 1. chemical composition of d. moldavica no compound ri rt % no compound ri rt % 1 1-octen-3-ol 982 7.14 0.2 46 trans-α-farnesene 1514 29.23 tr 2 3-octanone 990 7.38 0.2 47 italicene ether 1540 30.28 tr 3 dehydro-1,8-cineole 997 7.59 tr 48 spathulenol 1582 32.032 0.2 4 3-octanol 1000 7.65 0.1 49 caryophyllene oxide 1587 32.25 0.4 5 bergamal 1057 9.70 0.1 50 viridiflorol 1596 32.60 tr 6 cis-linalool oxide 1076 10.42 tr 51 salvial-4(14)-en-1-one 1598 32.67 tr 7 trans-linalool oxide 1093 11.04 tr 52 ni-12 1602 32.88 0.1 8 linalool 1104 11.47 0.4 53 ni-13 1610 33.17 0.1 9 ni-1 1109 11.65 0.1 54 humulene epoxide ii 1613 33.28 0.1 10 1-octen-3-yl acetate 1116 11.98 tr 55 1,10-di-epi-cubenol 1619 33.51 tr 11 ni-2 1142 13.10 0.1 56 ni-14 1630 33.93 0.1 12 exo-isocitral 1148 13.34 0.1 57 ni-15 1634 34.09 0.1 13 trans-chrysanthemal 1152 13.54 0.3 58 ni-16 1648 34.63 0.1 14 nerol oxide 1156 13.76 0.1 59 vulgarone b 1654 34.87 1.6 15 cis-isocitral 1166 14.18 0.6 60 ni-17 1659 35.02 0.1 16 ni-3 1175 14.59 0.2 61 cis-calamenen-10-ol 1663 35.20 0.1 17 rosefuran epoxide 1177 14.62 0.1 62 ni-18 1668 35.37 0.1 18 trans-isocitral 1184 14.96 0.9 63 ni-19 1675 35.67 0.2 19 α-terpineol 1193 15.34 tr 64 germacra-4(15),5,10(14)-trien-1-α-ol 1690 36.25 0.1 20 ni-4 1196 15.50 0.2 65 cyclocolorenone 1764 38.92 0.1 21 ni-5 1209 16.01 0.2 66 ni-20 1829 41.28 0.1 22 ni-6 1222 16.57 0.1 67 6,10,14-trimethyl-2-pentadecanone 1851 42.02 0.3 23 ni-7 1226 16.77 0.1 68 ni-21 1926 44.59 0.1 24 nerol 1232 17.04 0.4 69 ni-22 2011 47.47 0.1 25 ni-8 1236 17.22 0.2 70 ni-23 2038 48.29 0.1 26 neral 1248 17.76 19.4 71 manool 2068 49.18 0.1 27 piperitone 1258 18.21 1.8 72 ni-24 2082 49.60 0.1 28 geraniol 1262 18.38 5.4 73 ni-25 1997 50.02 0.1 29 ni-9 1264 18.48 0.2 74 ni-26 2116 50.62 0.1 30 geranial 1280 19.18 29.6 75 ni-27 2126 50.91 0.3 31 trans-anethole 1291 19.65 tr 76 ni-28 2156 51.80 0.2 32 thymol 1298 19.96 tr 77 ni-29 2164 52.05 0.2 33 carvacrol 1307 20.35 0.7 78 ni-30 2218 53.65 0.1 34 methyl geranate 1328 21.26 0.2 79 ni-31 2238 54.26 0.1 35 neryl acetate 1369 23.08 3.0 80 tricosane 2307 56.54 0.1 36 α-copaene 1380 23.58 0.1 81 pentacosane 2503 62.04 0.2 37 geranyl acetate 1393 24.15 27.2 82 hexacosane 2603 64.65 tr 38 decyl acetate 1412 25.07 0.1 83 heptacosane 2706 67.19 0.3 39 trans-caryophyllene 1425 25.49 0.2 84 2-methyloctacosane 2780 68.96 tr 40 α-humulene 1459 26.91 0.1 85 octacosane 2806 69.60 tr 41 ni-10 1464 27.11 0.1 86 nonacosane 2906 71.96 0.3 42 ni-11 1469 27.35 0.1 87 triacontane 3056 74.23 tr 43 germacrene d 1488 28.10 0.1 88 untriacontane 3105 76.45 0.1 44 ar-curcumene 1489 28.15 0.1 total identified 95.5 45 trans-β-ionone 1491 28.24 tr total ni 4.1 ri – retention index, rt – retention time, ni – not identified compounds (mass spectrum of these compounds, m/z (intensity) are shown at figures 2-32), tr – compound presented in traces (less than 0.1%) biologica nyssana ● 10 (1) september 2019: 23-28 aćimović et al. ● the chemical composition of the essential oil of dracocephalum moldavica l. from vojvodina province (serbia) 26 comprising 4.1% (tab. 1). a gc-fid chromatogram of d. moldavica essential oil is shown at fig 2. chemical composition of d. moldavica essential oil was previously studied in egypt (hussein et al., 2006; el-baky and el-bsroty, 2008; aziz et al., 2013; ahl et al., 2015; hegazy et al., 2016), iran (omidbaigi et al., 2010; maham et al., 2013, golparvar et al., 2016, ehsani et al., 2017; janmohammadi et al., 2017; fallah et al., 2018), turkey (eshan et al., 2014), ukraine (kotyuk and rakhmetov, 2017) and china (chu et al., 2011). it is established that chemical composition of essential oil from aerial parts of d. moldavica depends on many factors, among which origin, cropping system, fertilization, salt stress, weed management, etc. (aziz et al., 2013; janmohammadi et al., 2017; fallah et al., 2018). the principal compounds in almost all essential oils are neral, geranial, geranyl acetate and geraniol. however, the content of neral in essential oil varied between 10.25 and 43.49%, while geranial ranged between 9.10 and 42.45%. furthermore, geranyl acetate and geraniol content varied in larger scale, between 0.20%-40.40%, and 0.50 and 28.14%, respectively (aćimović et al., 2019). investigations show that the geranyl acetate, geranial and geraniol in essential oil reach their maximum levels during the flowering, while the content of neral, decreases during flowering. these observations indicate that the biosynthesis of geranyl acetate is dominant at the beginning of the vegetative period, but is superseded by biosynthesis of geranial and geraniol, from the early stage of flowering. it indicated that the optimal harvest time proved to be during the flowering stage, when the oil content is the highest and thus also the amount of the main terpenes is the highest (holm et al., 1988). conclusion dracocephalum moldavica from serbia contains geranial (29.6%), geranyl acetate (27.2%) and neral (19.4%) as the most abundant compounds. further investigations will be focused on the influence of weather conditions on essential oil composition, as well as on bioactive potential of this essential oil. acknowledgements. this investigation is supported by the ministry of education, science and technological development of the republic of serbia grant number tr31025. references aćimović, m., sikora, v., brdar-jokanović, m., kiprovski, b., popović, v., koren, a., puvača, n. 2019: dracocephalum moldovica: cultivation, chemical composition and biological activity. journal of agronomy, technology and engineering management, 2(1):153-167. ahl, h.a.h.s.a., sabra, a.s., gendy, a.n.g.e., aziz, e.e., tkachenko, k.g. 2015: changes in content and chemical composition of dracocephalum moldovica l. essential oil at different harvest dates. journal of medicinal plants studies, 3(2):6164. amirnia, r., manesh, m.f., danesh, y.r., najafi, s., seyyedi, n., ghiyasi, m. 2017: karyological study on teheran ecotype of moldavian balm (dracocephalum moldavica l.). journal of molecular biology and biotechnology, 1(1):29-31. aprotosoaie, a.c., mihai, c.t., vochita, g., rotinberg, p., trifan, a., luca, s.v., petreus, t., gille, e., miron, a. 2016: antigenotoxic and antioxidant activities of polyphenolic extract from european dracocephalum moldavica l. industrial crops and products, 79:248-257. aslanipour, b., heidari, r., farnad, n. 2017: phenolic combination and comparison of antioxidant activity in three different alcoholic extracts of dracocephalum moldavica l. turkish journal of agrifig. 2. a gc-fid chromatogram of d. moldavica essential oil biologica nyssana ● 10 (1) september 2019: 23-28 aćimović et al. ● the chemical composition of the essential oil of dracocephalum moldavica l. from vojvodina province (serbia) 27 culture food science and technology, 5(3):199206. aziz, e.e., hussein, m.s., wahba, h.e., razin, a.m. 2013: essential oil constituents of dracocephalum moldavica l. grown under salt stress and different sources of soil amendment. middle-east journal of scientific research, 16(5):706-713. chu, s.s., liu, s.l., liu, q.z., liu, z.l., du, s.s. 2011: composition and toxicity of chinese dracocephalum moldavica (labiatae) essential oil against two grain storage insects. journal of medicinal plants research, 5(18):4621-4626. ding, s.e., cixi, t., charles, p. 2015: synthesis and toxicity of chinese dracocephalum moldavica (labiatae) fundamental oil against two grain storage insect. african journal of insects, 2(2):77-81. ehsani, a., alizadeh, o., hashemi, m., afshari, a., aminzare, m. 2017: phytochemical, antioxidant and antibacterial properties of melissa officinalis and dracocephalum moldavica essential oil. veterinary research forum, 8(3):223-229. el-baky, h.h., el-bsroty, g.s. 2008: chemical and biological evaluation of the essential oil of egyptian moldavian balm (dracocephalum moldavica l.). international journal of integrative biology, 3(3):202208. eshan, k.a., bial, g., kiarash, a.p.r., mesut, u. 2014: gc/ms analysis of bioactive components of dracocephalum moldavica l., treated by boric acid doses. tarum bilimleri arastirma dergisi, 7(1):1921. fallah, s., rosttaei, m., lorigooini, z., surki, a.a. 2018: chemical compositions of essential oil and antioxidant activity of dragonhead (dracocephalum moldavica) in sole crop and dragonheadsoybean (glycine max) intercropping system under organic manure and chemical fertilizers. industrial crops and products, 115:158-165. golparvar, a.r., hadipanah, a., gheisari, m.m., khaliliazar, r. 2016: chemical constituents of essential oil of dracocephalum moldavica l. and dracocephalum kotschyi boiss. from iran. acta agriculturae slovenica, 107:25-31. hegazy, m.h., alzuaibr, f.m.a., mahmoud, a.a., mohamed, h.f.y., said-al ahl, h.a.h. 2016: the effects of zinc application and cutting on growth, herb, essential oil and flavonoids in three medicinal lamiaceae plants. european journal of medicinal plants, 12(3):1-12. holm, y., galambosi, b., hiltunen, r. 1988: variation of the main terpenes in dragonhead (dracocephalum moldavica l.) during growth. flavour and fragrance journal, 3:113-115. hussein, m.s., el-sherbeny, s.e., khalil, m.y., naguib, n.y., aly, s.m. 2006: growth characters and chemical constituents of dracocephalum moldavica l. plants in relation to compost fertilizer and planting distance. scientia horticulturae, 108:322331. janmohammadi, m., nouraein, m., sabaghnia, n. 2017: influence of different weed management technique on the growth and essential oils of dragonhead (dracocephalum moldavica l.). romanian biotechnological letters, 22(5):12950-12960. keikhaie, k.r., jahantigh, h.r., bagheri, r., kehkhaie, a.r. 2018: the effects of the ethanol extract of dracocephalum moldavica (badrashbu) against strains of antibiotic-resistant escherichia coli and klebsiella pneumoniae. international journal of infection, 5(1):e65295. kišgeci, j., adamović, d., janjatović, v., mijavec, a. 1982: introduction of medicinal and aromatic plant species in vojvodina province. bilten za hmelj, sirak i lekovito bilje, 39(1):5-24. kotyuk, l.a., rakhmetov, d.b. 2017: component composition of essential oil from dracocephalum moldavica l. grown in the ukrainian polissya. international scientific symposium “conservation of plant diversity”, 1-3, june, 2017, chisinau, moldova. maham, m., akbari, h., delazar, a. 2013: chemical composition and antinociceptive effect of the essential oil of dracocephalum moldavica l. pharmaceutical sciences, 18(4):187-192. maimaitiyiming, d., hu, g., aikemu, a., hui, s.w., zhang, x. 2014: the treatment of uygur medicine dracocephalum moldavica l on chronic mountain sickness rat model. pharmacognosy magazine, 10(40):477-482. martínez-vázquez, m., estrada-reyes, r., martínez-laurrabaquio, a., lópez-rubalcava, c., heinze, g. 2012: neuropharmacological study of dracocephalum moldavica l. (lamiaceae) in mice: sedative effect and chemical analysis of an aqueous extract. journal of ethnopharmacology, 141:908917. naderifar, m., sonboli, a., gholipour, a. 2015: pollen morphology of iranian dracocephalum l. (lamiaceae) and its taxonomic significance. bangladesh journal of plant taxonomy, 22(2):99-110. najafi, m., ghasemian, e., fathiazad, f., garjani, a. 2009: effects of total extract of dracocephalum biologica nyssana ● 10 (1) september 2019: 23-28 aćimović et al. ● the chemical composition of the essential oil of dracocephalum moldavica l. from vojvodina province (serbia) moldavica on ischemia/reperfusion induced arrhythmias and infarct size in the isolated rat heart. iranian journal of basic medical sciences, 11(4):229-235. omidbaigi, r., yavari, s., hassani, m.e., yavari, s. 2010: introduction of autoploidy in dragonhead (dracocephalum moldavica l.) by colchicines treatment. journal of fruit and ornamental plant research, 18(1):23-35. oniszczuk, t., wójtowicz, a., kocira, s., żelizko, k., oniszczuk, a., dib, a. 2017: the use of moldavian dragonhead bagasse waste in extruded products. ix international scientific symposium “farm machinery and processes management in sustainable agriculture”, lublin, poland. pak, z.h., abbaspour, h., karimi, n., fattahi, a. 2016: eco-friendly synthesis and antimicrobial activity of silver nanoparticles using dracocephalum moldavica seed extract. applied sciences, 6:69. sun, y., liu, t., dai, x., jiang, z., gao, z., zhang, m., wang, d., zheng, q. 2014: neuroprotective effect of dracocephalum moldavica l. total flavo28 biologica nyssana ● 10 (1) september 2019: 23-28 aćimović et al. ● the chemical composition of the essential oil of dracocephalum moldavica l. from vojvodina province (serbia) noids in transient cerebral ischemia in rats. annual research and review in biology, 4(12):1915-1926. weremczuk-jeżyna, i., grzegorczyk-karolak, i., frydrych, b., hnatuszko-konka, k., gerszberg, a., wysokińska, h. 2017: rosmarinic acid accumulation and antioxidant potential of dracocephalum moldavica l. cell suspension culture. notulae botanicae horti agrobotanici cluj-napoca, 45(1):215-219. wojtowicz, a., oniszczuk, a., oniszczuk, t., kocira, s., wojtunik, k., mitrus m., kocira a., widelski, j., skalicka-wozniak, k. 2017: application of moldavian dragonhead (dracocephalum moldavica l.) leaves addition as a functional component of nutritionally valuable corn snacks. journal of food science and technology, 54(10):3218-3229. zeng, c., jiang, w., yang, x., he, c., wang, w., xing, j. 2018: pretreatment with total flavonoid extract from dracocephalum moldavica l. attenuates ischemia reperfusion-induced apoptosis. scientific reports, 8:17491. anticancer compounds from medicinal plants biologica nyssana 4 (1-2)  december 2013: 65-69 nikolova et al.  comparison of flavonoid profiles of cultivated plants… 65 original article comparison of flavonoid profiles of cultivated plants of achillea asplenifolia, achillea collina and cultivar ”proa” milena nikolova 1* , antonina vitkova 1 , malina delcheva 1 , aglika edreva 2 , emiliya gesheva 2 1 department of applied botany, institute of biodiversity and ecosystem research 23, acad. g. bonchev, str., 1113 sofia, bulgaria 2 institute of plant physiology and genetics, 1113 sofia, bulgaria * e-mail: milena_n@bio.bas.bg abstract: nikolova, m., vitkova, a., delcheva, m., edreva, a., gesheva, e.: comparison of flavonoid profiles of cultivated plants of achillea asplenifolia, achillea collina and cultivar ”proa”. biologica nyssana, 4 (1-2), december 2013: 65-69. flavonoid aglycone profiles of cultivated plants of achillea collina, a. asplenifolia and cultivar "proa" presented with different origins were compared. the evaluation was undertaken to determine intraspecific variability of flavonoid profiles when the plants are grown in the uniform conditions. eight flavonoid aglycones were detected by thin layer chromatography and comparison with known compounds. highly methylated quercetagetin derivatives predominate in the exudates. the flavonoid profiles of a. colina consist mainly of quercetagetin 3,6,4'-trimethyl ether (centaureidin), 6-hydroxykaempferol 3,6,4'-trimethyl ether and quercetagetin 3,6,7,4'-tetramethyl ether (casticin) while the profiles of a. asplenifolia of quercetagetin 3,6,7,3',4'-pentamethyl ether (artemetin), casticin and scutellarein 6,4'-dimethyl ether. in the profiles of a. asplenifolia quercetagetin 3,6,4'-trimethyl ether was not found in the extracts of one of the two origins. the extracts of cultivar exhibited a less diversified flavonoid profile with main component centaureidin. no significant difference was found in the flavonoid profiles of the extracts of the both origins of the cultivar “proa” and a. collina. key words: chemotaxonomy, external flavonoids, intraspecific variability, tlc. introduction the species of achillea millefolium group are valuable plants with wide application in the phytotherapy and cosmetics. six achillea species of the group occur in bulgaria a. collina j. becker ex reichenb, a. millefolium l., a. setaceae waldst. et kit., a. asplenifolia vent., a. pannonica scheele, a. distans waldst. et kit. ex wild. as an extension of previous study of achillea genus in bulgaria (s a u k e l et al., 2003, v i t k o v a et al., 2005) in the present research we compare the profiles of surface flavonoid aglycones of a. collina, a. asplenifolia and cultivar "proa". the most widespread species is tetraploid a. collina (4x), which probably has a hybrid origin of a. setacea (2x) and a. asplenifolia (2x). it is spread from the sea level up to 1800 m altitude, grows both in xerothermal and the mesophilic places. recently diploid achillea asplenifolia was established for bulgaria in herbaceous habitats in antropogenic influenced phytocenoses from 500 to 600 m a.s.l. only two populations have been detected in the territory of the county – near the village of opizvet and village katina, sofia region. the plants of the last population characterized by a combination of characteristics of a. asplenifolia and. a. roseoalba (v i t k o v a et al., 2005). cultivar achillea collina 11 th sfses • 13-16 june 2013, vlasina lake 4 (1-2) • december 2013: 65-69 biologica nyssana 4 (1-2)  december 2013: 65-69 nikolova et al.  comparison of flavonoid profiles of cultivated plants… 66 "proa" represents a tetraploid, rich in proazulenes (k a s t n e r et al., 1992). the examined in the present study species have been objects of phytochemical analysis regarding different secondary metabolites – sesquiterpene lactones, flavonoids, essential oils (k o n a k c h i e v et al., 2005, t o d o r o v a et al., 2005, t r e n d a f i l o v a et al., 2006, t r e n d a f i l o v a et al., 2007). the species of achillea including a. asplenifolia and a. collina have been extensively studied for content of surface flavonoids (v a l a n t v e t s c h e r a & w o l l e n w e b e r 1988, i v a n c h e v a & s t a n c h e v a , 1996, v a l a n t v e t s c h e r a & w o l l e n w e b e r 1996, v a l a n t v e t s c h e r a & w o l l e n w e b e r , 2001a). external flavonoids attract attention except for their usefulness in taxonomic studies (w o l l e n w e b e r & s c h n e i d e r , 2000, v a l a n t v e t s c h e r a & w o l l e n w e b e r 2001b, v a l a n t v e t s c h e r a et al., 2003) but also with their ecological role (o n y i l a g h a & g r o t e w o l d , 2004). the aim of present study was to compare flavonoid aglycone profiles of cultivated plants of achillea collina, a. asplenifolia and cultivar "proa"presented with different origins. the evaluation was undertaken to determine intraspecific variability of flavonoid profiles when the plants are grown in the uniform conditions. material and methods plant material. the plant material included two wild species (achillea collina and a. asplenifolia) and cultivar “proa”. each species was presented by two populations. seeds of both species were collected from their natural habitats. the material of achillea collina were collected from vitosha mountain and village gorni lozen, sofia region and those of a. asplenifolia from village bezden, sofia region and village katina, sofia region. the seeds of cultivar proa have german and poland origin. seedlings were produced in a greenhouse and then transferred to the experimental field of the institute of plant physiology and genetics, near sofia, 570 m a.s.l., in 2009. all samples are collected at full flowering stage. preparation of acetone exudates. air-dried, but not ground (1g) plant material was briefly (2-3 min) rinsed with acetone at room temperature to dissolve the lipophilic components accumulated on the surface. the obtained acetone filtrate was then dried using a rotary-evaporator to give a crude extract which was suspended in meoh and then subjected on tlc. thin layer chromatographic analysis. the acetone exudates were screened for surface flavonoids by tlc analysis. three tlc sorbents and several mobile phases were used for the analysis of the flavonoid exudates. toluene-dioxan-acetic acid (95:25:4, v/v/v) was applied for the development of the aglycones mixture on silica gel plates kiselgel 60 f254 (10x20 cm, 0.2 mm layer). toluene-methylethylketone-methanol (60:25:15, v/v/v); toluene-petrol ether-methylethylketonemethanol (60:30:10:5, v/v/v/v) and toluenemethylethylketone-methanol (30:20:15, v/v/v) were used for dc-alufolien polyamid 11 f254 plates (10x20 cm, 0.15 mm layer). acetic acid–water (30:70, v/v) was used for cellulose plates dcalufolien cellulose 5552 (10x20 cm, 0.1 mm layer). chromatograms were viewed under uv light before and after spraying with ‘‘natural product reagent a”, 1% solution of diphenylboric acid 2-aminoethyl ester complex in methanol. the identification of the compounds was achieved by co-chromatography with authentic markers obtained from prof. eckhard wollenweber. results and discussion cultivated plants of a. collina, a. asplenifolia and cultivar "proa" were analyzed for their profiles of surface flavonoid aglycones. four individuals from each origin were studied. flavonoid profile of each individual was examined inflorescences and stem areas (leaves and stem) separately. total 48 extracts were comparatively analyzed for content of surface flavonoid aglycones. eight flavonoid aglycones were identified by thin layer chromatography and comparison with known compounds (fig. 1). polymethoxy derivatives of 6hydroxyflavonol and 6-hydroxyflavone in various combinations predominate in the exudates (table 1). the simple flavonoids (apigenin and luteolin) are rather accumulated in the inflorescences than in the exudates of stem areas. the extracts of cultivar "proa“ exhibited a less diversified flavonoid profile with the main flavonoid quercetagetin 3,6,4'trimethyl ether (5) in all studied parts. quercetagetin 3,6,7,4’-tetramethyl ether (6) was detected in the stem areas in trace. the main flavonoid of the extracts of a. colina was quercetagetin 3,6,4'-trimethyl ether (centaureidin) (5). this result indicates the closeness between cultivar proa and a. colina. additionally of the extracts of a. collina 6-hydroxykaempferol 3,6,4'-trimethyl ether (3), quercetagetin 3,6,7,3',4'pentamethyl ether (7) and scutellarein 6,4'-dimethyl ether (8) were detected. the flavonoid quercetagetin 3,6,7,4’-tetramethyl ether (6) that detected in trace amounts in the cultivar while in the extracts of a. collina is presented in large quantity. flavonoid biologica nyssana 4 (1-2)  december 2013: 65-69 nikolova et al.  comparison of flavonoid profiles of cultivated plants… 67 profiles of a. asplenifolia consisted predominantly of quercetagetin 3,6,7,4'-tetramethyl ether (casticin) (6), quercetagetin 3,6,7,3',4'-pentamethyl ether (artemetin) (7) and scutellarein 6,4'-dimethyl ether (8). the identified compounds are in accordance with the data previously reported for these species (v a l a n t -v e t s c h e t a & w o l l e n w e b e r , 1988, t r e n d a f i l o v a et al., 2007). ooh 2 1 r r r ho o 3 (1,2,8) r1=h, r2=oh apigenin (1) r1=oh, r2=oh luteolin (2) r1=h, r2=och3, r3=och3 scutellarein 6,4'-dimethyl ethers (8) r r rr 1 2 oh 3 4 o r 5 o (3-7) r1=h, r2=och3,r3=och3, r4=och3, r5=oh 6-hydroxykaempferol 3,6,4'-trimethyl ether (3) r1=oh, r2=och3,r3=och3, r4=och3, r5=och3 quercetagetin 3,6,7-trimethyl ether (4) r1=oh, r2=oh,r3=och3, r4=och3, r5=och3 quercetagetin 3,6,4'-trimethyl ether (5) r1=oh, r2=och3,r3=och3, r4=och3, r5= och3 quercetagetin 3,6,7,4'-tetramethyl ether (6) r1= och3, r2=och3,r3=och3, r4=och3, r5= och3 quercetagetin 3,6,7,3',4'-pentamethyl ether(7) fig. 1. structures of the identified flavonoid aglycones (1-8) individual variability of the flavonoid composition was observed regarding flavonoids that are presented in lesser amounts. no significant differences in the flavonoid profiles of the extracts of the both origins of cultivar proa. quantitative differences were observed in the accumulation of flavonoids in the profiles of a. collina and a. asplenifolia from different origins. furthermore in the profiles of a. asplenifolia from the population of village katina quercetagetin 3,6,4'-trimethyl ether was not detected as well as the flavonoid quercetagetin 3,6,7-trimethyl ether which was abundant in the same population was found in trace in the individuals of the population of village bezden. it is possible that these quality differences of flavonoid profiles are due to large quantitative differences that tlc analysis recognizes as qualitative. also must be taken into account that there are data that the population of v. katina showed combined features of a. asplenifolia and a. roseoalba (v i t k o v a et al., 2005). the results of flavonoid variability of a. asplenifolia were consistent with conclusions of dagnon et al., 2011. the authors reported on the basis of essential oils pattern high index of similarity (94.5%) for two populations of a. collina and less similar (82.3%) for a. asplenifolia populations. more data are needed to establish whether the plants of this population deserve an independent taxonomic rank. conclusion in conclusion the comparative analysis of flavonoid profiles of examined species showed that a. asplenifolia has more variable flavonoid composition in comparison to a. colina and cultivar "proa". the extracts of the last showed the most constant and simple flavonoid profile. the results obtained in this study indicate that surface flavonoids are a reliable taxonomic feature in the target species. references dagnon, s., konakchiev, a., vitkova, a., doncheva, m., edreva, a. 2011. application of pattern recognition methods to discrimination of achillea species of bulgarian origin. international conference “food science, engineering and technologies 2011”, 1415.10.2011, university of food technologies – plovdiv. 58: 403-408. ivancheva, s., stancheva, b. 1996: exudate flavonoid aglycones of achillea sp. sect. millefolium and sect. ptarmica. phytologia balcanica, 2: 102-105. kastner, u., glasl s., jurenitsch j., kubelka w., 1992: isolation and structure elucidation of the main proazulenes of the cultivar achillea collina “proa”. planta medica, 58:718 biologica nyssana 4 (1-2)  december 2013: 65-69 nikolova et al.  comparison of flavonoid profiles of cultivated plants… 68 table1. flavonoid aglycones in the samples of achillea species samples flavonoid aglycones 1 2 3 4 5 6 7 8 cultivar proa, origin germany inflorescences 1 x x xxx inflorescences 2 tr x xxx inflorescences 3 x x xxx inflorescences 4 x x xx stem areas 1 xxx tr stem areas 2 xxx tr stem areas 3 xxx tr stem areas 4 xx tr cultivar proa, origin poland inflorescences 1 xx xx xxx inflorescences 2 tr tr xxx inflorescences 3 xx xx xx inflorescences 4 xx xx xxx stem areas 1 tr xxx stem areas 2 tr xxx stem areas 3 x x xx stem areas 4 x x xxx a.collina, origin gorni lozen, sofia region inflorescences 1 xx x x x x inflorescences 2 xx xx x xx x inflorescences 3 x x x x tr inflorescences 4 xx xx x x tr tr stem areas 1 x x x stem areas 2 x xx x stem areas 3 tr tr x xx x stem areas 4 x xx x tr tr a.collina, origin vitosha mountain inflorescences 1 xx xx tr tr x x inflorescences 2 xx xx tr tr tr tr inflorescences 3 xx xx tr xx x x x inflorescences 4 xx xx tr xx tr tr tr stem areas 1 x tr xx x x x stem areas 2 x tr xxx x x x stem areas 3 x tr xx x x x stem areas 4 x x xxx x tr tr a. asplenifolia, origin bezden, sofia region inflorescences 1 tr x tr x tr xx xx inflorescences 2 tr x x tr xx xx inflorescences 3 x x xx xx xx inflorescences 4 xx xx tr xx tr tr stem areas 1 x x xx xx stem areas 2 tr x xx xx stem areas 3 x x tr tr stem areas 4 tr xx x xx xx a. asplenifolia, origin katina, sofia region inflorescences 1 xx xx xx x xx xx inflorescences 2 x x tr x xx xx inflorescences 3 x x x x xx xx inflorescences 4 xx xx tr x xx xx stem areas 1 xx x xx xx stem areas 2 x x xx xx stem areas 3 x x xx xx stem areas 4 x x xx xx legend: apigenin (1), luteolin (2), 6-hydroxykaempferol 3,6,4'-trimethyl ethers (3), quercetagetin 3,6,7-trimethyl ethers (4), quercetagetin 3,6,4'-trimethyl ethers (5), quercetagetin 3,6,7,4'-tetramethyl ethers (6) quercetagetin-3,6,7,3',4'-pentamethyl ethers (7), scutellarein 6,4'-dimethyl ethers (8), tr. – trace konakchiev, a., mikhova, b., todorova, m., najdenski, h., tzvetkova, i., vitkova, a., duddeck, h. 2005: composition of the esentiol oil of achillea asplenifolia vent. from bulgaria. jurnal of essential oilbearning plants, 8 (3):318-323 onyilagha, j.c., grotewold, e. 2004: the biology and structural distribution of surface flavonoids. biologica nyssana 4 (1-2)  december 2013: 65-69 nikolova et al.  comparison of flavonoid profiles of cultivated plants… 69 recent research developments in plant science, 2: 53-71. saukel, j., anchev, m., guo, y.p., vitkova, a., nedelcheva, a., goranova, v., konakchiev, a., lambrou, m., nejati, s., rauchensteiner f., erendorfer f. 2003: comments on the biosystematics of achillea (asteraceae – anthemideae) in bulgaria. phytologia balcanica, 9(3): 361-400; todorova, m., mikhova, b., trendafilova, a. vitkova, a., duddeck, h., anchev, m. 2006: sesquiterpene lactones from achillea asplenifolia. biochemical systematics and ecology, 34: 136-143. trendafilova, a., todorova, m., mikhova, b. & vitkova, a., duddeck, h. 2006: sesquiterpene lactones from achillea collina j.backer ex reichenb. phytochemistry, 67(8): 764-770 trendafilova, a., todorova, m., mikhova, b. duddeck h. 2007: flavonoids in flower heads of three achillea species belonging to achillea millefolium group. chemistry of natural compounds, 43(2): 212-213. valant-vetschera, k. m., wollenweber , e. 1988: leaf flavonoids of the achillea millefolium group part ii: distribution patterns of free aglycones in leaf exudates. biochemical systematics and ecology, 16: 605-614 valant-vetschera, k.m., wollenweber, e. 1996: comparative analysis of leaf exudate flavonoids in achillea sect. filipendulinae. biochemical systematics and ecology, 24: 435-446 valant-vetschera, k., wollenweber, e. 2001a: leaf flavonoids of the achillea millefolium group part ii: distribution patterns of free aglycones in leaf exudates. biochemical systematics and ecology, 16 (7-8): 605-614. valant-vetschera, k., wollenweber, e. 2001b: exudate flavonoid aglycones in the alpine species of achillea sect. ptarmica: chemosystematics of a. moschata and related species. biochemical systematics and ecology, 29: 149-159. valant-vetschera, k.m., fischer, r., wollenweber, e. 2003: exudate flavonoids in species of artemisia (asteraceae-anthemideae): new results and chemosystematic interpretation. biochemical systematics and ecology, 31: 487498 vitkova, a., anchev, m., goranova, v., todorova, m., konakchiev, a. 2005: achillea millefolium group (asteraceae) in bulgaria. pharmacie, 52(1-2): 60-63. wollenweber, e., schneider, h. 2000: lipophilic exudates of pteridaceae chemistry and chemotaxonomy. biochemical systematics and ecology, 28: 751-777. gc-ms metabolic profiling and free radical scavenging activity of micromeria dalmatica biologica nyssana 7 (2)  december 2016: 159-165 nikolova, m. et al.  gc-ms metabolic profiling and free radical… 159 original article received: 12 october 2016 revised: 01 november 2016 accepted: 29 november 2016 gc-ms metabolic profiling and free radical scavenging activity of micromeria dalmatica milena nikolova, ina aneva*, strahil berkov institute of biodiversity and ecosystem research, bulgarian academy of sciences, 1113 sofia, bulgaria * e-mail: ina.aneva@abv.bg abstract: nikolova, m., aneva, i., berkov, s.: gc-ms metabolic profiling and free radical scavenging activity of micromeria dalmatica. biologica nyssana, 7 (2), december 2016: 159-165. metabolite profile of acetone exudate and methanolic extract from aerial parts of micromeria dalmatica benth were analyzed by gc/ms. palmitic and linolenic acids, hentriacontane, amyrin, quercetagetin 3,6,7-trimethyl ether, sucrose were identified among the main components in the acetone exudate. in the methanolic extract more than 100 chromatographic peaks were detected including alkanes, fatty alcohols, fatty acids, organic acids, phenolic acids, saccharides, polyoles, phytosterols and other. most of the compounds were reported for the first time for the species. hydromethanolic extract of m. dalmatica was studied for in vitro antioxidant 2,2diphenyl-1-picrylhydrazyl (dpph) free radical-scavenging activity. the inhibitory concentration (ic50) of extract was calculated to be 21.36 µg/ml. the received result shows high antioxidant potential of micromeria dalmatica extract which provide scientific support for the use of the plant as herbs and spices. key words: alkanes, dpph, fatty acid, carbohydrate, flavonoid, lipid, phenolic apstrakt: nikolova, m., aneva, i., berkov, s.: gc-ms profilisanje metabolita i sposobnost hvatanja slobodnih radikala vrste micromeria dalmatica. biologica nyssana, 7 (2), decembar 2016: 159-165. profilisanje metabolita acetonskog eksudata i metanolnog ekstrakta iz nadzemnih delova vrste micromeria dalmatica benth. izvršeno je gc/ms analizom. palmitinska i linoleinska kiselina, hentriakontan, amirin, kvercetagetin 3,6,7-trimetil etar i saharoza identifikovane su kao glavne komponente u acetonskom eksudatu. u metanolnom ekstraktu detektovano je više od 100 hromatogramskih vrhova uključujući alkane, masne alkohole, masne kiseline, organske kiseline, fenolne kiseline, saharide, poliole, fitosterole i druge. većina jedinjenja pronađena je prvi put za ovu vrstu. antioksidantna aktivnost hidrometanolnog ekstrakta m. dalmatica proučavana je in vitro, analizom njegove sposobnosti hvatanja 2,2-difenil-1-pikrilhidrazil (dpph) slobodnih radikala. izračunata je inhibitorna koncentracija (ic50) ekstrakta od 21.36 μg/ml. dobijeni rezultati pokazuju visok antioksidantni potencijal ekstrakta micromeria dalmatica, koji daje naučnu potvrdu za upotrebu ove biljke kao medicinske biljke i začina. ključne reči: alkani, dpph, masne kiseline, ugljeni hidrati, flavonoidi, lipidi, fenoli 7 (2) • december 2016: 159-165 12th sfses • 16-19 june 2016, kopaonik mt doi: 10.5281/zenodo.200415 biologica nyssana 7 (2)  december 2016: 159-165 nikolova, m. et al.  gc-ms metabolic profiling and free radical… 160 introduction micromeria benth. (lamiaceae, nepetoideae) is widely distributed in the mediterranean region on rocky habitats. in bulgarian flora the genus is represented by four species that have relatively limited distribution but they are used widely as herbs and spices. micromeria species have been applied for the treatment of diseases of the cardiovascular system, digestive tract, respiratory system (asthma), skin inflammations (s a i d et al., 2002). the species are reported to have antimicrobial, antioxidant, gastroprotective, hepatoprotective, cytotoxic, antiinflammatory, anticholinesterase and analgesic activity (ö z t ü r k et al., 2011, v l a d i m i r k n e ž e v i ć et al., 2011, h e r k e n et al., 2012, s h e h a b & a b u -g h a r b i e h , 2012, a b u g h a r b i e h et al., 2013, b u k v i c k i et al., 2015). micromeria dalmatica benth. is balkan endemic distributed in bulgaria, greece, crit, crna gora (p e t r o v a & v l a d i m i r o v , 2010). populations of the species of bulgarian, serbian (montenegro) and greek origin have been well studied for essential oil composition (s l a v k o v s k a et al., 2005, k o s t a d i n o v a et al., 2007, k a r o u s o u et al., 2012). for the essential oil of the species has been reported to possess high antimicrobial efficacy against food spoilage microorganisms (b u k v i c k i et al., 2015). flavonoid profile of m. dalmadca is characterized by the presence of thymonin (5,6,4'-trihydroxy-7,8,3'trimethoxyflavone) as a major exudate flavonoid aglycone and acacetin (4'-methoxy-5,7dihydroxyflavone) derivatives as the most abundant flavonoid glycosides (t o m a s -b a r b e r a n et al., 1991, m a r i n et al., 2001). metabolite profiling is an analytical method for relative quantitation of a mixture of compounds from biological samples using chromatography and universal detection technologies (gc-ms, lcms). these techniques allow rapid identification of huge number of metabolites. metabolite profiling have been used in plant and fungi chemotaxonomy, for screening purposes, quality control and standardization purposes as well as for search of new natural products (f r i s v a d et al., 2008, f i s c h e d i c k et al., 2010, hill & r o e s s n e r , 2013, v r a n c h e v a et al., 2014, r o h l o f f , 2015). as a part of comprehensive survey of chemical composition and biological activity of bulgarian species of genus micromeria in the present study the metabolite profile and free radical scavenging activity of micromeria dalmatica benth were examined. material and methods plant material m. dalmatica was collected from vlahina mt, a part of west bulgarian frontier mts, in august, 2015. samples, upper flowering part of the plants were collected at full flowering phase without traces of soil, dust or parts of other plants. after that, samples were air-dried in place with good ventilation, away from direct sunlight. acetone exudate air-dried, but not ground (3g) plant material of aerial parts of m. dalmatica was briefly (2-3 min) rinsed with acetone at room temperature to dissolve the lipophilic components accumulated on the surface. the obtained acetone filtrate was then dried using a rotary-evaporator to give a crude extract. methanol extract 100 µg of plant material as well as internal standards of 50 µg of nonadecanoic acid, 50 µg of ribitol and 50 µg of 3,4 dichloro-4-hidroxy benzoic acid were placed in 2 ml ependorf tubes and extracted with 1 ml of meoh for 2 h at room temperature assisted by an ultrasonic bath for 15 min at 70 ˚c every 30 min, after that the sample was centrifuged. aliquot of 800 ul was transferred in other ependorf tubes and was added of 500 µl h2o and 500 µl of chcl3, vortexing for 2 min, and the mixture was centrifuged. the chloroform fraction was separated, evaporated and transmethylated with 2% of h2so4 in meoh at 60˚c for 18 h, than lipids were extracted with nhexane (2x500 µl) which was dried with anhydrous na2so4 and evaporated to obtain lipid fraction. an aliqote of 100 µl from the aqueous fraction was placed in glass vial and evaporated in a speed-vac to obtain polar fraction. the rest of aqueous fraction was hydrolyzed with 0.5 ml of 1n naoh for 18 h at 60˚c. after acidification to ph 1-2 with conc. hcl, the phenolic compounds were extracted with etoac (2x500 µl) which was dried with anhydrous na2so4 and evaporated to obtain phenolic fraction. the fractions of the methanolic extract as well as 20 mg of the acetone exudate were silylated with 50 μl of n,o-bis-(trimethylsilyl)trifluoro-acetamide (bstfa) in 50 μl of pyridine for 2 h at 50°c. metabolite analysis the gc–ms spectra were recorded on a termo scientific focus gc coupled with termo scientific dsq mass detector operating in ei mode at 70 ev. adb-5ms column (30 m x 0.25 mm x 0.25 m) was used. the temperature program was: 100-180 oc at 15 oc x min-1, 180-300 20 at 5 oc x min-1 and 10 min hold at 300 oc. the injector temperature was 250 oc. biologica nyssana 7 (2)  december 2016: 159-165 nikolova, m. et al.  gc-ms metabolic profiling and free radical… 161 table 1. identified compounds in the acetone exudate and methanolic extract of micromeria dalmatica by gc/ms compounds ri acetone exudate* methanol extract* lipid fraction polar fraction phenolic fraction alkanes (hydrocarbones) tridecane 1300 0,13 tetradecane 1400 3,42 pentadecane 1500 0,31 hexadecane 1600 2,84 heptadecane 1700 1,20 octadecane 1800 1,94 eicosane 2000 0,92 docosane 2200 0,37 tricosane 2300 0,11 pentacosane 2500 1,18 heptacosane 2700 4,5 2,10 octacosane 2800 0,89 3,45 nonacosane 2900 0,72 triacontane 3000 1,53 4,30 hentriacontane 3100 13,13 dotriacontane 3200 1,23 fatty alkohols 1-dodecanol 1560 39,96 4,23 1-tetradecanol 1757 0,64 0,69 1,52 1-hexadecanol 1955 3,55 10,61 1-octadec-9z-enol trimethylsilyl ether 2125 4,43 8,91 1-octadecanol 2152 0,13 1,72 5,72 1-eicosanol 2350 0,09 1-docosanol 2546 0,21 1-tetracosanol 2741 0,56 1-hexacosanol 2938 0,03 1-octacosanol 3133 0,04 0,17 fatty acids octanoic acid (caprylic acid, 8:0) 1575 0,17 tetradecanoic acid, methyl ester** (myristic acid, 14:0) 1721 0,62 hexadecanoic acid, methyl ether (palmitic acid, 16:0)** 1922 15,95 hexadecanoic acid (palmitic acid, 16:0) 2041 11,23 2,20 9,71 octadecadienoic acid 9,12(z,z),methyl ester (linoleic acid, 18:2)** 2090 4,16 octadecatrienoic acid 9,12,15-(z,z,z), methyl ether (α-linolenic acid 18:3) 2098 8,54 octadecanoic acid, methyl ester ** (stearic acid,18:0) 2124 3,52 octadecadienoic acid 9,12-(z,z) (linoleic acid, 18:2) 2204 2,42 octadecatrienoic acid 9,12,15-(z,z,z), methyl ether (α-linolenic acid 18:3) 2211 5,67 octadecanoic acid (stearic acid,18:0) 2238 0,10 octadecanoic acid, 2,3bis[(trimethylsilyl)oxy]propyl ester 2775 4,14 organic acids malic acid 1473 1,02 erythronic acid 1526 0,12 biologica nyssana 7 (2)  december 2016: 159-165 nikolova, m. et al.  gc-ms metabolic profiling and free radical… 162 table 1. continued glycerides glycerol 1258 2,88 1,03 15,25 hexadecanoylglycerol 2582 4,06 monooctadecanoylglycerol 2775 4,14 tocoferols α-tocopherol 3122 0,351 phytosterols β-sitosterol 3335 2,57 2,17 triternes amyrin 3415 13,58 polyoles erythritol 2,21 arabitol 1706 0,34 meso-erythritol 1711 5,87 7,35 myo-inositol 2080 1,22 13,18 mannose 1919 0,44 galactose 1926 0,14 2,3,4,5-tetrahydroxypentanoic acid1,4-lactone (arabinonic acid, 1,4lactone) 1624 1,53 monosaccharides arabinose 1683 2,39 monosaccharide 1 1792 7,78 fructose i 1800 1,58 6,69 fructose derivative 1805 0,83 3,99 fructose ii 1837 0,12 5,66 fructose iii 1855 0,04 6,47 glucose 1882 2,94 6,02 mannose 1918 7,16 monosaccharide 2 1969 2,95 12,40 monoaccharide 3 2539 0,25 disaccarides disacharide 1 2498 0,18 sucrose 2628 4,80 56,96 trisaccharides rafinnose 3420 0,68 amino acids 0,17 glycine 1121 0,21 flavonoid aglycones 2628 apigenin-4’-methyl ether 3040 0,17 quercetagein 3,6,7-trimethyl ether 3400 4,78 hydroxycinnamic acids quinic acid 1846 29,74 caffeic acid trans 2131 1,66 benzoates 3,5-bis(trimethylsiloxy)benzoic acid 1999 12,75 legend: *-data are expressed as percentage of the total peak area [%] **-due to the way of processing of the lipid fraction the fatty acid is detected as methyl ester the flow rate of carrier gas (helium) was 0.8 ml x min-1. the split ratio was 1:10 1 µl of the solution was injected. the metabolites were identified as tmsi derivatives comparing their mass spectra and kovats indexes (ri) with those of an on-line available plant specific database (the golm metabolome database; http://csbdb.mpimp-golm.mpg.de/csbdb/gmd/home/ gmd_sm.html), the nist 05 database and mass spectra available in the on-line lipid library (http://www. lipidlibrary.co.uk/ms/ms01/index.htm), nist 05 database and literature data as indicated in tab. 1. the measured mass spectra were deconvoluted by the automated mass spectral deconvolution and identification system (amdis), before comparison with the databases. then, the spectra of individual components were transferred to the nist mass spectral search program ms search http://www/ biologica nyssana 7 (2)  december 2016: 159-165 nikolova, m. et al.  gc-ms metabolic profiling and free radical… 163 2.0 where they were matched against reference compounds of the nist mass spectral library 2005 and the golm metabolome database. ri of the compounds were recorded with standard nhydrocarbon calibration mixture (c9-c36) (restek, cat no. 31614, supplied by teknokroma, spain) using amdis 3.6 software. free radical scavenging activity the stable 2,2-diphenyl-1-picryl hydrazyl radical (dpph) was used for determination of free radical scavenging activity of studied samples (stanojević et al., 2009). different concentrations (10, 20, 50 100 and 200 µg/ml in methanol) of m. dalmatica extract were added at an equal volume (2.5 ml) to methanol solution of dpph (0.3 mm, 1 ml). after 30 min at room temperature, the ab values were measured at 517 nm on a spectrophotometer (jenway 6320d) and converted into the percentage antioxidant activity using the following equation: dpph antiradical scavenging capacity (%) = [1–(absample–abblank)/abcontrol] × 100 methanol (1.0 ml) plus plant extract solution (2.5 ml) was used as a blank, while dpph solution plus methanol was used as a control.the ic50 values were calculated by software prizm 3.00. all of the experiments were carried out in triplicate. results and discussion metabolite analysis acetone exudate and methanolic extract from aerial parts of micromeria dalmatica were analyzed by gc/ms. in the acetone exudate of the sample 174 chemical peak signals were detected. a part of them was identified as representatives of aklanes, fatty alcohols, fatty acids, triterpenes, flavonoid aglycones, and other (tab. 1). eight n-aklanes were found and they ranged from 23 to 32 carbon numbers. among them the hentriacontane c31h64 was the most abundant (13,13%) that in accordance with previously reported data for micromeria cristata and m. juliana (r e d d y et al., 2000). seven fatty alcohols were identified as minor components in the acetone exudate. palmitic and linoleic acids were presented in the most significant relative amount. amyrin was detected as major triterpene in the acetone exudate. quercetagetin 3,6,7-trimethyl ether and apigenin 4'-methyl ether were identified as representatives of flavonoid aglycones. methyl derivatives of quercetagetin are rarely reported to lamiaceae family (g r a y e r et al., 2010), however this is the first time that quercetagetin derivative has been found in the genus micromeria. in the methanolic extract (lipid, polar and phenolic fractions) of m. dalmatica more than 100 chromatographic peaks were detected, including organic, fatty and phenolic acids, saccharides, polyoles, phytosterols, alkanes and other (tab. 1). unlike the acetone exudate in the lipid fraction of the methanolic extract alkanes with carbon number from 13 to 22 were found. alkanes with carbon number 30, 28 and 16 are dominant. six fatty alcohols were identified among them dodecanol was the most abundant (39,96%). seven fatty acids are found, the main being methyl ether of palmitic acid (16:0) followed by linolenic acid (18:3) and linoleic acid (18:2). this fatty acid composition confirms data previously reported for micromeria thymifolia and micromeria albanica (r i s t i ć et al., 1997). in the polar fraction the main carbohydrate was sucrose, approximately 50%. many monosaccharides and their derivatives were detected. among identified phenolic compounds quinic acid was the most abundant followed by 3,5-bis(trimethylsiloxy) benzoic acid. free radical scavenging activity the dpph assay has been widely used to evaluate the free radical scavenging effectiveness of various antioxidant substances and plant extracts (m a r i n o v a & b a t c h v a r o v , 2011). hydromethanolic extract of m. dalmatica was studied for in vitro antioxidant 2,2-diphenyl-1picrylhydrazyl (dpph) free radical-scavenging activity. the inhibitory concentration (ic50) of extract needed to inhibit 50% of the dpph radicals was calculated to be 21.36 µg/ml. the received value is similar to that obtained for micromeria croatica, m. juliana and m. thymifolia extracts and demonstrate considerable activity to scavenge dpph radicals (v l a d i m i r -k n e ž e v i ć et al., 2011). the commercial antioxidant butylated hydroxytoluene (bht) was used as positive control and its ic50 value was 12.6 µg/ml. conclusion the present study is first report on gc/ms based metabolite profiling of micromeria dalmatica. most of the compounds were reported here for the first time for the species. results obtained on the lipid composition appropriate complements the available literature data on other micromeria species and can be used in comparative chemotaxonomic analyzes. this is the first report on the occurrence of quercetagetin derivatives in genus micromeria. dpph assay shows high antioxidant potential of micromeria dalmatica extract. biologica nyssana 7 (2)  december 2016: 159-165 nikolova, m. et al.  gc-ms metabolic profiling and free radical… 164 acknowledgements. the authors are grateful to the financial support provided by program for career development of young scientists, bulgarian academy of sciences– grant № dfnp-67_a1. references abu-gharbieh, e., shehab, n.g., khan, s.a. 2013: anti-inflammatory and gastroprotective activities of the aqueous extract of micromeria fruticosa (l.) druce ssp serpyllifolia in mice. pakistan journal of pharmaceutical sciences, 26 (4): 799803. hill, c.b., roessner, u. 2013: metabolic profiling of plants by gc–ms. in: weckwerth w. & g. kahl, (eds.), the handbook of plant metabolomics, 1– 23, hoboken, nj: john wiley & sons. bukvicki, d., stojkovic, d., sokovic, m., nikolic ,m., vannini, l., montanari, c., marin, p.d. 2015: potential application of micromeria dalmatica essential oil as a protective agent in a food system. food science and technology, 63: 262-267. herken, e.n., celik, a., aslan, m., aydınlık, n. 2012: the constituents of essential oil: antimicrobial and antioxidant activity of micromeria congesta boiss. & hausskn. ex boiss. from east anatolia. journal of medicinal food, 15: 835–839. grayer, r. j., eckert, m.r., lever, a., veitch, n.c., kite, g.c., paton, a.j. 2010: distribution of exudate flavonoids in the genus plectranthus. biochemical systematics and ecology, 38: 335– 341. fischedick, j.t., hazekamp, a., erkelens, t., choi, y.h., verpoorte, r. 2010: metabolic fingerprinting of cannabis sativa l., cannabinoids and terpenoids for chemotaxonomic and drug standardization purposes. phytochemistry, 71: 2058–2073. frisvad, j.c., andersen, b., thrane, u. 2008: the use of secondary metabolite profiling in chemotaxonomy of filamentous fungi. mycological research, 112 (2): 231–240. karousou, r., hanlidou, e., lazari, d. 2012: essential oils of micromeria dalmatica benth., a balkan endemic species of section pseudomelissa. chemistry & biodiversity, 9: 27752783. kostadinova, e., alipieva, k., stefova, m., stafilov, t., antonova, d., evstatieva, l., matevski, v., kulevanova, s., stefkov, g., bankova, v. 2007: chemical composition of the essential oils of three micromeria species growing in macedonia and bulgaria. macedonian journal of chemistry and chemical engineering, 26 (1): 3-7. marin, p.d., grayer, r.j., veitch, n.c., kite, g.c., harborne, j.b. 2001: acacetin glycosides as taxonomic markers in calamintha and micromeria. phytochemistry, 58: 943-947. marinova, g., batchvarov, v. 2011: evaluation of the methods for determination of the free radical scavenging activity by dpph. bulgarian journal of agricultural science, 17: 11-24. öztürk, m., kolak, u., topçu, g., öksüz, s., choudhary, m. 2011: antioxidant and anticholinesterase active constituents from micromeria cilicica by radicalscavenging activity-guided fractionation. food chemistry, 126: 31–38. petrova, a., vladimirov, v. 2010: balkan endemics in the bulgarian flora. phytologia balcanica, 16 (2): 293 – 311. reddy, c.m., eglinton, t.i., palic, r., beniteznelson, b. c., stojanović, g., palić, i., djordjević, s., eglinton, g. 2000. even carbon number predominance of plant wax n-alkanes: a correction. organic geochemistry, 31, 331–336. ristić, n., palić, r., kitić, d, stojanović g. 1997: the fatty acids from some plants of micromeria genus. facta universitatis, 1 (4): 53-56. rohloff, j. 2015: analysis of phenolic and cyclic compounds in plants using derivatization techniques in combination with gc-ms-based metabolite profiling. molecules, 20: 3431-3462. said, o., khalil, k., fulder, s., azaizeh, h. 2002: ethnopharmacological survey of medicinal herbs in israel, the golan heights and the west bank region. journal of ethnopharmacology, 83: 251– 65. slavkovska, v., couladis, m., bojović, s., tzakou, o., pavlović, m., lakušić, b., jančić, r. 2005: essential oil and its systematic significance in species of micromeria bentham from serbia & montenegro. plant systematics and evolution, 255: 1-15. shehab, n.g., abu-gharbieh, e. 2012: constituents and biological activity of the essential oil and the aqueous extract of micromeria fruticosa (l.) druce subsp. serpyllifolia. pakistan journal of pharmaceutical sciences, 25: 687–92. stanojević, l., stanković, m., nikolić, v., nikolić, l., ristić, d., čanadanovic-brunet, j., tumbas, v. 2009: antioxidant activity and total phenolic and flavonoid contents of hieracium pilosella l. extracts. sensors, 9: 5702-5714. tomas-barberan, f.a., gil, m.i., marin, p.d., tomas-lorente, f. 1991: flavonoids from some yugoslavian micromeria species: chemotaxonomical aspects. biochemical systematics and ecology, 19: 697-698. biologica nyssana 7 (2)  december 2016: 159-165 nikolova, m. et al.  gc-ms metabolic profiling and free radical… 165 vladimir-knežević, s., blažeković, b., bival štefan, m., alegro, a., kőszegi, t., petrik j. 2011: antioxidant activities and polyphenolic contents of three selected micromeria species from croatia. molecules, 16: 1454–1470. vrancheva, r., ivanov, i., aneva, i., dincheva, i., badjakov, i., pavlov, a. 2014: gs-ms based metabolite profiling of five bulgarian fumaria species. journal of bioscience and biotechnology, 3(3): 195-201. morho-anatomical differentiation of the populations of daphne biologica nyssana 7 (1)  september 2016: 1-9 jušković, m. et al.  morpho-anatomical differentiation of the populations… 1 original article received: 28 june 2016 revised: 24 july 2016 accepted: 01 september 2016 morpho-anatomical differentiation of the populations of daphne cneorum l. (thymelaeaceae) from serbia marina jusković1*, perica vasiljević1, ana savić1, dragana jenačković1, branka stevanović2 1university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 nis, serbia 2 university of belgrade, faculty of biology, institute of botany and botanical garden “jevremovac”, takovska 43, 11000 belgrade, serbia * e-mail: marinaju@pmf.ni.ac.rs abstract: jušković, m., vasiljević, p., savić, a., jenačković, d., stevanović, b.: morho-anatomical differentiation of the populations of daphne cneorum l. (thymelaeaceae) from serbia. biologica nyssana, 7 (1), september 2016: 1-9. in this study was analyzed inter-population diffrentiation of the species daphne cneorum l. on the basis of morphoanatomical variability of its three different, geographically distant populations, two from eastern serbia and one from western serbia. the analyzed populations are situated in wide range of altitudes, as well as different geological substrates. the analysis of variance included 20 quantitative characters related to the leaf and stem anatomy and morphology. the results of morphoanatomical studies have shown the presence of general adaptive characteristics of the xeromorphic type. multivariate analysis have shown a clear distinction between the carbonate populations of d. cneorum from eastern serbia (mt. rtanj and mt. suva planina) and the serpentine populations from western serbia (mt. zlatibor). key words: daphne cneorum, anatomy, morphology, differentiation, serbia apstrakt: jušković, m., vasiljević, p., savić, a., jenačković, d., stevanović, b.: morfo-anatomska diferencijacija populacija vrste daphne cneorum l. (thymelaeaceae) iz srbije. biologica nyssana, 7 (1), septembar 2016: 1-9. u ovom radu analizirana je interpopulaciona diferencijacija vrste daphne cneorum na osnovu varijabilnosti morfoloških i anatomskih karakteristika listova i stabla, tri prostorno udaljene populacije, dve iz istočne i jedne iz zapadne srbije. analizirane populacije smeštene su u širokom rasponu nadmorskih visina i pokazuju preferencije prema različitim geološkim podlogama. statističkom analizom je obuhvaćeno 20 kvantitativnih karaktera anatomije i morfologije lista i stabla. analizom morfo-anatomskih karakteristika listova i stabla i staništnih uslova ustanovljeno je da d. cneorum pripada adaptivnom tipu kserofita. multivarijantne analize su pokazale da se analizirane populacije na osnovu ispitivanih karaktera klasifikuju u dve grupe. veća međusobna sličnost je prisutna između jedinki iz populacija koje rastu na karbonatu i na većoj nadmorskoj visini, u odnosu na jedinke populacije sa serpentinske podloge i niže nadmorske visine. key words: daphne cneorum, morfologija, anatomija, diferencijacija, srbija 7 (1) • september 2016: 1-9 doi: 10.5281/zenodo.158966 biologica nyssana 7 (1)  september 2016: 1-9 jušković, m. et al.  morpho-anatomical differentiation of the populations… 2 introduction daphne cneorum l. is one of seven species of the genus daphne l. native to serbia (b l e č i ć , 1972). it belongs to section daphnanthes c.a. mayer, subsection cneorum, together with d. arbuscula, d. juliae, d. petraea, d. striata (k e i s s l e r , 1898). species d. cneorum distributed in west, central and east europe, mediterranean region, south–west asia (w e b b & f e r g u s o n , 1968; m e u s e l et al., 1978). in serbia, d. cneorum is taxon with disjunct disribution and it is limited to the mountainous areas (b l e č i ć , 1972) (fig. 1). the species inhabits mountainous areas, between 700 and 1800 m a.s.l., prefers serpentinite substrate, but it was also recorded on limestone and on acid siliceous soil, usually on stony sites in the zone of seslerion rigidae, seslerio-festucion, euvaccinio-piceenion, orno-ericenion serpentinicum, poion violaceae, festucion rupicolae, ramondion nathaliae alliances (j u š k o v i ć , 2012). d. cneorum is an evergreen fruticose reptant chamaephytes (fo semp mi ch frut rept). the species d. cneroum has become one of the most popular perennial flowering shrubs, because of their desirable horticultural characteristics (b r i c k e l l & w h i t e , 1978; h a l d a , 2001). comparative morpho-anatomical studies have involved three different, geographically distant populations, two from eastern serbia and one from western serbia. the studied populations grew under different climate conditions. the habitats of populations from the area of western serbia (mt. zlatibor) are influenced by humid temperatecontinental climate (type 2.1 s t e v a n o v i ć & s t e v a n o v i ć , 1995; vi 2b w a l t e r & l e i t h , 1964), with mountain influences, or the transitional variant of temperate-continental and mountain climate of middle european type (2.1/4.1 s t e v a n o v i ć & s t e v a n o v i ć , 1995; vi 2b/x 1 w a l t e r & l e i t h , 1964). the populations that inhabit the eastern parts of serbia (mt. suva planina, mt. rtanj) are inflenced by the continental climate (2.2/4.2) this region is dominated by the sub-type of the semi-arid moderately-continental climate, also known as sub-continental climate (type 2.2 sensu s t e v a n o v i ć & s t e v a n o v i ć , 1995; vi 3 sensu w a l t e r & l e i t h , 1964). studies so far on the anatomy of thymelaeaceae and genus daphne have been limited. m e t c a l f e & c h a l k (1950) give a general description of the anatomical structure of vegetative organs from the family thymelaeaceae as well as the genus daphne. the species of genus daphne contains significant variation in native habitats, morphology and use (b r i c k e l l & w h i t e , 1978). the aim of this study was to determine variability of morpho-anatomical characteristics of the leaves and stems of the plants of species d. cneorum, as well as possible trends of differentiations among populations in serbia. material and methods the species of daphne cneorum were collected from western (mt. zlatibor -serpentinite) and from eastern serbia (mt. suva planina, mt. rtanj limestone), during the flowering season in 2008 (tab. 1). the collected plant material was either placed in the herbarium or fixed in 50% ethanol. a voucher specimen was deposited in the herbarium moesiacum niš, department of biology and ecology, faculty of sciencesand mathematics, university of niš, serbia (hmn), (tab. 1). fig. 1. distribution of the species d. cneorum in serbia. biologica nyssana 7 (1)  september 2016: 1-9 jušković, m. et al.  morpho-anatomical differentiation of the populations… 3 soil analysis soil samples were collected from 3 different sites of each of the three population localities. each analyzed sample contained 300 g of dried, sieved soil. the ph value was determined in a soil suspension with potassium chloride and in one with water (10g : 25cm3), using a potentiometer. the caco3 content was determined by the volumetric method, using the scheibler’s calcimeter. the humus content was determined after turin’s method, while that of nitrogen was calculated on the basis of humus values. the available phosphorus was measured by the method of a l l e n (1940) using a spectrophotometer, whereas the available potassium was determined according to the method of allen on a flame photometer. morpho-anatomical analysis the total of 20 quantitative characteristics subjected to statistical analysis was grouped into two categories: i morphometric characteristics: ii meristic characteristics. anatomical sections of leaves and stems was performed on permanent and temporary slides, prepared by the standard histological method for light microscopy (r u z i n , 1999). all measurements were done with on the microscope (leica dm 250-leica dfc490, leica qwin standard (leica microsystem, germany). leaf epidermis, trichomes and stomata were analyzed by a scanning electron microscope (sem, jeol 5300). statistical analysis the obtained results of measurements were analyzed in the statistical package systat 12 (systat software inc. 2007). for each quantitative character was performed basic descriptive statistics: mean, standard deviation, minimum and maximum values. one-way analysis of variance was used in order to calculate statistical significance for analyzed morpho-anatomical characters. principal component analysis (pca), canonical discriminant analysis (cda) and clustering method based on mahalanobis’ distances were used to determine the variability structure and level of importance of the anatomical differentiation of investigated populations. results soil analysis d. cneorum inhabits carbonate, silicate, as well as serpentine substrates (b l e č i ć , 1972). variations in edaphic features, the thickness of the soil, texture, ph values, concentration of minerals, are factors which table 1. ecological characteristics of the habitats and number of specimens of the analyzed populations d. cneorum. locality population biogeography coordinate habitat substratum altitude (m a.s.l.) voucher mt. rtanj, šiljak rtanj moesian province (ce) 42. 39 n, 19. 39 e subalpine pastures limestone 1450-1570 hmn-5515 mt. suva planina, devojački grob trem suva planina moesian province (ce) 43. 11 n, 22. 10 e rocky grounds limestone 16471765 hmn-5516 mt. zlatibor, šainovci zlatibor illirian province (ce) 43. 40 n, 19. 41 e pastures serpentinite 1000 hmn-5514 table 2. the results of physico-chemical analyses of the soil samples. the table contains detail informations on ph values, hardness and content of nutrients in soils on the studied habitats. locality ph caco3 (%) humus (%) n (%) p2o5 (mg/g) k2o (mg/g) mt. rtanj 6.65 15.81 11.56 0.59 12.4 >40 mt. suva planina 6.3 0.72 10.26 0.51 3.6 >40 mt. zlatibor 5.99 1.42 7.79 0.39 1.2 20.5 biologica nyssana 7 (1)  september 2016: 1-9 jušković, m. et al.  morpho-anatomical differentiation of the populations… 4 influence the distribution of plants (s t e v a n o v i ć & j a n k o v i ć , 2001). the results of soil analysis of the three different sites inhabited by d. cneorum are presented in tab. 2. the ph of soil varied from slightly acidic to neutral. soil is with low content of carbonates on mts. zlatibor and suva planina, and with high content on mt. rtanj. the greatest values of organic matter were recorded in the subalpine fig. 3. epidermal impressions of d. cneorum leaves: (a, d) mt. rtanj, (b, e) mt. suva planina, (c, f) mt. zlatibor: a-c adaxial surface, with various shapes of sinuous walls epidermal cells; d-f abaxial epidermal cells with straight or slightly sinuous anticlinal walls and anomocytic stomata (s). pastures soil in the mt. rtanj. chemical analysis shows that there is a sufficient amount of nitrogen. the amount of phosphorus in the soils from the studied localities varied from low to sufficient. concentration of potassium is sufficient. leaf shape and anatomy the leaves of all the analyzed plants species d. cneorum are evergreen, simple, narrow, oblanceolate, dark green, with a margin entire and densely arranged towards the end of the twigs. they are with a tapering base and broadly wedge-shaped apex, ending in a minute bristle-like tip. indumentum is absent from leaves (fig. 2). the leaf surface area of the analyzed plants from all localities varied, ranging between 28 mm2 and 67 mm2, whereas the leaf length was 12 and 22 mm, and the leaf width ranged between 2,5 mm and 4,6 mm (tab. 3). such microphyllous leaves reduce transpiration rate and represent an important adaptive response of the plants to the conditions of environmental water deficit (s t e v a n o v i ć & j a n k o v i ć , 2001). the highest leaf surface values were from mt. zlatibor, while the lowest were in plants from mt. suva planina (fig. 2.; tab. 3). fig. 4. cross section of the leaves of plants. (a) mt. rtanj, (b) mt. suva planina, (c) mt. zlatibor, (d) details of the transverse section of the upper leaf epidermis, (e) transverse section through the midrib, (f) details of the transverse section of the lower leaf epidermis. cut cuticule, mc mucilaginous cells, pp palisade parenchyma, vb vascular bundles, sp spongy parenchyma, s stomata, xy xylem, ph phloem. fig. 2. the leaf shapes of d. cneorum: (a) mt. rtanj, (b) mt. suva planina, (c) mt. zlatibor. biologica nyssana 7 (1)  september 2016: 1-9 jušković, m. et al.  morpho-anatomical differentiation of the populations… 5 the anticlinal cell walls of adaxial epidermis of leaves are wavy with shallow or deep amplitudes, while the anticlinal cell walls of abaxial epidermis are straight to slightly undulate (fig. 3). the undulating anticlinal walls represent a mesomorphic, while straight ones a xeromorphic characteristic (f a h n & c u t e r , 1992). the leaves are bifacial or of dorsiventral symmetry (fig. 4). their thickness in all the populations studied ranged between from 238 µm to 395 µm (tab. 3). the epidermis of the leaf is singlelayered, the adaxial epidermis is thicker than the abaxial epidermis and with well developed cuticule (fig. 4). table 3. results of one-way analysis of variance for comparing means of morphometric and meristic characteristics in three populations of d. cneorum. (* p<0.05). principal component analysis (pca) of measured parameters of the analyzed populations of d. cneorum. the first three principal components accounted for 63.28% of the variance. population mt. rtanj mt. suva planina mt. zlatibor anova p-value pca 1 pca 2 pca 3 character mean ± sd mean ± sd mean ± sd leaf thickness (µm) 324.81±45.33 316.24±27.82 319.69±32.43 0.65 0.50 0.75 -0.12 height of adaxial epidermal cells (µm) 35.27±4.66 34.98±5.49 33.70±5.11 0.45 0.44 0.74 -0.14 thickness of palisade tissue (µm) 132.75±25.19 126.18±21.28 122.33±19.23 0.19 0.39 0.69 0.03 thickness of spongy tissue (µm) 121.06±19.17 109.81±17.92 114.43±16.46 0.06 -0.39 0.22 0.12 height of abaxial epidermal cells (µm) 23.65±2.66 22.34±2.92 21.44±2.30 0.01* -0.44 0.08 0.38 surface area of adaxial epidermal cells (µm2) 2105.49±295.47 2003.01±234.59 2375.47±345.88 0.00* 0.37 0.16 -0.02 surface area of abaxial epidermal cells (µm2) 932.23±166.27 843.79±126.34 860.07±112.93 0.03* 0.47 -0.07 0.26 surface area of abaxial stomata (µm2) 1039.11±87.85 977.76±102.26 1079.10±86.38 0.00* 0.08 -0.13 0.10 leaf length (mm) 15.43±1.96 14.53±1.47 18.61±1.70 0.00* -0.07 0.44 0.37 distance between point of largest leaf width and leaf top (mm) 3.71±0.57 3.88±0.56 3.39±0.59 0.51 0.83 -0.37 0.16 largest width of leaf (mm) 3.45±0.46 3.51±0.61 3.62±0.35 0.38 0.81 -0.35 0.18 leaf surface area (mm) 43.67±9.31 39.28±7.93 51.01±7.92 0.00* 0.64 -0.22 -0.07 length of leaf nervature mm/mm2 9610.52±1162.67 9030.30±1135.57 9204.97±822.88 0.10 0.05 -0.20 -0.54 stem diameter (µm) 1405.49±202.91 1420.04±122.69 1396.40±130.73 0.84 0.19 0.24 -0.28 stem peridermis thickness (µm) 27.73±2.86 24.37±1.83 24.56±2.04 0.00* -0.04 0.36 0.44 stem cortex thickness (µm) 257.16±25.75 284.97±34.46 264.12±31.12 0.00* -0.05 0.06 -0.71 number of palisade layers 3.43±0.50 3.33±0.61 3.50±0.68 0.56 0.22 -0.31 0.41 number of abaxial stomata 131.28±18.57 163.18±23.41 163.12±22.59 0.00* 0.15 -0.41 -0.13 biologica nyssana 7 (1)  september 2016: 1-9 jušković, m. et al.  morpho-anatomical differentiation of the populations… 6 the epidermal cell walls are mucilaginous. this type of epidermis is characteristic of the family thymelaeaceae, as well as the genus daphne (m e t c a l f e & c h a l k , 1950) (fig. 4 a-d). mucilaginous epidermal cells are usually present mainly in the upper epidermis. thickened walls of the epidermal cells, well-developed cuticle and mucilaginous cells can prevent rapid water loss and are considered an ecological characteristic for plant species of sunny and dry sites (b r e d e n k a m p , 1999; b r e d e n k a m p & v a n w y k , 2001). those adaptive strategies limit water loss during periods of drought and such structural adaptations are of major importance to survival in the xerothermic habitat conditions. leaves of all studied populations are hypostomatic (fig. 4, 5). the number of stomata in leaves ranges from 95 to 223 per mm2 (tab. 3). the fig. 5. scanning electron micrographs showing epidermis of d. cneorum leaves, stomata on abaxial leaf surface. (a) mt. rtanj; (b) mt. suva planina (sem). fig. 6. cross section of the one-year old stem of d. cneorum (in primary state of growth) from mt. suva planina. (a) stem with leaves, (b) the cross section stem, (c, d) details of the transverse section of the stem. e epidermis, c stem cortex, p pith, sc sclerenchyma cells, vc vascular cylinder, xy xylem, ph phloem. biologica nyssana 7 (1)  september 2016: 1-9 jušković, m. et al.  morpho-anatomical differentiation of the populations… 7 stomata complexes are anomocytic, i.e., real subsidiary cells are absent (fig. 3). there is high wrinkled cuticule around the stomata (fig. 5). the same type of of stomata were found and described in other species of the genus daphne (j u š k o v i ć , 2010; j u š k o v i ć , 2012). the mesophyll is differentiated into spongy and palisade tissues. the palisade tissue is multilayered, composed of typically elongated, densely arranged cells, with very small intercellular spaces (fig. 4). incresead toughness of the leaves provides a considerably more efficient water transport and photosythetical light utilization. the cells of spongy parenchyma are usually irregularly shaped. the structure of leaves is involved in the general physiology of plant activity, it clearly explains the morphological adaptation of plants to specific environmental conditions (s t e v a n o v i ć & j a n k o v i ć , 2001). according to f a h n & c u t l e r (1992) reduced leaf size, increased thickness, deeply sunken stomata, and palisade developed at the expense of spongy mesophyll are common features of plants grown in xeric environments. fig. 7. the result of canonical discriminant analysis (cda). table 4. summary of canonical discriminant analysis. wilks’ lambda is the ratio of within-groups sums of squares to the total sums of squares. the f value for a variable indicates its statistical significance in the discrimination between groups. (* p<0.05) wilks's lambda lambda 0.09 df (18. 2. 87) approx. f-ratio 8.70 df (36. 140) p-value 0.00 variable f-to-remove tolerance leaf thickness 0.81 0.14 thickness of palisade tissue 0.34 0.22 thickness of spongy tissue 3.25 0.34 height of adaxial epidermal cells 0.31 0.80 height of abaxial epidermal cells 2.38 0.80 surface area of adaxial epidermal cells 2.15 0.84 surface area of abaxial epidermal cells 7.46 0.70 surface area of abaxial stomata 1.17 0.78 number of palisade layers 0.12 0.85 number of abaxial stomata 9.08 0.78 length of leaf nervature 1.41 0.73 leaf surface area 6.90 0.13 leaf length 27.38 0.18 largest width of leaf 2.03 0.35 distance between point of largest leaf width and leaf top 0.88 0.75 stem diameter 0.73 0.82 stem peridermis thickness 7.65 0.89 stem cortex thickness 2.66 0.81 biologica nyssana 7 (1)  september 2016: 1-9 jušković, m. et al.  morpho-anatomical differentiation of the populations… 8 stem shape and anatomy d. cneorum is a low-growing, evergreen shrub, with a great number of long, slender, downy branches and reddish brown bark (fig. 6). the cross-sections of one year-old stems ranged from rounded to elliptical in shape (fig. 6). stem diameter of analysis plants ranged between 1396 μm and 1420 μm (tab. 3). the epidermis is unilayered and covered by a conspicuous cuticle with unicellular trichomes (fig. 6a). underneath the epidermis, the collenchyma consists of several cell layers (fig. 6c). the cortical parenchyma consists of cells with a circular or polygonal shape and slightly thickened walls. the vascular tissue is well developed and forms a ring around pith. at the outer side of phloem, the sclerenchyma cells also form rings (fig. 6d). the parenchymatous pith has circular or polygonal shaped cells with thin walls. the correlation coefficient of the cortex and the whole stem diameter ranges from od 0,366 (mt. rtanj), 0,378 (mt. zlatibor) to 0,401 (mt. suva planina), which was within the usual values found in xeromorphic stems (f a h n & c u t l e r , 1992). these characteristics are in agreement with the data obtained from other studied daphne species (j u š k o v i ć , 2010; j u š k o v i ć , 2012). finally, leaf and stem anatomical features are closely linked to the habitat of species d. cneorum, indicating that they represent ecologically important adaptations. on the other hand, phenotypic plasticity among populations of the same plant species is one of the important mechanisms for their adaptation to heterogeneous habitats, and it is one of the major way by which plants are able to cope with environmental variability (j u š k o v i ć et al., 2010; l a k u š i ć et al., 2010; j a k o v l j e v i ć et al., 2013; g r a t a n i , 2014). there is statistical significant differences among populations according to some morpho-anatomical features: height of abaxial epidermal cells, surface area of adaxial epidermal cells, surface area of abaxial epidermal cells, surface area of abaxial stomata, leaf length, leaf surface area, stem peridermis thickness, stem cortex thickness and number of abaxial stomata among populations (tab. 3, 4). the results of pca analysis showed that the three principal components explain 63.28% of the total variability (tab. 3, 4). the largest contribution to the formation of variability provides the first principal component with 32.78%, followed second with 17.29% and the third with a 13.51% variation. the greatest significance in the formation of variability have characters that are related to leaf shape (leaf surface area, largest width of leaf, distance between point of largest leaf width and leaf top) and leaf anatomy (leaf thickness, height of adaxial epidermal cells, thickness of palisade tissue). stem cortex thickness is present in the structure of the variability in the third principal component. the results of canonical discriminant analysis of the population of the species d. cneorum show the existence of separation among the studied populations (fig. 7). population with mt. zlatibor is on the positive side of the first axis and is clearly separated from the population with mt.rtnja and mt. suva planina. less discrimination is present in populations from eastern serbia, which are the negative side of the first axis. the most important characters (tab. 4) in total discrimination among populations are characters related to the leaf shape, leaf epidermis and stem peridermis thickness. multivariate analysis (pca and cda) and cluster analysis (fig. 8) have shown that studied populations can be classified into two groups. one group includes populations (mt. rtanj and mt. suva planina) that grow on carbonate substrate, and at a higher altitude (1500-1700 m a.s.l.), while the second group includes plants from the population (mt. zlatibor), growing on serpetinite substrate and the lower altitude (1000 m a.s.l.). conclusion the investigated populations of the species daphne cneorum have shown certain differences in structure. the results of morpho-anatomical studies of the leaves and stems, and conditions on habitats, have shown the presence of general adaptive fig. 8. mahalnobius distances among three populations of daphne cneorum. biologica nyssana 7 (1)  september 2016: 1-9 jušković, m. et al.  morpho-anatomical differentiation of the populations… 9 characteristics of the xeromorphic type. the study provides useful information on the morphoanatomical and ecological characteristics of species d. cneorum. acknowledgements. the study was supported by the ministry of education, science and technological development of the republic of serbia (grant no. 173030). references blečić, v. 1972. daphne. in: josifović, m. (ed.), flora sr srbije 3: 570-578, srpska akademija nauka i umetnosti, beograd. bredenkamp, l.c. 1999: structure of mucilaginous epidermal cell walls in passerina (thymelaeaceae). botanical journal of the linnean society, 129: 223-238. bredenkamp, l.c., van wyk e.a. 2000: the epidermis in passerina (thymelaeaceae): structure, functionand taxonomic significance. bothalia, 3: 69-86. brickell, c.d., mathew, b. 1978: daphne, the genus in the wild and in cultivation. the alpine garden society, lye end link, st. john’s, woking gu21 1sw, surrey. 188 p. fahn, a., cutler, f.d. 1992: xerophytes. berlin, stuttgart, gebrüder borntraeger. gratani, l. 2014: plant phenotypic plasticity in response to environmental factors. advances in botany. halda, j.j. 2001: the genus daphne. eva kucerova sen dobre publikaca, prague, czeck republic. jakovljević, k., šinžar-sekulić, j., vukojičić, s., kuzmanović, n., lakušić, d. 2013: leaf anatomy of carex humilis (cyperaceae) from central and south eastern europe. botanica serbica, 37 (1): 3-11. jušković, m. 2012: morphological variability and ecological differentiation in populations of species from genus daphne l. (thymelaeaceae) in serbia and montenegro. phd thesis. biološki fakultet. beograd. jušković, m., vasiljević, p., ranđelović, v., stevanović, v., stevanović, b. 2010: comparative analysis of populations of the balkan endemic species daphne malyana blečić (thymeleaceae). archives of biological sciences, 62 (4): 1151-1162. keissler, k. 1898: die arten der gattung, daphne aus der section daphnanthes. botanische jahrbucher, 25: 29-124. lakušić, b., stevanović, b., jančić, r., lakušić, d. 2010: habitat-related adaptations in morphology and anatomy of teucrium (lamiaceae) species from the balkan peninsula (serbia and montenegro). flora, 205: 633-646. metcalfe, c.r., chalk, l. 1950: anatomy of dicotiledons. vol. 2, clarendon press, oxford, uk. meusel, h., jäger, e., weinert, e. 1965: vergleichende chorologie der zentraleuropäischen flora. 2. karten. gustav fischer, jena. ruzin, s.e. 1999: plant microtechnique and microscopy. oxford university press, new york. 322 p. stevanović, b., janković, m. 2001: ekologija biljaka sa osnovama fiziološke ekologije biljaka/ ecology of plants with the basics of physiological ecology of plants. belgrade: nnk international. stevanović, v., stevanović, b. 1995: osnovni klimatski, geološki i pedološki činioci biodiverziteta kopnenih ekosistema jugoslavije. in: stevanović, v. & vasić, v. (eds.), biodiverzitet jugoslavije sa pregledom vrsta od međunarodnog značaja, pp. 75-95. ecolibri, beograd, biološki fakultet, beograd. systat version 12 2007: systat software, inc, san jose california usa. walter, h., leith, h. 1964: klimadiagrammweltatlas. 2. liefernug, jena. webb, d. a., ferguson, i. k. 1968: daphne l. in: tutin, t. g., heywood, v. h., burges, n. a., moore, d. m., valentine, d. h., walters, s. m., webb, d. a. (eds.), flora europaea, vol. 2: 256– 258. cambridge university press, cambridge. a revision of the moss collection of the university of belgrade biologica nyssana 7 (1)  september 2016: 11-17 veljić, m. et al.  a revision of the moss collection… 11 original article received: 09 july 2015 revised: 19 mart 2016 accepted: 01 june 2016 a revision of the moss collection of the university of belgrade herbarium (beou) from the ostrozub mountain in serbia milan veljić * , nemanja rajčević, danka bukvički university of belgrade, faculty of biology, institute of botany and botanical garden “jevremovac”, takovska 43, 11000 belgrade, serbia * e-mail: veljicm@bio.bg.ac.rs abstract: veljić, m., rajčević, n., bukvički, d.: a revision of the moss collection of the university of belgrade herbarium (beou) from the ostrozub mountain in serbia. biologica nyssana, 7 (1), september 2016: 11-17. during the revision of moss collection of the university of belgrade herbarium from the ostrozub mountain which was collected in period from 1950 to 1953 it has been found that numerous specimens had not been determined. in total, 132 species were recorded: 23 liverwort, 109 moss species were found on the mt. ostrozub. out of those, 38 taxa (6, 25 and 7 species belonging to jungermanniopsida, bryopsida and sphagnopsida, respectively) represent new record for the mt. ostrozub. nineteen species belong to the endangered species, of different endangerment degree, on the red lists of european and serbian mosses. key words: liverworts, mosses, peat moss, conservation apstrakt: veljić, m., rajčević, n., bukvički, d.: revizija kolekcije mahovina iz herbarijuma univerziteta u beogradu (beou) sa planine ostrozub u srbiji. biologica nyssana, 7 (1), septembar 2016: 11-17. tokom revizije kolekcije mahovina iz herbarijuma univerziteta u beogradu prikupljene na planini ostrozub u periodu od 1950. do 1953. godine utvrđeno je da brojni primerci nisu određeni. ukupno je na ostrozubu pronađeno 132 vrste, odnosno 23 marchantiophyta i 109 bryophyta. u materijalu su zabeležena 38 taksona do sada nepoznata na ostrozubu, i to 6 jungermanniopsida, 25 bryopsida i 7 sphagnopsida. na crvenim listama mahovina evrope i srbije u različitim stepenima ugroženosti nalazi se 19 vrsta. key words: jetrenjače, mahovine, bele mahovine, konzervacija introduction this work includes the review of the research results on the mosses from the mt. ostrozub and the revision of the herbarium collection made by ilić, popović et al., 1950 and 1953. the material from 1950 was collected on localities: zeleničje (1), village ostrozub (2), near road predejane – ostrozub (3), the spring srebrni kladenac (4), near road ostrozub – ruplje (5), village ruplje (6), near the stream golema dolina (7) and peat near village stojimirovo (8). the collection from 1953 is mainly from peatlands from the following villages: selište (9), preslap (10), dobro polje (11), krstićevo (12), ruplje (6), stojimirovo (8), kolibište (13), čobanac (14) and zeleničje (1). only part of this collection 7 (1) • september 2016: 11-17 doi: 10.5281/zenodo.159099 biologica nyssana 7 (1)  september 2016: 11-17 veljić, m. et al.  a revision of the moss collection… 12 was published in the papers cited bellow, the rest remained undetermined. investigation of the mt. ostrozub moss flora was performed in parallel with phytocenological research. however, the only specified article on the bryoflora is the publication on mosses in reserves and protected areas where 84 taxa have been listed (15 liverworts and 69 mosses) in the community of prunus laurocerasus ( p o p o v i ć , 1966). m i š i ć et al. (1984) mentioned 38 (10 liverworts and 28 mosses), while il i ć (1951, 1952/53), mentioned several moss taxa within the community with cherry laurel. the mt. ostrozub is located in the southeast of serbia (fig. 1), within the rhodope mountain range (a v r a m o v i c et al., 2005). the area is linked to vlasina complex in the south and babička gora in the north. the peaks of čuklenik (1280 m.a.s.l.), ostrozubska čuka (1546 m.a.s.l.), čobanac (1496 m.a.s.l.) and title (1487 m.a.s.l.) extend in the southwest-northeast direction. hydrographic network consists of the rivers ostrozubska, rupska, kozačka and bistrička, and rivulets and streams srebrni, kladenac and zelenički. the largest area is covered with the silicate surface. the most widely spread of soil is skeletal brown forest soil, with a weak acid reaction. potential zonal vegetation is fagetum montanum b. jov. 1967. (j o va n o vi ć et al. 1997), i.e. subtype lauroceraso – fagetum b. jov. 1967. (j o v a n o v i ć , 1967). regular shift of vegetation zones was observed from the foothill to the peak of the mt. ostrozub. the mt. ostrozub drew attention of many botanists primarily because of cherry laurel (prunus laurocerasus l.) discovered by p a n č i ć (1887). after his discovery, a series of papers was created on this species and the beech forests, with which it forms a community lauroceraso-fagetum b. jov. (k o š a n i n , 1913; i l i ć , 1951; p o p o v i ć , 1952; j o v a n o v i ć , 1952/53, 1967, 1973; r a n đ e l o v i ć et al., 1983; m i š i ć , et al., 1984; j o v a n o v i ć et al., 1997). zeleničje area, a part of the mt. ostrozub, is now protected as a nature reserve. a rich hydrographic network and varied geological substrate enable formation of a large number of different microclimate habitats on the mt. ostrozub. all of the listed characteristics, as well as many others not mentioned here, suggest good conditions for the development of very diverse bryoflora on this mountain. material and methods the bryophyte samples were collected from fourteen localities in 1950 and 1953 in mt. ostrozub. for identification of taxa, the relevant bryology literature was used (p e t r o v , 1975, l a n d w e h r & b a r k m a n , 1966; l a n d w e h r & g r a d s t e i n , 1980; s m i t h , 1990, 2006). the nomenclature is in accordance with g r o l l e & l o n g (2000) and h i l l et al. (2006). the lists of the moss taxa are summarized according to classification by j o n a t h a n & k a r e n (2004) while genera are given in the alphabetical order. literature sources (*) and new data on localities (**) are given for each fig. 1. map of the mt. ostrozub with the study sites and its position. biologica nyssana 7 (1)  september 2016: 11-17 veljić, m. et al.  a revision of the moss collection… 13 table 1. list of bryophyte species on the mt. ostrozub with the literature and new data. species *literature data (1,2,3,4) **new data marchantiophyta marchantiopsida conocephalum conicum (l.) dum. (3) zeleničje marchantia polymorpha l. (3,4) zeleničje, peatland near stojimirova jungermanniopsida apometzgeria pubescens (schrank) kuwah. (3,4) bazzania trilobaia (l.) gray. (3,4) blepharostoma trichophyllum (l.) dum. (3) zeleničje calypogeia azurea stotler & crotz (4) •c. mulleriana (schiffn.) mull. frid. zeleničje, stream golema dolina chiloscyphus pallescens (ehrh. ex hoffm.) dum. (3) c. polyanthos (l.) corda var. polyanthus (3,4) near road ostrozub ruplje •frullania dilatata (l.) dum. zeleničje •jungermannia leiantha grolle zeleničje, srebrni kladenac lophocolea bidentata (l.) dum. var. bidentata (3) zeleničje metzgeria furcata (l.) dum. (3) •pedinophyllum inerruptum (nees.) kaal. zeleničje pellia epiphylla (l.) corda. (3,4) srebrni kladenac plagiochila asplenioides (l. emend. taylor) dum. (3,4) •p. porelloides (torrey ex nees) lindeb. zeleničje, stream golema dolina •porella cordaeana (hub.) moore zeleničje p. platyphylla (l.) pfeiff. (3,4) radula complanata (l.) dum. (3) near road ostrozub ruplje scapania nemorea (l.) grolle (4) zeleničje s. undulata (l.) dum. (3) zeleničje trichocolea tomentella (ehrh.) dum. (1,3,4) zeleničje bryophyta bryopsida abietinela abietina (hedw.) m. fleisch. (4) •amblistegium serpens (hedw.) schimp. near road predejane ostrozub antitrichia curtipendula (hedw.) brid. (3,4) zeleničje atrichum angustatum (brid.) bruch & schimp. (3) a. undulatum (hedw.) p. beauw. (4) zeleničje, near road predejane ostrozub barbula bicolor (bruch & schimp.) lindb. (3) b. convoluta hedw. (3) b. unguiculata hedw. (3) bartramia ityphylla brid. (3) stream golema dolina b. pomiformis hedw. (3,4) •brachythecium albicans (hedw.) schimp. peak of ostrozub •b. rivulare schimp. zeleničje, stream golema dolina b. rutabulum (hedw.) schimp. (3) near road predejane ostrozub bryum alpinum huds. ex with. (3) b. caespiticium hedw. (3) zeleničje, stream golema dolina b. capillare hedw. (3) near road predejane ostrozub, stream golema dolina b. archangelicum bruch & schimp. (3) b. intermedium (brid.) blandow (3) •b. pallescens schleich. ex schwarg stream golema dolina •b. pseudotriquetrum (hedw.) p. gaertn. & al. zeleničje calliergonella cuspidata (hedw.) loeske (3) peatland near stojimirovo ceratodon purpureus (hedw.) brid. (3) stream golema dolina climacium dendroides (hedw.) f. weber & d. mohr. (3,4) zeleničje •cratoneuron filicinum (hedw.) spruce zeleničje ctenidium molluscum (hedw.) mitt. (3,4) zeleničje, near road predejane ostrozub dichodontium palustre (dicks.) m. stech (3) dicranella crispa (hedw.) schimp. (3) zeleničje d. heteromalla (hedw.) schimp. (3) zeleničje, stream srebrni izvor dicranum scoparium hedw. (2,3,4) zeleničje, near road predejane ostrozub, ruplje, stream golema dolina biologica nyssana 7 (1)  september 2016: 11-17 veljić, m. et al.  a revision of the moss collection… 14 d. viride (sull. & lesq.) lindb. (3) diphyscium foliosum (hedw.) d. mohr (3) ditrichum flexicaule (schwagr.) hampe (3) •d. pallidum (hedw.) hampe stream golema dolina drepanocladus aduncus (hedw.) warnst. (3) zeleničje •eurhynchium angustirete (broth.) t. j. kop. zeleničje e. striatum (hedw.) schimp. (3,4) fissidens adianthoides hedw. (3) fontinalis antipyretica hedw. (3,4) funaria hygrometrica hedw. (3,4) zeleničje, stream golema dolina •grimmia hartmanii schimp. peak of ostrozub g. ovalis (hedw.) lindb. (3) •g. pulvinata (hedw.) sm. village ostrozub hedwigia ciliata (hedw.) p. beauv. (3) •herzogiella seligeri (brid.) z. iwats. stream golema dolina homalothecium lutescens (hedw.) h. rob. (3,4) •h. philippeanum (spruce) schimp. zeleničje hylocomium splendens (hedw.) schimp. (2,3,4) zeleničje, ruplje, stream golema dolina •hymenoloma crispulum (hedw.) ochyra stream golema dolina hypnum cupressiforme hedw. (3,4) zeleničje, stream golema dolina isothecium alopecuroides (lam. ex dubois) isov. (4) zeleničje, near road predejane ostrozub, stream golema dolina •kindbergia praelonga (hedw.) ochyra zeleničje •leskea polycarpa hedw. near road predejane ostrozub, stream golema dolina leucobrium glaucum (hedw.) ĺngstr. (2) leucodon sciuroides (hedw.) scwaegr. (3) zeleničje mnium marginatum (dicks.) p. beauv. (3) neckera crispa hedw. (4) stream golema dolina orthothecium rufescens (dicks. ex brid.) schimp. (4) orthotrichum pulchellum brunt. (3) •o. speciosum nees. village ostrozub •oxyrrhynchium schleicheri (r. hedw.) roll near road predejane ostrozub palustriela commutata (hedw.) ochyra var. commutate (3) zeleničje philonotis fontana (hedw.) brid. (3) peatland near stojimirovo plagiomnium cuspidatum (hedw.) t. j. kop. (3,4) near road predejane ostrozub p. undulatum (hedw.) t. j. kop. (1,3,4) zeleničje, near road predejane ostrozub, stream golema dolina plagiothecium denticulatum (hedw.) schimp. (3,4) zeleničje p. nemorale (mitt.) a. jaeg. (3) platyhypnidium riparioides (hedw.) dixon (3) pleurozium schreberi (willd. ex brid.) mitt. (3) pogonatum aloides (hedw.) p. beauv. (3) zeleničje p. urnigerum (hedw.) p. beauv. (3) zeleničje, stream golema dolina polytrichastrum formosum (hedw.) g. l. sm. (3) zeleničje polytrichum commune hedw. (2,3,4) zeleničje, near road predejane ostrozub, peatland near stojimirovo, selište, kolibište p. juniperinum hedw. (3) zeleničje p. piliferum hedw. (3) psedoleskea incurvata (hedw.) loeske (3) zeleničje •pseudoleskeella nervosa (brid.) nyholm stream golema dolina pseudoscleropodium purum (hedw.) m. fleich. (3,4) zeleničje pterigynandrum filiforme hedw. (3) zeleničje rhizomnium punctatum (hedw.) t. j. kop. (1,3,4) zeleničje, stream golema dolina rhytidiadelphus loreus (hedw.) warnst. (3,4) zeleničje •r. squarrosus (hedw.) warnst. zeleničje, stream golema dolina r. triquetrus (hedw.) warnst. (2,4) zeleničje, stream golema dolina, ruplje •sanionia uncinata (hedw.) loeske stream golema dolina schistidium apocarpum (hedw.) bruch & schimp. (3) zeleničje, stream golema dolina •sciuro-hypnum populeum (hedw.) ignatov & huttunen zeleničje •s. reflexum (hedw.) ignatov & huttunen zeleničje syntrichia latifolia (bruch ex hartm.) huebener (3) biologica nyssana 7 (1)  september 2016: 11-17 veljić, m. et al.  a revision of the moss collection… 15 s. montana nees (3) s. ruralis (hedw.) f. weber & d. mohr (3) tetraphis pellucida hedw. (3) •thamnobryum alopecurum (hedw.) gangulee stream golema dolina •thuidium recognitum (hedw.) lindb. near road predejane ostrozub, stream golema dolina t. tamariscinum (hedw.) schimp. (4) zeleničje tortella humilis (hedw.) jenn. (3) t. tortuosa (hedw.) limpr. (3) tortula lanceola r. h. zander (3) t. muralis hedw. (3) •t. subulata hedw. stream golema dolina, near road predejane ostrozub warnstorfia fluitans (hedw.) loeske (4) sphagnopsida •sphagnum angustifolium (c. e. o. jens. ex russ.) c. e. o. jens. selište •s. capillifolium (ehrh.) hedw. stojimirovo s. cuspidatum ehrh. ex hoffm. (3) ruplje •s. fallax (h. klinggr.) h. klinggr. selište, stojimirovo •s. flexuosum dozy & molk. selište, stojimirovo •s. inundatum russ. selište, under preslapa, čobanac, krstićevo, zeleničje, dobro polje, s. palustre l. (3) selište, under preslap, kolibište, dobro polje, krstićevo, stojimirovo •s. squarrosum crome under preslap, kolibište s. subsecundum nees (3) zeleničje, čobanac, krstićevo •s. teres (schimp.) angstr. golema dolina * literature data: 1 (i l i ć , 1951, 1952/53), 2 (jovanović , 1952/53), 3 (p o p o v i ć , 1966), 4 (m i š i ć et al., 1984) ** data from the herbarium material collected in 1950 and 1953 • new species in the flora of the mt. ostrozub taxon. new species on the mt. ostrozub are marked with the dot (•) in tab. 1. herbarium collection has been deposited at the university of belgrade – faculty of biology herbarium (beou). results and discussion in this paper, we report new data on the bryoflora of the mt. ostrozub obtained during the revision of the material collected in the mt. ostrozub in 1950 and 1953, as well as available literature data (tab. 1). in total, 132 taxa of mosses were recorded, which makes this small area bryologically very important for serbia. twenty-three taxa were recorded from the marchantiophyta: two from classis marchantiopsida and twenty-one from classis jungermanniopsida. six of this species were recorded for the first time on the mt. ostrozub: calypogeia mulleriana, frullania dilatata, jungermannia leiantha, pedinophyllum inerruptum, plagiochila porelloides and porella cordaeana. bryophyta (class bryopsida and sphagnopsida) is represented with 109 taxa. the class bryopsida includes 99 taxa of which 25 species are recorded for the first time for the bryoflora of the mt. ostrozub: amblistegium serpens, brachythecium albicans, b. rivulare, bryum pallescens, b. pseudotriquetrum, cratoneuron filicinum, ditrichum pallidum, eurhynchium angustirete, grimmia hartmanii, g. pulvinata, herzogiella seligeri, homalothecium philippeanum, hymenoloma crispulum, kindbergia praelonga, leskea polycarpa, ortotrichum speciosum, oxyrrhynchium schleicheri, pseudoleskeella nervosa, rhytidiadelphus squarrosus, sanionia uncinata, sciuro-hypnum populeum, s. reflexum, thamnobryum alopecurum, thuidium recognitum, tortula subulata. ten peat moss (class sphagnopsida) species were recorded, out of which seven were recorded for the first time on the mt. ostrozub (s. angustifolium, s. capillifolium, s. fallax, s. inundatum, s. flexuosum, s. squarrosum, s. teres). the genera with the highest number of species for mosses this study were sphagnum (10 taxa), bryum (7 taxa) and barbula, brachythecium, grimmia, polytrichum, rhytidiadelphus, syntrichia, tortula (3 taxa each). the most diverse bryoflora (54 taxa) is recorded in the reserve area, which includes zeleničje locality (36). in locality number 7 (stream biologica nyssana 7 (1)  september 2016: 11-17 veljić, m. et al.  a revision of the moss collection… 16 golema dolina), great number of taxa (30) was also recorded, making this locality a bryologycally rich area. significantly lower diversity was recorded in all other localities, possibly due to the less intense research in these localities by the researchers. it is remarkable that on the mt. ostrozub peatlands, ten sphagnum species were recorded. it is also one of the localities with the greatest diversity of this group. on the mt. ostrozub, nineteen recorded taxa are classified in the red book of europe (iucn, 1994) and serbia (s a b o v l j e v i ć et al., 2004) at different levels of vulnerability (tab. 2). bern convention annex 1 (council of europe, 1979) declares the species dicranum viride as a strictly protected species. according to the ordinance on the proclamation and protection of the wild species of plants, animals and fungi, sphagnum species are strictly protected by local legislation (sl. glasnik rs, 2009). calypogeia muelleriana and bazzania trilobata are also protected according to this document. a great number of species on the red lists justifies preserving maximum protection of habitats on the mt. ostrozub. it is also necessary to put the special emphasis on the protection of the peatlands outside the reserve in order to maintain the diversity of the genus sphagnum. acknowledgements. this research was supported by a grant from the ministry of education, science and development of serbia (project no. 173029). references avramović, d., zlatković, b., ranđelović, n., 2005: zaštićena prirodna dobra jugoistone srbije. viii symposium on flora of southeasteren serbia and neighbouring regions, niš. proceding, 223-227. council of europe, 1979: convention on the conservation of european wildlife and natural habitats. bern. grolle, r., & long, d. g., 2000: an annotatated check-list of the hepaticae and anthocerotae of europe and macronesia. journal of bryoogy, (22), 103-140. hill, m. o., bell, n., bruggeman-nannenga, m. a., brugues, m., cano, m. j., enroth, j., flatberg, k. i., frahm, j. p., gallego, m. t., garilleti, r., guerra, j., hedenäs, l., holyoak, d. t., hyvönen, j., ignatov, m. s., lara, f., mazimpaka, v., muñoz, j., and l. söderström., 2006: an annotated checklist of the mosses of europe and macronesia. journal of bryoogy, (28), 198-267. ilić, e., 1951: prilog poznavanju ekologije prunus laurocerasus l. na ostrozubu u srbiji. srpska akademija nauka, zbornik radova 11, institut za ekologiju i biogeografiju (2): 253-258. ilić, e. 1952/53: edafski uslovi u bukovim šumama u rezervatu na ostrozubu. srpska akademija nauka, zbornik radova 29, institut za ekologiju i biogeografiju (3): 113-128. iucn, 1994: iucn red list categories. iucn, gland, switzerland. jonathan, s., & karen, r., 2004: phylogeny and diversification of bryophytes. american journal of botany, 91(10): 1557–1581. jovanović, b., 1952/53: o dvema fitocenozama istočne srbije. quercetum montanum i fagetum muscetum, zbornik radova 29, institut za ekologiju i biogeografiju (3): 1-44. jovanović, b., 1967: fitocenoza sa zeleničetom na ostrozubu (lauroceraso-fagetum). in: josifović, m (ed.), pančićev zbornik u spomen 150. godišnjice njegovog rođenja, 127-137, srpska akademija nauka i umetnosti, odeljenje prirodno-matematičkih nauka, beograd. jovanović, b., 1973: prilog poznavanju fitocenoze bukve na ostrozubu. glasnik prirodnjačkog muzeja, c (7), 5-27. jovanović, b., mišić, v., dinić, a., diklić, n., vukićević, e., 1997: zajednica planinske bukve na neutralnim ili slabo kiselim zemljištima. in: sarić, m (ed.), vegetacija srbije ii1, 190-198, srpska akademija nauka i umetnosti, odeljenje prirodno-matematičkih nauka, beograd. table 2. endangered species in the mt. ostrozub bryoflora category taxon critically endangered calypogeia muelleriana endangered bazzania trilobata, trichocolea tomentella vulnerable barbula bicolor, dicranum viride, tetraphis pellucid, sphagnum capillifolium, s. cuspidatum, s. fallax, s. flexuosum, s. palustre, s. squarrosum, s. subsecundum and s. teres low risk diphyscium foliosum, ditrichum pallidum, fontinalis antipyretica, orthotrichum pulchellum data deficient sciuro-hypnum reflexum biologica nyssana 7 (1)  september 2016: 11-17 veljić, m. et al.  a revision of the moss collection… 17 košanin, n., 1913: život zeleničeta na ostrozubu. glasnik srpske kraljevske akademije, 89. prirodno-matematičke nauke, 37: 228-278. landwehr, j., barkman, j. j., 1966: atlas van de nederlandse bladmossen. koninklijke nederlandse natuurhistorische vereniging, amsterdam. landwehr, j., gradstein r.s., 1980: atlas van de nederlandse levermossen. koninklijke nederlandse natuurhistorische vereniging, amsterdam. mišić, v., popović, m., čolić, d., 1984: varijabilitet i ekologija zeleničeta (prunus laurocerasus l.) na ostrozubu (istočna srbija). zaštita prirode, (37): 49-79. pančić, j., 1887: der kirschlorbeer im süd-osten von serbien. petrov, s. (1975): bryophyta bulgarica clavis diagnostica, 536 pp. academia scientiarum bulgarica. sofia. popović, m., 1966: prilog poznavanj mahovina u rezervatima i zaštićenim područjima u srbiji. zaštita prirode, (33): 219-228. ranđelović, n., martinović, z., sotirov, s., jovanović, v., ružić, m., stamenković, v., hill, d. a., krivošej, z., ranđelović, v., 1983: prunus laurocerasus l. na ostrozubu cveta. glasnik prirodnjačkog muzeja, b (38): 73-80. sabovljević, m., cvetić, t., stevanović, v., 2004: bryophyte red list of serbia and montenegro. biodiversity and conservation, (13): 1781-1790. službeni glasnik rs, 2009: pravilnik o proglašenju i zaštiti strogo zaštićenih i zaštićenih divljih vrsta biljaka, životinja i gljiva. br. 5/2010 i 47/2011. smith, a. j. e., 1990: the liverworts of britain and ireland. university press, cambridge. smith, a. j. e., 2006: the moss flora of britain and ireland. university press. cambridge. the effect of satureja montana l. aqueous extract on soybean seedlings biologica nyssana 7 (2)  december 2016: 125-129 šućur, j. et al.  the effect of satureja montana l. aqueous… 125 original article received: 01 july 2016 revised: 18 october 2016 accepted: 24 november 2016 the effect of satureja montana l. aqueous extract on soybean seedlings jovana šućur1, dejan prvulović1, goran anačkov2, đorđe malenčić1 1 department of field and vegetable crops, faculty of agriculture, university of novi sad, trg dositeja obradovića 8, novi sad, serbia 2 department of biology and ecology, faculty of science, university of novi sad, trg dositeja obradovića 3, novi sad, serbia * e-mail: jovana.sucur@polj.edu.rs abstract: šućur, j., prvulović, d., anačkov, g., malenčić, đ.: the effect of satureja montana l. aqueous extract on soybean seedlings. biologica nyssana, 7 (2), december 2016: 125-129. the aim of this study was to examine the impact of satureja montana l. aqueous extract on soybean antioxidant properties so as to assess its possible side effects when applied as biohebicide in soybean organic production. the effects of two concentrations (0.1% and 0.2%) of s. montana aqueous extract on the activity of the antioxidant enzymes superoxide dismutase (sod) and catalase (cat) in leaves and roots of soybean (glycine max l.) seedlings were examined 24, 72 and 120 h after the treatment. our results showed that the significant increase in the catalase activity was recorded in roots of soybean treated with both concentrations of the extract used. on the other hand, both concentrations of s. montana aqueous extract stimulated the significant increase of the superoxide dismutase activity in leaves and roots of soybean. higher activity of the antioxidant enzymes in the roots of soybean compared with activity of the antioxidant enzymes in leaves showed that roots were more affected than leaves. key words: allelopathy, glycine max (l.) merr., satureja montana l. apstrakt: šućur, j., prvulović, d., anačkov, g., malenčić, đ.: efekat vodenog ekstrakta satureja montana l. na sadnice soje. biologica nyssana, 7 (2), decembar 2016: 125-129. cilj ovog rada je ispitivanje uticaja vodenog ekstrakta satureja montana l. na parametre oksidativnog stresa u sadnicama soje kako bi se procenile eventualne nuspojave nakon primene ovog ekstrakta kao bioherbicida u organskoj proizvodnji soje. ispitan je uticaj dve koncentracije (0,1% i 0,2%) vodenog ekstrakta s. montana na aktivnost antioksidantnih enzima superoksid-dismutaze (sod) i katalaze (cat) u listu i korenu sadnica soje (glycine max l.) 24, 72 i 120 časova nakon tretmana. dobijeni rezultati su pokazali statistički značajno povećanje aktivnosti enzima katalaze u korenu soje, kao i statistički značajno povećanje aktivnosti superoksiddismutaze u listu i korenu soje, u tretmanima za obe primenjene koncentracije ekstrakata. veća aktivnost antioksidantnih enzima u korenu soje, u poređenju sa aktivnošću u listu, ukazuje na veću senzitivnost korena od lista prema primenjenom ekstraktu. ključne reči: alelopatija, glycine max (l.) merr., satureja montana l. 7 (2) • december 2016: 125-129 12th sfses • 16-19 june 2016, kopaonik mt doi: 10.5281/zenodo.200409 biologica nyssana 7 (2)  december 2016: 125-129 šućur, j. et al.  the effect of satureja montana l. aqueous… 126 introduction satureja montana l. (winter savory or mountain savory), belonging to the lamiaceae family, native to the mediterranean regions, is a well-known aromatic plant which contains various biologically active constituents such as essential oils, triterpenes, flavonoids and rosmarinic acid (h a s s a n e i n et al., 2014; d u d a š et al., 2013). it is found throughout europe, russia and turkey, growing in sunny, stony and rocky regions (t r i f a n et al., 2015). owing to the active components and pleasant aroma, winter savory is often used as a medicinal plant and a spice (d u d a š et al., 2013; t r i f a n et al., 2015). the plant releases many bioactive chemicals from its various parts (leaves, stems, roots) into its surrounding environment, which are called allelochemicals (m o n d a l et al., 2015). allelopathy is an interference mechanism by which plants release allelochemicals which affect the growth of other plants (b a j a l a n et al., 2013). allelopathic interaction is either positive or negative (m o n d a l et al., 2015). released allelochemicals become stressful only when they are toxic or when they affect the growth and development of surrounding plants (c r u z – o r t e g a et al., 2007). increasing attention has been given to the role and potential of allelopathy as a management strategy for crop protection against weeds and other pests (b a j a l a n et al., 2013). the aim of this study was to examine the effect of aqueous extract of s. montana on soybean (glycine max (l.) merr.) antioxidant properties so as to assess its possible side effects when applied as biohebicide in soybean organic production. material and methods the aromatic plant, s. montana, was collected at localities near the adriatic coast in montenegro, in june, 2012. voucher specimens of collected plant was confirmed and deposited at the herbarium of the department of biology, faculty of natural sciences, university of novi sad (buns 2-1544). the air– dried plant material was ground into powder. the powdery material (10 g) was extracted with 100 ml distilled water. after 24 h, the extract was filtered through filter paper and kept at 4 °c until application. the experiment was performed at the laboratory of biochemistry, faculty of agriculture, novi sad and conducted under controlled conditions (temperature 28 °c, 60% relative humidity, 18 h photoperiod, a light intensity of 10.000 lx). soybean seedlings (g. max) cv. viktorija were grown for 30 days in plastic pots (500 ml) containing sterile sand, after which they were transferred on plastic pots (700 ml) containing the hoagland’s solution and 7 and 14 ml of s. montana aqueous extract, while 7 and 14 ml of the hoagland’s solution were added in pots of control. seedlings were harvested for determining the investigated biochemical parameters 24, 72 and 120 h after the treatments. fresh leaves and roots of soybean plants (2 g each) were homogenized in 10 ml of phosphate buffer (0.1 m, ph 7.0). homogenates were centrifuged for 20 min at 10.000 x g and filtered. the supernatants were used to test activity of the antioxidant enzymes superoxide dismutase (sod) and catalase (cat). the catalase (cat) (ec 1.11.1.6) activity was determined according to sathya & bjorn (2010). the decomposition of h2o2 was followed as a decrease in absorbance at 240 nm. the enzyme extract was added to the assay mixture containing 50 mm potassium phosphate buffer (ph 7.0) and 10 mm h2o2. the activity of the enzyme is expressed as u per 1 g of protein (u mg–1 protein). superoxide dismutase (sod) (ec 1.15.1.1) activity was assayed according to a slightly modified method of m a n d a l et al. (2008) by measuring its ability to inhibit photochemical reduction of nitro blue tetrazolium (nbt) chloride. the reaction mixture contained 50 mm phosphate buffer (ph 7.8), 13 mm l– methionine, 75 μm nbt, 0.1 mm edta, 2 μm riboflavin and 0.02 ml of the enzyme extract. it was kept under a fluorescent lamp for 30 min, and then the absorbance was read at 560 nm. one unit of the sod activity is defined as the amount of enzyme required to inhibit the reduction of nbt by 50%. the activity of the enzyme is expressed as u per 1 mg of protein (u mg–1 protein). values of the biochemical parameters were expressed as means ± standard error (se) of determinations made in triplicates and tested by anova followed by comparison of the means by the duncan multiple range test (p < 0.05). data were analyzed using statistica for windows, version 11.0. results and discussion allelochemicals can cause oxidative stress in target plants and therefore activate the antioxidant mechanism (c r u z – o r t e g a et al., 2007). accordingly, the response of plants to damaging adverse circumstances is closely related to their enzyme activity (s u n m o n u & v a n s t a d e n , 2014). enzymes, such as superoxide dismutases (sod), catalases (cat) and peroxidases, play important roles in protecting cells against reactive oxygen species (k u t h a n et al., 1986). therefore, the activity of those enzymes can be used as indicators of oxidative stress in plants (a n et al., 2005). biologica nyssana 7 (2)  december 2016: 125-129 šućur, j. et al.  the effect of satureja montana l. aqueous… 127 our results showed that a significant increase in the catalase activity was recorded in roots of soybean seedlings treated with both concentrations of the extract used 72 h after the treatment (fig. 1). on the other hand, both concentrations of s. montana aqueous extract stimulated a significant increase of the superoxide dismutase activity in roots of soybean seedlings 72 and 120 h after the treatment (fig. 2). the increases in activity of antioxidant enzymes probably occur in response to stress. a higher activity of the antioxidant enzymes in the roots of soybean seedlings compared with the activity of the antioxidant enzymes in leaves showed that roots were more affected than leaves. the activity of examined antioxidant enzymes showed a downward trend with the duration of the experiment, thus the highest activity of the catalase and superoxide dismutase was observed after 72 h. this could point to the fact that scavenging effects of superoxide dismutase and catalase could prevent an oxidative burst and the induction of lipid peroxidation process. conclusion in conclusion, our results showed that s. montana aqueous extract did not stimulate an increase of the catalase and superoxide dismutase activity in the leaves of soybean seedlings, while both tested concentrations caused a significant increase in fig. 1. activity of catalase in leaves and roots of soybean seedlings 24, 72 and 120 h after treatment with different concentrations (%) of s. montana aqueous extracts (v/v) and in control (c). fig. 2. activity of superoxide dismutase in leaves and roots of soybean seedlings 24, 72 and 120 h after treatment with different concentrations (%) of s. montana aqueous extracts (v/v) and in control (c). 0,00 5,00 10,00 15,00 20,00 25,00 30,00 c 0.1 0.2 c 0.1 0.2 c 0.1 0.2 24 h 72 h 120 h u / m g p ro te in extract concentration (%) roots leaves 0 10 20 30 40 50 60 70 80 90 100 c 0.1 0.2 c 0.1 0.2 c 0.1 0.2 24 h 72 h 120 h u /m g p ro te in extract concentration (%) roots leaves biologica nyssana 7 (2)  december 2016: 125-129 šućur, j. et al.  the effect of satureja montana l. aqueous… 128 soybean roots. this may be attributed to the permeability of allelochemicals to root tissues arising from direct contact with the phytotoxic compounds present in the extract. acknowledgements. this study was carried out within a project of the ministry of education, science and technological development, republic of serbia, grant no tr-31022. references assyov, b., denchev, c. 2015: verbascum davidoffii murb. in: biserkov, v. et al. (eds.), digital edition of red data book of republic of bulgaria. bas & moew, sofia. http://e-ecodb.bas.bg/rdb/en/vol1/verdavid.html. biserkov, v. et al. (eds.) 2015: digital edition of red data book of republic of bulgaria. bas & moew, sofia. http://e-ecodb.bas.bg/rdb/en/. camacho, f.j., liston, a. 2001: population structure and genetic diversity of botrychium pumicola (ophioglossaceae) based on inter-simple sequence repeats (issr). american journal of botany, 88 (6): 1065-1070. doyle, j.j., doyle, j.l. 1987: a rapid dna isolation procedure for small quantities of fresh leaf tissue. phytochemical bulletin, 19: 11-15. fang, d.q., roose, m.l. 1997: identification of closely related citrus cultivars with inter-simple sequence repeat genetics. theoretical and applied genetics, 95 (3): 408-417. firat, m. 2015: verbascum kurdistanicum (scrophulariaceae), a new species from hakkari, turkey. phytokeys, 52: 89-94. gonzalo-turpin h., hazard, l. 2009: local adaptation occurs along altitudinal gradient despite the existence of gene flow in the alpine plant species festuca eskia. journal of ecology, 97 (4): 742-751. hamrick, j.l., godt, m.j.w. 1996: conservation genetics of endemic plant species. in: avise, j.c., hamrick, j.l. (eds.), conservation genetics: case histories from nature. chapman and hall, new york, pp. 281-304. helenurm, k. 2001: high levels of genetic polymorphism in the insular endemic herb jepsonia malvifolia. journal of heredity, 92 (5): 427-432. huang, y., zhang, c.q., li, d.z. 2009: low genetic diversity and high genetic differentiation in the critically endangered omphalogramma souliei (primulaceae): implications for its conservation. journal of systematics and evolution, 47 (2): 103–109. idrees, m., irshad, m. 2014: molecular markers in plants for analysis of genetic diversity: a review. european academic research, 2 (1): 1513-1540. jia, j., zeng, l., gong, x. 2016: high genetic diversity and population differentiation in the critically endangered plant species trailliaedoxa gracilis (rubiaceae). plant molecular biology reporter, 34 (1): 327-338. li, y.y., guan, s.m., yang s.z., luo, y., chen, x.y. 2012: genetic decline and inbreeding depression in an extremely rare tree. conservation genetics, 13 (2): 343-347. nybom, h. 2004: comparison of different nuclear dna markers for estimating intraspecific genetic diversity in plants. molecular ecology, 13 (5): 1143-1155. peakall, r., smouse, p.e. 2012: genalex 6.5: genetic analysis in excel. population genetic software for teaching and research an update. bioinformatics, 28 (19): 2537-2539. petrova, g., dzhambazova, t., moyankova, d., georgieva, d., michova, a., djilianov, d., moeller, m. 2014: morphological variation, genetic diversity and genome size of critically endangered haberlea (gesneriaceae) populations in bulgaria do not support the recognition of two different species. plant systematics and evolution, 300 (1): 29-41. rao, v.r., hodgkin, t. 2002: genetic diversity and conservation and utilization of plant genetic resources. plant cell, tissue organ culture, 68 (1): 1-19. stefanova-gateva, b. 1995: verbascum l. in: kožuharov, s. (ed.), flora reipublicae popularis bulgaricae 10: 26-100, acad. "prof. m. drinov", serdicae, sofia, bulgaria (in bulgarian). turchetto, c., segatto, a.l.a., mäder, g., rodrigues, d.m., bonatto, s.l., freitas, l.b. 2016: high levels of genetic diversity and population structure in an endemic and rare species: implications for conservation. aob plants 8: plw002; doi:10.1093/aobpla/plw002. walther, g.-r., beißner, s., burga, c.a. 2005: trends in the upward shift of alpine plants. journal of vegetation science, 16 (5): 541-548. willi, y., van buskirk, j., hoffmann, a.a. 2006: limits to the adaptive potential of small populations. annual review of ecology, evolution and systematics, 37: 433-458. wolfe, a.d., liston, a. 1998: contributions of pcrbased methods to plant systematics and evolutionary biology. in: soltis, p.s., soltis, d.e., doyle, j.j. (eds.), molecular systematics of plants: dna sequencing. kluwer, new york, pp. 43-86 xue, d.-w., ge, x.-j., hao, g., zhang, c.-q. 2004: high genetic diversity in a rare, narrowlu endemic http://e-ecodb.bas.bg/rdb/en/ biologica nyssana 7 (2)  december 2016: 125-129 šućur, j. et al.  the effect of satureja montana l. aqueous… 129 primrose species: primula interjacens by issr analysis. acta botanica sinica, 46 (10): 11631169. zawko, g., krauss, s.l., dixon, k.w., sivasithamparam, k. 2001: conservation genetics of the rare and endangered leucopogon obtectus (ericaceae). molecular ecology, 10 (10): 23892396. zhu, y., geng, y., tersing, t., liu, n., wang, q., zhong, y. 2009: high genetic differentiation and low genetic diversity in incarvillea younghusbandii, an endemic plant of qinghaitibetan plateau, revealed by aflp markers. biochemical systematics and ecology, 37 (5): 589-596. zietkiewicz, e., rafalski, a., labuda, d. 1994: genome fingerprinting by simple sequence repeat (ssr)-anchored polymerase chain reaction amplification. genomics, 20 (2): 176-183. milošević ilić et al., 2019, biologica nyssana 10(1) 10 (1) september 2019: 49-57 doi: 10.5281/zenodo.3464008 diversity of aphids (homoptera: aphididae) in southeastern serbia original article marijana ilić milošević department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, 18000 niš, serbia marijana183@gmail.com (corresponding author) vladimir žikić department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, 18000 niš, serbia vzikic@pmf.ni.ac.rs darija milenković department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, 18000 niš, serbia darija.velickovic@pmf.edu.rs saša s. stanković department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, 18000 niš, serbia ssasa@pmf.ni.ac.rs olivera petrović-obradović institute of phytomedicine, faculty of agriculture, university of belgrade, nemanjina 6, 11080 belgrade-zemun, serbia petrovic@agrif.bg.ac.rs received: august 2, 2019 revised: september 11, 2019 accepted: september 19, 2019 abstract: aphids represent one of the most significant pest species in agroecosystems. in this study we investigated aphid diversity in southeastern serbia. we recorded 132 species, mostly infesting host plants from the families asteraceae and rosaceae. the most diverse genus was aphis linnaeus with 34 species. within this genus, the most frequently sampled species was a. fabae scopoli. genera uroleucon mordvilko, brachycaudus van der goot and dysaphis börner were also represented by a large number of species. key words: aphids, southeastern serbia, species richness apstract: raznovrsnost biljnih vaši (homoptera: aphididae) u jugoistočnoj srbiji biljne vaši predstavljaju jednu od najznačajnijih grupa štetočina u agroekosistemima. u ovoj studiji istraživali smo raznovrsnost biljnih vaši u jugoistočnoj srbiji. zabeležili smo 132 vrste, koje uglavnom zaražavaju biljke domaćine iz porodice asteraceae i rosaceae. najraznovrsniji rod je aphis linnaeus sa 34 vrste. u ovom rodu, najčešće uzorkovana vrsta je a. fabae scopoli. rodovi uroleucon mordvilko, brachicaudus van der goot i disaphis borner takođe su predstavljeni velikim brojem vrsta. ključne reči: biljne vaši, jugoistočna srbija, bogatstvo vrsta introduction aphids (homoptera: aphididae) are one of the most economically important insect group in agriculture. they are significant plant pests, both in cultivated and self-seeding plants. apart from causing direct damage by sucking plant juices, they also transmit many plant viruses, such as the bean yellow mosaic virus, bean common mosaic virus, cucumber mosaic virus (gálvez & morales, 1989). aphids are distributed worldwide, but usually most common in temperate areas of the northern hemisphere. number of described species in world to this date is about 4700, of which about one third have been recorded from europe (coeur d’acier et al., 2010). within the territory of serbia, 374 species were found. the research on aphid fauna in serbia was most contributed by petrović (1998), petrovićobradović (2003), as well as poljaković-pajnik et al. (2002), vučetić et al. (2014) and petrović-obradović et al. (2007, 2010, 2018). the primary aim of this study was to explore the diversity of aphid fauna during the ten-year research period in the region of southeastern serbia. the secondary aim was to determine the number of trophic associations between aphid species and their host plants. materials and methods this study was based on material collected during the last ten years in different types of habitats © 2019 ilić-milošević et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 49 of southeastern serbia. aphids were collected from plant parts by using a fine thin brush and were transferred to plastic tubes filled with 90% ethanol. the specimens were identified according to keys prepared by blackman & eastop (1994, 2000, 2006). all analyzed material has been deposited at the faculty of agriculture, university of belgrade, serbia. plants were identified using josifović, ed. (19701976) and deposited in the herbarium of the faculty of sciences and mathematics, university of niš, serbia (hmn). results aphid-host plant associations species of aphididae are listed alphabetically, with the following order: host plant, host plant family in parenthesis, collection site, date of collection and legator (vž-vladimir žikić, dv-darija veličković, sss-saša s. stanković, mr-milica ristić, mimmarijana ilić milošević, mđ-maja đorđević, ml-maja lazarević, ns-nikola stanković, asaleksandra stojiljković, ms-milan stanisavljević, bz-bojan zlatković, nm-nikola mladenović, mmmajda mehanović, dn-danijela nikolić, zk-zorana kojičić, ljm-ljubomir mihajlović). acyrthosiphon caraganae (cholodkovsky, 1908) on: coronilla emerus (fabaceae), niš, niška banja, 19.5.2015, vž; pčinja canyon, prohor pčinjski, 10.6.2014, vž acyrthosiphon malvae (mosley, 1841) on: petunia sp. (solanaceae), prokuplje, 6.6.2017, dv; geranium dissectum (geraniaceae), niš, 26.4.2014, vž. acyrthosiphon pisum (harris, 1776) on: medicago sativa (fabaceae), lebane, konjino, 3.5.2014, sss; pisum sativum (fabaceae), prokuplje, gornja konjuša, 11.6.2017, mr; vicia angustifolia (fabaceae), niš, popovac, 27.4.2014, vž; vicia sp., vlasinsko jezero (lake), 6.8.2015, vž acyrthosiphon sp. on: coronilla varia, niš, 3.6.2016, vž anoecia corni (fabricius, 1775) on: cornus mas (cornaceae), niš, donji matejevac, 26.4.2018, mim; sićevačka klisura (gorge), ostrovica, 28.5.2013, mđ aphis acetosae linnaeus, 1761 on: rumex acetosella (polygonaceae), vlasinsko jezero (lake), 19.6.2018, vž aphis affinis del guercio, 1911 on: mentha aquatica (lamiaceae), bela palanka, divljana, 6.6.2014, vž; grdelička klisura (gorge), 5.6.2014, sss; m. longifolia, niš, niška banja, 23.5.2014, sss; pčinja canyon, gornji starac, 10.6.2014, sss; sićevačka klisura (gorge), sićevo, 24.5.2018, ml; vlasinsko jezero (lake), 6.8.2015, sss; vranje, 18.6.2016, ns; m. piperita, niš, bubanj, 5.6.2013, sss aphis balloticola szelegiewicz, 1968 on: ballota nigra (lamiaceae), niš, 31.5.2011, vž aphis confusa walker, 1849 on: knautia sp. (dipsacaceae), niš, niška banja, 31.5.2017, dv; sićevačka klisura (gorge), ostrovica, 28.5.2013, mđ aphis craccae linnaeus, 1758 on: vicia cracca, radan planina (mt.), 12.6.2016, vž; vlasinsko jezero (lake), 30.6.2016, sss aphis craccivora koch, 1854 on: cichorium intybus (asteraceae), leskovac, 30.5.2017, as; prokuplje, gornja konjuša, 11.6.2017, mr; coronilla varia, niš, 3.6.2015, vž; crepis biennis (asteraceae), niš, donji matejevac, 5.6.2013, vž; c. foetida, niš, 1.6.2013, vž; crepis sp., niš, 5.6.2013, vž; lactuca sp. (asteraceae), lebane, 1.6.2013, sss; medicago sativa, aleksinac, moravac, 18.6.2015, ms; lebane, 19.6.2011, sss; leskovac, 19.5.2017, as; melilotus alba (fabaceae), leskovac, 12.6.2017, as; robinia pseudoacacia (fabaceae), dukat (mt.), 7.8.2011, vž; leskovac, 25.5.2018, as; niš, 19.5.2013, vž; vranje, 17.6.2016, ns; vlasinsko jezero (lake), 7.8.2015, vž; salvia sp. (lamiaceae), niš, 5.6.2013, vž; sophora japonica (fabaceae), niš, niška banja, 6.6.2013, vž; trifolium lagopus (fabaceae), pčinja canyon, prohor pčinjski, 10.6.2014, bz; vicia cordata, niš, 21.5.2013, sss; pčinja canyon, gornji starac, 10.6.2014, sss; v. cracca, lebane, konjino, 31.5.2014, sss; prokuplje, beloljin, 14.5.2016, mr; prokuplje, bresničić, 15.6.2014, vž; vlasinsko jezero (lake), 8.8.2015, vž; vranje, 8.6.2016, ns; v. pannonica, radan planina (mt.), 12.6.2016, vž aphis crepidis (börner, 1940) on: crepis foetida, vlasinsko jezero (lake), 21.7.2013, sss aphis davletshinae hille ris lambers, 1966 on: malva sylvestris (malvaceae), prokuplje, gornja konjuša, 11.6.2017, mr aphis epilobii kaltenbach, 1843 on: epilobium montanum (onagraceae), grdelička klisura (gorge), 5.6.2014, sss aphis fabae scopoli, 1763 50 biologica nyssana ● 10 (1) september 2019: 49-57 ilić-milošević et al. ● diversity of aphids (homoptera: aphididae) in southeastern serbia 51 on: achillea millefolium asteraceae), prokuplje, gornja konjuša, 15.6.2016, mr; aegopodium podagraria (apiaceae), niš, 11.6.2014, vž; amaranthus retroflexus (amaranthaceae), niš, 1.7.2011, vž; prokuplje, gornja konjuša, 15.6.2016, mr; anthriscus sp. (apiaceae), niš, gornji matejevac, 25.5.2014, vž; prokuplje, 8.6.2017, dv; vlasinsko jezero (lake), 30.6.2016, sss; a. sylvaticus, prokuplje, gornja konjuša, 23.4.2016, mr; a. sylvestris, niš, niška banja, 14.5.2017, vž; radan planina (mt.), 6.6.2015, vž; arctium lappa (asteraceae), sićevačka klisura (gorge), ostrovica, 28.5.2013, mđ; vranje, 18.5.2018, nm; atriplex patula (chenopodiaceae), vranje, 17.7.2016, ns; ballota nigra, vranje, 4.5.2016, ns; beta vulgaris (chenopodiaceae), leskovac, 26.5.2017, as; bifora radians (apiaceae), niš, 22.5.2013, mđ; carduus acanthoides (asteraceae), leskovac, 25.5.2018, as; prokuplje, gornja konjuša, 29.5.2016, mr; centaurea cyanus (asteraceae), lebane, konjino, 31.5.2014, sss; chenopodium album (chenopodiaceae), lebane, 1.6.2013, sss; niš, 8.6.2015, sss; prokuplje, 6.6.2017, dv; prokuplje, gornja konjuša, 21.5.2017, mr; sićevačka klisura (gorge), 4.6.2011, vž; sićevačka klisura (gorge), sićevo, 17.7.2013, vž; vranje, 30.10.2016, ns; prokuplje, bresničić, 15.6.2014, sss; cichorium intybus, niš, 8.6.2015, sss; cirsium acanthoides (asteraceae), lebane, konjino, 31.5.2015, sss; c. arvense, leskovac, 18.6.2017, as; merošina, oblačinsko jezero (lake), 15.6.2014, sss; niš, 25.5.2014, sss; niš, kamenički vis, 4.6.2017, dv; sićevačka klisura (gorge), ravni do, 24.5.2015, vž; vlasinsko jezero (lake), 15.6.2013, sss; vranje, 20.6.2016, ns; c. vulgare, suva planina (mt.), bojanine vode, 7.7.2015, vž; vlasinsko jezero (lake), 6.8.2015, vž; coronilla varia, niš, 10.7.2014, vž; digitalis lanata (plantaginaceae), sićevačka klisura (gorge), sićevo, 24.5.2018, vž; euonymus japonicus (celastraceae), lebane, konjino, 31.5.2014, sss; fumaria officinalis (papaveraceae), niš, 22.5.2013, mđ; galium aparine (rubiaceae), lebane, konjino, 16.5.2015, sss; niš, 5.6.2013, mđ; niš, gornji matejevac, 25.5.2014, vž; niš, kamenički vis, 4.6.2017, dv; prokuplje, 8.6.2017, dv; sićevačka klisura (gorge), 24.5.2015, vž; sićevačka klisura (gorge), ravni do, 29.5.2014, vž; vlasinsko jezero (lake), 30.6.2016, sss; hedera helix (araliaceae), niš, 16.5.2015, vž; malva sylvestris, niš, 21.5.2018, nm; matricaria chamomilla (asteraceae), grdelička klisura (gorge), 5.6.2014, sss; lebane, 1.6.2013, sss; prokuplje, gornja konjuša, 11.6.2017, mr; sićevačka klisura (gorge), ostrovica, 28.5.2013, mđ; onopordum acanthium (asteraceae), niš, lalinac, 8.6.2013, sss; orlaya grandiflora (apiaceae), niš, gornji matejevac, 25.5.2014, vž; prokuplje, gornja konjuša, 13.6.2017, mr; papaver dubium (papaveraceae), niš, gornji matejevac, 25.5.2014, vž; p. rhoeas, niš, 5.6.2013, vž; niš, gornji matejevac, 25.5.2014, vž; sićevačka klisura (gorge), sićevo, 24.5.2018, ml; pastinaca hirsuta (apiaceae), vlasinsko jezero (lake), 6.8.2015, vž; philadelphus coronarius (hydrangeaceae), niš, 20.5.2014, sss; plantago major (plantaginaceae), niš, 6.6.2017, mr; ranunculus sp. (ranunculaceae), sićevačka klisura (gorge), ostrovic, 28.5.2013, mđ; rumex acetosella, leskovac, 6.5.2018, as; r. patientia, prokuplje, gornja konjuša, 23.4.2016, mr; vranje, 20.5.2018, nm; rumex sp., lebane, konjino, 31.5.2014, sss; niš, 22.5.2013, mđ; radan planina (mt.), 6.6.2015, vž; sićevačka klisura (gorge), sićevo, 24.5.2018, mim; suva planina (mt.), bojanine vode, 24.5.2018, ml; vranje, 7.6.2016, ns; solanum nigrum (solanaceae), niš, 8.10.2010, vž; vranje, 21.8.2016, ns; tamarix sp. (tamaricaceae), niš, niška banja, 29.5.2014, vž; tanacetum sp. (asteraceae), sićevačka klisura (gorge), sićevo, 24.5.2018, ml; tordylium maximum (apiaceae), niš, 20.5.2015, vž; torilis sp. (apiaceae), sićevačka klisura (gorge), sićevo, 7.7.2015, mim; valeriana officinalis (valerianaceae), vlasinsko jezero (lake), 21.7.2013, sss; viburnum lantana (viburnaceae), niš, 18.5.2013, mđ; xeranthemum annuum (asteraceae), prokuplje, gornja konjuša, 29.5.2016, mr; x. cylindraceum, sićevačka klisura (gorge), brljavski kamen, 7.7.2015, vž; yucca filamentosa (asparagaceae), niš, 2.7.2014, mm aphis galliscabri schrank, 1801 on: galium aparine, niš, 6.6.2017, mr; sićevačka klisura (gorge), sićevo, 24.5.2018, ml aphis gossypii glover, 1877 on: ballota nigra, niš, 18.5.2016, mr; prokuplje, gornja konjuša, 1.5.2016, mr; chrysanthemum indicum (asteraceae), lebane, konjino, 22.11.2014, sss; hibiscus syriacus (malvaceae), niš, 12.6.2011, vž; vranje, 9.5.2016, ns aphis grossulariae kaltenbach, 1843 on: lythrum salicaria (lythraceae), sićevačka klisura (gorge), sićevo, 7.7.2015, vž aphis hederae kaltenbach, 1843 on: hedera helix, niš, 1.6.2013, vž; prokuplje, gornja konjuša, 21.5.2017, mr aphis hieracii schrank, 1801 biologica nyssana ● 10 (1) september 2019: 49-57 ilić-milošević et al. ● diversity of aphids (homoptera: aphididae) in southeastern serbia 52 on: pilosella bauhini (asteraceae), sićevačka klisura (gorge), sićevo, 24.5.2018, vž aphis idaei van der goot, 1912 on: rubus ideus (rosaceae), suva planina (mt.), bojanine vode, 21.5.2014, vž aphis intybi koch, 1855 on: cichorium intybus, lebane, konjino, 31.5.2014, sss; leskovac, 28.5.2018, as; niš, 23.6.2016, mr; vranje, 28.5.2018, nm aphis nasturtii kaltenbach, 1843 on: jovibarba sp. (crassulaceae), niš, 20.6.2018, dn aphis nerii boyer de fonscolombe, 1841 on: nerium oleander (apocynaceae), leskovac, 5.6.2018, as aphis newtoni theobald, 1927 on: iris germanica (iridaceae), niš, 30.5.2013, vž aphis polygonata (nevsky, 1929) on: polygonum aviculare (polygonaceae), prokuplje, gornja konjuša, 29.5.2016, mr aphis pomi de geer, 1773 on: crataegus monogyna (rosaceae), vlasinsko jezero (lake), 15.6.2013, sss; cydonia oblonga (rosaceae), gadžin han, ovsinjinac, 20.5.2015, mr; malus pumila (rosaceae), niš, 30.5.2013, vž; niš, donji matejevac, 5.6.2013, vž; niš, pasi poljana, 27.5.2013, vž; prokuplje, gornja konjuša, 23.4.2016, mr; vranje, 28.5.2018, nm; vranje, golemo selo, 21.6.2017, nm; vranje, kopanjane, 1.7.2017, nm; prunus cerasifera (rosaceae), niš, 18.5.2016, mr; pyrus communis (rosaceae), prokuplje, 6.6.2017, dv; spiraea japonica (rosaceae), niš, niška banja, 3.5.2015, sss aphis ruborum (börner, 1932) on: rubus discolor, pčinja canyon, prohor pčinjski, 10.6.2014, vž; preševo, slavujevac, 10.6.2014, sss; r. fruticosus, niš, novo selo, 1.6.2015, mr; prokuplje, gornja konjuša, 23.5.2015, mr; rubus sp., lebane, konjino, 31.5.2014, sss; niš, 21.5.2013, vž; niš, niška banja, 23.5.2014, vž; sićevačka klisura (gorge), 4.6.2011, sss; sićevačka klisura (gorge), sićevo, 28.5.2013, vž; vranje, 4.5.2016, ns; r. ulmifolium, pčinja canyon, prohor pčinjski, 10.6.2014, sss aphis sambuci linnaeus, 1758 on: sambucus nigra (viburnaceae), merošina, oblačinsko jezero (lake), 19.4.2018, vž; niš, 15.5.2015, ml; prokuplje, 6.6.2017, dv; prokuplje, gornja konjuša, 21.5.2017, mr; sićevačka klisura (gorge), 12.5.2013, sss; suva planina (mt.), bojanine vode, 24.5.2018, ml aphis sanguisorbae (schrank, 1801) on: sanguisorba minor (rosaceae), niš, donji matejevac, 5.6.2013, vž; pčinja canyon, gornji starac, 10.6.2014, sss; prokuplje, gornja konjuša, 11.6.2017, mr aphis sedi kaltenbach, 1843 on: jovibarba heuffelii, niš, 13.6.2017, dn aphis spiraecola patch, 1914 on: chaenomeles japonica (rosaceae), niš, 30.5.2013, vž; malus pumila, leskovac, 7.5.2018, as; scabiosa argentea (dipsacaceae), pčinja canyon, prohor pčinjski, 10.6.2014, vž; campsis radicans (bignoniaceae), niš, 14.5.2013, sss aphis spiraephaga f.p. muller, 1961 on: spiraea japonica, niš, niška banja, 3.5.2015, sss aphis taraxacicola (börner, 1940) on: taraxacum officinale (asteraceae), prokuplje, gornja konjuša, 11.6.2017, mr aphis ulmariae schrank, 1801 on: filipendula ulmaria (rosaceae), vlasinsko jezero (lake), 27.5.2018, vž aphis umbrella (börner, 1950) on: malva sylvestris, sićevačka klisura (gorge), ostrovica, 28.5.2013, mđ; grdelička klisura (gorge), 5.6.2014, sss; niš, 17.6.2016, mr; vranje, 26.4.2016, ns aphis urticata j.f. gmelin, 1790 on: urtica dioica (urticaceae), niš, niška banja, 31.5.2017, dv; niš, 25.5.2014, sss; lebane, 1.6.2013, sss; niš, kamenički vis, 26.4.2018, mim; pčinja canyon, gornji starac, 10.6.2014, sss; prokuplje, gornja konjuša, 1.5.2016, mr; sićevačka klisura (gorge), 24.5.2015, vž; sićevačka klisura (gorge), ostrovica, 29.5.2014, vž; sićevačka klisura (gorge), sićevo, 28.5.2013, zk; vlasinsko jezero (lake), 6.6.2014, vž; vranje, 4.5.2016, ns aphis veratri walker, 1852 on: veratrum album (melanthiaceae), vlasinsko jezero (lake), 19.6.2018, vž aphis verbasci schrank, 1801 on: verbascum sp. (scrophulariaceae), niš, popovac, 8.6.2013, vž; vlasinsko jezero (lake), 6.6.2014, sss aulacorthum solani kaltenbach, 1843 on: lathyrus tuberosus (fabaceae), prokuplje, bresničić, 16.6.2014, sss; vinca minor (apocynaceae), niš, 1.5.2014, vž biologica nyssana ● 10 (1) september 2019: 49-57 ilić-milošević et al. ● diversity of aphids (homoptera: aphididae) in southeastern serbia 53 brachycaudus cardui (linnaeus, 1758) on: carduus acanthoides, leskovac, 25.5.2018, as; merošina, oblačinsko jezero (lake), 15.6.2014, sss; niš, 7.7.2013, vž; pčinja canyon, prohor pčinjski, 10.6.2014, vž; prokuplje, gornja konjuša, 11.6.2017, mr; carduus sp., pčinja canyon, prohor pčinjski, 10.6.2014, vž; cirsium acanthoides, lebane, konjino, 31.5.2014, sss; c. arvense, vlasinsko jezero (lake), 6.6.2014, sss; c. canddelabrum, sićevačka klisura (gorge), sićevo, 7.7.2015, mim; c. eriophorum, vlasinsko jezero (lake), 21.7.2013, sss; c. palustre, vlasinsko jezero (lake), 21.7.2013, sss; hibiscus syriacus, niš, 25.5.2013, vž; onopordum acanthium, niš, 3.6.2015, vž; niš, popovac, 8.6.2013, vž; preševo, slavujevac, 10.6.2014, sss; sićevačka klisura (gorge), ravni do, 24.5.2015, sss; prunus cerasifera, grdelička klisura (gorge), 5.6.2014, sss; niš, 22.5.2018, vž; prokuplje, gornja konjuša, 23.4.2016, mr; vlasinsko jezero (lake), 22.5.2016, vž; p. domestica, sićevačka klisura (gorge), 1.5.2013, vž; p. spinosa, niš, 23.5.2014, vž brachycaudus helichrisy (kaltenbach, 1843) on: matricaria chamomilla (asteraceae), prokuplje, gornja konjuša, 11.6.2017, mr; cephalaria transsilvanica (dipsacaceae), niš, 15.5.2013, vž; erigeron annuus (asteraceae), leskovac, 19.5.2017, as brachycaudus persicae passerini, 1860 on: prunus persica, niš, 20.5.2014, vž brachycaudus prunicola (kaltenbach, 1843) on: prunus cerasifera, niš, 3.6.2015, vž brachycaudus schwartzi (börner, 1931) on: prunus persica, niš, pasi poljana, 27.5.2013, vž brachycaudus sp. on: arctium lappa, merošina, oblačinsko jezero (lake), 15.6.2014, vž; prunus cerasifera, merošina, vlasinsko jezero (lake), 15.6.2013, vž brachycaudus tragopogonis (kaltenbach, 1843) on: scorzonera sp. (asteraceae), radan planina (mt.), 6.6.2015, vž; tragopogon dubius (asteraceae), niš, 24.5.2017, mr; prokuplje, gornja konjuša, 15.6.2016, mr; t. major, lebane, konjino, 31.5.2014, sss; niš, 23.5.2014, vž brevicoryne brassicae (linnaeus, 1758) on: alliaria petiolata (brassicaceae), gadžin han, ovsinjinac, 13.4.2016, mr calaphis flava mordvilko, 1928 on: betula pubescens (betulaceae), vlasinsko jezero (lake), 21.7.2013, sss capitophorus hippophaes walker, 1852 on: polygonum lapathifolium, niš, 13.10.2013, vž cavariella aegopodii (scopoli, 1763) on: aegopodium podagraria, niš, 2.6.2017, mim; chaerophyllum aureum (apiaceae), sićevačka klisura (gorge), ravni do, 24.5.2015, vž; foeniculum vulgare (apiaceae), sićevačka klisura (gorge), 6.6.2013, vž; sićevačka klisura (gorge), sićevo, 28.5.2013, zk cavariella theobaldi (gillette & bragg, 1918) on: pastinaca hirsuta, niš, 3.6.2015; vlasinsko jezero (lake), 15.6.2013, vž chaitophorus populeti (panzer, 1804) on: populus alba (salicaceae), leskovac, 19.5.2017, as chaitophorus sp. on: salix caprea (salicaceae), vlasinsko jezero (lake), 15.6.2013, sss; s. matsudana, niš, niška banja, 6.6.2013, vž chromaphis juglandicola (kaltenbach, 1843) on: juglans regia (juglandaceae), leskovac, 1.6.2013, vž; niš, 17.7.2011, vž; niš, popovac, 8.6.2013, vž; sićevačka klisura (gorge), ostrovica, 28.5.2013, mđ cinara cuneomaculata (del guercio, 1909) on: larix sp. (pinaceae), niš, 6.6.2017, mr cinara juniperi (de geer, 1773) on: juniperus oxicedrus (cupressaceae), pčinja canyon, gornji starac, 10.6.2014, sss cinara pilicornis (hartig, 1841) on: picea pungens (pinaceae), niš, 6.6.2017, mr cinara sp. on: pinus nigra (pinaceae), niš, gornji matejevac, 25.5.2014, vž; p. sylvestris, vlasinsko jezero (lake), 6.6.2014, sss corylobium avellanae (schrank, 1801) on: corylus avellana (corylaceae), niš, 6.6.2017, mr; niš, novo selo, 1.6.2015, mr ctenocallis dobrovljanskyi klodnitsky, 1924 on: chamaecytisus heuffelii (fabaceae), vlasinsko jezero (lake), 28.9.2017, vž drepanosiphum platanoidis (schrank, 1801) on: acer platanoides, vlasinsko jezero (lake), 6.8.2015, vž dysaphis crataegi (kaltenbach, 1843) on: orlaya grandiflora, radan planina (mt.), 12.6.2016, sss; vlasinsko jezero (lake), 15.6.2013, sss biologica nyssana ● 10 (1) september 2019: 49-57 ilić-milošević et al. ● diversity of aphids (homoptera: aphididae) in southeastern serbia 54 dysaphis devecta (walker, 1849) on: malus pumila, niš, novo selo, 17.4.2016, mr dysaphis plantaginea (passerini, 1860) on: malus pumila, niš, donji matejevac, 5.6.2013, vž; niš, pasi poljana, 27.5.2013, vž; vranje, 9.5.2016, ns; vranje, golemo selo, 3.5.2017, nm; vranje, katun, 8.5.2017, nm dysaphis pyri (boyer de fonscolombe, 1841) on: pyrus communis, lebane, konjino, 24.4.2016, sss; pyrus sp., vranje, 9.5.2016, ns dysaphis reaumuri (mordvilko, 1928) on: pyrus spinosa, preševo, slavujevac, 10.6.2014, sss dysaphis sp. on: foeniculum vulgare, sićevačka klisura (gorge), sićevo, 6.6.2013, vž; malus pumila, leskovac, 13.5.2018, as eucallipterus tiliae (linnaeus, 1758) on: tilia sp. (malvaceae), niš, niška banja, 6.6.2013, vž eulachnus sp. on: pinus nigra, vlasinsko jezero (lake), 7.8.2015, sss; p. sylvestris, vlasinsko jezero (lake), 6.8.2015, sss hyadaphis coriandri (b. das, 1918) on: foeniculum vulgare, niš, gornji matejevac, 25.5.2014, vž hyalopterus pruni (geoffroy, 1762) on: prunus cerasifera, niš, 26.4.2014, vž; vranje, 9.6.2016, ns; p. cerasus, vranje, 8.6.2016, ns; p. domestica, prokuplje, gornja konjuša, 13.6.2017, mr; vranje, 30.5.2017, ns; p. persica, niš, novo selo, 5.6.2013, vž; p. spinosa, niš, 7.5.2014, vž; vranje, 8.6.2016, ns hyperomyzus lactucae (linnaeus, 1758) on: sonchus sp. (asteraceae), grdelička klisura (gorge), 5.6.2014, sss; niš, 28.5.2014, sss; niš, gornji matejevac, 25.5.2014, vž liosomaphis berberidis (kaltenbach, 1843) on: berberis sp. (berberidaceae), vranje, 19.6.2016, ns macrosiphoniella artemisiae boyer de fonscolombe, 1841 on: artemisia absinthium (asteraceae), sićevačka klisura (gorge), 4.6.2011, vž; vladičin han, 2.6.2011, ljm; a. vulgaris, leskovac, 25.5.2018, as; niš, kamenički vis, 4.6.2017, nm macrosiphoniella sanborni (gillette, 1908) on: chrysanthemum sp., leskovac, 13.5.2018, as; niš, 3.11.2017, mr macrosiphoniella sp. on: achillea millefolium, vlasinsko jezero (lake), 8.8.2015, sss; centaurea rhenana, vlasinsko jezero (lake), 15.6.2013, vž macrosiphum euphorbiae (thomas, 1878) on: euphorbia esula (euphorbiaceae), niš, 26.4.2014, vž; malva sylvestris, vranje, 9.6.2016, ns macrosiphum funestum (macchiati, 1885) on: rubus sp., radan planina (mt.), 6.6.2015, vž macrosiphum rosae (linnaeus, 1758) on: dipsacus sp. (dipsacaceae), leskovac, 25.5.2018, as; rosa canina, niš, 30.5.2010, vž; prokuplje, gornja konjuša, 23.4.2016, mr; rosa sp., leskovac, 8.6.2018, as; niš, 31.5.2011, vž; niš, donji matejevac, 5.6.2013, vž; radan planina (mt.), 6.6.2015, vž; vranje, 7.6.2016, ns; scabiosa columbaria, vlasinsko jezero (lake), 19.6.2018, vž melanaphis pyraria (passerini, 1862) on: pyrus communis, leskovac, 1.6.2013, vž; prokuplje, gornja konjuša, 21.5.2017, mr metopeurum fuscoviride stroyan, 1950 on: tanacetum sp., sićevačka klisura (gorge), sićevo, 24.5.2018, ml; vranje, 20.6.2016, ns; t. vulgare, suva planina (mt.), bojanine vode, 7.7.2015, ml; svodje, 6.6.2014, vž; vlasinsko jezero (lake), 30.6.2016, sss metopolophium dirhodum (walker, 1849) on: triticum aestivum (poaceae), leskovac, 1.6.2013, vž microlophium carnosum (buckton, 1876) on: urtica dioica, niš, 18.5.2016, mr myzocallis carpini (koch, 1855) on: carpinus betulus (corylaceae), niš, niška banja, 29.5.2013, vž myzocallis coryli (goeze, 1778) on: corylus avellana, niš, novo selo, 1.6.2015, mr; c. colurna, niš, 21.5.2013, sss myzus cerasi (fabricius, 1775) on: prunus avium, leskovac, 1.6.2013, vž; niš, 30.5.2013, vž; niš, pasi poljana, 27.5.2013, vž; vranje, 4.5.2016, ns; p. cerasifera, niš, 18.5.2016, mr; vranje, 22.8.2016, ns; p. cerasus, leskovac, 18.5.2017, as; niš, 15.5.2013, sss; vranje, 9.5.2016, ns myzus linariae holman, 1965 on: linaria genistifolia (plantaginaceae), niš, gornji matejevac, 10.5.2015, sss; (plantaginaceae), niš, 2.7.2015, vž biologica nyssana ● 10 (1) september 2019: 49-57 ilić-milošević et al. ● diversity of aphids (homoptera: aphididae) in southeastern serbia myzus lythri (schrank, 1801) on: lythrum salicaria, niš, 2.7.2013, vž; sićevačka klisura (gorge), 28.5.2013, zk; sićevačka klisura (gorge), ostrovica, 29.5.2014, vž; vlasinsko jezero (lake), 15.6.2013, sss myzus persicae sulzer, 1776 on: capsicum annuum (solanaceae), lebane, 17.10.2011, sss; niš, 18.1.2019, vž; niš, popovac, 26.5.2013, vž; viola cornuta (violaceae), niš, 10.11.2011, vž; niš, popovac, 26.10.2011, vž myzus varians davidson, 1912 on: prunus persica, niš, 25.5.2014; vranje, 28.5.2018, nm nasonovia sp. on: pilosella officinarum (asteraceae), vlasinsko jezero (lake), 21.7.2013, sss ovatus insitus (walker, 1849) on: cydonia oblonga, gadžin han, ovsinjinac, 20.5.2015, mr panaphis juglandis (goeze, 1778) on: juglans regia, niš, 24.5.2017, mr; sićevačka klisura (gorge), 28.5.2013, mđ periphyllus aceris (linnaeus, 1761) on: acer campestre, niš, kamenički vis, 4.6.2014, sss; niš, gornji matejevac, 10.5.2015, vž; a. monspessulanum, niš, gornji matejevac, 26.4.2018, mim; niš, kamenički vis, 26.4.2018, mim periphyllus sp. on: acer campestre, niš, niška banja, 11.5.2014, sss; a. monspessulanum, niš, niška banja, 19.5.2015, vž; a. platanoides, niš, 22.5.2010, vž; a. pseudoplatanus, suva planina (mt.), bojanine vode, 21.5.2018, ml phorodon humuli (schrank, 1801) on: prunus cerasifera, lebane, konjino, 31.5.2014, sss; niš, niška banja, 23.5.2014, vž; prokuplje, gornja konjuša, 1.5.2016, mr; niš, 21.5.2013, vž; p. domestica, lebane, 1.6.2013, sss; niš, 28.5.2015, mr; niš, popovac, 20.5.2013, vž pterocomma pilosum buckton, 1879 on: salix alba, niš, donji matejevac, 3.6.2010, vž; s. caprea, vlasinsko jezero (lake), 18.6.2010, vž pterocomma populeum (kaltenbach, 1843) on: populus alba, sićevačka klisura (gorge), 29.5.2010, sss; p. nigra, lebane, konjino, 3.5.2014, sss; sićevačka klisura (gorge), 29.5.2010, sss pterocomma salicis (linnaeus, 1758) on: salix caprea, vlasinsko jezero (lake), 15.6.2013, vž pterocomma sp. on: populus tremula, niš, 25.5.2010, mim; salix caprea, stara planina (mt.), babin zub, 4.7.2010, vž rhopalosiphum maidis (fitch, 1856) on: zea mays (poaceae), sićevačka klisura (gorge), sićevo, 17.7.2013, vž rhopalosiphum nymphaeae (linnaeus, 1761) on: typha latifolia (typhaceae), bela palanka, divljana, 6.6.2014, vž; niš, niška banja, 23.7.2013, vž rhopalosiphum padi (linnaeus, 1758) on: hordeum murinum (poaceae), grdelička klisura (gorge), 5.6.2014, sss schizaphis graminum (rondani, 1852) on: zea mays, sićevačka klisura (gorge), sićevo, 17.7.2013, vž semiaphis pastinacae börner, 1950 on: pastinaca hirsuta, vlasinsko jezero (lake), 19.6.2018, vž sipha maydis passerini, 1860 on: elytrigia repens (poaceae), sićevačka klisura (gorge), 28.5.2013, zk; arrhenatherum elatius (poaceae), radan planina (mt.), 6.6.2015, vž; zea mays, niš, lalinac, 8.6.2013, sss sitobion avenae (fabricius, 1775) on: avena sativa (poaceae), lebane, 19.6.2011, sss; prokuplje, gornja konjuša, 11.6.2017, mr; triticum aestivum, leskovac, 1.6.2013, vž smiela sp. on: berteroa sp., leskovac, 21.5.2017, as tetraneura sp. on: ulmus campestris (ulmaceae), lebane, 1.6.2013, sss; ulmus sp. (ulmaceae), sićevačka klisura (gorge), 24.5.2018, vž thelaxes dryophilus (schrank, 1801) on: quercus robur (fagaceae), radan planina (mt.), 12.6.2016, sss tuberculatus sp. on: quercus cerris, niš, niška banja, 29.5.2013, sss uroleucon cichorii (koch, 1855) on: cichorium intybus, leskovac, 31.5.2017, as; crepis biennis, prokuplje, 6.6.2017, dv; c. foetida, leskovac, 19.5.2017, as; niš, kamenički vis, 4.6.2017, dv; prokuplje, gornja 55 biologica nyssana ● 10 (1) september 2019: 49-57 ilić-milošević et al. ● diversity of aphids (homoptera: aphididae) in southeastern serbia konjuša, 11.6.2017, mr; vlasinsko jezero (lake), 15.6.2013, sss; crepis sp., bela palanka, divljana, 6.6.2014, vž; vlasinsko jezero (lake), 19.6.2018, vž; lapsana communis (asteraceae), prokuplje, 6.6.2017, dv uroleucon jaceae (linnaeus, 1758) on: carthamus creticus (asteraceae), preševo, slavujevac, 10.6.2014, sss; c. lanatus, merošina, oblačinsko jezero (lake), 15.6.2014, vž; prokuplje, bresničić, 15.6.2014, sss; centaurea jacea, radan planina (mt.), 12.6.2016, sss; vlasinsko jezero (lake), 19.6.2018, vž; c. rhenana, radan planina (mt.), 12.6.2016, sss; vlasinsko jezero (lake), 15.6.2013, vž; c. salonitana, niš, 3.6.2015, vž; c. solstitialis, prokuplje, bresnčić, 15.6.2014, sss uroleucon picridis (fabricius, 1775) on: cichorium intybus, vlasinsko jezero (lake), 21.7.2013, sss; crepis paludosa, vlasinsko jezero (lake), 21.7.2013, sss uroleucon sonchi (linnaeus, 1767) on: sonchus oleraceus, prokuplje, gornja konjuša, 11.6.2017, mr uroleucon sp. on: chondrilla juncea (asteraceae), niš, 21.5.2013, vž; inula sp. (asteraceae), lebane, 1.6.2013, sss; lapsana communis, suva planina (mt.), bojanine vode, 24.5.2018, vž uroleucon tussilaginis (walker, 1850) on: tussilago farfara (asteraceae), banja topilo, 21.4.2018, vž discussion in this study we sampled 211 plant species, which were attacked by 118 aphid species within 46 genera and 9 subfamilies, formed 436 bi-trophic associations. most of the aphid species were collected on plants from the family asteraceae (105 associations) belonging to 27 genera, followed by the family rosaceae (88 associations) from 11 genera. among the aphid genera, aphis linnaeus was at the same time the most common (211 associations) and the most diverse aphid genus (34 species). within this genus, species a. fabae scopoli established the largest number of trophic associations (88), usually with plants from families asteraceae and rosaceae. aphis fabae has an almost cosmopolitan distribution and represents one of the most important agricultural pests (blackman & eastop, 2000, minks & harrewijn, 1989). also, a. craccivora koch formed 27 associations. as well as a. fabae, a. craccivora is a polyphagous species. however it prefers plants from family fabaceae (blackman & eastop, 2007). after the genus aphis, the next most diverse genera were brachycaudus van der goot (7 species), dysaphis börner (6 species) and uroleucon mordvilko (6 species). within the genus brachycaudus, the most frequently recorded species was b. cardui. this aphid altered the hosts between various fruit trees of genus prunus l. and usually carduus l. and cirsium mill. species (influentialpoints. 2018). within the genera dysaphis and uroleucon, d. plantaginea (linnaeus), u. cichorii (passerini) and u. jaceae (linnaeus) formed the largest number of associations. the primary hosts for d. plantaginea were species from genus malus mill (influentialpoints. 2018). in our study, we noticed that this aphid caused damage on species m. pumila mill. species u. cichorii mordvilko was usually collected on crepis l., while u. jaceae (linnaeus) on carthamus l. and centaurea l. species macrosiphum rosae (linnaeus) and myzus cerasi (fabricius) formed nine associations. all other recorded aphid species appeared in lower frequencies. this research contributes to a better knowledge about diversity of aphids in the territory of serbia. acknowledgements. this study was supported by the ministry of education, science and technological development of the republic of serbia (iii43001). references blackman, r.l., eastop, v.f. 1994: aphids on the world’s trees: an identification and information guide. cab international, wallingford, uk. 987p. blackman, r.l., eastop, v.f. 2000: aphids on the world’s crops: an identification and information guide. john wiley & sons, chichester, uk. 466p. blackman, r.l., eastop, v.f. 2006: aphids on the world’s herbaceous plants and shrubs, 1 volume set. john wiley & sons, chichester, uk. 1024p. blackman, r.l., eastop, v.f. 2007: taxonomic issues. in: van emden, h.f., harrington, r. (eds.), aphids as crop pests, 1-29, cab international, uk. coeur d’acier, a., perez hidalgo, n., petrovicobradovic, o. 2010: aphids (hemiptera, aphididae) chapter 9.2. in: roques, a., rabitsch, w., rasplus, j.y., lopez-vaamonde, c., nentwig w., kenis, m. (eds), terrestrial invertebrate invasions in europe, biorisk 4(1): 435–474. gálvez, g.e., morales, f. j. 1989: aphidtransmitted viruses. in: schwartz, h.f., pastorcorrales, m.a. (eds.), bean production problems in the tropics, 2nd. ed., 333-361, international center for tropical agriculture (ciat), cali, colombia. 56 biologica nyssana ● 10 (1) september 2019: 49-57 ilić-milošević et al. ● diversity of aphids (homoptera: aphididae) in southeastern serbia influentialpoints. 2018: influentialpoints – services for ecologists, medics and veterinarians. aphids identifications, characteristics of genera. available on: https://influentialpoints.com/gallery/aphid_ genera.htm josifović, m. (ed.) 1970-1977: flora sr srbije, i-ix. sanu. beograd. minks, a.k., harrewijn p. 1989: aphids—their biological, natural enemies and control, vol. c. elsevier science publishers b.v., amsterdam. petrović, o. 1998: check-list of aphids (homoptera: aphididae) in serbia. acta entomologica serbica, 3 (1/2): 9-42. petrović-obradović, o. 2003. biljne vaši (homoptera: aphididae) srbije. poljoprivredni fakultet univerziteta u beogradu, beograd – zemun. 152p. petrović-obradović, o., tomanović, ž., poljaković-pajnik, l., vučetić, a. 2007: an invasive species of aphid, prociphilus fraxinifolii (hemiptera, aphididae, eriosomatinae) found in serbia. archives of biological sciences, 59 (1): 9-10. petrović-obradović, o., tomanović, ž., poljaković-pajnik, l., hrnčić, s., vučetić, a, radonjić, s. 2010: new invasive species of aphids (hemiptera, aphididae) in serbia and montenegro. archives of biological sciences, 62 (3): 775-780. petrović-obradović, o., radonjić, a., jovičić, i., petrović, a., kocić, k., tomanović, ž. 2018: alien species of aphids (hemiptera: aphididae) found in serbia, new to the balkan peninsula. phytoparasitica, 46 (5): 653-660. poljaković-pajnik, l., petrović, o. 2002: bowlegged fir aphid cinara curvipes (patch) (aphididae, homoptera) new pest of abies concolor in serbia. acta entomologica serbica, 7 (1/2): 147-150. vučetić, a., jovičić, i., petrović-obradović, o. 2014: several new and one invasive aphid species (aphididae, hemiptera) caught by yellow water traps in serbia. phytoparasitica, 42 (2): 247-257. 57 biologica nyssana ● 10 (1) september 2019: 49-57 ilić-milošević et al. ● diversity of aphids (homoptera: aphididae) in southeastern serbia tošić, s. et al.  micropropagation of micromeria juliana (l.) nenth. ex rchb.... biologica nyssana 6 (1)  september 2015: 17-23 tošić, s. et al.  micropropagation of micromeria juliana (l.) nenth. ex rchb.... 17 original article received: 14 july 2015 revised: 11 august 2015 accepted: 20 august 2015 micropropagation of micromeria juliana (l.) benth. ex rchb. (lamiaceae) svetlana tošić*, sanja nikolić, marija jovanović, bojan zlatković, dragana stojičić university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia * e-mail: tosicsvetlana59@yahoo.com abstract: tošić, s., nikolić, s., jovanović, m., zlatković, b., stojičić, d.: micropropagation of micromeria juliana (l.) benth. ex rchb. (lamiaceae). biologica nyssana, 6 (1), september 2015: 17-23. micromeria juliana belongs to family lamiaceae, whose representatives are characterized by a significant level of essential oils and antioxidant components. several species of genus micromeria contain biologically active substances and are therefore used in folk medicine, food industry and cosmetic industry. methods of their tissue culture may provide rapid mass multiplication of plants for various purposes, including research on production, accumulation and metabolism of important secondary metabolites. the goal of this paper was to determine the protocol for regeneration of m. juliana plants through use of nodal explants on nutritive substrate with various growth regulators. the greatest number of axillary buds was formed in explants grown on ms nutritive medium with 3 μm benzyladenine (ba) and 0.57 μm indole-3-acetic acid (iaa). the explants grown at the medium without any growth regulators and the medium with auxin have shown spontaneous root formation. key words: axillary bud induction, shoot culture, biomass production apstrakt: tošić, s., nikolić, s., jovanović, m., zlatković, b., stojičić, d.: micropropagacija vrste micromeria juliana (l.) benth. ex rchb. (lamiaceae). biologica nyssana, 6 (1), septembar 2015: 17-23. micromeria juliana (l.) benth pripada familiji lamiaceae koja se odlikuje značajnim sadržajem etarskih ulja i antioksidativnih komponenti. vrste roda micromeria sadrže biološke aktivne supstance zbog čega se koriste u narodnoj medicini, kao i u prehrambenoj i kozmetičkoj industriji. metode kulture tkiva mogu da obezbede brzu i masovnu multiplikaciju biljaka za različite svrhe kao što je proučavanje produkcije, akumulacije i metabolizma značajnih sekundarnih metabolita. cilj ovog rada je da ustanovi protokol za regeneraciju biljaka m. juliana korišćenjem nodalnih eksplantata, na hranljivoj podlozi sa različitim regulatorima rastenja. najveći broj aksilarnih pupoljaka formiran je na eksplantatima gajenim na ms hranljivoj podlozi sa 3 μm benziladenina (ba) i 0,57 μm indol-3-sirćetne kiseline (iaa). eksplantati gajeni na podlozi bez regulatora rastenja i na podlozi sa auksinom su spontano ožiljavali. key words: indukcija aksilarnih pupoljaka, kultura izdanaka, produkcija biomase 6 (1) • september 2015: 17-23 biologica nyssana 6 (1)  september 2015: 17-23 tošić, s. et al.  micropropagation of micromeria juliana (l.) nenth. ex rchb.... 18 introduction in relation to the morphological characteristics and phylogenetic relationships, species of genus micromeria are grouped into three sections: cymularia, micromeria and pseudomelissa. the representatives of this genus in the flora of serbia (d i k l i ć , 1974) belong to micromeria (m. croatica, m. juliana, m. cristata and m. parviflora) and pseudomelissa (m. thymifolia, m. albanica and m. pulegium). m. juliana is comparatively rare species in serbia, recorded only in the vicinity of kačanik in kosovo and metohija province (d i k l i ć , 1974). according to c h a t e r & g u i n e a (1973) and š i l i ć (1979) the native range of the species includes countries of mediterranean region (montenegro, croatia, herzegovina, macedonia, greece, bulgaria, albania, france, portugal, italy, turkey, northwestern africa, as well as the western part of asia minor and crete). it inhabits dry, sunny sandbased habitats, stony ground and crevices in limestone rocks (fig. 1). this is a perennial, dwarf shrubby plant, with many erect, mostly simple stems, up to 40 cm in height. the leaves are narrowly ovate, upper linear-lanceolate, entire, with revolute margins and opposite. the root and the basal part of stems are somewhat fruticose. flowers are comparatively small, purple in sessile or shortly pedunculate verticillasters, organized into long, common inflorescences (d o r o s z e n k o , 1986). the representatives of genus micromeria are characterized by low morphological diversity and their growth and development are influenced by numerous ecological factors, leading to high diversity of their secondary metabolites. due to presence of secondary metabolites, species of genus micromeria show antioxidant (g ü l l ü c e et al., 2004; s t o j a n o v i ć & p a l i ć , 2008; v l a d i m i r k n e ž e v i ć et al., 2011;), antibacterial (t a b a n c a et al., 2001; d u r u et al., 2004; s a r a c & u g u r , 2007; s t o j a n o v i ć & p a l i ć , 2008), antifungal (m a r i n k o v i ć et al., 2003; a b o u j a w d a h et al., 2004; ö z c a n , 1999), antiphytoviral (b e z i ć et al., 2013), insecticidal (a s l a n et al., 2005), bioherbicidal (d u d a i et al., 1999) and allelopathic (d u d a i et al., 2009) biological activity. due to their pharmacological properties, plant species of genus micromeria are used in folk medicine for treating gastrointestinal and respiratory tract, skin infections and wounds, as well as for soothing pain and preventing insomnia (š a r i ć k u n d a l i ć et al., 2011). they are also used in cooking in order to improve taste and aroma of food, as additives and spices (t a b a n c a et al., 2001; g ü l l ü c e et al., 2004). fig. 1. micromeria juliana, canyon of river morača (montenegro) the high individual variability characterizing species of genus micromeria is a limiting factor for the intensity of their use in pharmaceutical purposes. the solution to this problem may include use of plant tissue cultures in vitro (s a h a et al., 2012). the methods of culture in vitro are important in reproduction of endemic and rare plants, contributing to preservation of biodiversity (f a y 1992; r e e d et al., 2011). growing in culture in vitro decreases the exploitation pressure on natural populations and in a short period of time may yield several thousand plants from a small amount of plant samples. the only species of genus micromeria previously grown in culture in vitro used to be m. pulegium (t o š i ć et al., 2015). material and methods plant material and source of explants the surface sterilization included treating seeds with 30% solution of na-hypochlorite with 4% of active chlorine for 25 minutes. after three rinsing in sterile distilled water, the seeds were treated with 5% solution of nystatin for 24 hours in order to eliminate possible fungal infections. next step included rinsing seeds in sterile distilled water three times, and then they were placed individually in test tubes on nutritive medium in aseptic conditions in order to germinate. after the seeds have germinated, the resulting plants were used for preparing explants – nodal segments. in order to introduce m. juliana into in vitro culture, seeds were biologica nyssana 6 (1)  september 2015: 17-23 tošić, s. et al.  micropropagation of micromeria juliana (l.) nenth. ex rchb.... 19 collected from plants in their natural habitat in canyon of river morača (montenegro). a voucher specimens, under the acquisition number 10850, are deposited at the herbarium collection of department of biology and ecology, faculty of science and mathematics in niš (hmn). culture medium and culture condition the nutritive medium used in culture in vitro was m u r a s h i g e & s k o o g (1962) – ms medium, including appropriate macroand micro mineral salts and organic additions. all medium have included 100 mg l-1 myo-inositol, 30 g l-1 sucrose and 7 g l-1 agar. the ph value of each medium was checked immediately before autoclaving and if necessary adjusted to ph 5.8 by addition of 0.1 n naoh. the sterilized nodal segments of m. juliana of approximately same size (5 mm) were placed on nutritive medium: without any growth regulators, with cytokinin ba (0.1, 0.3, 1, 3, 10 µm), with auxin iaa (0.57µm), and with combination of ba and iaa. each medium was used for 30 explants, 10 in each of three jars. the plants were maintained at the temperature 23˚c ± 3˚c and photoperiod of 16 hours of light and 8 hours of dark. the density of light flux was 47 µmol s-1m-2. measured parameters the explants were grown for four weeks until they formed axillary buds. the explants reacting positively to treatment were recorded and following parameters were measured: number of axillary buds per explant, length of each bud, fresh and dry mass of each explant. buds shorter than 1 mm were disregarded. after the fresh mass was measured, explants were dried in separate paper containers and dry mass was then measured by using the same analytic scales. shoot forming capacity (sfc) index was calculated according to martinez-pulido et al. (1992) as follows: sfc index = % explant with shoots x mean number of shoot per explants / 100. statistical methods data processing was performed by using the statistically-graphic package statgraphics, procedure anova and test lcd at the significance level p<0.05. results and discussion culture of m. juliana was formed by germinating seeds on ms nutritive medium. use of ms nutritive medium produces favorable results for in vitro germinating of numerous species (m o l i a & k w a p a t a , 2000), including many species of lamiaceae. nodal segments were isolated from in vitro germinated seedlings and placed in inductive medium. the initiation of the culture was achieved according to the same procedure, using nodal segments of seedlings, in clinopodium odorum (d i a z et al., 2012). use of nodal segments for regeneration of plants with the goal of mass production is considered a simple method for many species of lamiaceae, and at the same time it is the most reliable method for achieving uniform plant material (g e o r g e & s h e r r i n g t o n , 1984; d o d e et al., 2003). all explants on the ms nutritive medium without regulators (100%) have developed axillary buds (tab. 1). the smallest ratio of explants with axillary buds (63.3%) was recorded at the medium with 0.57 μm iaa. at the explants grown on medium without growth regulators there were on average 4.97 buds per explant. most buds were formed on explants grown on medium with 3 μm ba and 0.57 μm iaa. their number is statistically significantly greater than the number of buds formed on explants on all other medium. explants from this medium are characterized with the greatest multiplication index of 8.73. after three months, one explant may produce 3665 buds which potentially may produce new plants. low-concentration auxin combined with cytokinin often assists development of buds on explants (t e j a v a t h i & i n d i r a , 2011). the greatest average length of axillary buds was developed on explants grown at medium with 0.57 μm iaa (tab. 1). all concentrations of ba used in this study with or without auxin have inhibited elongation of axillary buds in m. juliana. the inhibitory effect of high cytokinin concentrations on elongation of buds was recorded in mentha piperita (g h a n t i et al., 2004) and melissa officinalis (t a v e r s et al., 1996). cytokinins may show inhibitory activity on elongation of axillary buds in spite of promoting their proliferation (v a n s t a d e n et al., 2008). the explants with the greatest average mass were grown on ms medium with 3 μm ba and 0.57 μm iaa (tab. 2). on that medium they formed the greatest number of buds, contributing to the greatest biomass. the explants from these medium were healthy, with good branching, green, quite uniform in stature, with shorter internodes and almost bushlike habitus (fig. 2). combination of cytokinin and auxin was used by numerous authors in order to induce bud formation. for example in agastache rugosa the greatest number and best quality of buds was achieved on ms medium with this particular combination of hormones (z i e l i n s k a et al., 2011). the comparison of explants of m. juliana biologica nyssana 6 (1)  september 2015: 17-23 tošić, s. et al.  micropropagation of micromeria juliana (l.) nenth. ex rchb.... 20 table 1. effect of plant growth regulators on micromeria juliana shoot proliferation, number of shoots per explants and shoot length after 28 days of culture. pgr (μm) explants producing shoots (%) shoots per explant shoot length (mm) sfc index ba iaa 100 4,97 ± 0,50abc 3,95 ± 0,31bc 5,86 0.1 96.7 5,86 ± 0,40bcd 3,25 ± 0,27ab 6,97 0.3 100 6,00 ± 0,36de 3,18 ± 0,24ab 5,72 1 96.7 7,21 ± 0,43e 3,19 ± 0,20ab 3,82 3 83.3 5,72 ± 0,38cd 2,72 ± 0,19a 4,14 10 70.0 3,95 ± 0,49ab 2,46 ± 0,23a 3,28 0.57 63.3 4,68 ± 0,33abcd 6,47 ± 0,79d 2,96 0.1 0.57 100 5,73 ± 0,42d 4,12 ± 0,37 c 5,73 0.3 0.57 93.3 3,68 ± 0,49a 4,18 ± 0,42 c 3,43 1 0.57 96.7 4,52 ± 0,42abc 3,94 ± 0,36 bc 4,37 3 0.57 93.3 9,39 ± 0,65f 3,84 ± 0,20 bc 8,73 10 0.57 100 4,37 ± 0,38ab 2,56 ± 0,18 a 4,37 the values are means ± standard error. means within the column of each factor followed by the same letter are not significantly different according to lsd multiple range test (p  0.05) table 2. effect of plant growth regulators on micromeria juliana biomass production (using nodal shoot segments as explants) after 28 days of culture. pgr (μm) biomass fresh weight per explant (mg) biomass dry weight per explant (mg) ba iaa 0.017 ± 0.0019a 0.0028 ± 0.00030a 0.1 0.022 ± 0.0016a 0.0031 ± 0.00019a 0.3 0.021 ± 0.0013a 0.0031 ± 0.00021a 1 0.022 ± 0.0016a 0.0034 ± 0.00024ab 3 0.015 ± 0.0010a 0.0022 ± 0.00015a 10 0.014 ± 0.0014a 0.0020 ± 0.00021a 0.57 0.046 ± 0.0079cd 0.0056 ± 0.00066bcd 0.1 0.57 0.027 ± 0.0019ab 0.0043 ± 0.00027abc 0.3 0.57 0.044 ± 0.0053c 0.0057 ± 0.00066cd 1 0.57 0.051 ± 0.0013cd 0.0060 ± 0.00057cd 3 0.57 0.061 ± 0.0042d 0.0070 ± 0.00041de 10 0.57 0.038 ± 0.0034bc 0.0085 ± 0.00074e the values are means ± standard error. means within the column of each factor followed by the same letter are not significantly different according to lsd multiple range test (p  0.05). fig. 2. m. juliana on ms medium with 0,57 µm iaa biologica nyssana 6 (1)  september 2015: 17-23 tošić, s. et al.  micropropagation of micromeria juliana (l.) nenth. ex rchb.... 21 grown on medium with ba and explants grown on medium with combination of cytokinin and auxin has shown an obvious prominent increase in biomass yield in medium supplemented with auxin. low-concentration auxin and ba have synergic effect contributing to formation of biomass in explants of m. juliana. ba and iaa also had positive effect on bud induction in species ocimum killimandscharicum l. (s h a r m a et al., 2013), ocimum citriodorum vis. (j a n a r t h a n a m & s u m a t h i , 2012), and in species mentha viridis l. (r a h m a n et al., 2013). medium with combination of auxin and cytokinin was effective for regeneration of ocimum basilicum (n i r m a l & s e h g a l , 1999) and salvia brachyodon (m i s i c et al., 2006). of the explants grown on medium without growth regulators, a small number has spontaneously developed adventive roots, but almost all explants grown on medium with auxin and without cytokinin have developed adventive roots, indicating strong role of auxin in development of adventive roots (fig. 2). conclusion micromeria juliana (l.) benth. ex rchb. was successfully introduced into in vitro culture. results of this study have shown that the maximum number of axillary buds (9.39) was formed in explants grown on nutritive medium with 3 μm ba and 0.57 μm iaa. the nutritive medium with 0.57 μm iaa was the most efficient in elongation of axillary buds, and the average bud length was 6.47 mm. combination of auxin and high concentrations of ba (3 and 10 μm, respectively) induced formation of the greatest fresh and dry mass, respectively. root formation in nodal explants took place in medium without growth regulators and in medium that contained both auxin and low concentration of ba. with the increase in concentration of ba there was a decrease of the number of explants developing adventive roots. this study has shown that the described micropropagation protocol enabled production of a substantial number of plantlets, starting with a small quantity of plant material. in addition, micropropagation of m. juliana may be considered as an alternative method for production of secondary metabolites. acknowledgements. the ministry of education, science and technological development of the republic of serbia (grant 173015, 173030) supported this research. references abou-jawdah, y., wardan, r., sobh, h., salameh, a. 2004: antifungal activities of extracts from selected lebanese wild plants against plant pathogenic fungi. phytopathologia mediterranea, 43: 377–386. aslan, i., calmasur, o., sahon, f., caglar, o. 2005: insecticidal effects of essential plant oils against ephestia kuehniella (zell.), lasioderma serricorne (f.) and sitophilus granarius (l.). journal of plant diseases and protection, 112 (3): 257–267. bezić, n., dunkić, v., vuko, e. 2013: antiphytoviral activity of essential oils of some lamiaceae species and there most important compounds on cmv and tmv. in: méndez-vilas, a. (ed.), microbial pathogens and strategies for combating them: science, technology and education.2: 982-988.formatex research center, spain. chater, a.o., guinea e. 1973: micromeria bentham. in:tutin, t.g., j.r., heywood, v.h., moore, valentine, s.m., webb, d.a. (eds.), flora europaea. 3:167-170. the university press, cambridge. diaz, m.s., palacio, l., figueroa, a.c., goleniowski, m.e. 2012: in vitro propagation of muna-muna (clinopodium odorum (griseb.) harley). biotechnology research international, 2012: 1-6. diklić, n. 1974: micromeria benth. in: josifović, m. (ed.), flora srbije 6:458-462. srpska akademija nauka i umetnosti, beograd. dode, l.b., seixas, f.k., schuch, m.w., braga, e.j.b., bobrowski, v.l. 2003: in vitro clonal propagation of ocimum basilicum l. (lamiaceae). acta scientiarum. biological sciences, 25 (2): 439-441. doroszenko, a. 1986: taxonomic studies on the satureja complex (labiatae). ph.d. thesis, university of edinburgh, edinburgh (manuscr.). dudai, n., poljakoff-mayber, a., mayer, a.m., putievsky, e., lerner, h.r. 1999: essential oils as allelochemicals and their potential use as bioherbicides. journal of chemical ecology, 25(5): 1079-1089. dudai, n., chaimovitsh, d., larkov, o., fischer, r., blaicher, y., mayer, a.m. 2009: allelochemicals released by leaf residues of micromeria fruticosa in soils, their uptake and metabolism by inhibited wheat seed. plant soil, 314: 311–317. duru, m.e., öztürk, m., ugur, a., ceylan, o. 2004: the constituents of essential oil and in vitro antimicrobial activity of micromeria cilicica biologica nyssana 6 (1)  september 2015: 17-23 tošić, s. et al.  micropropagation of micromeria juliana (l.) nenth. ex rchb. 22 from turkey. journal of ethnopharmacology, 94: 43–48. fay, m.f. 1992: conservation of rare and endangered plants using in vitro methods. in vitro cellular and developmental biology. plant, 28: l-4. george, e.f., sherrington, p.d. 1984: plant propagation by tissue culture. exgetics ltd. basingstoke, england. ghanti, k., kaviraj, c.p., venugopal, r.b., jabeen, f.t.z., rao, s. 2004: rapid regeneration of mentha piperita l. from shoot tip and nodal explants. indian journal of biotechnology, 3: 594-598. güllüce, m., sökmen, m., şahin, f., sökmen, a., adigüzel, a., özer, h. 2004: biological activities of the essential oil and methanolic extract of micromeria fruticosa (l.) druce ssp. serpyllifolia (bieb.) p.h. davis plants from the eastern anatolia region of turkey. journal of the science of food and agriculture, 84(7): 735– 741. janarthanam, b., sumathi, e. 2012: plantlet regeneration from nodal explants of ocimum citriodorum vis.. bangladesh journal of scientific and industrial research, 47: 433 – 436. marinković, b., marin, p.d., knežević-vukčević, j., soković, m.d., brkić, d. 2002: activity of essential oils of thee micromeria spesies (lamiaceae) againd micromycetes and bacteria. phytoterapy research, 16 (4): 336-339. martinez-pulido, c., harry, i.s., thorpe, t.a. 1992: optimization of bud induction in cotyledonary explants of pinus canariensis. plant cell tissue and organ culture, 29:247–255. misic, d., grubisic, d., konjevic, r. 2006: micropropagation of salvia brachyodon through nodal explants. biologia plantarum, 50 (3): 473476. molia, m.f.a., kwapata, m.b. 2000: apomictic embryo development and survival in uapaca kirkiana under in vitro and in vivo seed germination. scientia horticulturae, 83: 139– 147. murashige, t., skoog, f. 1962: a revised medium for rapid growth and bioassays with tobacco tissue cultures. plant physiology, 15: 473-497. nirmal, k.s., sehgal, c.b. 1999: micropropagation of „holy basil‟ (ocimum sanctum linn.) from young inflorescences of mature plants. plant growth regulation, 29: 161-166. özcan, m. 1999: antifungal effects of micromeria myrtifolia boiss and hohen in boiss. and prangos uechtritzii (boiss.) hawsskn decoctions. acta alimentaria, 28: 355–360 rahman, m.m., ankhi, u.r., biswas, a. 2013: micropropagation of mentha viridis l.: an aromatic medicinal plant. international journal of pharmacy and life sciences, 4 (9): 29262930. reed, b.m., sarasan, v., kane, m., bunn, e., pence, v.c. 2011: biodiversity conservation and conservation biotechnology tools. in vitro cellular and developmental bioogy. plant, 47: 1–4. saha, s., kader, a., sengupta, c., ghosh, p. 2012: in vitro propagation of ocimum gratissimum l. (lamiaceae) and its evaluation of genetic fidelity using rapd marker. american journal of plant sciences, 3: 64-74. sarac n., ugur a. 2007: antimicrobial activites and usage in folkloric medicine of some lamiaceae species growing in mugla turkey. eurasian journal of biosciences, 4: 28-37. šarić-kundalić, b., dobes, c., klatte-asselmeyer, v., saukel, j. 2011: ethnobotanical survey of traditionally used plants in human therapy of east, north and north-east bosnia and herzegovina. journal of ethnopharmacology, 133: 1051–1076. sharma, d.k., sharma, t. 2013: biotechnological approaches for biodiversity conservation. indian journal of scientific research, 4 (1): 183-186. šilić, č. 1979: monografija rodova satureja l., calamintha miller, micromeria bentham, acinos miller i clinopodium l. u flori jugoslavije. zemaljski muzej, sarajevo. stojanovic, g., palic, i. 2008: antimicrobial and antioxidant activity of micromeria bentham species. current pharmaceutical design, 14 (29): 3196-3202. tabanca, n., kirimer, n., demirci, b., demirci, f., baser, k.h.c. 2001: composition and antimicrobial activity of the essential oils of micromeria cristata subsp. phrygia and the enantiomeric distribution of borneol. journal of agricultural and food chemistry, 49: 43004303. tavers, a.c., pimenta, m.c., goncalves, m.t. 1996: micropropagation of melissa officinalis through prokiferation af axillary shoots, plant cell reports, 15: 441-444. tejavathi, d.h., indira, m.n. 2011: in vitro regeneration of multiple shoots from the nodal explants of drymaria cordata (l.) wild. ex. roem. and schult. the bioscan, 6(4): 657-660. tošić, s., stojičić, d., stankov-jovanović, v., mitić, v., mihajilov-krstev, t., zlatković, b. 2015: chemical composition, antioxidant and antimicrobial activities of micropropagated and native micromeria pulegium (lamiaceae) biologica nyssana 6 (1)  september 2015: 17-23 tošić, s. et al.  micropropagation of micromeria juliana (l.) nenth. ex rchb.... 23 extracts. oxidation communications, 38 (1): 5566. van staden, e., zazimalova, e., george, e.f. 2008: plant growth regulators ii: cytokinins, their analogues and antagonists. in: george, e.f., hall, m.a., de klerk, g.-j. (eds.): plant propagation by tissue culture, (3rd edition). 1: 205–226. springer, bachground, dordrecht, netherlands. vladimir-knežević, s., blažeković, b., bival štefan, m., alegro, a., köszegy, t., petrik, j. 2011: antioxidant activities and polyphenolic contents of three selected micromeria species from croatia. molecules, 16: 1454-1470. zielinska, s., piatczak, e., kalemba, d., matkowski, a. 2011: influence of plant growth regulators on volatiles produced by in vitro grown shoots of agastache rugosa (fischer & c.a.meyer) o. kuntze. plant cell, tissue and organ culture, 107: 161–167. biologica nyssana 6 (1)  september 2015: 17-23 tošić, s. et al.  micropropagation of micromeria juliana (l.) nenth. ex rchb. 24 valchev, v., georgiev, v., tsoneva, s.: communities of phragmites australis (cav.) trin.ex steud. in the fluvial terrace of lower danube: their diversity and structure of the above-ground phytomass. biologica nyssana, 8 (1), september 2017 53 5 3 original article received: 27 june 2017 revised: 12 august 2017 accepted: 30 august 2017 communities of phragmites australis (cav.) trin.ex steud. in the fluvial terrace of lower danube: their diversity and structure of the above-ground phytomass vladimir valchev*, valeri georgiev, sonya tsoneva institute of biodiversity and ecosystem research at the bulgarian academy of sciences, “akad. g. bonchev str.” bl. 21 bas, sofia, bulgaria * e-mail: vlado@bio.bas.bg abstract: valchev, v., georgiev, v., tsoneva, s.: communities of phragmites australis (cav.) trin.ex steud. in the fluvial terrace of lower danube: their diversity and structure of the above-ground phytomass. biologica nyssana, 8 (1), september 2017: 53-60. determination of stocks of aboveground phytomass of reed communities in bulgaria is not well studied yet. this survey is the most detailed study on the stocks of the aboveground phytomass of the reed communities (phragmites australis (cav.) trin. ex steud.) in bulgaria. the studied reed beds are formed in the floodplain part of the danube river. they are located in the fluvial terrace of the river between archar village (west) and srebarna village (east). as a result of the survey, it was found that the average stocks of the aboveground phytomass of the reed communities reached to 19.78 t/ha. moreover, 54.04% of the aboveground phytomass is concentrated in the stems of the reed plants. almost 42% of it is produced by the leaves, and the inflorescences produce less than 5% of the total amount of the phytomass. this study once again demonstrates the important role of reed beds for the normal functioning and development of riparian ecosystems. key words: reed communities, primary production, above-ground phytomass, danube apstrakt: valchev, v., georgiev, v., tsoneva, s.: zajednice phragmites australis (cav.) trin. ex steud. na fluvijalnim terasama donjeg dunava: diverzitet i struktura nadzemne biomase. biologica nyssana, 8 (1), septembar 2017: 53-60. količina nadzemne biomase zajednica trske u bugarskoj još uvek nije dovoljno proučavana. ovo istraživanje je najdetaljnija studija nadzemne biljne mase zajednice trske (phragmites australis (cav.) trin. ex steud.) u bugarskoj. ispitivane rečne zajednice su formirane u plavnom području dunava. locirane su na fluvijalnim terasama reke između sela arčar (zapad) i srebarna (istok). kao rezultat studije, utvrđeno je da su prosečne količine nadzemne biomase zajednice trske do 19,78 t/ha. štaviše, 54,04% nadzemne biomase koncentrisano je u drškama trske. skoro 42% nje je produkovano od strane lišća, a cvetovi proizvode manje od 5% ukupne količine biljne biomase. ovo istraživanje još jednom demonstrira značajnost uloge tršćaka za normalno funkcionisanje i razvoj riparijalnih ekosistema. ključne reči: zajednice trske, primarna produkcija, nadzemna biljna biomasa, dunav 8 (1) • september 2017: 53-60 doi: 10.5281/zenodo.963885 biologica nyssana 8 (1)  september 2017: 53-60 valchev, v. et al.  communities of phragmites australis… 54 introduction determination of the above-ground phytomass stocks of reed communities is a problem still pending proper investigation in bulgaria, so there are only few publications with data on this subject in the bulgarian scientific literature (k o c h e v & y o r d a n o v , 1981; k o c h e v & y u r u k o v a , 1984). in 19861987, g e r g i n a b a e v a (1994) determined the primary biological production of two species, phragmites australis (cav.) trin.ex steud. and typha angustifolia l., which formed comparatively pure associations in the so-called “reed belt” of srebarna reserve applying a method for determination of the annual growth of plant biomass (root biomass production). k o c h e v & y o r d a n o v (1981) devoted modest attention to the issues of biomass, biological productivity and total stocks of the above-ground phytomass of reed communities in their large-scale survey of the flora and vegetation of water bodies in bulgaria. however, it was not clear from their results how the dry mass was determined as a component of the primary biological production of these coenoses. some data on the bioproduction and energy stocks of common reed (phragmites australis) and lesser bulrush (typha angustifolia) in bulgaria were also published by k o c h e v & y u r u k o v a (1984), a few years latter. and these were about bulgarian publications on the surveys of reed and its communities as phytomass producers. material and methods reed coenoses (phragmitetum communis) that participate in the structure of the vegetation cover on the territory of the danube fluvial terrace are investigated in this study. dominant species of that stands, phragmites australis, was chosen for survey because its phytocoenoses are some of the widest distributed in the river valley and are the greatest producer of above-ground phytomass among the components of grassy vegetation. when growing in river shallows, they develop most powerfully at water depth between 0.2 and 0.6 m and form different in width belts. the transect method was applied, with organized sample plots, in line with the requirements of european standard en 14184 (cen, 2003a) for running waters and international standard cen tc 230/tg 3/n72 (cen, 2003b) for standing water bodies. macrophytes (higher plants) were described for each sampling plot (a n d r e e v et al., 1992). plant abundance was assessed according to the 5grade scale of braun-blanquet (g u i n o c h e t , 1973). some objects got more than one description, in order to acquire a better idea of the phytocoenotic specificities of the reed communities formed along the danube (tab. 1). determination of the above-ground phytomass stocks of the reed communities (in this case it coincides with their biological productivity) follows the quantifying method of m i l n e r & h u g h e s (1968). field samples were taken from plots sized 50×50 cm (0.25 m²), after the reed has finally formed its inflorescences. reed stems were cut close up to the soil. they were divided then into fractions (stems, leaves, inflorescences) and their fresh weight was measured, as quickly as possible. an average sample was taken from each fraction to determine the absolute dry weight (adw) in laboratory conditions. the obtained data were recalculated in kg/m², and then into t/ha of absolute dry matter. the absolute dry weight (adw) of the vegetation samples was determined following a standard method: by drying at 60 ºc in the course of 24 hours. the number of cut stems at the sampling plot and the number of inflorescences were recorded for each sample. data obtained on the quantity and distribution of the above-ground phytomass per stem and per community are published in table 2. the same table gives summarized data on the distribution of the above-ground phytomass by fractions and their quantitative participation in the formation of aboveground phytomass. similarly were determined the stocks of above-ground phytomass of the accompanying species participating in the formation of reed coenoses (tab. 3). the latin names of the plants are given according to the field guide to the vascular plants in bulgaria (a n d r e e v et al., 1992). results and discussion strong reproductive capacity is a specific characteristic of the phragmites australis (cav.) trin. ex steud. (common reed). common reed propagates by seeds and vegetatively, owing to a very good root system (of the rhizome type), which forms typical root suckers, several meters long. they give rise to the young stems and thus new, so far reed-free areas are colonized. the very mode of vegetative propagation of the species, and probably some of its allelopathic properties, precondition the creation of coenoses, which usually are monodominant and with a comparatively poor species composition of usually 6 to 10 species (tab. 1). common reed often forms mixed communities with other macrophytic species with kindred biology, namely typha latifoliа l., typha angustifolia l., bolboschoenus maritimus palla, 5 5 table 1. species composition of the common reed communities in the fluvial terrace of lower danube sites orsoya zagrazhden kaikusha kalimok malak preslavets garvan marsh srebarna frequency of occurrence species/number of transect 1 2 3 1 2 1 2 1 2 3 4 5 6 7 phragmites communis 5 5 5 4 5 5 4 4 1 5 5 5 5 4 5 3 3 17 ceratophyllum demersum + 1 + + 1 1 1 1 1 1 10 calystegia sepium + + 1 + + 1 1 1 1 9 lycopus europaeus + + + + 1 1 1 + 8 hydrocharis morsus-ranae + + + 2 1 + 6 typha angustifolia 1 + 1 1 1 + 6 urtica dioica + + 1 1 + 5 epilobium hirsutum + + + + + 5 lemna trisulca + + + 3 + 5 thelipteri spalustris 1 1 1 1 1 5 bolboschoenus maritimus + + + + 4 lemna minor + 1 + 3 4 typha latifolia + + 1 1 4 berula erecta + 1 1 1 4 mentha aquatica + + + 3 alisma plantago-aquatica + + + 3 lysimachia vulgaris + + + 3 potamogeton crispus + 1 1 3 salvinia natans 2 + 1 3 stachys palustris + + 2 cirsium arvense + + 2 sambucus ebulus + + 2 myosotis palustris + + 2 persicaria hydropiper + + 2 schoenoplectus lacustris + + 2 veronica beccabunga gr. + + 2 bidens tripartita + + 2 sparganium erectum + + 2 lythrum salicaria + 1 2 rumex hydrolapathum 1 1 2 solanum dulcamara + + 2 symphytum officinale + 1 b i o l o g i c a n y s s a n a 8 (1 )  s e p te m b e r 2 0 1 7 : 5 3 -6 0 v a lc h e v , v . e t a l.  c o m m u n itie s o f p h ra g m ite s a u s tra lis … 56 5 6 potentilla reptans + 1 bidens cernua + 1 myriophyllum spicatum + 1 centaurea stenolepis + 1 convolvulus arvensis + 1 euphorbia lucida + 1 lycopus exaltatus + 1 lapa major + 1 sanicula europaea 1 1 butomus umbellatus + 1 elodea nuttallii 1 1 spirodella polyrhiza 1 1 mentha pulegium + 1 potamogeton lucens + 1 utricularia vulgaris + 1 bidens frondosa + 1 persicaria lapathifolia 1 1 inula germanica + 1 carex sp. + 1 species richness 4 7 4 17 7 6 16 12 13 7 7 7 6 10 11 10 8 table 2. summarised data of the grass-stand samplings of phragmites australis coenoses fractions orsoya o r y a h o v o gulyantsi belene k a li m o k m .a la k p r e sl a v e ts g a r v a n srebarna a v e r a g e v a lu e s 1 2 3 milko -vitsa dolni vit kaiku sha dekov kochka island bryag 2010 stems fresh weight (g/0.25 m²) 409.7 1075.7 905.7 279.1 336.7 1400.7 168.3 424.7 669.1 1005.7 824.1 1797.1 433.1 748.4 absolute dry weight (g/0.25 m²) 307.0 461.1 450.9 150.5 193.7 749.5 84.3 228.3 312.7 509.3 344.3 812.1 225.4 371.5 absolute dry matter (%) 74.9 42.9 49.8 53.9 57.5 53.5 50.1 46.3 53.3 49.4 58.2 54.8 48.0 53.3 relative participation of the fraction (%) 82.9 57.4 51.2 46.6 46.8 64.7 74.5 53.9 54.9 69.1 67.1 58.6 52.0 58.8 leaves fresh weight (g/0.25 m²) 207.6 575.6 683.7 269.9 290.7 774.7 62.8 350.7 528.9 414.7 225.9 934.9 353.9 436.4 absolute dry weight (g/0.25 m²) 63.3 332.4 412.9 138.6 172.8 361.1 27.8 173.3 239.8 227.3 168.7 565.1 207.9 237.8 b i o l o g i c a n y s s a n a 8 (1 )  s e p te m b e r 2 0 1 7 : 5 3 -6 0 v a lc h e v , v . e t a l.  c o m m u n itie s o f p h ra g m ite s a u s tra lis … 5 7 absolute dry matter (%) 30.5 57.8 60.4 51.4 59.4 46.6 44.3 49.4 45.4 54.8 74.7 60.5 58.8 53.4 relative participation of the fraction (%) 17.1 41.4 46.9 42.9 41.8 31.2 24.6 40.9 42.1 30.9 32.9 40.8 48.0 37.6 inflorescences fresh weight (g/0.25 m²) 16.4 26.3 58.4 74.0 82.4 1.4 32.8 32.4 11.7 37.3 absolute dry weight (g/0.25 m²) 10.0 16.0 33.6 47.3 48.0 1.1 21.8 17.2 6.8 22.4 absolute dry matter (%) 61.2 60.8 57.5 63.9 58.3 76.1 66.4 53.1 57.8 62.1 relative participation of the fraction (%) 1.2 1.9 10.5 11.4 4.1 0.9 5.2 3.0 0.6 3.6 number of stalks per m² 36.0 40.0 96.0 32.0 56.0 56.0 20.0 44.0 28.0 44.0 36.0 44.0 16.0 42.2 number of inflorescences per m² 12.0 32.0 24.0 36.0 44.0 8.0 32.0 24.0 16.0 17.5 total weight for the plot (kg/m²) 1.5 2.2 3.5 1.3 1.7 4.6 0.5 1.7 2.3 2.9 2.1 5.5 1.7 2.4 yields of above-ground phytomass (t/ha) 14.8 22.2 35.2 12.9 16.6 46.3 4.5 16.9 22.8 29.5 20.5 55.4 17.3 24.2 2011 stems fresh weight (g/0.25 m²) 619.1 353.7 454.1 239.7 269.7 212.0 661.0 591.7 620.7 446.9 absolute dry weight (g/0.25 m²) 286.7 204.5 231.9 99.7 99.9 102.7 267.6 217.7 188.8 188.8 absolute dry matter (%) 46.3 57.8 51.1 41.6 37.0 48.4 40.5 36.8 30.4 54.0 relative participation of the fraction (%) 64.4 55.2 46.4 36.6 43.0 49.2 59.2 35.5 53.0 49.3 leaves fresh weight (g/0.25 m²) 395.5 320.1 471.4 282.1 224.7 211.7 381.7 545.7 472.7 367.3 absolute dry weight (g/0.25 m²) 140.8 124.2 222.6 144.9 108.5 87.9 184.2 392.4 167.7 174.8 absolute dry matter (%) 35.6 38.8 47.2 51.4 48.3 41.5 48.3 71.9 35.5 36.5 relative participation of the fraction (%) 31.6 33.5 44.6 53.2 46.7 42.1 40.8 64.1 47.1 45.6 inflorescences fresh weight (g/0.25 m²) 29.0 68.0 77.1 36.7 33.0 27.0 4.3 30.6 absolute dry weight (g/0.25 m²) 17.7 41.7 44.9 27.6 24.2 18.2 2.6 19.6 absolute dry matter (%) 61.0 61.3 58.3 75.1 73.3 67.4 60.2 50.7 relative participation of the fraction (%) 4.0 11.3 9.0 10.1 10.4 8.7 0.4 5.1 number of stalks per m² 48.0 32.0 32.0 32.0 20.0 24.0 28.0 28.0 20.0 29.3 number of inflorescences per m² 24.0 32.0 24.0 16.0 12.0 24.0 8.0 15.6 total weight for the plot (kg/m²) 1.8 1.5 2.0 1.1 0.9 0.8 1.8 2.5 1.4 1.5 yields of above-ground phytomass (t/ha) 17.8 14.8 20.0 10.9 9.3 8.4 18.1 24.5 14.3 15.3 b i o l o g i c a n y s s a n a 8 (1 )  s e p te m b e r 2 0 1 7 : 5 3 -6 0 v a lc h e v , v . e t a l.  c o m m u n itie s o f p h ra g m ite s a u s tra lis … biologica nyssana 8 (1)  september 2017: 53-60 valchev, v. et al.  communities of phragmites australis… 58 5 8 schoenoplectus lacustris palla and etc. mention deserves the fact that, as a rule, species from the reed floor participate to a much lesser extent in the formation of its grass stand. owing to the fact that reed coenoses have been studied in a relatively large region (the valley of the danube between archar and srebarna), the total species diversity of these plant communities registers 53 higher plant species, which is quite impressive floristic richness (table 1). these were reed phytocoenoses formed in marshy places in the river valleys and in other open water bodies (b a r d a t et al., 2001). they are a major component to aquatic vegetation and of vegetation around the waterbodies, attached to the floor of the fresh-waterbasins. in many places, belts and patches mainly of common reed (phragmites australis) have been formed, with the participation of rush (typha sp.), bulrush (schoenoplectus lacustris), high sedges (carex sp.), and others in the process of shallowing out, drying and filling up of the various water bodies with plant remains. this slow natural process could be stepped up by anthropogenic activity. these facts contribute to the environmental and nature-protection importance of rush coenoses. when these coenoses are formed in the river shallows or in the near-by shallow water bodies, the composition of reed communities is joined also by submerged or water floating species, such as ceratophyllum demersum, lemna minor, lemna trisulca, myriophyllum spicatum, frequently hydrocharis morsus-ranae, as well as by some representatives of the sedges (genus carex, schoenoplectus lacustris) and grasses. quite telling is the fact that ceratophyllum demersum species figures in ten out of 17 descriptions altogether. the other species identified in the reed coenoses have much lower frequency of occurrence: usually between one and four to five times (tab. 2). there is a difference in species composition in the studied descriptions of reed communities, determined by the specific conditions of the biotope. a common trait is the very strong quantitative presence of the dominant species, which forms almost pure onefloor grass stands, where the projection cover of dominant species often reaches 85-90% and over. the reed floor is 3 m to 5 m high and even higher. its characteristics (height and projection cover) determine very strongly the specific ecological conditions in these coenoses: temperature, humidity, light and air movement. the other species are mostly present on the periphery of these grass stands or in their thinned-out areas. special mention deserves a very important factor for the floristic enrichment of these phytocoenoses, namely, the presence of residual water on the surface of the terrain occupied by them, where a second (and even third) floor could be formed of the species developing in it or on its surface (lemna minor, myriophyllum spicatum, ceratophyllum demersum and others). about the formed above-ground phytomass and its distribution, the obtained data have shown that the absolutely dry matter of the different fractions of reed above-ground phytomass exceed by half its fresh weight: 53-54% for the stems, 35-53% for the leaves and 50-60% for the inflorescences (tab. 3). therefore, the plants stems contain the greatest amounts of water, which is due to the fact that most conductive tissues, which carry out the water and assimilates needed for normal existence of the plant organism, are situated there. the main part of the reed above-ground phytomass is formed by its stems: somewhere between 82.90% and 35.53%. second comes the leaves fraction: from 64.05% to 24.60%. relative participation of inflorescences does not exceed table 3. average values of fractions from different parts of reeds for the two years of investigation fractions average values stems fresh weight (g/0.25 m²) 597.64 absolute dry weight (g/0.25 m²) 280.14 absolute dry matter (%) 43.55 relative participation of the fraction (%) 54.04 leaves fresh weight (g/0.25 m²) 401.86 absolute dry weight (g/0.25 m²) 206.29 absolute dry matter (%) 44.94 relative participation of the fraction (%) 41.6 inflorescences fresh weight (g/0.25 m²) 33.93 absolute dry weight (g/0.25 m²) 21.04 absolute dry matter (%) 56.39 relative participation of the fraction (%) 4.36 number of stalks per m² 35.74 number of inflorescences per m² 16.54 total weight for the plot (kg/m²) 1.98 yields of above-ground phytomass (t/ha) 19.78 biologica nyssana 8 (1)  september 2017: 53-60 valchev, v. et al.  communities of phragmites australis… 59 11.40%, i.e. the reproductive organs of this species form less than one-twentieth of the plant aboveground phytomass (tab. 2). data obtained from the samples cut in the field have permitted calculation of the stocks of aboveground phytomass of the surveyed reed communities. apparently, the lowest stocks came from the sampling plot at belene (kaikusha locality) – 4.53 t/ha, and the highest came from the sampling plot on srebarna island – 55.36 t/ha (tab. 2). comparison of the stocks of above-ground phytomass of reed conenoses shows clearly higher data for 2010– 24.22 t/ha, against 15.33 t/ha in 2011. this is quite explicable, considering the fact that the vegetation season in 2011 was much drier than in the previous year. averaged results from the different sites during the two years of survey (tab. 3) have shown the following: ■ somewhat above half of the above-ground phytomass was concentrated in the reed stems (54.04 %). almost 42% of that mass was formed by the leaves, while the generative organs (inflorescences) contained less than 5% of the general amount of the phytomass. further more, the surveyed reed coenoses formed a main floor with a very dense grass stand: on the average, almost 36 reed stalks grew per square meter, which prompted the conclusion that reed communities make relatively very good use of the ecotope conditions. ■ the table 3 also shows that nearly half (47%) of the reed stalks in the model plot did not develop inflorescences. this could be used as a proof of the importance of vegetative propagation of this plant species. ■ the average stocks of above-ground phytomass of the plant communities amount to 19.78 t/ha, or about 4.6 times less than the data (91.7 t/ha) published by k o c h e v & y o r d a n o v (1981) from a survey of the primary biological production of reed associations in bulgaria. the figures reported by k o c h e v & y o r d a n o v (1981) were probably so high due to the fact that the authors had measured both green and dry mass in the reed floor and the latter comprised the plant substance formed during the previous vegetation season. according to k o c h e v & y u r u k o v a (1984), reed communities in the aldomir marsh form bioproduction to the tune of 36.7 t/ha, which is only twice higher than the present results. the samples taken to determine the stocks of above-ground phytomass formed by the other plant species participating in the formation of reed coenoses have shown that their proportion of the above-ground phytomass varies between 0.49 t/ha and 4.41 t/ha,or on the average is1.46 t/ha (tab. 4). thus the total stocks of above-ground phytomass of the surveyed reed coenoses amount on the average to 21.24 t/ha. ecosystematically, besides being a major phytomass producer, reed communities also play an important part in the formation of local micro climate. suitable micro climatic conditions predetermine a greater diversity of the faunistic component of these specific bio-geocoenoses: periphyton (mollusks, insects), ornithofauna, amphibians and reptiles, as well as fish stock, where there is water. table 4. data for absolute dry weight of the accompanying species in reed communities measured in 2011 sites absolute dry weight (g/0.25 m²) absolute dry weight(t/ha) orsoya 1 15.73 0.63 orsoya 2 12.24 0.49 lower vit 16.93 0.68 kaikusha 67.34 2.69 kalimok 110.24 4.41 garvan marsh 16.53 0.66 srebarna 16.16 0.65 average 36.45 1.46 conclusion the total stocks of above-ground phytomass of the surveyed reed communities averaged 21.24 t/ha. only about 8% of that amount was formed by the accompanying plant species. the average stocks of above-ground phytomass of the plant communities accounted for 19.78 t/ha. little more than half of the above-ground phytomass was concentrated in the stems of the reeds (54.04%). about 40% of it was formed by the leaves, while less than 5% of the general phytomass amount was due to the inflorescences. nearly half of the reed stalks in a model plot did not develop inflorescences, which comes to prove the importance of vegetative propagation of this plant species. considering the fact that they are among the widest distributed components along the river valley and the greatest producer of phytomass among the other components of grassy vegetation in the river’s biologica nyssana 8 (1)  september 2017: 53-60 valchev, v. et al.  communities of phragmites australis… 60 fluvial terrace, and also that reed massifs play a major part in the normal functioning and development of riparian ecosystems, it becomes clear that they are very important and determinant for the processes of mineralization of the produced phytomass and its depositing on the soil or floor of the water bodies. references andreev, n., anchev, m., kozhuharov, s., markova, m., peev, d., petrova, a., 1992: field guide to the vascular plants in bulgaria. naouka & izkoustvo, sofia (in bulgarian). 788 p. baeva, g., 1994. investigation on the overground phytomass of phragmites australis (cav.) trin. ex steud. and typha angustifolia l. in the srebarna biosphere reserve. godishnik na sofiyskiya universitet “st kl. ohridski”, biologicheski fakultet, kniga 2 – botanika, 84: 103-109. bardat, j., bioret, f., botineau, m., boullet, v., delpech, r., géhu, j. m., haury, j., lacoste, a., rameau, j. c., royer, j. m., roux, g., touffet, j. 2004: prodrome des végétations de france. muséum national d'histoire naturelle. 171 p. guinochet, m. 1973: phytosociologie. masson. paris. 227 p. cen, 2003a: water quality-guidance standard for the surveying of aquatic macrophytes in running waters, en 14184. comité européen de normalisation. bruxelles. cen, 2003b: water quality-guidance standard for the surveying of macrophytes in lakes complementary element, cen tc 230/tg 3/n72.comité européen de normalisation. bruxelles. kochev, h., yordanov, d. 1981: vegetation of bulgarian water bodies. ecology, protection and economic importance. bulgarian academy of sciences. sofia. (in bulgarian). kochev, h., yurukova, l. 1984: primary biological production and energy value of the vegetation in the aldomirovo marsh, sofia district. contemporary theoretical and applied aspects of plant ecology, 1: 166-174. (in bulgarian). milner, c., hughes, r. e. 1968: methods for the measurement of the primary production of grasslands. oxford and edinburg. abingdon, berkshire.70 p. anticancer compounds from medicinal plants biologica nyssana 4 (1-2)  december 2013: 75-79 žabar, a. et al.  larvicidal activity and in vitro … 75 original article larvicidal activity and in vitro effects of green tea (camellia sinensis l.) water infusion andrea žabar* 1 , vladimir cvetković 1 , jelena rajković 1 , jovana jović 1 , perica vasiljević 1 , tatjana mitrović 1 department of biology and ecology, faculty of science and mathematics,university of niš, višegradska 33,18000 niš * e-mail: andreazabar@yahoo.com abstract: žabar, a., cvetković, v., rajković, j., jović, j., vasiljević, p., mitrović, t.: larvicidal activity and in vitro effects of green tea (camellia sinensis l.) water infusion. biologica nyssana, 4 (1-2), december 2013: 7579. in this study green tea water infusion was tested on drosophila melanogaster wild-type larvae in vivo, also an in vitro antihemolytic and hemolytic tests were performed. three different concentrations were used 7.5 mg/ml, 37.5 mg/ml and 75 mg/ml, the lowest dose representing the recommended dose followed by five times and ten times higher doses. effect of these three concentrations was monitored and tested in vivo on drosophila melanogaster (meigen, 1830) wt (wild type) larval development and surviving. all three concentrations showed toxic effect for larvae, with toxicity being increased in dose – depended manner. the time needed for larvae to fully develop was delayed. this decrease of developmental time was in dose – dependent manner, too. amount of hemolysis caused by the lowest concentration was very small when compared with the percent of spontaneous hemolysis. other two higher concentrations, 37.5 mg/ml and 75 mg/ml, showed higher hemolytic effect. during the four hour incubation period percent of hemolysis grew in time – dependent manner. the highest hemolytic effect was recorded for the concentration of 37.5 mg/ml. antihemolytic test showed that the lowest concentration had the highest inhibitory effect to h2o2 induced hemolysis. the 37.5 mg/ml and 75 mg/ml concentrations had lower inhibitory effect when compared with the dose of 7.5 mg/ml. according to our study it can be concluded that the high concentrations of green tea water infusion exhibit larvicidal activity against d. melanogaster larvae, don't have protective effect to rbc membrane and cause greater hemolysis. key words: green tea; drosophila melanogaster wt; larvicidal activity; rbc; hemolytic test; antihemolytic test. introduction one of the most consumed beverages in the world is green tea. caffeine is one of the active compounds present in tea, and its content is around 3 to 4 % (w i l l s o n et al., 1992). caffeine may play an important role against some pathogens (van b r e d a et al., 2012). green tea is also a source of polyphenol antioxidants such as epicatechin, epigallocatechin, epicatechin gallate and epigallocatechin gallate (z h a n g et al., 1997, h o d g s o n , 2008). catechins have similar backbone but differ in number and location of hydroxyl groups. catechins are water – soluble, colorless compounds that make up to 30% of the dry leaf weight (g r a h a m , 1992). these natural antioxidants have been reported to have the ability of preventing cancer (l i et al., 2008), are effective scavengers of free radicals (n a n j o et al., 1996), inhibit hbv in vitro (x u et al., 2008). it has been 11 th sfses • 13-16 june 2013, vlasina lake 4 (1-2) • december 2013: 75-79 biologica nyssana 4 (1-2)  december 2013: 75-79 žabar, a. et al.  larvicidal activity and in vitro … 76 reported that one cup of tea can provide 150 – 200 mg of flavonoids. also, lower risk of heart disease and stroke are related to the flavonoids intake (h o d g s o n , 2008). isolated flavonoids at high physiological concentrations can reduce platelet aggregation and markers of platelet activation (r e i n et al., 2000). green tea extract can delay and inhibit oxidation of low-density lipoprotein ldl catalyzed by copper in vitro (y o k o z a w a et al., 1997). some authors report that drinking green tea may result in lower level of total triglyceride and cholesterol in serum together with deceased atherogenic index (i m a i et al., 1995). in view of variety of reports considering green tea, we have conducted a study to evaluate the effects of high concentrations of green tea water infusion. drosophila melanogaster is frequently used in larvacidal and insecticidal studies (m i y a z a w a et al., 1998; m i y a z a w a et al., 2000). our aim was to see whether these high doses have beneficial effects on rbc membrane and on drosophila melanogaster wt larvae development. to evaluate green tea infusion effect rbc membrane the hemolytic test with drabkin's reagent was used. also, to see whether high concentrations of green tea water infusion could inhibit lipid peroxidation caused by h2o2, the antihemolytic method was employed. materials and methods test organisms in this experiment, the drosophila melanogaster reference type 5 wt, originated from „bloomington drosophila stock centre”, indiana university, usa was used. fruit flies were cultivated on a standard drosophila medium (9% sugar, 10% corn meal, 2% agar, 2% yeast, with addition of fungicide nipagin ® ), in a standard cultivating 200 ml flasks on 25 o c, 60% humidity, with 12h/12h day/night cycle. adults were allowed to oviposit eggs during the eight hour period, after that they were removed from the flasks. then, after three days larvae which were 72±4h old, have been removed from the cultivating medium, washed with distilled water, and placed on the new medium containing green tea infusion. concentrations of green tea in medium were 7.5 mg/ml, 37.5 mg/ml and 75 mg/ml. experiment was done in triplicate. there were 15 larvae from the same hatch in each replicant flask, and there were 12 replicant flasks including the water control (negative control). replicants were incubated in standard way, already described. alive specimens were collected 10-16 days after the egg laying. rbc preparation a modified method from noudeh et al. (2009) was used. briefly, blood was collected in heparinized tubes from male wistar rats via cardiopuncture. whole blood was centrifuged at 2000 rpm for 10 minutes at 4 ºc. plasma and the buffy coat were removed and an equal volume of pbs (ph 7.4) was added. this was repeated three times, to obtain washed erythrocytes. at the end packed erythrocytes were diluted with pbs to obtain 4% suspension. rbc prepared in this way were used both for hemolytic and antihemolytic test. solutions of green tea infusion and pbs (0.9%, ph=7.4) were made to give the final concentrations of 7.5mg/ml, 37.5mg/ml and 75mg/ml. hemolytic test the following method was employed: 200 μl of green tea water infusions (concentrations 7.5 mg/ml, 37.5 mg/ml, 75 mg/ml) were incubated with 200 μl of rbc suspension at room temperature for one, two, three and four hours. after each hour of incubation samples were centrifuged at 1500 rpm for 10 minutes at 4 ºc. then, 200 μl of resulting supernatant was added to 3 ml of drabkin's reagent and the absorbance was measured at 540 nm on spectrophotometer (shimadzu uv-vis 1650, tokyo, japan). positive control consisted of 200 μl of distilled water and 200 μl rbc suspension and negative control were 200 μl of pbs and 200 μl rbc suspension. everything was done triplicate. percentage of hemolysis was calculated by the equation: % hemolysis = [(ab(sample) ab(negative control)) / ab(positive control)] * 100% (gould et al., 2000). antihemolytic test antihemolytic test was done by modified method previously described (ebrahimzadeh et al, 2010.) 2 ml of rbc suspension was pre-incubated with 0.5 ml of green tea water infusion. the final volume was made to be 5 ml with pbs, and the final concentrations of green tea infusions were 7.5 mg/ml, 37.5 mg/ml and 75 mg/ml. this mixture was incubated for 15 minutes at 37 ºc. then 0.5 ml of h2o2 dissolved in pbs was added to the mixture. concentration of h2o2 was made to give 90% of hemolysis after 4 hours. at the end of the incubation period of 4 hours, different concentration samples were spun at 1500 rpm for 10 min at 4 ºc. then 200 μl of supernatant was added to 3 ml of drabkin's reagent and the absorbance was measured at 540 nm. positive control consisted only of pbs and biologica nyssana 4 (1-2)  december 2013: 75-79 žabar, a. et al.  larvicidal activity and in vitro … 77 h2o2 without green tea infusion. experiments were done in triplicate. percentage of inhibition was calculated by equation: %inhibition = [(ab (h2o2) ab(sample) ) / ab (h2o2) ] *100% . results and discussion the absolute mortality was monitored number of hatched adult specimens that were scored and compared to total transferred larval specimens. it was observed that the mortality increases with the increase in green tea concentration in the medium (fig. 1). fig. 1. absolute mortality of d. melanogaster larvae in presence of green tea infusion development was also observed and number of pupae that were scored from ninth to sixteenth day of larval life (data not shown). in comparison with water control, developmental time from larvae to adults prolongs if the concentration of green tea infusion is higher. one of the active compounds of green tea is caffeine. according to the literature, content of the caffeine is around 3 to 4% (w i l l s o n et al., 1992). it is supposed that caffeine, localized in the vascular bundles of young camellia sinensis l. leaves plays a crucial role in the defense against various pathogens (v a n b r e d a et al., 2012.). itoyama and b i c u d o (1992 and 1997) showed harmful effects of caffeine on drosophila prosaltans d., related to fecundity, egg lying capacity and longevity which is consistent with our results. on the other hand, some studies show that camellia sinensis l., inhibits the accumulation of iron and thus extends the life span of drosophila melanogaster (m a s s i e et al., 1993). also, these results are consistent with previous studies of other authors who showed that polyphenols as antioxidants affect larval development in drosophila causing its time delay (r i c e -e v a n s et al., 1996; a r u o m a , 2003; k i m et al., 2004). results for both antihemolytic and hemolytic test are accordant. in both experiments the lowest doses of green tea infusion exibited slightly beneficial effect on rbc membrane (fig. 2 and 3). fig. 2. percentage of rbc hemolysis in presence of green tea water infusion http://www.ncbi.nlm.nih.gov/pubmed?term=massie%20hr%5bauthor%5d&cauthor=true&cauthor_uid=8326745 biologica nyssana 4 (1-2)  december 2013: 75-79 žabar, a. et al.  larvicidal activity and in vitro … 78 percentage of hemolysis was the lowest for the concentration of 7.5 mg/ml, and it grew as the time of incubation prolonged (after first hour it was 4.99% ± 3.28, second 4.56% ± 0.4, third 5.41% ± 2.22, fourth 5.8 % ± 1.43). other two higher doses showed an increase in rbc hemolysis also in time dependant manner. hemolysis was highest for the 37.5 mg/ml concentration. percent of hemolysis for the 37.5 mg/ml concentration was 15.16% ± 2.4 after first hour, and for the second, third and fourth hour of incubation it was 20.96 % ± 3.55, 20.86 % ± 1.27 and 20.52 % ± 3.3 respectively. percent of hemolysis for the concentration of 75 mg/ml was 8.45 % ± 3.79, 10.79 % ± 0.95, 11.63 % ± 1.33, 12.96 % ± 2.69 after first, second, third and fourth hour respectively. fig. 3. antihemolytic effect of green tea infusion according to the fick's law, diffusion flux from a membrane is proportional to concentration difference of both sides. so, an increase in concentration of green tea infusion in outer membrane leads to diffusion to internal membrane until it gets to specific concentration which can promote membrane disruption and induce hemolysis (k l e s z c z y n s k a et al., 2005.). lipid peroxidation of rat rbc induced by h2o2 creates membrane damage and hemolysis. the highest percentage of inhibition was recorded for the 7.5 mg/ml concentration (94.96 % ± 0.1). other two concentrations had lower inhibitory effect, and for 37.5 mg/ml dose it was 90.39 % ± 0.29, while for the 75 mg/ml it was 85.42 % ± 0.45. certain polyphenols may interact with membrane, leading to the decrease in its fluidity and the diffusion of free radical into the rbc (c o s t a et al., 2009.). in higher doses effects differ (fig. 3.), percentage of hemolysis inhibition is lower. high concentrations of green tea infusion don't have ameliorative effect against rbc free radical hemolysis. conclusion high concentrations of green tea water infusion exibit toxic effect to drosophila larvae. the time of development was prolonged. this is probably due to high concentration of active compounds present in green tea water infusion such as caffeine and catechins. also these results indicate that high doses of green tea infusion can cause a change in tonicity of the erythrocyte membrane. change in tonicity is probably due to the interactions of polyphenols with the outer lipid monolayer of the erythrocyte membrane. therefore, it is necessary to continue the research further. acknowledgements. the authors acknowledge the financial support of the ministry of education, science and technological development of the republic of serbia (grant no.: 172047). references aruoma, o.i. 2003: methodological considerations for characterizing potential antioxidant actions of bioactive components in plant foods. mutation research, 523–524: 9-20. costa, r.m., magalhaes, a.s., pereira, a.j., andrade, p.b., valentao, p., carvalho, m., silva, b.m., 2009: evaluation of free radicalscavenging and antihemolytic activities of quince (cydonia oblonga) leaf: a comparative study with green tea (camellia sinensis). food and chemical toxicology 47: 860-865. ebrahimzadeh, m.a., nabavi, s.f., nabavi, s.m., eslami, b., 2010: antihemolytic and antioxidant activities of allium paradoxum. central european journal of biology, 5(3): 338-345. gould, l.a., lansley, a.b., brown, m.b., forbes, b., martin, g.p., 2000: mitigation of surfactants erythrocyte toxicity by egy phosphatidylcholine. journal pharmacy and pharmacology, 52(10): 1203-1209. graham, h.n. 1992: green tea consumption, composition and polyphenol chemistry. preventive medicine, 21:334-350. hodgson, j.m., 2008: tea flavonoids and cardiovascular disease. asia pacific journal of clinical nutrition 17(s1): 288-290. imai, k., nakachi, k., 1995: cross sectional study of effects of drinking green tea on cardiovascular and liver disease. bmj 310:693-696. itoyama, m.m., bicudo, h.e.m.c., 1992: effects of caffeine on fecundity, egg laying capacity, development time and longevity in drosophila prosaltans. revista brasileira de genética 15: 303-321. biologica nyssana 4 (1-2)  december 2013: 75-79 žabar, a. et al.  larvicidal activity and in vitro … 79 itoyama, m.m., bicudo, h.e.m.c., 1997: effects of caffeine on mitotic index in drosophila prosaltans (diptera). revista brasileira de genética 20: 655-658. kim, d.o., lee, c.y., 2004: comprehensive study on vitamin c equivalent antioxidant capacity (vceac) of various polyphenolics in scavenging a free radical and its structural relationship. critical reviews in food science and nutrition, 44: 253 273. kleszczynska, h., bonarska, d., luczvnski, j., witek, s., sarapuk, j., 2005: hemolysis of erythrocytes and erythrocyte membrane fluidity change by new lysosmtropic compounds. journal of fluorescence, 15(2): 137-141. li, f., wang, f., yu, f., fang, y., xin, z., yang, f., xu, j., zhao, l., hu, q., 2008: in vitro antioxidant and anticancer activities of ethanolic extract of selenium-enriched green tea. food chemistry, 111: 165-170. massie, h.r., aiello, v.r., williams, t.r., 1993: inhibition of iron absorption prolongs the life span of drosophila. mechanisms of ageing and development, 67(3):227-37. miyazawa, m., nakamura, y., ishikawa, y., 2000: insecticidal sesquiterpene from alpinia oxyphylla against drosophila melanogaster. journal of agricultural and food chemistry, 48: 3639-3641. miyazawa, m., yoshio, k., ishikawa, y., kameoka, h., 1998, insecticidal alkaloids against drosophila melanogaster from nuphar japonicum dc. journal of agricultural and food chemistry, 46: 1059-1063. nanjo, f., goto, k., seto, r., suzuki, m., sakai, m., hara, y., 1996: scavenging effects of tea catechins and their derivatives on 1,1-diphenyl2-picylhydrazyl radical. free radical biology and medicine, 21: 895-902. noudeh, g.d., sharififar, f., khatib, m., behravan, e., afzadi, m.a., 2009: study of aqueous extract of three medicinal plants on cell membranepermeabilizing and their surface properties. african journal of biotechnology, 9(1): 110-116. rein, d., paglieroni, t.g., pearson, d.a., wun, t., schmitz, h.h., gosselin, r., keen, c.l. 2000: cocoa and wine polyphenols modulate platelet activation and function, journal of nutrition 130:2120s-6s. rice-evans, c.a., miller, n.j., paganga, g., 1996: structure-antioxidant activity relationships of flavonoids and phenolic acids. free radical biology and medicine, 20: 933 956. van breda, s.v., van der merwe, c.f., robbertse, h., apostolides, z., 2012. immunohistochemical localization of caffeine in young camellia sinensis (l.) o. kuntze (tea) leaves. planta, doi: 10.1007/s00425-012-1804-x willson k.c., clifford, m.n., 1992: tea. cultivation to consumption. chapman and hall, london. xu, j., wang, j., deng, f., hu, z., wang, h., 2008: green tea and its major component epigallocatechin gallate inhibits hepatitis b virus in vitro. antiviral research 78: 242-249. yokozawa, t., dong, e., 1997: influence of green tea and its three major components upon lowdensity lipoprotein oxidation. experimental and toxicologic pathology, 49(5): 329-335. zhang, a., zhu, q.y., luk, y.s., ho, k.y., fung, k.p., chen, z.y., 1997: inhibitory effect of jasmine green tea epicatechin isomers on free radical induced lysis in red blood cells. life science, 61(4): 383-394. cvetković, v. et al.  toxicity of dimethyl sulfoxide against drosophila… biologica nyssana 6 (2)  december 2015: 91-95 cvetković, v. et al.  toxicity of dimethyl sulfoxide against drosophila… 91 original article received: 11 july 2015 revised: 17 august 2015 accepted: 01 october 2015 toxicity of dimethyl sulfoxide against drosophila melanogaster vladimir j. cvetković1*, tatjana lj. mitrović1, boris jovanović2, slaviša s. stamenković1, miljana todorović1, miljana đorđević3, niko radulović3 1university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia 2chair for fisheries biology and fish diseases, ludwig maximilian university of munich, kaulbachstrasse 37, 80539, munich, germany 3university of niš, faculty of sciences and mathematics, department of chemistry, višegradska 33, 18000 niš, serbia * e-mail: biovlada@yahoo.com abstract: cvetković, v.j., mitrović, t.lj., jovanović, b., stamenković, s.s., todorović, m., đorđević, m., radulović, n.: toxicity of dimethyl sulfoxide against drosophila melanogaster. biologica nyssana, 6 (2), december 2015: 91-95. dimethyl sulfoxide (dmso) is commonly used as a solvent for organic compounds but its toxic properties can affect both in vitro and in vivo studies. thus, evaluation of dmso toxicity must be performed on the model organism before its application as a solvent in the target studies. present study aimed to determine the lethal concentration required to kill 50% of the population in a given period of time (lc50), no observed effect concentration (noec), and lowest observed effect concentration (loec) values for dmso on drosophila melanogaster which is frequently used as a model for toxicity studies. twelve different concentrations of dmso were tested on three-day old d. melanogaster larvae for 12 days. at the end of the life cycle, number of live hatched adults and un-hatched pupae were counted. based on probit analysis 12 days loec was 0.04% v/v, 12 days noec was ˂ 0.04% v/v and 12 days lc50 was 0.42% v/v. the results indicate that dmso may be more toxic to d. melanogaster than it was initially considered. key words: drosophila melanogaster, noec, loec, lc50, dmso, toxicity apstrakt: cvetković, v.j., mitrović, t.lj., jovanović, b., stamenković, s.s., todorović, m., đorđević, m., radulović, n.: toksičnost dimetil sulfoksida na modelu drosophila melanogaster. biologica nyssana, 6 (2), december 2015: 91-95. dimetil sulfoksid (dmso) se često koristi kao organski rastvarač ali njegova toksičnost može da utiče na ishode in vitro i in vivo istraživanja. zato se procena toksičnosti dmso-a mora ispitati na model organizmu pre njegove primene kao rastvarača u željenom istraživanju. ovo istraživanje ima za cilj da se za dmso ustanovi letalna koncentracija koja ubija 50% populacije u posmatranom vremenskom periodu (lethal concentration 50 lc50), koncentracija koja ne uzrokuje detektabilne promene (no observed effect concentration noec) i koncentracija koja uzrokuje najmanje detektabilne promene (lowest observed effect concentration loec) na modelu drosophila melanogaster koji se veoma često koristi u toksikološkim 6 (2) • december 2015: 91-95 biologica nyssana 6 (2)  december 2015: 91-95 cvetković, v. et al.  toxicity of dimethyl sulfoxide against drosophila… 92 istraživanjima. larve d. melanogaster stare tri dana tretirane su sa dvanaest različitih koncentracija dmso-a u periodu od 12 dana. na kraju životnog ciklusa, prebrojani su adulti koji su se izlegli i lutke iz kojih se adulti nisu izlegli. prema probit analizi, za 12 dana tretmana loec je 0.04% v/v, noec je ˂ 0.04% v/v i lc50 je 0.42% v/v. rezultati ukazuju da je dmso toksičniji za d. melanogaster nego što se prvobitno smatralo. key words: drosophila melanogaster, noec, loec, lc50, dmso, toksičnost introduction dmso originates from 19th century as a by-product in the process of paper production from a wood pulp (c a p r i o t t i & c a p r i o t t i , 2012; h o r i t a & w e b e r , 1964). since then, colorless liquid dmso is characterized as a good aprotic solvent which can easily dissolve various non-polar and polar molecules (c a p r i o t t i & c a p r i o t t i , 2012; h o r i t a & w e b e r , 1964). the concern regarding the toxicity of solvents is always a topical issue in toxicology studies. thus, the pre-testing of solvents on certain model organism, which is planned to be used in further toxicology studies, is an imperative. one of the widest and frequently used model organisms in toxicity and genotoxicity studies is fruit fly, drosophila melanogaster (s t a m e n k o v i ć r a d a k et al., 2008; p a t e n k o v i ć et al., 2009; v a l e s et al., 2013). drosophila is a remarkable model for in vivo assays in toxicology because of high number of progeny and its short lifespan. in addition, culturing and maintaining of drosophila is more economical compared to other model organisms. it was demonstrated that administration of the test substances through the feeding medium is simple (n a z i r et al., 2003; s t a m e n k o v i ć r a d a k et al., 2008) making toxicology assays on drosophila even easier. the adverse effects of dmso in drosophila were noticed in the 1980's (m a s s i e et al., 1985; b r o d b e r g et al., 1987) when the authors recommended that dmso must be used carefully as a solvent. therefore, present study aimed to determine the lethal concentration required to kill 50% of the population in a given period of time (lc50), no observed effect concentration (noec), and lowest observed effect concentration (loec) values for dmso on a three-day-old d. melanogaster larvae of the wild-type strain. material and methods test organism in this experiment the d. melanogaster oregon-rc (wild-type flies, stock no. 5) ("bloomington drosophila stock center", indiana university, usa) were used. flies were cultivated on a standard feeding medium for drosophila (9% sugar, 10% corn meal, 2% agar, 2% yeast) with addition of fungicide 2.50 mg ml-1 methyl 4-hydroxybenzoate (nipagin®, alfa aesar gmbh & co kg, germany) diluted in 95% etoh (ethanol). the flies were cultivated in a standard cultivating 200 ml vials on 25 oc, 60% humidity, with 12 h/12 h day-night cycle. adults were allowed to lay eggs during the eight hour period, after that they were removed from the vials. after three days larvae which were 72±4 h old, have been removed from the cultivating medium, washed with distilled water and used in further procedure. test substance and treatment schedule twelve concentrations of dmso (carl roth, germany) were tested in duplicates and each experimental group consisted of 30 three-day-old larvae. therefore, final concentrations of dmso in 3ml of standard feeding medium were: 0.16%, 0.32%, 0.48%, 0.64%, 0.80%, 0.97%, 1.13%, 1.29%, 1.45%, 1.61%, 2.42% and 3.23% v/v. negative, water control, was also included. at the end of incubation time, which lasted up to 12 days, numbers of live hatched units (adults) and un-hatched units (at pupal stadium) were counted. statistical analysis mean values were calculated and used for further analysis. risk assessment software tool (ra v1.0; pensacola, florida, usa) and probit analysis were utilized in order to calculate values for lc50, loec, and noec. also, the confidence intervals (95%) were calculated. results and discussion the toxic effects of dmso, particularly antimicrobial and virucidal properties, were noticed in the 1960's. dmso was shown to inhibit bacterial and fungal growth at concentration of 30-50% and at concentration of 80% inactivated infectivity of some rna and dna viruses (b a s c h & g a d e b u s c h , 1968; c h a n & g a d e b u s c h , 1968; w a g n e r et al., 2002). even nowadays, the adverse properties of dmso, which is used as a solvent in toxicology studies of aquatic model organisms, was noticed (m á c h o v á et al., 2009). m á c h o v á et al. (2009) tried to establish general value for the non-toxic concentration of dmso as a solvent that could be applied across different conditions in aquatic model organism such as fish. but they found that effect of biologica nyssana 6 (2)  december 2015: 91-95 cvetković, v. et al.  toxicity of dimethyl sulfoxide against drosophila… 93 dmso on fish model depends on fish age, species, type of test and indicators observed so they concluded that general value for the non-toxic concentration of dmso can't be found and every experiment should include solvent control (m á c h o v á et al., 2009). it implies that dmso must be pre-tested on certain model organism if it is planned to be used in experiment as solvent. the fig. 1a represents survival plot of d. melanogaster units exposed to different concentration of dmso which unambiguously shows that number of hatched flies decreased in dosedepended manner. fig. 1b represents probability of d. melanogaster units mortality exposed to dmso which was calculated by risk assessment software tool. 12 days lc50 value is 0.42% v/v (tab. 1) and total number of adults hatched from pupal stadium drastically decreased from 0.32% v/v where almost every unit developed into adult to 0.97% v/v where none of the adults hatched. in addition, pupal mortality (un-hatched units) increased drastically in this concentration range, respectively (fig 2). concentration of dmso in substrate, from 0.97% v/v up to the 3.23%, v/v caused increase of larval mortality (fig. 2). concentrations above 0.32% v/v decreased hatchability of adults. these observations are in accordance with similar study (n a z i r et al., 2003). in genotoxicity and toxicity studies larvae, or adults, of d. melanogaster, as a model organisms, are widely used (n a z i r et al., 2003; a d a m s k i et al., 2009; c a s t a ñ e d a p a r t i d a et al., 2011; m i y a z a w a et al., 1998; 2000; g r a f et al., 1984). there are many references on dmso toxicity, but only a few papers are on dmso toxicity in drosophila. for instance, g r a f et al. (1984) used dmso in final concentration of 2% in feeding medium for drosophila in somatic mutation and recombination test (g r a f et al., 1984). but recent research by n a z i r et al. (2003) showed that dmso should be used very cautiously as solvent because it could results with false conclusions in the screening fig 1. survival plot of d. melanogaster units (a) and probability of the units mortality exposed to dmso with 95% confidence intervals (b). table 1. probit analysis: loec, noec and lc50 values with confidence intervals after 12 days of exposure. % of population response concentration in % (v/v) lower limit 95% upper limit 95% loec 12 days 0.01% 0.04555 0.00780 0.09803 0.10% 0.06642 0.01461 0.12884 1.00% 0.10502 0.03122 0.18004 5.00% 0.15806 0.06122 0.24374 10.00% 0.19656 0.08741 0.28734 20.00% 0.25598 0.13387 0.35252 30.00% 0.30967 0.18096 0.41093 40.00% 0.36435 0.23254 0.47149 lc50 12 days 50.00% 0.42404 0.29133 0.54068 biologica nyssana 6 (2)  december 2015: 91-95 cvetković, v. et al.  toxicity of dimethyl sulfoxide against drosophila… 94 fig 2. percentage of hatched (alive adults), un-hatched (non-survived units at pupal stadium) and non-survived units at larval stage (larval mortality) influenced with different concentration of dmso (showed in % v/v) in feeding media and in water (negative) control. of test substances because of its toxicity to d. melanogaster (n a z i r et al., 2003). our results confirmed observation that dmso is toxic and can impact normal larval development in concentration greater than 0.04% v/v (loec value) in feeding medium. the present study was performed with twelve concentrations which provided better resolution of obtained results than in the study by n a z i r et al. (2003) where six concentrations were tested. also, we treated three-day-old larvae of the wild-type strain of d. melanogaster which differs from the study by n a z i r et al. (2003) whose treatment started at the egg stadium of the transgenic strain of d. melanogaster and lasted for 10-14 days. finally, probit analysis was performed as a common analysis in toxicology, in order to determine noec, loec and lc50. n a z i r et al. (2003) considered dmso at 0.3% v/v as noael (no observed adverse effect level) value which can be used as dietary concentration in toxicity studies with d. melanogaster. however, according to present research 12 days loec is 0.04% v/v so the absolutely safe dietary concentration of dmso in feeding medium for d. melanogaster larvae of wildtype strain, should be less than 0.04% v/v. one of the reasons for higher toxicity (and lower value of loec) observed in the present study compared to the n a z i r et al. (2003) may be the lower sensitivity of d. melanogaster eggs to dmso since the eggs do not actively feed on the exposure media thus the cumulative toxicity during same exposure time period is lower. therefore, a more realistic scenario would be to start the exposure during the larval stage, as it was performed in the present study. conclusion the probit analysis showed that 12 days loec was 0.04% v/v, noec was ˂ 0.04% v/v and lc50 was 0.42% v/v. pupal mortality reached maximum at 0.97% v/v of dmso in substrate while concentration greater than 0.32% v/v reduced hatchability of adults at the end of lifespan. these results indicate that dmso is more toxic to d. melanogaster larvae than it was initially thought. obtained lc50, loec and noec values for dmso in this experiment should be considered seriously in further toxicity testing with d. melanogaster larvae as experimental model. acknowledgements. this work was supported by the ministry of education, science and technological development of the republic serbia during activity on project oi171025 and iii41018. also, authors want to thank dr. aleksandra patenković and dr. zorana kurbalija-novičić (institute of biological research, university of belgrade, serbia) who kindly provided drosophila melanogaster oregon-rc strain which was used in this experiment. references adamski, z., błoszyk, j., musiał, j., łysiak, m., urbaniak, l., ziemnicki, k. 2009: diflubenzuron inhibits reproduction of different strains of drosophila melanogaster. insect science, 16 (4): 305-309. 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% p e rc e n ta g e o f u n it s concentration of dmso in treatments hatched un-hatched (pupal mortality) larval mortality biologica nyssana 6 (2)  december 2015: 91-95 cvetković, v. et al.  toxicity of dimethyl sulfoxide against drosophila… 95 aguilar, j.s., roy, d., ghazal, p., wagner, e.k. 2002: dimethyl sulfoxide blocks herpes simplex virus-1 productive infection in vitro acting at different stages with positive cooperatively. bmc infectious diseases, 2 (9): 1–10. basch, h. & gadebusch, h.h. 1968: in vitro antimicrobial activity of dimethyl sulfoxide. applied microbiology, 16: 1953-1954. brodberg, r.k., mitchell, m.j., smith, s.l., woodruf, r.c. 1987: specific reduction of n,ndimethylnitrosamine mutagenicity in drosophila melanogaster by dimethyl sulfoxide. environmental and molecular mutagenesis, 10: 425–432. capriotti, k. & capriotti, aj. 2012: dimethyl sulfoxide: history, chemistry, and clinical utility in dermatology. journal of clinical and aesthetic dermatology, 5 (9): 24–26. castañeda-partida, l., heres-pulido, m.e., guzmánrincón, j., hernández-portilla, l.b., dueñasgarcía, i.e., durán-díaz, á., delfín-alcalá, i. 2011: lead acetate does not inhibit dimethylnitrosamine activation and interacts with phenobarbital which is genotoxic in the st cross of the drosophila wing spot test. food and chemical toxicology, 49 (9): 2172-2179. chan, j.c. & gadebusch, h.h. 1968: virucidal properties of dimethyl sulfoxide. applied microbiology, 16: 1625-1626. graf, u., würgler, f., katz, a., frei, h., juon, h., hall, c., kale, pg. 1984: somatic mutation and recombination test in drosophila melanogaster. environmental mutagenesis, 6 (2): 153-188. horita, a. & weber, lj. 1964: skin penetrating property of drugs dissolved in dimethyl sulfoxide (dmso) and other vehicles. life sciences, 3: 1389-1395. máchová, j., prokeš, m., kroupová, h., svobodová, z., mácová, s., doleželová, p., velíšek., j. 2009: early ontogeny, growth and mortality of common carp (cyprinus carpio) at low concentrations of dimethyl sulfoxide. acta veterinaria brno, 78: 505-512. massie, h.r., williams, t.r., lodice, a.a. 1985. influence of antiinflammatory agents on the survival of drosophila. journal of gerontology, 40: 257–260. miyazawa, m., nakamura, y., ishikawa, y. 2000: insecticidal sesquiterpene from alpinia oxyphylla against drosophila melanogaster. journal of agricultural and food chemistry, 48 (8): 36393641. miyazawa, m., yoshio, k., ishikawa, y., kameoka, h. 1998: insecticidal alkaloids against drosophila melanogaster from nuphar japonicum dc. journal of agricultural and food chemistry, 46 (3): 1059-1063. nazir, a., mukhopadhyay, i., saxena, d., chowdhuri, d.k. 2003: evaluation of the no observed adverse effect level of solvent dimethyl sulfoxide in drosophila melanogaster. toxicology mechanisms and methods, 13 (2): 147-52. patenković, a., stamenković-radak, m., banjanac, t., andjelković, m. 2009: antimutagenic effect of sage tea in the wing spot test of drosophila melanogaster. food and chemical toxicology, 47 (1): 180-183. stamenković-radak, m., kalajdzić, p., savić, t., savić, m., kurbalija, z., rasić, g., andjelković, m. 2008: the effect of lead on fitness components and developmental stability in drosophila subobscura. acta biologica hungarica, 59 (1): 47-56. vales, g., demir, e., kaya, b., creus, a., marcos, r. 2013: genotoxicity of cobalt nanoparticles and ions in drosophila. nanotoxicology, 7: 462-468. http://www.sciencedirect.com/science/article/pii/s0278691511002456 biologica nyssana 6 (2)  december 2015: 91-95 cvetković, v. et al.  toxicity of dimethyl sulfoxide against drosophila… 96 karadžić, b.: chasmophytic forests of ostrya carpinifolia in west-serbian canyons. biologica nyssana, 8 (1), september 2017 biologica nyssana 8 (1)  september 2017: 73-81 karadžić, b. et al.  chasmophytic forests of ostrya carpinifolia… 73 original article received: 22 july 2017 revised: 17 august 2017 accepted: 30 august 2017 chasmophytic forests of ostrya carpinifolia in west-serbian canyons branko karadžić institute for biological research of belgrade university, bul. despota stefana 142, 11000 belgrade, serbia * e-mail: branko@ibiss.bg.ac.yu abstract: karadžić, b.: chasmophytic forests of ostrya carpinifolia in west-serbian canyons. biologica nyssana, 8 (1), september 2017: 73-81. variability patterns and biodiversity components of chasmophytic forests of ostrya carpinifolia in westserbian ravine habitats were analyzed in this article. investigations were performed in four gorges along gornja trešnjica, gradac, ljutina and lim rivers. chasmophytic hop hornbeam forests occur on steep slopes (25° to 50°) at elevations ranging from 260 m to 1200 m, on all aspects. these forests grow on screes, rocky clifs, colluvial gravel, on shallow soils on dolomites, limestone and serpentine. canonical correspondence analysis indicates that moisture, light and temperature gradients are the main factors affecting diversification of investigated forests. the greatest alpha diversity was detected in brodarevo and gostun gorges, along the lim river. key words: alpha diversity, beta diversity, ostrya carpinifolia, chasmophytes, canonical correspondence analysis apstrakt: karadžić, b.: hazmofitske šume sa ostrya carpinifolia u zapadnoj srbiji. biologica nyssana, 8 (1), septembar 2017: 73-81. u ovom radu ispitivani su obrasci varijabilnosti i komponente biodiverziteta hazmofitskih šuma ostrya carpinifolia u klisurskim habitatima zapadne srbije. istraživanja su sprovedena u četiri klisure duž reka gornja trešnjica, gradac, ljutina i lim. hazmofitske šume crnog graba se javljaju na strmim padinama (25° do 50°) i visinama od 260 m do 1200 m. ove šume rastu na siparima, kamenitim liticama, koluvijalnom šljunku, plitkim zemljištima na dolomitu, krečnjaku i serpentinima. kanonska korespodentna analiza ukazala je da gradijenti vlage, svetlosti i temperature predstavljaju glavne faktore koji utiču na diverzifikaciju ispitivanih šuma. najveći alfa diverzitet uočen je u klisurama brodareva i gostuna duž reke lim. ključne reči: alfa diverzitet, beta diverzitet, ostrya carpinifolia, hazmofite, kanonijska korespodentna analiza 8 (1) • september 2017: 73-81 doi: 10.5281/zenodo.964117 biologica nyssana 8 (1)  september 2017: 73-81 karadžić, b. et al.  chasmophytic forests of ostrya carpinifolia… 74 introduction the chasmophitic vegetation is very complex, since it involves communities on calcareous, serpentine and silicate rocks, at different altitudes, from colline (usually ravine) habitats to nival regions (l a k u š i ć & k a r a d ž i ć , 2010). these communities are intrazonal, since they occur in all vegetation zones. chasmophytes in ravine habitats usually belong to the group of (paleo) endemic rare species. in ravine habitats, these species found a refuge from unfavourable climatic conditions, stronger competitors from neighbouring non-ravine communities, grazing and human-induced disturbances. this article is focused on the chasmophytic forests, dominated by european hop hornbeam (ostrya carpinifolia scop.). the forests are located in ravine habitats along the gradac, gornja trešnjica, ljutina and lim rivers. ostrya carpinifolia scop. is a species adapted to warm to moderate climate. this species has a wide tolerance limits with respect to both, the light and moisture gradients (popović, et al., 1996). therefore it (co)dominates in a wide spectrum of communities that belong to ecologically different alliances (t r i n a j s t i ć & c e r o v e č k i , 1978, p u n c e r & z u p a n č i č , 1982, t o m i ć , 1994, 2000). distribution of ostrya carpinifolia comprises apennines, tyrol, western parts of the balkan peninsula, asia minor and lebanon (m e u s e l et al., 1965). the closest aliens of this species occur in east asia and north as well as central america (t r i n a j s t i ć & c e r o v e č k i , 1978). such distribution clearly indicates a tertiary disjunction of the genus ostrya scop. european hop hornbeam codominates with other (sub)mediterranean xeric trees (carpinus orientalis mill., quercus pubescens willd., paliurus spina-christi mill.) in xeric communities of the alliance carpinion orientalis horvat 1954, along the adriatic sea and in regions where the maritime influence extends deep into the continent, along the rivers una, neretva, drim and vardar. in more humid habitats, these forests are replaced by the amphiadriatic mesic calcareous climte-zonal alliance fraxino orni-ostryion carpinifoliae tomažič 1940 = ostryo-carpinion orientalis horvat 1959 (h o r v a t et al., 1974, č a r n i et al., 2009, m u c i n a et al., 2016). hop hornbeam also occurs on relict pinus nigra forests on dolomite and ultramafic substrates of the dinarides (alliance erico carneae-fraxinion orni horvat 1959). due to a broad ecological amplitude with respect to moisture, ostrya carpinifolia forms mesic intrazonal forests in inland ravine habitats. these forests are included into the alliance tilio platyphylli acerion pseudoplatani klika 1955 (b o r h i d i et al., 1999, k o š i r et al., 2008, d a k s k o b l e r et al., 2013). broad-leaved ravine tilio-acerion forests specify an eu priority type of ecosystems (r o d w e l l et al., 1998). compared with tilio platyphylli acerion pseudoplatani forests, the hop hornbeam forests in ravine habitats of inland balkan aea are more diverse (k a r a d ž i ć et al., 2001, 2015). the aim of present study is to describe structure of the mixed black hornbeam forests that are located in west-serbian canyons, to relate their floristic composition with environmental factors and to detect patterns of and -diversity. material and methods the study area is located in western parts of the central serbia. investigations were performed in four ravine habitats along the gradac, gornja trešnjica, ljutina and lim rivers. the altitudinal range and geographic coordinates of investigated localities are presented in table 1. the eastermost and northernmost locality is the gorge of the gradac river. this gorge is exposed to the influence of panonnian plane. the vegetation samples (relevés) were collected seasonally, from early spring to late autumn. combined cover-abundance values of b r a u n b l a n q u e t ’s (1965) alphanumeric scale were replaced by a nine-degree numeric values (w e s t h o f f & v a n d e r m a a r e l , 1973). the taxonomic nomenclature was harmonized with the plant list database that involves accepted latin name for vascular plants and bryophytes (http://www.theplantlist.org). differentiation of communities with respect to environmental variables was assessed using the table 1. geographic position and altitudinal range of investigated localities rivers locality altitudinal range angular coordinates gornja trešnjica trešnjica 315-855 44.08447 n 19.32165 e lim brodarevo, gostun 500-1080 43.10567 n 19.46642 e ljutina ljutina 619-1200 43.28807 n 19.24945 e gradac (sušaja) lastva 261-819 44.11722 n 19.51642 e biologica nyssana 8 (1)  september 2017: 73-81 karadžić, b. et al.  chasmophytic forests of ostrya carpinifolia… 75 canonical correspondence analysis (t e r b r a a k , 1986). environmental variables within analyzed communities were assessed using the weighted average of ecological indicator values (k o j i ć et al., 1997). alpha diversity was calculated using the shanon function    n i ii pph 1 log , where pi is the proportion for species within community. according to h i l l (1973) and p i e l o u (1974), the shanon’s function is a special case of renyi's general entropy function. beta diversity was detected according to the r o u t l e d g e (1977) method: 1 2 2    sr s  , where s is the total number of species, and r is the number of species pairs whose distributions overlap. above equation specifies the changing rates of compositional turnover along an environmental gradient (w i l s o n & s h m i d a , 1984). all statistical analyses were performed using “flora” software package (k a r a d ž i ć , 2013). results and discussion floristic differences among analyzed canyons ravine forests in serbian gorges and canyons occur on screes, rocky clifs, colluvial gravel and also on developed soils, mainly shallow rankers, rendzinas and brown calcareous soils (p a v l o v i ć et al., 2017). screes were recorded in all investigated ravines. they are covered by sparsely distributed individuals of ostrya carpinifolia scop., fraxinus ornus l., frangula rupestris (scop.) schur. and cotinus coggygria scop. on more favourable soils, the hop hornbeam occurs in combination with carpinus orientalis l., rhamnus saxatilis jacq., r. cathartica l., r. alpina subsp. fallax (boiss.) maire & petitm., tilia platyphyllos scop., acer campestre l., juglans regia l., carpinus betulus l., viburnum lantana l., quercus cerris l., q. petraea (matt.) liebl., q. pubescens willd., fagus sylvatica l. etc, forming complex polydominant communities. in order to detect patterns of floristic variability of analyzed forests a set of multivariate statistical methods was performed. correspondence analysis (ca) and principal components analysis (pca) with their canonical forms are the most frequently used ordination methods in ecology (j o n g m a n et al., 1987; l e g e n d r e & l e g e n d r e , 2003; g r e e n a c r e , 2010). principal axes obtained by either ca or pca are mutually uncorrelated, but not necessarily independent. a fig 1. correspondence analysis (a) and discriminant correspondence analysis (b) of analyzed forests. analyses were performed on a hypercorrelated matrix, in order to avoid undesirable guttman effect biologica nyssana 8 (1)  september 2017: 73-81 karadžić, b. et al.  chasmophytic forests of ostrya carpinifolia… 76 spurious polynomial relation between ordination axes (the arch effect or guttman effect) is a wellknown drawback of ca and pca. compared to ca, pca is more sensitive to the arch effect, especially in the case when beta diversity (species turnover) along spatial or environmental gradients is high (j o n g m a n et al., 1987, k a r a d ž i ć & p o p o v i ć , 1994, k a r a d ž i ć et al., 1999). k a r a d ž i ć et al. (2014) revealed that the hypercorrelated matrices significantly reduce the guttman effect. therefore, all multivariate analyses were performed on hypercorrelated matrices. the results of correspondence analysis (fig. 1) clearly indicate that the forests continuously intergrade. this is a consequence of polydominant structure and a great number of species with overlapping distribution. contrary to ordinary correspondence analysis, the discriminant correspondence analysis (g r e e n a c r e , 2000), clearly separates chasmophytic forests of ostrya carpinifolia in different gorges. first principal axis separates analyzed forests according to the altitudinal gradient (from the lowermost gorges along the gradac and gornja trešnjica to mountainous communities in ljutina and brodarevo gorges). environmental conditions specific topography of gorges and canyons significantly modifies microclimate conditions. solar rays hit the opposite slopes of canyons at different angles. amount of solar energy per unit surface is much greater on southern than on northern slopes. moreover, the evaporation is much greater on southern than on northern slopes. such conditions form strong environmental gradients. effects of environmental variables on floristic differentiation of analyzed forests were detected using canonical correspondence analysis (cca). the canonical ordination axes maximize floristic variability of a data set that can be explained by a set of environmental variables. the results of cca (fig. 2) clearly indicate that the forests continuously intergrade. this is a consequence of polydominant structure and a great number of species with overlapping distribution. the main factors that affected floristic differentiation of analyzed forests are moisture, light and temperature. thermophylous communities covering the gorge of the gradac river are clearly separated from other communities. fig. 3. components of alpha diversity in chasmophytic hop hornbeam communities fig 2. canonical correspondence analysis of chasmophytic hop hornbeam forests in investigated gorges biologica nyssana 8 (1)  september 2017: 73-81 karadžić, b. et al.  chasmophytic forests of ostrya carpinifolia… 77 diversity components of analyzed forests biological diversity is the term, which in its broadest sense means variability of the living world. such general definition needs further elaboration, since biological variability is a multi-component, complex parameter that involves intraspecies (population) and interspecies (cenotic) variety (w h i t t a k e r , 1972; k a r a d ž i ć & m a r i n k o v i ć , 2009). intraspecies or population variability is the result of genetic variability, the variability induced by influence of environmental factors and the variability which arises from the interaction of genetic background and fig 4. beta diversity of hop hornbeam forests along the gradients of moisture (a), temperature (b), light (c), soil acidity (d) and soil nitrogen (e) biologica nyssana 8 (1)  september 2017: 73-81 karadžić, b. et al.  chasmophytic forests of ostrya carpinifolia… 78 environmental conditions. interspecies or coenotic variability can be divided into within-community variability (alpha diversity), variability between communities (beta diversity) and the total variability in a given region (gamma diversity). alpha diversity (within-community diversity) depends on species richness and equitability of species abundances in a community. both, species richness and entropy are the greatest in brodarevo (and nearby gostun) gorges along the lim river. a high species richness and shanon’s entropy were recorded also in communities that inhabit the ljutina river gorge. the lowermost values of alpha diversity components were recorded in communities covering the gorge of the gradac river (fig. 3). a n d e r s o n et al. (2015) distinguish two types of beta diversity: directional turnover along a gradient and nondirectional between-community variation. directional beta-diversity (or extent of species replacement along environmental gradients) indicates the degree to which habitats have been partitioned by species. replacement of species (species turnover) is a function of the habitat heterogeneity and ecological tolerance of species (w h i t t a k e r , 1972; k a r a d ž i ć et al., 2003). beta diversity was calculated for each adjacent pairs of sites that are ordered along an environmental gradient (fig. 4). irrespective on environmental gradient, the greatest values of beta diversity were observed in communities that inhabit gorges along the lim river. endemism of hasmophytic taxa according to v e l č e v et al. (1992), the balkan endemics may be divided into two main categories: paleo-endemics and neo-endemics. the first group involves tertiary relics, with low-genetic variability, diploid or low-ploid chromosome number and fig 5. important endemic chasmophytes in analyzed forests: onosma stellulatum wald. et kit (a), lathyrus binatus pančić (b), aquilegia grata zimmeter (c), achillea ageratifolia (sibth. & sm.) boiss. (d), daphne malyana blečić (e), athamanta turbith (l.) brot. subsp. haynaldii (borbas & uechtr.) tutin. (f), campanula secundiflora vis. & pančić (g), corydalis ochroleuca koch. (h) and hieracium waldsteinii tausch (i). biologica nyssana 8 (1)  september 2017: 73-81 karadžić, b. et al.  chasmophytic forests of ostrya carpinifolia… 79 apparent disjunct distribution with its closest relatives. due to long time scale isolation and absence of genetic contacts with their relatives, these species formed stable genotypes under particular conditions. neo-endemics are species that appeared during quartenary climatic stresses. these species are characterized by polyploidy (tetraploid or high ploid chromosome numbers). tertiary relics comprise species that dominated in europe at the beginning of the so-called quaternary period. repeated swings of glacial and interglacial periods affected significant reduction of floristic diversity in europe. periodic latitudinal (northward and southward) migrations of flora were obstructed by the mountain barriers. the east–west orientation of european main mountain massifs prevented latitudinal migration of tertiary relics. therefore, the remnants of tertiary flora persisted in three south-european peninsulas (iberia, apennines and the balkans), mostly in ravine habitats, where specific topography attenuated climate oscillations. the most important endemorelics in analyzed communities are onosma stellulatum wald. et kit, lathyrus binatus pančić, aquilegia grata zimmeter, achillea ageratifolia (sibth. & sm.) boiss., daphne malyana blečić, athamanta turbith (l.) brot. subsp. haynaldii (borbas & uechtr.) tutin., campanula secundiflora vis. & pančić, corydalis ochroleuca koch., hieracium waldsteinii tausch, cardamine glauca sprengel, dianthus petraeus waldst. & kit. seseli rigidum waldst. & kit., edraianthus graminifolius (l.) a. subsp. jugoslavicus lakušić. some (steno)endemic taxa that inhabit investigated forests are presented in fig. 5. most of endemic species in serbia belong to the group of paleo-endemics and high-mountain endemics. the third group of the endemics belongs to the serpentinophytes. the serpentine soils are characterized by low levels of the essential plant elements (nitrogen, phosphorus, potassium and calcium), as well as high levels of iron, magnesium, and manganese, and toxic levels of chromium, cobalt and nickel. due to the “serpentine stress” (toxic effects of heavy metals, nutrient shortages and droughts), most plant species avoid serpentine soils. a small percent of serpentine-tolerant taxa has evolved morpho-anatomical and physiological adaptations that allow them to survive in extremely unfavourable conditions. strong selective pressures of serpentine soil and spatial isolation of serpentine regions resulted with high percent of endemic serpentinophyte taxa in the balkans (j a k o v l j e v i ć et al., 2011). the most important serpentinophytes that inhabit analyzed communities are euphorbia glabriflora vis., scrophularia tristis maly and asplenium cuneifolium viv. these species occur on patches of serpentinous soil in gorges along the lim river. the chasmophitic vegetation is distributed on rocky habitats of paleo-temperate region (p i g n a t t i et al., 1995). despite the wide distribution, chasmophitic vegetation is fragmented and isolated. gene flow between population in fragmented and geographically isolated habitats is obstructed. even within the same saxatile region the effective gene flow between chasmophytic populations may be considerably inhibited through the frequent failure of seed to establish new individuals (d a v i s , 1951). the first step on the colonization of bare-rock must be the establishment of seedlings. this occurs in microsites with finely divided material, especially crevices, in which rootsystems can find anchorage. most of the holes suitable for ecesis are already occupied in saxatile habitats, so that it is extremely difficult for the individual to produce progeny. due to a low effective population size, the effects of genetic drift and inbreeding depression may lead to the accident elimination of alleles and increased proportion of homozygous (often nonadaptive) alleles. ecological plasticity of rare chasmophytes is narrowed because of reduced genetic variability. therefore, most of rare chasmophytes belong to the group of critically endangered taxa. reduced gene flow, in combination with both, the strong natural selection in adverse saxatile habitats, together with genetic drift due to reduced effective population size, resulted in amazing diversification of chasmophytic flora. most chasmophytic species are endemics. strong selective pressures and reduced gene flow due to both, geographical isolation of saxatile areas and ecessis problems resulted in fast speciation of chasmophytic taxa with relatively narrow distribution. conclusion polydominant forest vegetation in west-serbian canyons represents a valuable pool of species diversity. a great heterogeneity of environmental conditions and specific history of biota in the canyons resulted with complex communities that represent significant resource of rare taxa. additional research of these forests is required, in order to elucidate syntaxonomic status of these communities. acknowledgements. this work was supported by the ministry of education, science and technological development of the republic of serbia (grant. no.173018). i would like to thank two anonymous referees for valuable comments on the chasmophytic endemism. biologica nyssana 8 (1)  september 2017: 73-81 karadžić, b. et al.  chasmophytic forests of ostrya carpinifolia… 80 references anderson, m.j., crist, t.o., chase, j.m., vellend, m., inouye, b.d., freestone, a.l., sanders, n.j., cornell, h.v., comita, l.s., davies, k.f., harrison, s.p., kraft, n.j.b., stegen, j.c., swenson, n.g. 2010: navigating the multiple meanings of b diversity: a roadmap for the practicing ecologist. ecology letters, 14: 19–28 borhidi, a., kevey, b., varga, z. 1999: checklist of the higher syntaxa of hungary. annali di botanica, 57: 159-166. dakskobler, i., košir, p., kutnar, l. 2013: gozdovi plemenitih listavcev v sloveniji: združbe gorskega javorja, gorskega bresta, velikega jesena, ostrolistnega javorja, lipe in lipovca. silva slovenica, and zveza gozdarskih društev slovenije, ljubljana. davis, h.p. 1951: cliff vegetation in the eastern mediterranean. journal of ecology, 39, 63-93 čarni, a., košir, p., karadžić, b., matevski, v., redžić, s., škvorc, ž. 2009: thermophilous deciduous forests in southeastern europe. plant biosystems 143:1–13. greenacre, m.j. 1984: theory and applications of correspondence analysis. academic press, london. greenacre, m.j. 2010: biplots in practice. fundación bbva, barcelona. hill, m.o. 1973: diversity and evenness: a unifying notation and its consequences. ecology, 54: 427432. horvat, i., glavač, v., ellenberg, h. 1974: vegetatin sudosteuropas. gustav fischer: jena. jakovljević, k., lakušić, d., vukojičić, s., tomović, g., šinžar-sekulić, j., stevanović, v. 2011: richness and diversity of pontic flora on serpentine of serbia. cent. eur. j. biol. 6: 260274. jongman, r.h., ter braak, c.j.f., van tongeren, o.f.r. 1987: data analysis in community and landscape ecology. pudoc, wageningen. karadžić, b., jarić, s., pavlović, p., mitrović, m. 2015: aquatic and wetland vegetation along the sava river. pp. 249-316. in: milačič. r., ščančar, j., paunovič, m. (eds), the sava river. springer heidelberg. karadžić, b., jarić, s., pavlović, p., marinković, s., mitrović, m. 2014: application of hypercorrelated matrices in ecological research. computational biology and bioinformatics 2: 5762. karadžić, b., marinković, s. 2009: kvantitativna ekologija. ibiss, beograd, p 489. karadžić, b., marinković, s., kataranovski, d. 2003: use of the -function to estimate the skewness of species responses. journal of vegetation science, 14: 799-805. karadžić, b., mijović, a., popović, r., perišić, s., marinković, s. 2001: forest vegetation in west serbian canyons: biodiversity hotspots. in: ragodlou, k. (ed), forest research: a challenge for an integrated european approach. nagrefforest research institute, thessaloniki, greece, pp 513–518. karadžić, b., popović, r. 1994: the generalized standardization procedure in ecological ordination: tests with "metric" ordination methods. journal of vegetation science, 5: 259262. karadžić, b., šašo-jovanović, v., jovanović, z., karadžić, d. 1999: on detrending in correspondence analysis and principal components analysis. ecoscience, 6: 110-116. karadžić, b. 2013: flora: a software package for statistical analysis of ecological data. water research and management, 3: 45-54. kojić, m., popović, r., karadžić, b. 1997: vaskularne biljke srbije kao indikatori staništa. ibiss: beograd. 160 p. košir, p., čarni, a., di pietro, r. 2008: classification and phytogeographical differentiation of broadleaved ravine forests in southeastern europe. journal of vegetation science, 19: 331–342. lakušić, d., karadžić, b. 2010: new associations of serpentine chasmophitic vegetation (asplenietea trichomanis br.-bl. 1934 corr. oberd. 1977) on kopaonik mt in serbia. botanica serbica, 34: 6779. legendre, p., legendre, l. 2003: numerical ecology. elsevier, amsterdam. 853 p. meusel, h., jager, a., weinert, a. 1965: vergleichende chorologie der ceatraleuropischea flora. gustav fisher: jena. pavlović, p., kostić, n., karadžić, b., mitrović, m. 2017: the soil of serbia. springer. heidelberg. 225 p. pianka, e. 1984: evolutionary ecology. harper & row: new york. pielou, e.c. 1974: population and community ecology. gordon and breach, new york. popović, r., kojić, m., karadžić, b. 1996: ecological characcteristics of six important submediterranean tree species in serbia. bocconea 5: 431-438. puncer, i., zupančič, m. 1982: die ökologische und wirtschaftliche bedeutung der ostrya carpinifolia scop. in slowenien. studia geobotanica, 2: 2532. rodwell, j.s., schamine´e, j.h.j., mucina, l., pignatti, s., dring, j., moss, d., 1998: the scientific basis of the eunis habitat biologica nyssana 8 (1)  september 2017: 73-81 karadžić, b. et al.  chasmophytic forests of ostrya carpinifolia… 81 classification. report to the european topic centre on nature conservation, unit of vegetation science, lancaster. routledge, r.d. 1977: on whittaker's components of diversity. ecology, 58, 1120-1127. shmida, a., wilson, m.v. 1983: biological determinants of species diversity. journal of biogeography, 12: 1-20. ter braak, c.j.f. 1986: canonical correspondence analysis: a new eigenvector technique for multivariate direct gradient analysis. ecology, 67: 1167-1179. tomić, z. 1994: cenoareal crnog graba (ostrya carpinifolia scop.) u srbiji. glasnik instituta za botaniku botaničke bašte univerziteta u beogradu, 28: 173-182. tomić, z. 2000: zajednica orno-ostryetum aich. 1933 u refugijumima zapadne srbije. glasnik šumarskog fakulteta, beograd, 82: 177-189. trinajstić, i., cerovečki, z. 1978: o cenoarealu crnog graba, ostrya carpiaifolia scop. (corylaceae) u hrvatskoj. biosistematika 4: 57-65. westhoff, v., van der maarel, e. 1973: the braunblanquet approach. in: handbook of vegetation science v, pp 617-726. edited by r. h. whittaker. junk: the hague. whittaker, r.h. 1970: communities and ecosystems. macmillan: new york. wilson, m., shmida, a. 1984: measuring  diversity with presence-absence data. journal of ecology 72: 1055-1064. velčev, v.i., kožuharov, s.i., ančev, m.e. 1992: atlas of endemic plants in bulgaria. bulgarian academy of sciences, sofia. acar, hacıoğlu doğru, 2019, biologica nyssana 10(2) 10 (2) december 2019: 175-179 doi: 10.5281/zenodo.3600199 effects of drought stress factors on antibacterial activity of two triticum aestivum l. varieties original article okan acar department of biology, faculty of arts and sciences, çanakkale onsekiz mart university, çanakkale, turkey oacar@comu.edu.tr nurcihan hacıoğlu doğru department of biology, faculty of arts and sciences, çanakkale onsekiz mart university, çanakkale, turkey nurcihan.n@gmail.com (corresponding author) received: july 10, 2019 revised: september 10, 2019 accepted: september 13, 2019 abstract: triticum aestivum l. (wheat grass), one of the members of poaceae family, has been considered to be a very efficient therapeutic drug. in the present study, we propose to evaluate antibacterial effects of the two varieties of t. aestivum l. [cv. tosunbey (drought tolerant) and cv. sultan 95 (drought-sensitive)] which grown in three different stress conditions [(1) drought stress; (2) pre-treatment of seeds with acetyl salicylic acid; (3) drought stress and pre-treatment of seeds with acetyl salicylic acid]. the antibacterial activity of the ethanol extracts was assayed against five pathogens (pseudomonas aeruginosa atcc 27853, proteus vulgaris atcc 13315, escherichia coli nrrl b-3704, staphylococcus aureus atcc 25923 and bacillus subtilis atcc 6633) by agar disc diffusion and micro broth dilution methods. the results showed that the ethanol extracts from the different studied treatments showed antibacterial activities, with the diameters of the inhibition zone ranging from 8 to 15 mm and minimum inhibitory concentration ranging from 2.5 to 20.0 µg/ml, respectively. the highest antibacterial activity, against b. subtilis atcc 6633, was demonstrated by the extract of t. aestivum cv. sultan 95, which grown in conditions where drought stress and pre-treatment of seeds with acetyl salicylic acid were combined. key words: antibacterial activity, t. aestivum cv. tosunbey, t. aestivum cv. sultan 95 apstract: antibiofilm aktivnost etanolnog ekstrakta verbascum pinnatifidum vahl. triticum aestivum l. (pšenica), jedan od članova familije poaceae, smatra se za veoma efikasnu terapeutsku drogu. u ovom istraživanju, utvrđivan je antibakterijski efekat dva varijeteta t. aestivum l. [cv. tosunbey (tolerantna na sušu) i cv. sultan 95 (osetljiva na sušu)] koji su uzgajani u tri različita uslova stresa [(1) sres suše; (2) semena pretretirana acetil salicilnom kiselinom; (3) sres suše i pretretman semena acetil salicilnom kiselinom]. antibakterijska aktivnost etanolnih ekstrakata ispitivana je u odnosu na pet patogenih vrsta (pseudomonas aeruginosa atcc 27853, proteus vulgaris atcc 13315, escherichia coli nrrl b-3704, staphylococcus aureus atcc 25923 i bacillus subtilis atcc 6633) korišćenjem disk difuzije na agaru i mikrodilucione metode. rezultati su pokazali da etanolni ekstrakti dobijeni iz ispitivanih različitih uslova gajenja poseduju antimikrobnu aktivnost, sa dijametrima inhibicionih zona koji su varirali od 8 to 15 mm i minimalnim inhibitornim koncentracijama od 2,5 do 20,0 µg/ml. najveća antibakterijska aktivnost, utvrđena u odnosu na b. subtilis atcc 6633, pokazana je od strane ekstrakta t. aestivum cv. sultan 95, koji je rastao u uslovima kombinacije sušnog stresa i pretretmana semena acetil salicilnom kiselinom. ključne reči: antibakterijska aktivnost, t. aestivum cv. tosunbey, t. aestivum cv. sultan 95 introduction plants contain numerous of constituents and are valuable sources of new and biologically active molecules having antimicrobial properties that are important for drug designing against diseases (das et al., 2010; bhattacharjee and islam, 2015; saha et al., 2018). wheat as one of the members of gramineae family, has been known for very efficient medical values and several therapeutic drugs that include bran and germ. it is used as a protection against various diseases such as constipation, heart diseases, appendicitis, obesity, diabetes and condition of the colon called diverticulum (hadjivassiliou et al., © 2019 acar, hacıoğlu doğru. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work noncommercially under the same license as the original. 175 13th symposium on the flora of southeastern serbia and neighboring regions 2003; saha et al., 2018). triticum aestivum has also been shown to be a rich source of vitamins a, c, e and b complex, including b12 (ashok, 2011; sundaresan et al., 2015). it contains a multitude of minerals like calcium, phosphorus, magnesium, alkaline earth metals, potassium, zinc, boron, and molybdenum. other compounds that are making this grass therapeutically effective are the indole compounds, choline and laetrile (amygdalin) (padalia et al., 2011; sundaresan et al., 2015). antibiotic resistance has become a global concern (westh et al., 2004; saha et al., 2018) and clinical efficiency of many existing antibiotics is being threatened by the emergence of multidrug-resistant pathogens as reported by saha et al. (2018). so, there is an urgent need to develop new antimicrobial compounds which are more active against novel and re-emerging infectious diseases (rojas et al., 1992; saha et al., 2018). duke and ayensu (1985) reported that wheat is an easily grown plant in the world and that the young stems had proven effective for the treatment of biliousness, intoxication and removing skin blemishes, antipyretic, antihydrotic and sedative. triticum aestivum has also been used against cough, sore throat, malaise, spasmic pain, abdominal coldness and constipation (ashok, 2011). wheat is also known to have anticancer and antimicrobial properties (gregorova et al., 2015). drought is the most critical limiting factor for crop production and is becoming an increasingly severe problem in many regions of the world. abiotic stress conditions such as drought, salinity, cold stress can induce the synthesis of pathogen-associated proteins (prs) in plants (van loon et al., 2006; sehgal and mohamad, 2018). in severe drought conditions, prs such as chitinase and glucanase have been activated in wheat (gregorova et al., 2015). in addition, tdltp4, a pr protein, was found to be higher in wheat variety with salt and drought tolerance (safi et al., 2015). on the other hand, prs induced by the effect of salicylic acid (sa) has been shown to have antimicrobial activity in vitro (van loon et al., 2006). also, sa applications have been reported to increase antimicrobial activity in turnip (thiruvengadam et al., 2016) and sesame plants (hossein et al., 2016). it is known that antifungal and antimicrobial activity increases due to oxidative stress induced by environmental stresses (sharma et al., 2018; schmidt et al., 2019). the relationship between antimicrobial activity and prs, produced by plants under environmental stress conditions such as defensins (schmidt et al., 2019) and phytoalexins (ejike et al., 2013), has been demonstrated earlier (li et al., 2011). in this study, the effects of drought and salicylic acid (sa) priming combinations were investigated on antibacterial activity in 21-day-old seedlings of two t. aestivum varieties. material and methods plant materials the seeds were sterilized by washing with 5% sodium hypochlorite solution for 5 min (1 time) and with sterile distilled water for 2.5 min (3 times). two tablets of 500 mg aspirin were thoroughly dissolved in 500 ml of distilled water and sterile seeds were kept in this solution for 2 hours for ascorbic acid (asa) priming. at the end of the period, the seeds were placed on moist filter paper and germinated for three days. seedlings were planted in perlite:peat mixture (1:3) containing pots. plants were watered with hoagland nutrient solution (100%; steward, 1983) during 21 days in growth chamber (22-24 oc, photoperiod 16/8 day/night). drought stress application was carried out with water scarcity for 5 days starting from the 21st day. seedlings of t. aestivum l. cv. tosunbey (drought tolerant) and cv. sultan 95 (drought-sensitive) were divided into four groups: (1) control (2) drought stress; (3) pretreatment of seeds with acetyl salicylic acid; (4) drought stress and pre-treatment of seeds with acetyl salicylic acid. leaf sampling was done on 21-day old plants. preparation of plant extracts air-dried samples of two wheat varieties were grounded into a fine powder in a grinding mill. pulverized plant samples (1 g) were extracted with 10 ml of 80% ethanol, (1:10 w/v) using an orbital shaker for 8 h at room temperature. the extract was separated from the solids by filtration with whatman no. 1 filter paper. the remaining solids were extracted twice with the same solvent and extracts combined. extracts were stored in a refrigerator (4 oc) until analyzed (sultana et al., 2007). test microorganisms gram-negative bacteria (pseudomonas aeruginosa atcc 27853, proteus vulgaris atcc 13315, escherichia coli nrrlb-3704), gram-positive bacteria (staphylococcus aureus atcc 6538p, bacillus subtilis atcc 6633) were used for determining the antibacterial activities of two t. aestivum varieties. screening for antimicrobial activities disc diffusion method was used for qualitative analyses of two t. aestivum species extracts (collins et al., 1989). studies were performed in triplicate. treatments with penicillin (p10) served as positive controls, respectively and treatments with ethanol without plant extracts served as negative controls. 176 biologica nyssana ● 10 (2) december 2019: 175-179 acar, hacıoğlu doğru ● effects of drought stress factors on antibacterial activity of two triticum aestivum l. varieties minimum inhibitory concentration assay minimum inhibitory concentration (mic) was investigated as recommended by the clinical and laboratory standards institute (clsi, 2006). the lowest concentration of extracts inhibiting visible growth of each test microorganisms was taken as the mic. the medium, 0.1% (w/v) streptomycin (st), nystatin (nys100) and 10% dmso were used as the non-treated, positive and negative controls, respectively (teanpasian et al., 2017). results and discussion tab. 1 indicated antimicrobial activity of two varieties of t. aestivum plant extracts and the inhibition zones formed by standard antibiotic disks and mic ratios. the results of antimicrobial testing showed that the ethanol extracts from the different treatments studied showed variable antibacterial activities, with the diameters of the inhibition zone ranging from 8 to 15 mm and 2.5 to 20.0 µg/ml, respectively. the highest antibacterial activity was demonstrated by the extract of t. aestivum cv. sultan 95, grown in drought conditions (s3 in tab.1), against b. subtilis atcc 6633. triticum aestivum plant varieties used in this study showed higher antibacterial activity against gram-positive bacteria (b. subtilis atcc 6633) than gram-negative test bacteria. the results in present study are parallel to those reported in the previous investigations. in general, the distinctive feature of gram-negative bacteria is the presence of a double membrane surrounding each bacterial cell. although all bacteria have an inner cell membrane, gramnegative bacteria have a unique outer envelope. this outer membrane excludes certain drugs and antibiotics from penetrating the cell, partially accounting for why gram-negative bacteria are generally more resistant to antibiotics than grampositive bacteria (dülger and dülger, 2018). in the literature there are many investigations about wheat grass antimicrobial activity (pallavi et al., 2011; das et al., 2012; sundaresan et al., 2015). however, there is no data effect of drought stress on wheat grass antibacterial activity in the literature. drought and salinity conditions can cause oxidative stress due to osmotic stress. this may induce the emergence of prs such as phytoalexin and defensin (van loon et al., 2006; sehgal and mohamad, 2018). our results showed the highest antibacterial activity in drought-sensitive sultan 95 with sa and drought treatments together. accordingly, our results are consistent with studies in which severe drought stress activates prs in wheat (gregorova et al., 2015), and increases a pr which causes higher fungal resistance in drought-tolerant wheat (safi et al., 2015). in addition, the increases in 177 biologica nyssana ● 10 (2) december 2019: 175-179 acar, hacıoğlu doğru ● effects of drought stress factors on antibacterial activity of two triticum aestivum l. varieties ta bl e 1. d is c d iff us io n, m ic ra tio s of th e tw o va rie tie s of t . a es tiv um e xt ra ct s te st b ac te ri a p la nt e xt ra ct s *d is c d iff us io na (m m ) m ic (µ g/ m l) cv . t os un be y cv . s ul ta n 95 cv . t os un be y cv . s ul ta n 95 t1 t2 t3 t4 s 1 s 2 s 3 s 4 t1 t2 t3 t4 s 1 s 2 s 3 s 4 e .c ol i n r r lb -3 70 4 8. 0 11 .0 10 .0 7. 0 12 .0 11 .0 7. 0 7. 0 16 .0 20 .0 2. 5 10 .0 20 .0 2. 5 2. 5 20 .0 20 .0 4. 0 p. a er ug in os a at c c 2 78 53 10 .0 9. 0 7. 0 7. 0 11 .0 7. 0 9. 0 9. 0 8. 0 5. 0 5. 0 20 .0 20 .0 2. 5 20 .0 10 .0 2. 5 1. 0 p. v ul ga ris a tc c 1 33 15 13 .0 10 .0 13 .0 8. 0 12 .0 9. 0 11 .0 10 .0 13 .0 2. 5 5. 0 2. 5 20 .0 2. 5 10 .0 10 .0 20 .0 4. 0 s . a ur eu s at c c 6 53 8p 7. 0 8. 0 7. 0 9. 0 11 .0 9. 0 10 .0 12 .0 14 .0 20 .0 20 .0 20 .0 5. 0 2. 5 10 .0 5. 0 5. 0 4. 0 b . s ub til is a tc c 6 63 3 11 .0 9. 0 10 .0 11 .0 10 .0 8. 0 15 .0 13 .0 15 .0 2. 5 5. 0 10 .0 5. 0 5. 0 20 .0 2. 5 20 .0 4. 0 t: t . a es tiv um c v. t os un be y s : t . a es tiv um c v. s ul ta n 95 t1 : c on tro l; t2 : t re at m en t w ith a ce ty l s al ic yl ic a ci d; t 3: tr ea tm en t w ith d ro ug ht s tre ss ; t 4: d ro ug ht s tre ss a nd p re -tr ea tm en t o f s ee ds w ith a ce ty l s al ic yl ic a ci d s 1: c on tro l; s 2: tr ea tm en t w ith a ce ty l s al ic yl ic a ci d; s 3: tr ea tm en t w ith d ro ug ht s tre ss ; s 4: d ro ug ht s tre ss a nd p re -tr ea tm en t o f s ee ds w ith a ce ty l s al ic yl ic a ci d in hi bi tio n zo ne (m m ); a in cl ud es d ia m et er o f d is k (6 m m ); p 10 = p en ic ill in (1 0 ug /d is c) ; s t: s tre pt om yc in antimicrobial activity by sa priming were similarly shown to increase antimicrobial activity in sa applications in sesame (hossein et al., 2016) and turnip (thiruvengadam et al., 2016). as a result, sa and drought application showed high antibacterial activity in drought-sensitive variety. this may indicate different prs synthesized in this variety compared to the tolerant one. the occurrence of lower antimicrobial activity in the tolerant variety compared to the sensitive variety suggests that some other stress factor is needed due to its tolerance to drought. conclusion this study was conducted to evaluate the antibacterial activity of two t. aestivum varieties under three different stress conditions. natural products are important source of new drugs which are having importance in modern medicine. the results pointed that the ethanol extracts from plants treated with combined stress conditions had significant antibacterial activities against p. vulgaris atcc 13315 (t3) and b. subtilis atcc 6633 (s3). the need to increase food production and exhaustion of wheat genetic resources has increased interest in alternative approaches for wheat improvement, including the use of different stress conditions. it is well known that abiotic stress leads to a series of morphological, physiological, biochemical, and molecular changes that adversely affect plant growth, productivity and antagonistic activity (hayat et al., 2013; ripa et al., 2019). therefore, selection, screening, and application of the stress-tolerant t. aestivum plant varieties for better farming would considerably facilitate the farming community by overcoming such extreme climate changes. this is the first report on the bioactivities of two varieties of t. aestivum grown in different drought conditions and pre-treatment of seeds with acetyl salicylic acid. this approach may also allow new kind of research in medicinal usage or development of drug research. acknowledgments. this paper was presented at the symposium on the flora of southeastern serbia and neighboring regions, june, 20-23 2019, serbia. references ashok, s.a. 2011: phytochemical and pharmacological screening of wheat grass juice (triticum aestivum l.). international journal of pharmaceutical sciences review and research, 9: 159-164. bhattacharjee, b., islam, s.m.s. 2015: assessment of antibacterial and antifungal activities of the extracts of rhynchostylis retusa. blumea medici178 nal orchid. world journal of pharmacy and pharmaceutical sciences, 4: 74-87. clsi, 2006: methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard-seventh edition. m07a7. collins, c.h., lyne, p.m., grange, j.m. 1989: microbiological methods, 6th ed. london, butterworths, 395-410. das, k., tiwari, r.k.s., shrivastava, d.k. 2010: techniques for evaluation of medicinal plant products as antimicrobial agent: current methods and future trends. journal of medicinal plants research, 4: 104-111. das, a., raychaudhuri, u., chakraborty, r. 2012: antimicrobial effect of edible plant extract on the growth of some foodborne bacteria including pathogens. nutrafoods, 12: 83-88. duke, j.a., ayensu, e.s. 1985: medicinal plants of the world. algonac, mi (usa): reference publications. dülger, b., dülger, g. 2018. antibacterial activity of verbascum antinori. konuralp tıp dergisi, 10(3): 395-398 ejike, c.e.c.c., gong, m., udenigwe, c.c. 2013: phytoalexins from the poaceae: biosynthesis, function and prospects in food preservation. food research international, 52(1), 167-177. gregorova, z., kovacik, j., klejdus, b., maglovski, m., kuna, r., hauptvogel, p., matusíkova, i. 2015: drought-induced responses of physiology, metabolites, and pr proteins in triticum aestivum. journal of agricultural and food chemistry, 63(37): 8125-8133. hadjivassiliou, m., grunewald, r.a., sharrack, b., sanders, d., lobo, a., williamson. c. woodroofe, n., wood, n., davies-jones, a. 2003: gluten ataxia in perspective: epidemiology, genetic susceptibility and clinical characteristics. brain, 126: 685-691. hayat, r., khalid, r., ehsan, m., ahmed, i., yokotaand, a., ali, s. 2013: molecular characterization of soil bacteria for improving crop yield in pakistan. pakistan journal of botany, 45: 1045–1055. hossein, s., hosseini, s.m., azari, a., rafsanjani, m. h. 2018: effects of seed priming with aba and sa on seed germination and seedling growth of sesame (sesamum indicum l.) under saline condition. australian journal of crop science, 12(9), 1385-1392. biologica nyssana ● 10 (2) december 2019: 175-179 acar, hacıoğlu doğru ● effects of drought stress factors on antibacterial activity of two triticum aestivum l. varieties li, h., goodwin p.h., han, q., huang, l., kang, z. 2011: microscopy and proteomic analysis of the non-host resistance of oryza sativa to the wheat leaf rust fungus, puccinia triticina f. sp. tritici. plant cell reports, 31(4): 637-650. padalia, s., drabu, s., raheja, i., gupta, a., dhamija, m. 2010: multitude potential of wheatgrass juice (green blood): an overview. chronicles of young scientists, 1(2): 23-28. pallavi, k., kumarswammy, g., shruthi. b. 2011: pharmacognostic investigation and antibacterial activity of triticum aestivum. journal of pharmacy research, 4: 3355-3359. ripa, f.a., cao, w., tong, s., sun, j. 2019: assessment of plant growth promoting and abiotic stress tolerance properties of wheat endophytic fungi. biomed research international, article id 6105865: 1-12. rojas, a., hernandez, l., pereda-miranda, r., mata, r. 1992: screening for antimicrobial activity of crude drug extracts and pure natural products from mexican medicinal plants. journal of ethnopharmacology, 35: 275-83. safi, h., saibi, w., alaoui, m.m., hmyene, a., masmoudi, k., hanin, m., brini, f. 2015: a wheat lipid transfer protein (tdltp4) promotes tolerance to abiotic and biotic stress in arabidopsis thaliana. plant physiology and biochemistry, 89, 64-75. saha, s., islam, z., islam, s., hossain, s., islam, s.m.s. 2018: evaluation of antimicrobial activity of wheat (triticum aestivum l.) against four bacterial strains. skuast journal of research, 20(1): 58-62. schmidt, m., arendt, e.k., thery, t.l.c. 2019: isolation and characterisation of the antifungal activity of the cowpea defensin cp-thionin ii. food microbiology, 82: 504–514. sehgal, o.p., mohamad, f. 2018: pathogenesis related proteins. in: mandahar, c.l. (ed.) plant viruses vol. ii: pathology: 65-84. crc press, boca raton. sharma, a., sharma, d., verma, s.k. 2018: in silico study of iron, zinc and copper binding proteins of pseudomonas syringae pv. lapsa: emphasis on secreted metalloproteins. frontiers in microbiology, 9: 1838. sultana, b., anwar, f., przybylski, r. 2007: antioxidant activity of phenolic components present in barks of barks of azadirachta indica, terminalia arjuna, acacia nilotica, and eugenia jambolana lam. trees. food chemistry, 104: 1106-1114. sundaresan, a., selvi, a., manonmani, h.k. 2015: the anti-microbial properties of triticum aestivum (wheat grass). international journal of biotechnology for wellness industries, 4:(3): 84-91. van loon, l.c., rep, m.p. 2006: significance of inducible defense-related proteins in infected plants. annual review of plant pathology, 44: 135-162. thiruvengadam, m., baskar, v., kim, s.h., chung, i.m. 2016: effects of abscisic acid, jasmonic acid and salicylic acid on the content of phytochemicals and their gene expression profiles and biological activity in turnip (brassica rapa ssp. rapa). plant growth regulation, 80(3): 377-390. westh, h., zinn, c.s., rosdahl v.t. 2004: an international multicenter study of antimicrobial consumption and resistance in staphylococcus aureus isolates from 15 hospitals in 14 countries. microbial drug resistance, 10: 169-76. 179 biologica nyssana ● 10 (2) december 2019: 175-179 acar, hacıoğlu doğru ● effects of drought stress factors on antibacterial activity of two triticum aestivum l. varieties jogan, n.: morphometric recognition of hordeum murinum l. subspecies in slovenia. biologica nyssana, 8 (1), september 2017 biologica nyssana 8 (1)  september 2017: 23-30 jogan, n.  morphometric recognition of hordeum murinum… 23 original article received: 28 june 2017 revised: 26 july 2017 accepted: 08 september 2017 morphometric recognition of hordeum murinum l. subspecies in slovenia nejc jogan university of ljubljana, biotechnical faculty, department of biology, večna pot 111, ljubljana, slovenia * e-mail: nejc.jogan@bf.uni-lj.si abstract: jogan, n.: morphometric recognition of hordeum murinum l. subspecies in slovenia. biologica nyssana, 8 (1), september 2017: 23-30. morphometric analysis of slovenian material belonging to hordeum murinum ssp. leporinum and type subspecies confirmed some of already reported distinguishing characters, but majority of them were not measured precisely before. after revision of herbarium material based on results, about 9% of sheets are still somehow »intermediate«, h. murinum ssp. leporinum is more common in submediterranean part with some scaterred populations in continental slovenia and the type subspecies occurr all over slovenia in lowland ruderal places. detailed determination key is provided based on results. key words: hordeum murinum, subspecies delimitation, slovenia apstrakt: jogan, n.: morfometrijsko raspoznavanje podvrsta hordeum murinum l. u sloveniji. biologica nyssana, 8 (1), septembar 2017: 23-30. morfometrijske analize materijala vrste hordeum murinum sa područja slovenije potvrdile su prisutnost dve podvrste: h. murinum ssp. leporinum i tipske podvrste. potvrđeni su neki već ranije upotrebljeni karakteri za razlikovanje i pored toga preciznije su obrađeni neki od karaktera, koje su prethodni autori pretežno navodili samo deskriptivno. između ostalog, revizija herbarijumskog materijala je jedan od rezultata, koji potvrđuje veliku prisutnost h. murinum ssp. leporinum u obalnom submediteranskom području slovenije sa nekoliko lokalnih populacija i u unutrašnjosti, a tipska podvrsta se javlja po čitavoj sloveniji na ruderalnim mestima u nizijama. oko 9% materijala nije identifikovano. . prikazan je ključ za determinaciju pomenute dve podvrste. ključne reči: hordeum murinum, razdvajanje podvrsta, slovenija introduction grasses themselves are often neglected even by serious field botanists, particularly so when they are part of ruderal vegetation. this is also the case of hordeum murinum complex, which is probably one of the best studied groups in the genus, however, the taxonomic delimitation of subordinated taxa has always been controversial (n e v s k i , 1941; c u a d r a d o et al., 2013). in central and se europe, hordeum murinum complex represents a taxonomically interesting polyploid group with 4 8 (1) • september 2017: 23-30 doi: 10.5281/zenodo.963149 biologica nyssana 8 (1)  september 2017: 23-30 jogan, n.  morphometric recognition of hordeum murinum… 24 taxa recognized at the level of subspecies (or sometimes species). two of them are rare in the mentioned territory, h. murinum ssp. glaucum (steud.) tzvelev confined to the warmest areas of southern europe (e.g. greece, cyprus, s italy, spain) and a peculiar h. murinum ssp. setariurum h. scholz & raus known only from few localities in n greece and s macedonia (s c h o l z & r a u s , 1997; j o g a n , 2005). hordeum murinum ssp. leporinum (link) arcang. and the type subspecies are reported in much wider area with "leporinum" being more thermophilous and particularly common in mediterranean europe and the type subspecies widespread but with overlapping range reaching as far south as peloponnese and n aegean (s t r i d , 2016). there are three ploidy levels recognized in the group, diploids (2n=14), tetraploids (2n=28) and hexaploids (2n=42) but the taxonomic delimitation is not congruent with ploidy levels so at least two different ploidy levels are reported for each subspecies, e.g. mostly 2n=28 but also 2n=42 or 2n=14 for both widespread subspecies (c v e l e v , 1976; k a n k a n p a a et al., 1996; t i s o n & f o u c a u l t , 2014; c u a d r a d o & al., 2013). there is no completely reliable morphological characteristic that distinguishes the recognized subspecies, so several authors refer to the "murinum complex" (c u a d r a d o & al., 2013). despite several studies evolution of the polyploid h. murinum complex is still not well understood (ibid.) and we can still agree with n e v s k i (1941), that h. leporinum is an ancestor of h. murinum s. str., whose spread towards north had been connected to man made ruderalization. in the territory of slovenia (and also neighbouring contries) only the the two widespread subspecies are reported, namely h. murinum ssp. murinum (further hmm) and h. murinum ssp. leporinum (hml). in the past, their distribution had been simply recognized as allopatric: hml reported only for the coastal submediterranean region of slovenia and hmm as being excluded from that region and scaterred in all other parts (m a r t i n č i č , 1984). but some field records blurred that picture with hml records in the continental slovenia and also hmm recorded in some localities in the coast (j o g a n & al., 1999; j o g a n , 2007). using determination keys it is not always easy to distinguish the two subspecies so our aim was to test the usefulness of morphometric characters for delimitation. in european floristic works approach in delimitation of the studied two taxa is slightly diverse, either taxa are recognized as independent species (n e v s k i , 1941; p i g n a t t i , 1983; m a r t i n č i č , 1984; l a m b i n o n & al., 1992; j o g a n , 2007) or mostly as subspecies (a m a r a l f r a n c o & r o c h a a f o n s o , 1998; b o l o s & v i g o , 2001; c i n c o v i ć & k o j i ć , 1976; c i o c i r l a n , 1990; c o n e r t , 2000; c s i k y , 2009; c u a d r a d o & al., 2013; f i s c h e r & al., 2008; i l i j a n i ć & t o p i ć , 2000; l a u b e r & w a g n e r , 1998; p o l d i n i & al., 2002; s t o h r , 2002; t i s o n & f o u c a u l t , 2014). both were mentioned in countries with at least some submediterranean influence, but in central and w europe hml mostly as only casual plant of ruderal sites (c o n e r t , 2000; l a m b i n o n & al., 1992). determination keys are mostly using 1-3 characters for delimitation between hmm and hml, only some up to 5 characters (n e v s k i , 1941; b o l o s & v i g o , 2001; j o g a n , 2007; l a u b e r & w a g n e r , 1998). in majority of keys difference in spikelet (and awn) lenght between central and lateral spikelets of triple is mentioned and quite often also differences in shape of both glumes of lateral spikelet. often the only mentioned distinguishing measure is lenght of central spikelet's "stalk". this stalk is a peculiar characteristic in some hordeum species and is developed above glumes and below lemma, so in fact this is a prolonged internode of spikelet axis. reported gap of this measurment between the subspecies is between 0.6 and 1 mm, in hmm central spikelet stalk is shorter and in hml longer, mostly with no overlapping reported (n e v s k i , 1941; a m a r a l f r a n c o & r o c h a a f o n s o , 1998; b o l o s & v i g o , 2001; c i o c i r l a n , 1990; f i s c h e r & al., 2008; j o g a n , 2007; s t o h r , 2002; t i s o n & f o u c a u l t , 2014). in practice it is often not so easy to use this character. other distinguishing characters used in some of the determination keys were: inflorescence width (b o l o s & v i g o , 2001; l a u b e r & w a g n e r , 1998) and colour (l a u b e r & w a g n e r , 1998; t i s o n & f o u c a u l t , 2014), lateral lemma width (n e v s k i , 1941; j o g a n , 2007) and hairiness (c v e l e v , 1976; j o g a n , 2014). material and methods herbarium material available in herbarium lju (university of ljubljana, biotechnical faculty) and personal herbarium collection of the author (hsnj, hortus siccus n. jogan) enriched by some more systematic sampling for h. murinum have been taken as the main source for selection of representative sample of well preserved specimens, together 86 sheets served as operational taxonomic unis (otus). in each selected herbarium sheet one well preserved and complete plant served as an otu and all the measurements taken on that plant. in the measuring phase, one inflorescence was broken apart biologica nyssana 8 (1)  september 2017: 23-30 jogan, n.  morphometric recognition of hordeum murinum… 25 so that all spikelet measurements were done on the triple of spikelets from the central part of inflorescence. remaining spikes are enclosed in herbarium capsulae attched to the sheet. macroscopic measurements were done by a ruler and all smaller parts measured under stereo microscope euromex at 20x magnification and with an ocular measuring scale divided to 1/20 mm. all the distinguishing characters used in above mentioned literature were measured (one measurement per each character per otu) and in addition to them about as big number of other potentially useful characters as follows: vsn: total plant height (dm), cpp: palea length of central spikelet (cm), cr: length of cental lemma's awn (cm), cpc: length of central "stalk" (mm), ckp: width of central lemma (mm), ant: length of ripe anther (mm), lpp: palea length of lateral spikelet (cm), lr: length of lateral lemma's awn (cm), lpc: length of lateral "stalk" (mm), lkp: width of lateral lemma (mm), dpp: central palea hairy (y/n), dstr: margin of outer lateral spikelet glume ciliate (y/n), dnot: margin of inner lateral spikelet glume ciliate (y/n), kod: length of central spikelet axis prolongation (mm), kos: width of central spikelet axis prolongation (mm), kob: axis prolongation yellow (y/n). palea lengths were taken as equal to lemma lengths, so the lemma's awn lenghts were measured from the palea tip to the end of awn. some peculiar character measurements are shown in fig. 1. input matrix of 86 otus with 12 measured characters were used for all subsequent analyses starting with simple univariate statistics and further on different multivariate approaches (using past 1.74, http://palaeo-electronica.org/2001_1/past/) to recognize texonomic structure. a priori formed groups of 2 taxa (hmm and hml) were slightly modified after resuls of numerical analyses so at the end a small "transitional" group of otus with unclear taxonomic position had been recognized as the third entity. after removing that group from analyses, the taxonomic structure is much more clear-cut, but in the field we have to be aware of such populations. studied material is listed in supplementum with locality, mtb grid code (n i k l f e l d , 1971), author, date, herbarium acronym and accession number. results and discussion majority of otus was determined using the mentioned determination keys but some of them (9%) remain at the level of h. murinum s. lat. those were temporarily excluded from the data matrix for first univariate statistical analyses. from all measured characters, some that showed a potential usefulness for distinguishing studied taxa are represented by box plots in fig. 2. usefulness of already reported distinguishing characters has mostly been confirmed for following characters: central stalk length (fig. 2a), central awn length (fig. 2b), lateral awn length (fig. 2c), lateral palea length (fig. 2d), lateral lemma width (fig. 2e) and plant height (fig. 2f). several of them are reported in determination keys without measurements, only describing their relative length. some of the recorded atributive characters were more randomly distributed among studied taxa and results are not presented here because of limited usability for distiguishing taxa. several conducted multivariate analyses helped us to recognize the most useful distinguishing characters and also to recognize the overall taxonomic structure. with exclusion of mentioned small group of «intermediates« separation of two clusters representing both subspecies was clear with only slight overlapping as shown in fig. 3. obviously delimitation of subspecies will remain uneasy in some populations. interesting results turned to be comparison of total lengths of spikelets (fig. 4), quite often roughly mentioned as »lateral awn exceeding central awn« or »... not exceeding ...«. total lengths were calculated from 3 measurements for each spikelet (cpc+cpp+cr and lpc+lpp+lr respectively). indeed in hmm central awn is mostly slightly fig. 1. three of the mentioned characters that are specific for hordeum spikelet triple: cpc (length of central "stalk"), lpc (length of lateral "stalk") and kod (length of central spikelet axis prolongation) biologica nyssana 8 (1)  september 2017: 23-30 jogan, n.  morphometric recognition of hordeum murinum… 26 fig. 2. box plots of some of the useful distinguishing characters. box comprising 2nd and 3rd quartile, whiskars 1st and 4th quartile, some outliers marked with *, hmm on the left (grey), hml on the right (white). a) central stalk length; b) central awn length; c) lateral awn length; d) lateral palea length; e) lateral lemma width; f) plant height fig. 3. results of pca. input data matrix with 12 characters and 86 otu grouped in hmm (green squares), hml (red crosses) and intermediate group (blue triangles) mur lep 0,0 0,2 0,4 0,6 0,8 1,0 1,2 1,4 m m central "stalk" length mur lep 0 2 4 6 c m central awn length mur lep 0 2 4 6 c m lateral awn length mur lep 0,0 0,2 0,4 0,6 0,8 1,0 1,2 1,4 1,6 1,8 2,0 c m lateral palea length mur lep 0,0 0,2 0,4 0,6 0,8 1,0 1,2 1,4 1,6 1,8 2,0 m m lateral lemma width mur lep 0 2 4 6 8 10 d m plant height -0,24 -0,16 -0,08 0 0,08 0,16 0,24 0,32 co o rdinate 1 -0,25 -0,2 -0,15 -0,1 -0,05 0 0,05 0,1 0,15 c o o rd in a te 2 biologica nyssana 8 (1)  september 2017: 23-30 jogan, n.  morphometric recognition of hordeum murinum… 27 exceeding the laterals, but in hml where it is often stated, that laterel awns are exceeding central one, situation is more diverse with only about half of otu having laterel awns reaching or exceeding the central one. but mostly dimensions of spikelets are distinctly bigger than in hmm, so probably better distinguishing character for quick orientation in the field would be: total length of spikelets 3-5 cm vs. 57 cm in hmm and hml respectively. in fact all the average measured values of hmm and hml differ mostly 20-35%, an interesting situation that is often observed in polyploids when compared to ancestral diploids, but in discussed group both subspecies are predominantly tetraploid so interpretation must be different. our results can be compiled in a determination key which use much more characters than the others mentioned above and some of the measurements are now more precisely measured. but of course we have to bear in mind that slovenia is a tiny territory where distribution range of both discussed taxa overlap and so populations far from studied area can be slightly different. statistics of the useful distinguishing characters are presented as minimum and maximum (in brackets) and an interval between 1st and 3rd quartile. 1 total spikelet lengths (including awns) (2.5) 3.54.6 (5.9) cm, central spikelet of a triple with »stalk« (0.3) 0.6-1 (1.4) mm long, palea (0.8) 0.85-1 (1.2) cm long and lemma's awn (1.8) 2.4-3.5 (4) cm long, lateral spikelets with lemma's awn (1.4) 2-3 (3.5) cm long, palea (0.75) 0.9-1.1 (1.2) cm long and lemma (1) 1.2-1.6 (1.8) mm wide. anthers (0.6) 0.7-1 (1.4) mm, plant (20) 25-45 (60) cm ........................ h. murinum ssp. murinum 1* total spikelet lengths (including awns) (4) 5-6.5 (8) cm, central spikelet of a triple with »stalk« (0.5) 0.9-1.2 (1.5) mm long, palea (0.9) 1-1.15 (1.3) cm long and lemma's awn (2.8) 3.4-4.7 (5.9) cm long, lateral spikelets with lemma's awn (2.8) 3.5-4.8 (5.7) cm long, palea (1.1) 1.2-1.4 (1.6) cm long and lemma (1.2) 1.6-1.9 (2.1) mm wide. anthers (0.7) 0.9-1.3 (1.6) mm, plant (25) 40-50 (90) cm ...................... h. murinum ssp. leporinum and finally here is the updated distribution map of the studied taxa in slovenia (fig. 5). with light grey dots not only "intermediate" otus were represented but also other reported occurrences of h. murinum complex (cf. j o g a n et al., 2001) for which no herbarium material had been available so it is better to recognize them on that taxonomic level. obviously the group is represented only in lowland slovenia mostly below 300 m a.s.l., only few scattered localities can reach up to 600 m a.s.l. along the main highways. in the extreme sw part along the adriatic coast and in vipava valley hml is the common taxon, but also some populations of hmm can be found. in central and e slovenia hmm is present in majority of sampling sites, but there are also some records of hml populations. as it has been expected that both studied taxa are mostly tetraploids, possible hybridization could not be excluded as a cause for "intermediate" populations, but detailed study of those "intermediate" otus was beyond our scope. it is important to stress that in the discussed territory both studied taxa are sympatric with slighly bigger frequency of hml populations in the sw coastal part of slovenia and hmm present in all lowland parts of the country. in interpreting »intermediates« also a hypothesis of n e v s k i (1941), that hml is an ancestral relative of hmm can be roughly taken into consideration, but it seems that the evoultion of the »murinum complex« had been more complicated (c u a d r a d o & al., 2013) distribution pattern matches quite well pattern in adjacent friuli-venezia giulia (ne italy, fig. 4. comparison of total spikelet lengths of central vs. lateral spikelet of triple. hmm represented with grey, hml with empty symbols. equal lengths at dashed diagonale, longer central spikelet below total spikelet lengths 0 2 4 6 8 0 2 4 6 8 10 central spikelet (cm) la te ra l s p ik e le t (c m ) hmm hml biologica nyssana 8 (1)  september 2017: 23-30 jogan, n.  morphometric recognition of hordeum murinum… 28 p o l d i n i 2002) but maybe also in mentioned region hml can be expected with some local populations further from the coast. in croatia situation is similar, hml reported in the coastal region with only one locality in continental part and hmm scattered (n i k o l i ć , 2015): in continental adjacent countries, austria and hungary, hml ocurrence is very local to ephemeral (c s i k y , 2009; f i s c h e r et al., 2008). in the future presence of hml in continental slovenia is to be studied in detail as it would not be possible to exclude the probability of mostly ephemeral occurrence of this taxon in extremely dry ruderal places as e.g. along the railways or in abandoned gravel sites in bigger cities where "urban heat island" phenomenon can impact local mesoclimatic conditions. on the other hand it would be interesting to study populations of hmm in the coastal part of slovenia. at least the impression after the sampling is that hmm is more linked to shadowy, slightly wet ruderal places whereas hml is common in very dry ruderal communities. conclusion as a result we can confirm, that both previously mentioned subspecies of h. murinum are present in slovenia, there are more useful distinguishing characters than reported before but still it is not always easy to recognize the subspecies. hordeum murinum ssp. murinum is present all-over slovenian lowland and h. murinum ssp. leporinum predominantly in the coastal region, but with some scaterred populations also inland. ecological preferences in the areas where both co-ocurr remained to be studied. references amaral franco, j. do, rocha afonso, m. 1998: nova flora de portugal 3/ii, gramineae. escolar editora, lisboa. 283 p. bolos, o. de, vigo, j. 2001: flora dels paisos catalans iv. editorial barcino, barcelona. 750 p. cincović, t., kojić, m. 1976: poaceae. in: josifović, m. (ed.), flora sr srbije 8: 259-472, sanu, beograd. ciocirlan, v. 1990: flora ilustrata a romaniei ii. editura ceres, bucuresti. 598 p. conert, h. j. 2000: pareys graeserbuch. parey bucherlag, berlin. 592 p. csiky, j. 2009: hordeum l. arpa. in: g. kiraly (ed.), uj magyar fuveszkonyv 522-523 aggteleki nemzeti park igazgatosag, josvafo. cuadrado, a., carmona, a., jouve, n. 2013: chromosomal characterization of the three subgenomes in the polyploids of hordeum murinum l.: new insight into the evolution of this complex. plos one, 8 (12). fig. 5. distribution of discussed taxa in slovenia: green squares hmm, black dots hml, only revised records presented, grey circles: h. murinum s. lat. partly as a result of revision, mostly based on literature and unpublished field records hordeum murinum 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 s. lat. ssp. murinum ssp. leporinum © n e jc j o g a n 46°n 15°e a i cro h biologica nyssana 8 (1)  september 2017: 23-30 jogan, n.  morphometric recognition of hordeum murinum… 29 cvelev, n.n. 1976: zlaki sssr. nauka, leningrad. 788 p. fischer, m.a., adler, w., oswald, k. 2008: exkursionsflora. österreich, liechtenstein, suedtirol. biologiezentrum der oberoesterreichischen landesmuseen, linz. 1380 p. ilijanić, l., topić, j. 2000: poaceae. in: nikolić, t. (ed.), index florae croaticae 3, natura croatica, 9 (suppl. 1): 130-149. jogan, n. (ur.), bačič, t., frajman, b., leskovar, i., naglič, d., podobnik, a., rozman, b., strgulc krajšek, s., trčak, b. 2001: gradivo za atlas flore slovenije. center za kartografijo favne in flore, miklavž na dravskem polju. 443 p. jogan, n. 1999: skupina mišjega ječmena (hordeum murinum) v sloveniji. in: jogan, n. (ed.), flora in vegetacija slovenije 1999, zbornik izvlečkov referatov simpozija. botanično društvo slovenije, ljubljana. jogan, n. 2005: some interesting records of grasses in the flora of r macedonia. in: ranđelović, n. (ed.): symposium on the flora of se serbia and neighbouring regions, niš 20.-24.06.2005. abstracts. niš: university of niš, faculty of sciences and mathematics. p. 46. jogan, n. 2007: poaceae (gramineae) trave. in: martinčič, a., t. wraber, n. jogan, a. podobnik, b. turk, b. vreš, v. ravnik, b. frajman, s. strgulc krajšek, b. trčak, t. bačič. m. a. fischer, k. eler & b. surina: mala flora slovenije. 4 izd. 826-932, tehniška založba slovenije, ljubljana. jogan, n. 2014: poaceae barnhart – suesgraeser. in: rottensteiner, w. k.: exkursionsflora fuer istrien. naturwissenschaftlicher verein fuer kaernten. 679-751. jogan, n., bačič, t., vreš, b. 1999. prispevek k poznavanju flore okolice ormoža. natura sloveniae, ljubljana 1(1): 5-27. kankanpää, j, schulman, a. h, & mannonen, l., 1996: the genome sizes of hordeum species show considerable variation. genome, 39 (4): 730-735. lambinon, j., de langhe, j. e., delvosalle, l., duvigneaud j. & al. 1992: nouvelle flore de la belgique, du grand-douche de luxembourg, du nord de la france et des regions voisines. edition du jardin botanique national de belgique, meise. lauber, k., wagner, g. 1998: flora helvetica. haupt verlag, bern, stuttgart, wien. martinčič, a. 1984: poaceae trave. in: martinčič, a. & f. sušnik: mala flora slovenije: praprotnice in semenke. dzs, ljubljana. nevskij, s. a. 1941: materiali k poznaniju dikorastuščih jačmenej v svjazi s voprosom proishoždenija hordeum vulgare l. i hordeum distichon l. flora i sistematika vyšsih rastenij, 5: 64-255. moskva, leningrad. niklfeld, h., 1971: bericht über die kartierung der flora mitteleuropas. taxon, 20: 545–571. nikolić, t. (ed.), 2015: flora croatica baza podataka; http://hirc.botanic.hr/fcd. prirodoslovno-matematički fakultet, sveučilište u zagrebu; access: 21.06.2017 pignatti, s. 1983: flora d'italia 1-3. edagricole, bologna. poldini, l. 2002: nuovo atlante corologico delle piante vascolari nel friuli venezia giulia. regione autonoma friuli venezia giulia, azienda parchi e foreste regionali & universita degli studi di trieste. scholz, h., raus, t. 1997: zwei neue unterarten des hordeum murinum (gramineae) aus griechenland und spanien. feddes repertorium, 108: 527-531. stohr, g. 2002: poales (graminales). in: rothmaler, w. & al.: exkursionsflora. spektrum akademischer verlag, berlin. strid, a. 2016: atlas of the aegean flora. bgbm, berlin. 700-878. tison, j. m., de foucault, b. 2014: flora gallica. biotope editions, meze. 1196 p. supplemetal data: specimina visa: hordeum murinum ssp. leporinum sežana (10249/3), leg. r. justin, 16.3.1905, lju 10130753 (17315); strunjan (10447/4), leg. t. wraber, 21.7.1972, lju 10130751 (33548); koper (10448/3), leg. t. wraber, 19.5.1973, lju 10130750 (24142); biljenski griči (10148/1), leg. g. seljak, 22.5.1987, lju 10130766 (120321); prade (10448/4), leg. b. mozetič, 5.5.1989, lju 10130785 (121541); sežana-vrhovlje (10249/3), leg. v. debevec, 10.6.1989, lju 10130784 (121095); dane pri sežani (10249/3), leg. m. frelih, 10.6.1989, lju 10130786 (121751); piran, mogoron (10447/3), leg. g. planinc, 9.5.1993, lju 10130772 (124994); osp (10449/1), leg. n. dolenc, 16.4.1994, lju 10130743 (59976); strunjan (10447/4), leg. s. toth, 24.4.1994, lju 10130748 (59992); škocjan (10448/3), leg. a. zajko, 18.6.1994, lju 10130747 (60006); koper, norbedi (10448/2), leg. b. toškan, 20.6.1994, lju 10130749 (60051); sežana, kopriva (10249/1), leg. biologica nyssana 8 (1)  september 2017: 00-00 jogan, n.  morphometric recognition of hordeum murinum… 30 m. vrabec, 20.5.1995, lju 10130757; avber pri sežani (10249/1), leg. m. pegan žvokelj, 22.5.1995, lju 10130774 (126998); lozice (10250/1), leg. m. žvanut, 15.6.1995, lju 10130759; brkini, sv. pavel (10551/1), leg. n. jogan, 29.7.1996, lju 10130760; vrtovin, šateji (10048/4), leg. u. jelenc, 25.5.1997, lju 10130768 (127748); dane (10249/3), leg. m. šebart, 31.5.1997, lju 10130767 (127788); turjak (10153/2), leg. p. presetnik, 26.6.1997, lju 10130770 (127743); panovec, strelišče (10047/2), leg. n. jogan, 19.7.1997, lju 10130761 (63481); vel. žablje (10149/1), leg. k. vodopivec, 19.6.1999, lju 10130763 (63403); haloze, borl (9662/1), leg. n. jogan, 12.7.2002, lju 10143040; sp. škofije (10448/2), leg. p. glasnović, 2.5.2003, lju 10134768; sp. škofije, bonifika (10448/2), leg. p. glasnović, 8.5.2004, lju 10134770; bertoki (10448/2), leg. p. glasnović, 15.5.2004, lju 10134769; ankaran (10448/1), leg. p. glasnović, 20.5.2005, lju 10134771; (9957/4), leg. n. jogan, hsnj l1028; (9952/2), leg. n. jogan, hsnj l993; (10447/4), leg. n. jogan, hsnj p1134; (10449/3), leg. n. jogan, hsnj p1277; (10449/1), leg. n. jogan, hsnj p1293; (10148/2), leg. n. jogan, hsnj p1296; (10447/4), leg. n. jogan, hsnj p1350; (10447/4), leg. n. jogan, hsnj p1379; (10448/1), leg. n. jogan, hsnj p1423; (10448/1), leg. n. jogan, hsnj p1424; (10449/1), leg. n. jogan, hsnj p1459; (10447/4), leg. n. jogan, hsnj p1505; (10447/4), leg. n. jogan, hsnj p1623; (10447/4), leg. n. jogan, hsnj p1633; (10447/4), leg. n. jogan, hsnj p1763; (10149/4), leg. n. jogan, hsnj p562; (10349/2), leg. n. jogan, hsnj p578; (10448/1), leg. n. jogan, hsnj p879; hordeum murinum ssp. murinum vremski britof (10350/1), leg. r. justin, 30.6.1905, lju 10130752 (17314); gornje ležeče (10350/1), leg. r. justin, 2.7.1905, lju 10130746 (17313); ljubljana (9953/1), leg. r. justin, 30.5.1918, lju 10130745 (17310); šmarna gora (9852/4), leg. r. justin, 6.7.1936, lju 10130744 (17308); ljubljana, ilirska cesta (9953/1), leg. b. turk, 15.9.1985, lju 10130739; ljubljana, bežigrad (9953/1), leg. n. jogan, 24.5.1988, lju 10130756; solkan (10047/2), leg. l. merljak, 16.6.1989, lju 10130783 (121108); novo mesto (10257/1), leg. d. kovačec, 10.6.1993, lju 10130778 (125060); lendava, sv. trojica (9464/2), leg. n. jogan, 12.7.1994, lju 10130755; preserje pri domžalah (9853/2), leg. t. bačič, 1.6.1995, lju 10130776 (126581); belveder-ronek (10447/4), leg. j. france, 17.7.1995, lju 10130775 (126532); ormož (9562/4), leg. n. herga, 8.6.1996, lju 10130782 (127498); pobegi (10448/4), leg. k. sedmak, 17.6.1996, lju 10130780 (127448); sežana (10249/3), leg. b. tavčar, 18.6.1996, lju 10130781 (127524); vel. badin (10549/2), leg. n. jogan, 11.5.1997, lju 10130769; ormož, ž. p. (9562/4), leg. n. jogan, 29.7.1997, lju 10130758; grosuplje (10053/2), leg. d. simonič, 18.5.1998, lju 10130762 (54571); metlika, radovica (10358/1), leg. b. frajman, 1.8.2001, lju 10130864; valdoltra (10448/1), leg. p. glasnović, 21.4.2003, lju 10134767; izola (10447/4), leg. u. & w. starmuhler, 24.4.2006, lju 10135027; portorož (10447/4), leg. v. mikolaš & al., 15.5.2010, lju 10142708; dovje (9549/2), leg. v. plemel, 4. 8. 1873, lju 10130740; (9848/1), leg. n. jogan, hsnj a910; (9852/4), leg. n. jogan, hsnj l1033; (9953/1), leg. n. jogan, hsnj l1035; (9953/1), leg. n. jogan, hsnj l818; (9953/2), leg. n. jogan, hsnj l867; (10349/1), leg. n. jogan, hsnj p1241; (10249/1), leg. n. jogan, hsnj p1266; (10547/2), leg. n. jogan, hsnj p1346; (10449/3), leg. n. jogan, hsnj p1385; (10448/2), leg. n. jogan, hsnj p1408; (10447/4), leg. n. jogan, hsnj p1477; (10448/3), leg. n. jogan, hsnj p1630; (10349/4), leg. n. jogan, hsnj p1820; (10548/1), leg. n. jogan, hsnj p27; (10349/2), leg. n. jogan, hsnj p571; (9363/1), leg. n. jogan, hsnj š769; (9462/2), leg. n. jogan, hsnj š902; (9359/2), leg. n. jogan, hsnj š917; (9360/3), leg. n. jogan; hordeum murinum s. lat. celje (9757/4), leg. a. knap, 7.8.1938, lju 10130741; žalec (9757/1), leg. s. grobelnik, 14.6.1993, lju 10130777 (125058); ljubljana, bs3 (9953/1), leg. h. krečič, 19.6.1994, lju 10130742 (60076); lendava, železnica (9464/2), leg. n. jogan, 12.7.1994, lju 10130754; dane-šmarje (10249/3), leg. a. lisjak, 30.6.1995, lju 10130779 (126939); hrvatinski hrib (10448/2), leg. k. marc, 25.7.1996, lju 10130773 (127831); ig-pod strahom (10053/1), leg. j. zajc, 26.6.1997, lju 10130764 (63775); zg. hrušica (9953/3), leg. m. kukec, 31.7.1997, lju 10130771 (127911); tolmin (9848/1), leg. š. glišovič, 22.5.1999, lju 10130765 (63422); (10547/2), leg. n. jogan, hsnj p1345; jakšić, p., zlatković, b.  distribution of burnets & foresters species in... biologica nyssana 6 (1)  september 2015: 33-39 jakšić, p., zlatković, b.  distribution of burnets & foresters species in... 33 original article received: 11 april 2015 revised: 15 june 2015 accepted: 01 july 2015 distribution of burnets & foresters species in plant associations and habitats of special nature reserve „jelašnička klisura gorge“ (serbia) (lepidoptera: zygaenidae) predrag jakšić*, bojan zlatković university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia * e-mail: jaksicpredrag@gmail.com abstract: jakšić, p., zlatković, b.: distribution of burnets & foresters species in plant associations and habitats of special nature reserve „jelašnička klisura gorge“ (serbia) (lepidoptera: zygaenidae). biologica nyssana, 6 (1), september 2015: 33-39. general geographic and biogeographic features of special nature reserve „jelašnička klisura gorge“ in eastern serbia are represented. the lists of plant communities and habitat types for the area are given. faunistic account of zygaenidae species recorded in period 2009-2014. is analised. distribution of zygaenidae species within plant assotiations and habitat types is presented for the first time. key words: zygaenidae, habitats, jelašnička klisura gorge apstrakt: jakšić, p., zlatković, b.: distribucija vrsta familije zygaenidae (lepidoptera) u biljnim zajednicama i staništima specijalnog rezervata prirode „jelašnička klisura“ (srbija). biologica nyssana, 6 (1), septembar 2015: 33-39. prikazane su opšte geografske i biogeografske odlike specijalnog rezervata prirode „jelašnička klisura“, smeštenog u istočnoj srbiji. navedene su liste biljnih zajednica i tipova staništa konstatovanih na podrućju klisure. sumirani su rezultati faunističkog istraživanja predstavnika familije zygaenidae realizovanog u periodu od 2009. do 2014. godine. po prvi put je prikazana distribucija utvrđenih vrsta familije zygaenidae na primeru biljnih zajednica i staništa specijalnog rezervata prirode „jelašnička klisura“. key words: zygaenidae, staništa, jelašnička klisura introduction adopting rio declaration on environment and development (1992), serbia significantly initiated activities in protection of biodiversity and nature in general (a n o n y m o u s , 1992). within numerous protected natural resources there are 67 nature reserves in the country. one of them is jelašnička klisura gorge, situated 15 km se of the town of niš, between jelašnica village and čukljenik village (fig. 1). geographic location, geological, geomorphological, climatic and other natural features have made jelašnička klisura gorge 6 (1) • september 2015: 33-39 biologica nyssana 6 (1)  september 2015: 33-39 jakšić, p., zlatković, b.  distribution of burnets & foresters species in... 34 one of the most interesting areas in eastern serbia from the aspect of biodiversity and nature conservation. the main hidrological phenomena among several, periodical streams of the area is studena river, that forms small but magnificent gorge, with narrowed sometimes closed sides. geological base is made of limestone. the gorge is strongly influenced by a temperate continental climate (semi-arid temperate continental (subcontinental) climate) with ethesian influences from sub-mediterranean region. however, largest part of the gorge is situated at lower altitudes (ranged from 293 to 580 m.a.s.l.) and therefore opened to the influence of the continental and southern climate that strongly influenced its plant life and species composition in general. typical climate type is additionally underlined by characteristic of limestone ground and prominent karst geomorphology, making the special mesoclimatic conditions in the gorge. the climate of the area is depicted by climate diagram according to w a l t e r & l e i t h (1967) for niška banja spa as the nearest location (fig. 2). from the phytogeographycal point of view investigated area belongs to the west-moesian floristic province of the balkan sub-region, and central european floristic region. due to a strong presence of steppe floristic region to the gorge, some of its parts can be understood as an enclave of pontic-south-sibirian floristic region. the role of refugia that have gorges and canyons of the central balkan peninsula explains species richness, originality and the antiquity of their flora and vegetation (m i š i ć , 1981). the most important elements of flora are tertiary relicts ramonda serbica pančić and r. nataliae pančić & petrović, as well as balkan endemic and sub-endemic taxa such as satureja kitaibelii wierzb. ex heuff., dianthus noeanus boiss., parietaria lusitanica subsp. serbica (pančić) p. w. ball, micromeria cristata (hampe) griseb., ect. (l a z a r e v i ć et al., 2007). the protected area of of the nature reserve, established in 1995., is around 116 ha (a n o n y m o u s , 1995). investigated area is positioned in the close vicinity of large sićevačka klisura gorge and surrounded by large massif of mt. suva planina, also important from the point of biodiversity and nature conservation. material and methods during the period of six years of field work (2009.-2014.) representatives of burnets & foresters (family zygaenidae) were notified in certain habitat types of jelašnička klisura gorge. the adult specimens has been collected on the field using fig. 1. jelašnička klisura gorge, habitat detail, photo p. jakšić fig. 2. climatic diagram of niška banja spa according to w a l t e r & l e i t h (1967) biologica nyssana 6 (1)  september 2015: 33-39 jakšić, p., zlatković, b.  distribution of burnets & foresters species in... 35 entomological net. after preparing, we determined the specimens by the wing-patterns and in all cases the identification has been also carried out by an examination of the male genitalia. the preparations were carried out following the well known standard procedure: maceration by boiling in potash, dissecting and cleaning, clearing in xylolum and mounting in canada balsam. the photos of genital parameters were taken using the "leica dm 1000" microscope with the "camera leica dec 290"; photos terrain, habitats and in situ specimen were taken using “olympus” sp-510uz (7.1 megapixel and 25x optical zoom). all the material (specimens and genitalia slides) are deposited in the first author's collection. the list of plant communities is based on field survey as well as relevant literature sources given in the article. the list of syntaxonomic units is aligned with j o v a n o v i ć et al. (1986), k o j i ć et al. (1998). habitat types are defined by immediate field study. the habitat types are in accordance with “natura 2000“ classification system (a n o n y m o u s , 2000). their equivalents for the territory of serbia according to l a k u š i ć (2005) are shown in the tab. 2. and 3. fieldwork on protected areas was done on the basis of permits provided by the ministry of environment, mining and spatial planing, republic of serbia, no. 353-01-1559-2011-03, dated from 8. 6. 2011; no. 353-01-1070/2012-03, dated from 12. 06. 2012. and no. 353-01-916/2014-08, dated from 29.05.2014. results and discussion on the base of the literature data given by several authors (j o v a n o v i ć d u n j i ć , 1955; j o v a n o v i ć , 1980; m i š i ć , 1981; s t e v a n o v i ć et al., 1987; l a z a r e v i ć et al., 2007) as well as field survey, the list of plant communities in jelašnička klisura gorge has been established (tab. 1). the presence of 23 plant associations belonging to 16 alliances and 9 classes is recorded. table 1. syntaxonomic review of vegetation in jelašnička klisura gorge syntaxonomic unit (classis, order, alliantia, assotiation) querco-fagetea br.-bl. et vlieger 1937 quercetalia pubescentis br.-bl. 1932 ostryo-carpinion orientalis horvat 1954 ass.: carpinetum orientalis serbicum rudski 1940 em. b. jov. 1953 quercion frainetto ht. 1954 ass.: quercetum frainetto-cerris rudski (1940) 1949 subass. carpinetosum orientalis (knapp) b. jovanović 1953 syringo-carpinion orientalis jakucs 1959 ass.: carpino orientalis-quercetum mixtum mišić 1967 ass.: syringo-carpinetum orientalis (greb. 1950.) mišić 1967 prunetalia spinosae r. tx. 1952 prunion spinosae soó (1930) 1940 ass.: pruno spinosae-crataegetum (soó 1927) hueck 1931 populetalia albae br.-bl. 1931 salicion albae soó (1930) 1940 ass.: salicetum albae-fragilis soó (1933) 1958 paliuretea trinajstić 1978 paliuretalia trinajstić 1978 paliurion moesiacum b. jov. 1985 ass.: botryochloo-paliuretum b. jov. 1973 asplenietea trichomanis br.-bl. 1926 potentilletalia caulescentis br.-bl. 1926 edraiantho graminifolii-erysimion comatae mucina et al. 1990 ass.: ceterachi-ramondetum serbicae r. jovanović 1953 subass.: ramondetosum nathaliae v. stevanović et al. ass.: parietarietum serbicae niketić 1986 micromerion cristatae n. & v. ranđ. 1998 ass.: campanulo velebiticae-micromerietum cristatae v. et n. ranđ. 1999 festuco-seslerietea barbero et bonim 1969 seslerietalia juncifoliae ht. 1930 seslerion rigidae zolyomy 1939 ass.: seslerietum filifoliae zolyomy 1939 subass. scabiosetsoum fumarioides niketić et lakušić 1988 ass.: anthyllo-seslerietum rigidae r. jovanović 1955 biologica nyssana 6 (1)  september 2015: 33-39 jakšić, p., zlatković, b.  distribution of burnets & foresters species in... 36 festuco-brometea br.-bl. et r. tx. 1943 scorzonero-chrysopogonetalia horvatić et horvat (1956) 1958 chrysopogoni-satureion horvat et horvatić 1934 ass.: euphorbio myrsiniti-bothryochloetum r. jovanović 1955 festucetalia valesiacae br.-bl. et tx. 1943 festucion valesiacae klika 1933 ass.: poterio-festucetum valesiacae danon 1960 ass.: potentillo-caricetum humilis r. jovanović 1955 ass.: galio-festucetum valesiacae r. jovanović 1955 ass.: andropogono-danthonietum calycinae danon et blaženčić 1975 ass.: festuco-agrostidetum vulgaris danon et blaženčić 1978 satureion kitaibelii horvat 1962 ass.: sedo-potentilletum arenariae ružić 1978 phragmitetea communis r. tx. et preising 1942 magnocaricetalia pignatti 1953 caricion gracilis-vulpinae e. bálatová-tulačková 1963 ass.: junco articulatae-caricetum vulpinae danon et blaž. 1978 bidentetea tripartiti tx., lohm. et prsg. 1950 bidentetalia tripartitii br.-bl. ex tx. 1943 bidention tripartiti nordh 1940 ass.: polygono-rumicetum conglomerati v. ranđ. 1990 plantaginetea majoris tx. et preising 1950 plantaginetalia majoris tx.et preising 1950 polygonion avicularis br.bl.1931 ass.: lolio-plantaginetum majoris beger 1930 artemisietea vulgaris lohm., preising et r. tx. 1950 artemisietalia vulgaris lohm. apud r.tx.1947 arction lappae tx.(1937) 1942 em. gutte 1972 ass: arctio-artemisietum vulgaris (r.tx.1942) oberd. et al.1967 table 2. survey of “natura 2000“ habitat types in jelašnička klisura gorge. codes for their equivalents according to lakušić (2005) given in the brakets temperate heath and scrub 40a0 *subcontinental peri-pannonian scrub (f3.24) semi-natural dry grasslands and scrubland facies 6210 semi-natural dry grasslands and scrubland facies on calcareous substrates (festuco-brometalia) (*important orchid sites) (e1.21, e1.22, e1.55) rocky slopes with chasmophytes vegetation 8210 calcareous rocky slopes with chasmophytic vegetation (h3.2a) forests of temperate europe 9180 *tilio-acerion forests of slopes, screes and ravines (g1.a4) 91e0 *alluvial forests with alnus glutinosa and fraxinus excelsior (alno-padion, alnion incanae, salicion albae) (g1.11) 91m0 pannonian-balkanic turkey oak-sessile oak forests (g1.76) *priority habitat summarizing literature data (l a k u š i ć et al., 2005; s e k u l i ć et al., 2010), as well as personal terrain insight, the list of most important habitats for species of family zygaenidae has been made (tab. 2). field research established that fauna of burnets and foresters in special nature reserve “jelašnička klisura gorge” are represented with total number of 8 species: adscita (1 species), jordanita (2 species), and zygaena (5 species). following species have been notified: adscita mannii (8 records), jordanita chloros (2 records), j. graeca (4 records), zygaena carniolica (1 record), z. ephialtes (1 record), z. filipendulae (1 record), z. loti (1 record) and z. purpuralis (6 records), listed in tab. 3. the largest number of representatives, actually the presence of entire 8 identified species have been notified at habitats type 6210 in accordance with classification “natura 2000”. at habitat type 8210 in accordance with previously mentioned classification, only 3 species have been notified (a. mannii, j. graeca and j. chloros), while at the rest of habitat types in the gorge, only 1 species of burnets and foresters has been presented and notified. biologica nyssana 6 (1)  september 2015: 33-40 jakšić, p., zlatković, b.  distribution of burnets & foresters species in... 37 table 3. distribution of zygaenidae species within the main habitats of jelašnička klisura gorge and presence of their larval foodplants species, date of collection and genitalia slides number habitat type according to „natura 2000“, (eunis); larval foodplants and related plant species adscita (tarmannita) mannii (lederer, 1853) 5.vi 2013., 1♂ (prep. no. sr-2556); 14. v 2014., 1♂ (prep. no. sr-2571); 20. v 2014., 2♂♂ (prep. no. sr-2578 and sr-2589) and 27.v 2014., 1♂ (prep. no. sr-2576) 6210 (e1.21, e1.22, e1.55), 8210 helianthemum spp. (including h. nummularium, h. canum and h. salicifolium) jordanita (jordanita) graeca (jordan, 1907) 6. vii 2010., 1♂; (prep. no. sr-2464); 2. vi 2011., 2♀♀. 6210 (e1.21, e1.22, e1.55), 8210 centaurea spp. (including c. salonitana, centaurea scabiosa subsp. apiculata and c. cyanus) jordanita (jordanita) chloros (hűbner, [1813]) 6. vii 2010., 2♂♂, 1♀. (prep. no. sr-2473 and sr-2474). 6210 (e1.21, e1.22, e1.55), 8210 centaurea spp. (including c. salonitana, centaurea scabiosa subsp. apiculata and c. cyanus) zygaena (mesembrynus) purpuralis (brünnich, 1763) 2. vi 2011., 1♂; 14. v 2014., 3♂♂ (prep. no. anz-164 and anz-165); 20.v 2014., 1♂ (prep. no. anz-166); 5.vi 2014., 1♂ (prep. no. anz167). 6210 (e1.22), 9180 (g1.a4), 91m0 (g1.76) thymus sl. (including thymus praecox subsp. jankae, and t. glabrescens); eryngium spp. as larval foodplants of z. minos (e. palmatum, e. campestre) zygaena (agrumenia) carniolica (scopoli, 1763) 6. vii 2010, 1♀. 6210 (e1.21, e1.22, e1.55) onobrychis arenaria (including o. alba) zygaena (zygaena) loti ([denis & schifferműller], 1775) 6. vii 2010., 1♂. 6210 (e1.22, e1.55) onobrychis spp., (including onobrychis arenaria and o. alba) coronilla varia (including c. elegans), lotus corniculatus zygaena (zygaena) ephialtes (linnaeus, 1767) 6. vii 2010., 1♂, 1♀. 6210 (e1.22, e1.55) coronilla varia (including c. elegans) zygaena (zygaena) filipendulae (linnaeus, 1758) 23. vi 2011., 1♂. 6210 (e1.22) dorycnium pentaphyllum (including d. pentaphyllum subsp. germanicum and d. pentaphyllum subsp. herbaceum) semi-natural dry grassland and scrubland facies on calcareous substrates (6210) are developed throughout research area, so the fact that they have a great significance in terms of species diversity of family zygaenidae, is not unexpected. they are mainly dry, termophilic steppe alike habitats, occupying large areas on shallow soil of eroded and moderately steep slopes of the gorge. importance of this habitat type is also multiple, according to the presence of several protected orchid species e.g. orchis purpurea hudson and himantoglossum hircinum (l). sprengel on the slopes of the gorge. in terms of vegetation point of view they belong to a vegetation of dry pastures of rocky continental which is characterized by extreme presence of steppe and submediterranian plant species. in syntaxonomic sense, these are associations of class festuco-brometea, presented in jelašnička klisura gorge with large numbers of phytocoenoses, where by the most significant are communities of festucion valesiacae and satureion kitaibelli alliances. supported by anthropogenic activities this vegetation type has been widespread throughout east and south-east serbia. it is of secondary character and it is developed under subaride climate conditions on destroyed forest communities, mainly oak forest communities with termophilic character (d i k l i ć & n i k o l i ć , 1964). calcareous rocky slopes with chasmophytic vegetation (8210) habitat types have far less importance compared to represents of family zygaenidae in terms of distribution and diversity of the research area. although in terms of spatial representation, this habitat type represents one of the area characteristic, and it is one of the most interesting in the gorge, from the phytogeographical point of view, but has secondary role in terms of diversity of burnets and foresters. this type of habitat includes rocky, limestone cliffs, that are overgrown by vegetation from the class asplenietea biologica nyssana 6 (1)  september 2015: 33-39 jakšić, p., zlatković, b.  distribution of burnets & foresters species in... 38 fig. 3. position of examined habitat types on topographic map (sheet 583_1_3_bela palanka, 1: 25000) fig. 4. male genitalia slide of adscita (tarmannita) mannii (lederer, 1853), jelašnička klisura gorge, 27.v 2014., genitalia slide no. sr-2576, x25 fig. 5. zygaena purpuralis (brünnich, 1763), jelašnička klisura gorge, 14. v 2014., (genitalia slide prep. no. anz-164), photo p. jakšić trichomanis on slopes and canyon parts of the gorge. the greatest significance in the context of this type of vegetation on the research area are phytocoenoses of alliances edraiantho graminofolii-erysmion comatae and micromerion cristatae, comprising community of endemo-relict species of the genus ramonda, as well as community of parietaria lusitanica subsp. serbica, which represents specific vegetation of this part of europe. related to two mentioned habitat types, all other types of habitats, listed in tab. 2, as well as plant communities, listed in tab. 1, have much smaller role in terms of distribution and diversity of family zygaenidae. conclusion after several years of monitoring of distribution of the species of zygaenidae family in jelašnica gorge we have established their priority habitats and shed light to their diversity in this area (tab. 3). analysing distribution of certain types we can conclude that main criteria in selection of habitats was presence of their larval foodplants, followed by the number of zygaenidae species recorded. from that point of view, the most important habitats in the gorge are semi-natural dry grasslands and scrubland facies on calcareous substrates and calcareous rocky slopes with chasmophytic vegetation. according to the parameters that calculated richness of zygaenidae in the gorge those habitats are particularly important, diserving special attention in further aspects of conservation within deserving area of the gorge. acknowledgements. we express our thanks to ivan gnjatović for the photo of genitalia and to ana nahirnić for photo treatment in photoshop and determination of zygaena purpuralis specimens. the fieldwork was supported by the scientific society biodat alpin (innsbruck, austria). references anonymous, 1992: agenda 21, rio declaration, united nations. anonymous, 1995: uredba o zaštiti specijalnog rezervata prirode „jelašnička klisura“. službeni glasnik rs, 9/95. anonymous, 2000: reference list of habitat types and species present in the region. continental region. natura 2000. european environmental agency, european topic centre of nature conservation. doc. cont/b/fin. 3. anonymous, 2010: the rulebook on habitat types, the criteria for the selection of habitat types, biologica nyssana 6 (1)  september 2015: 33-39 jakšić, p., zlatković, b.  distribution of burnets & foresters species in... 39 on sensitive, endangered, rare and priority for protection habitat types and on the protection measures for their conservation. official gazette of rs 35/2010. beograd diklić, n., nikolić, v., 1964: o nekim zajednicama pašnjaka i livada na svrljiškim planinama. glasnik prirodnjačkog muzeja u beogradu. b18: 65-88. jakšić, p., 2014: pregled faune dnevnih leptira na širem području jelašničke klisure (lepidoptera: hesperioidea i papilionoidea). univerzitet u nišu, prirodno-matematički fakultet. niš. jovanović, b., 1980: šumske fitocenoze i staništa suve planine. šumarski fakultet u beogradu. 216 p. jovanović-dunjić r. 1955: tipovi pašnjaka i livada suve planine. zbornik radova instituta za ekologiju i biogeografiju san, beograd 6(2): 545. jovanović, b., lakušić, r., rizovski, r., trinajstić, i., zupančić, m.. 1986: prodromus phytocoenosum jugoslaviae, ad mappam vegetationis m 1:200000. naučno veće vegetacijske karte jugoslavije, bribir-ilok. kojić, m., popović, r., karadžić, b., 1998: sintaksonomski pregled vegetacije srbije. institut za biološka istraživanja “siniša stanković“. beograd. 220 pp. lakušić, d. (ed.)., 2005: staništa srbije. rezultati projekta “harmonizacija nacionalne nomenklature u klasifikaciji staništa sa standardima međunarodne zajednice”. institut za botaniku i botanička bašta “jevremovac”, biološki fakultet, univerzitet u beogradu, ministarstvo za nauku i zaštitu životne sredine republike srbije. http://www.ekoserb.sr.gov.rs/projekti/stanista/. lazarević, p., mitrović, v., zlatković, b., 2007: flora and vegetation of the sićevo and jelašnica gorges. in: trajković, s. and branković, s. (eds.): sićevo and jelašnica gorges environment status monitoring. institute for nature conservation of serbia and faculty of civil enginiering and architecture niš. pp.: 27-35. niš. mišić, v., 1981: the forest vegetation of gorges and canyons in eastern serbia. institut za biološka istraživanja „siniša stanković“, pp.: 1-328, beograd (in serbian with english summary). petrović, s., 1882. flora okoline niša – flora agri nyssani. kraljevsko-srpska državna štamparija. beograd. sekulić, n., šinžar-sekulić, j., 2010: emerald ecological network in serbia. institute for nature conservation of serbia. beograd. simonov, n., 1993: predlog za zaštitu prirodnog dobra „jelašnička klisura“ kao specijalnog rezervata prirode. zavod za zaštitu prirode srbije. beograd. stevanović, v., niketić, m., stevanović, b., 1987: fitocenološke karakteristike simpatričkih staništa endemo-reliktnih vrsta ramonda serbica panč. i r. nathaliae panč. et petrov. glasnik instituta za botaniku i botaničke bašte univerziteta u beogradu, 21: 17-26. walter, h., leith, h., 1967: klimadiagramm veb gustav fischer, jena. zimmermann, f., 2005: beschreibung der biotoptypen auf der grundlage der liste der biotoptypen brandenburgs (stand 2004) und der erläuterungstexte (stand 1994). berlin. biologica nyssana 6 (1)  september 2015: 33-39 jakšić, p., zlatković, b.  distribution of burnets & foresters species in... 40 kajevska, i., kuštera, m., cvijetan, i.: a contribution to the knowledge of ascomycetes in eastern serbia. biologica nyssana, 9 (2). december, 2018 biologica nyssana 9 (2) ⚫ december 2018: 77-88 kajevska et al. ⚫ a contribution to the knowledge of ascomycetes... 77 original article received: 7 jene 2018 revised: 28 august 2018 accepted: 24 september 2018 a contribution to the knowledge of ascomycetes in eastern serbia iskra kajevska1, marjan kuštera2, ivana cvijetan3 1faculty of sciences and mathematics, university of niš, višegradska 33, 18000 niš, serbia 2mycological society naissus, dvanaeste srpske brigade 7, 18000 niš, republic of serbia 3mycological society of niš, bul. svetog cara konstantina 37/34, 18000 niš, republic of serbia * e-mail: iskra.kajevska@pmf.edu.rs abstract: kajevska, i., kuštera, m., cvijetan, i.: a contribution to the knowledge of ascomycetes in eastern serbia. biologica nyssana, 9 (2). december, 2018: 77-88. mycological field research conducted from 2015 2018 in the eastern part of serbia resulted with a registration of a new data on 9 insufficiently known or new ascomycetes for the country: cordyceps militaris, ophiocordyceps gracilis, peziza phyllogena, neottiella vivida, scutellinia legalie, s. trechispora, plectania melastoma, pseudoplectania nigrella and urnula mediterranea. all species are presented with color photos of fresh specimens, microscopic morphology and notes on their taxonomy, ecology and distribution in the country. key words: ascomycetes, serbia apstrakt: kajevska, i., kuštera, m., cvijetan, i.: prilog poznavanju askomiceta istočne srbije. biologica nyssana, 9 (2). decembar, 2018: 77-88. mikološko terensko istraživanje obavljeno u periodu od 2015. do 2018. godine u istočnom delu srbije rezultiralo je registracijom novih podataka o 9 nedovoljno poznatih ili novih askomiceta za područje ove države: cordiceps militaris, ophiocordiceps gracilis, peziza phillogena, neottiella vivida, scutellinia legalie, s. trechispora, plectania melastoma, pseudoplectania nigrella i urnula mediterranea. sve vrste su predstavljene fotografijama svežih uzoraka, mikroskopskom morfologijom i podacima o njihovoj taksonomiji, ekologiji i distribuciji u srbiji. ključne reči: askomicete, srbija introduction although the interest in mycology, especially regarding fungal diversity is steadily increasing in serbia, the knowledge of the distribution of ascomycetes in serbia is scarce and limited. the eastern part of serbia is characterized with biologically rich land ecosystems but in terms of 9 (2) • december 2018: 77-88 doi: 10.5281/zenodo.2538598 biologica nyssana 9 (2) ⚫ december 2018: 77-88 kajevska et al. ⚫ a contribution to the knowledge of ascomycetes... 78 fungi, especially in ascomycota diversity, so far is the least explored area. therefore, the main aim of this study is to provide a contribution to the mycological knowledge of some ascomycetes collected from the eastern part of serbia which are insufficiently known or represent new species for the country. in scientific literature, a small number of mycological papers is dealing with species diversity of ascomycota in serbia. significant contribution to the research on ascomycota was made on np fruška gora, situated in the northern part of serbia, representing a checklist and a database of fungi (savić, 2016; http://www.naturefg.com/pages/fuascomycota.htm). some valuable published data regarding on distribution of this fungal phylum are as follows: ranojević (1910, 1914), ivančević & beronja (2004), marjanović et al. (2010), karaman et al. (2012); sadiković & kuštera (2013); ivančević (2016), milosavljević (2016), savić et al. (2016), savić & karaman (2016), vukojević et al. (2016). material and methods sources of information for this paper were available as unpublished data of ascomycetes registered during field surveys conducted in various habitats of selected locations in the eastern part of serbia. informations of research notes, revised exsiccates fig. 1. map of vicinity of the city of niš in serbia with marked sites where species have been registered biologica nyssana 9 (2) ⚫ december 2018: 77-88 kajevska et al. ⚫ a contribution to the knowledge of ascomycetes... 79 and references of those previously reported taxa from the country are also provided. collection of fungi was made from 2015 2017 by members of the mycological society of niš and mycological society naissus, niš. laboratory analyses were obtained with the use of optika n 400m and leica dm 1000 microscopes, and by examination and measuring of spores and other elements of fresh and dry material in water, 5% koh, lugol, cotton blue and melzer’s reagents. the descriptions of macroand micro characters are based exclusively on the collected material. all specimens are preserved in the private fungal collection of the society (m.s.n.). the location of the sites was determined using a gps receiver and all examined localities are provided with geographical coordinates. the sites where the taxa were registered are marked on a map (fig. 1). specimens were identified according to the following taxonomical books and keys: dennis (1960), breitenbach & kränzlin (1981), moser (1983), table 1. taxonomic review of investigated species of ascomycetes in eastern serbia class order family species sordariomycetes hypocreales clavicipitaceae cordyceps militaris ophiocordycipitaceae ophiocordyceps gracilis pezizomycetes pezizales pezizaceae peziza phyllogena pyrenomataceae neottiella vivida scutellinia legaliae scutellinia trechispora sarcosomataceae plectania melastoma pseudoplectania nigrella urnula mediterraneaea fig. 2. in situ macro-photos of the examined species. a) scutellinia legalie, ph.: i. kajevska; b) peziza phyllogena, ph.: s. lazarević; c) s.trechispora, ph.:i. kajevska; d) ophiocordyceps gracilis; ph.: s. lazarević; e) pseudoplectania nigrella, ph.: i. kajevska; f) plectania melastoma, ph.: s. lazarević, v. lilić; g) cordyceps militaris, ph.: g.taskov, s. lazarević; h) urnula mediterranea, ph.: i. kajevska; i) neottiella vivida, ph.: s.lazarević. biologica nyssana 9 (2) ⚫ december 2018: 77-88 kajevska et al. ⚫ a contribution to the knowledge of ascomycetes... 80 medardi (2006), hansen & knudsen (2000). names of the species have been modified according to index fungorum (http://www.indexfungorum.org) and mycobank (http://www.mycobank.org). the following abbreviations are used: mt. mountain, r. river, v. village, * new species for serbia. results and discussion the biodiversity of ascomycota in serbia is unknown for the most part. field research on mycodiversity conducted from 2015 2018 in the eastern part of serbia resulted with a registration of the following new and noteworthy taxa for the country: cordyceps militaris, ophiocordyceps gracilis, peziza phyllogena, neottiella vivida, scutellinia legalie, s. trechispora, plectania melastoma, pseudoplectania nigrella and urnula mediterranea (tab. 1, fig. 2). macroand microscopic descriptions, notes on ecology and diversity in the country are provided exclusively on the collected material. data on presence in the country of cordyceps militaris, peziza phyllogena, scutellinia trechispora and plectania melastoma hasn’t been reported in scientific literature up to now and scattered information were known only from web, macroscopic photos and personal communications. neottiella vivida, ophiocordyceps gracilis, scutellinia legalie and pseudoplectania nigrella are reported for serbia for the first time. a second country record is confirmed for the rare european species urnula mediterranea. fam.: clavicipitaceae (lindau) earle cordyceps militaris (l.) link description: stroma 10 70 x 2 10 mm wide, solitary or in small groups, club-shaped, cylindrical, bright orange to orange-red with abundant perithecia. stem 10 25 x 3 5 mm, smooth, wavy, paler and whitish at base (fig. 2.g). perithecia 500 720 x 300 480 μm, ovoid, usually 2/3 submersed in the stroma, paler in colour. asci 300 510 x 3.5 5 μm, narrow, cylindrical, not staining blue in iodine. ascospores elongate, filiform, multiseptate, breaking into bacilliform cell fragments with dimension of 2 4.5 x 1 1.5 μm (fig. 3). specimen examined: gabrovac v., near st. trojica monastery (niš), seličevica mt., deciduous forest, on a lepidopteran pupae half buried in soil, among moss, 316 m, n 43°15’55”, e 21°55’28”, 25/12/2015, m. kuštera, m.s.n. 25/12/15 104; gabrovac v., above st. trojica monastery (niš), seličevica mt., quercus cerris and carpinus orientalis forest, on a lepidopteran pupae, 400 m, n 43°16’13”, e 21°55’12”, 09/5/2017, n. pavković, m.s.n. 09/05/17 – 195; gadžin han town, above, suva planina mt. (niš), quercus cerris forest with carpinus orientalis, on larvae, 631 m, n 43°12’25”, e 22°03’05”, 01/06/2017/18, i. kajevska, m.s.n. 01/06/17 190. claviceps militaris is a type species of the genus with a worldwide distribution. this entomopathogenic species parasites insect larvae or pupae of several genera of lepidoptera and hymenoptera order (webster & weber, 2007). there are several species of the genus recorded in the northern part of serbia (more on this in savić et al., 2016). however, the cosmopolitan species c. militaris has not been reported in scientific literature for the mycobiota of serbia thus far. unpublished data with exsiccate and in situ photographs from the eastern part of serbia, were also kindly provided from grozdan taskov (pers.comm.): four well developed fruiting bodies found on lepidopteran pupae in pinus nigra plantings, near the city of pirot (n 43°10’18” e 22°36’56”; 11/2016). our material was collected in deciduous forests, all found on a lepidopteran pupae half buried in soil, except for one collection with small fruiting bodies emerging from larvae. as indicated above, we do not consider it as a rare species. fig. 3. microscopic photographs of cordyceps militaris, a) perithecia in melzer’s reagent (mlz); b) perithecium in lugol’s solution (iki); c) asci in iki; d) asci in mlz; e) asci tips in iki; f) spores in mlz (x400); g) spores in iki (x1000). photo – i. kajevska. biologica nyssana 9 (2) ⚫ december 2018: 77-88 kajevska et al. ⚫ a contribution to the knowledge of ascomycetes... 81 fam.: ophiocordycipitaceae g. h. sung, j.m. sung, howel-jones & spatafora *ophiocordyceps gracilis (grev.) g. h. sung, j.m. sung, howel-jones & spatafora (syn: cordyceps gracilis (grev.) durieu & mont.) description: stroma 8 x 4 mm wide, solitary, capitate, dark yellow to orange with abundant dark orange perithecial ostioles; flesh thick and paler than surface. stem 35 x 2.5 mm unfertile part of the stroma, cylindric, faintly floccose, somewhat wavy, sulphur yellow at base, paler and whitish near the fertile part of the stroma (fig. 2.d). perithecia (550)630 750 x 210 270 μm, ovoid, completelly immersed in the stroma, paler in colour. asci 370 500 x 3 5 μm, narrow, cylindrical, not staining blue in iodine, swollen at the apex. ascospores elongate, filiform, multiseptate, breaking into bacilliform cell fragments with dimension of (3)5 8 x 1 1.5 μm (fig. 4). specimen examined: niška banja city municipality (niš), suva planina mt., deciduous forest (populus tremula, p. nigra, salix alba), on lepidopteran larva, humid place, 200 m, n 43°18’14”, e 21°59’52”, 13/04/2018, s. lazarević (leg. d. vučić), m.s.n. 13/04/18 228. ophiocordyceps gracilis is a cosmopolitan but rarely reported species throughout the world. no occurrence for serbia is cited for this species, commonly known as cordyceps gracilis and this record is possibly the first for the country. a single stroma of o. gracilis has been found on lepidopteran larva emerging from the host completely covered by sulphur yellow mycelial strands in deciduous forest, predominantly polars. macro and microscopically the examined material is in accordance with the description given by mains (1951, 1958), eckblad (1967), allard (1998), breitenbach & kränzlin (1984), hansen and knudsen (eds.) (2000). fam.: pezizaceae dumort. peziza phyllogena cooke description: apothecia 5 45 mm in diameter, cup-shaped to disk-shaped, often irregularly shaped when mature; hymenium smooth, wrinkled in the center, brown with violet tinge; the outer surface is granular, brown to lilac, purplish at base; margin with purplish granules, slightly involute, and undulate; flesh thick and pale violet (fig. 2.b). asci 220 300 x 12 16 μm, cylindrical, amyloid, operculate, 8 spored, apex obtuse or truncate, ascus base with croziers. paraphyses 310 370 x 3 4 μm, cylindrical, septate, slightly enlarged at apex 4 6 μm. ascospores (13)17 19(21) x 6 11 μm, ellipsoid to narrowly ellipsoid, hyaline, structure with fine warts sometimes forming small ridges. medullarly excipulum of textura intricata, with fig. 4. microscopic photographs of ophiocordyceps gracilis, a) perithecia in (iki); b) asci in congo red (cr); c) asci tips (x1000) in cr; d) ascus and spores (x400) in cr; e) spores (x1000) in cr. photo – i. kajevska. fig. 5. microscopic photographs of peziza phyllogena. a) ascus in mlz and lpcb; b) ascus apex in lpcb; c) spores (x400) in lpcb; d) excipulum in mlz; e) ascus base in lpcb; f) hymenium (asci and spores) in lpcb. photo – i. kajevska. biologica nyssana 9 (2) ⚫ december 2018: 77-88 kajevska et al. ⚫ a contribution to the knowledge of ascomycetes... 82 hyaline cylindrical cells. ectal excipulum of textura globulosa-angularis with cells of 20 40 μm wide (fig. 5). specimen examined: popova glava hill, between hum v. and rujnik v. (niš), kalafat mt., quercus sp. and carpinus orientalis forest, on soil and wood debris, 700 m, n 43°23’25”, e 21°53’44”, 09/05/2016/7/8, i. kajevska (leg. s. lazarević), m.s.n. 09/05/17 116. no published record is reported from serbia for this species and so far, we managed to register only one locality of p. phyllogena (popova glava hill) in this part of the country. on this locality, in 2016 large quantity of this species was observed, but during the next two years these populations were with scattered number of fruiting bodies. this humus saprotroph may be confused with p. badia but its occurrence in late autumn and its reticulate spores removes any suspicion (pfister, 1987; elliot & kaufert, 1974). fam.: pyronemataceae corda *neottiella vivida (nyl.) dennis description: apothecia 2 13 x 3 8 mm in diameter, disk – shaped to cup-shaped; hymenium smooth or wrinkled with maturity, bright orange to dark orange-red when dry; excipulum concolorous with the hymenium or paler, villose with white soft and tender (delicate) hairs (up to 3.5 mm) which are mostly concentrate at the margin; margin entire or lobed, somewhat eroded; flesh thick and white. stem central if present, villose and thick (fig. 2.i). asci 150 270 x 15 18 μm, cylindrical, 8 spored, uniseriate, apex obtuse or truncate, not staining blue in iodine, ascus base with croziers. paraphyses 170 300 x 2 3 μm, narrowly cylindrical, swollen at the apex (5 6 μm) and often wry, filled with orange granules vivid in cotton blue or becoming greenish in melzer’s reagent. ascospores 21 25(-27) x 14 15(-17) μm, hyaline, thick-walled, ellipsoid, usually contains 1 2 large globose oil drops (7 10(18) μm); ornamentation verrucose on oil immersion, with small isolated warts but some are connected and form also isolated ridges. hairs 170 300 x 4 8 μm, hyaline or greenish in melzer’s reagent, usually with 2 3 septa, thickened walls, base of hair with somewhat clavate widening but mostly wry and widened (4 10 μm) with median constriction, tips obtuse. ectal excipulum of textura angularisintricata, with cells of 20 40 μm wide. medullarly excipulum of textura intricata, with hyphae 3 7 μm wide, hyaline, with thin walls (fig. 6). specimen examined: grbavče v., svrljiške planine mt. (svrljig), deciduous forest (carpinus orientalis, quercus spp., on moss rhizoids of polytrichum spp., 550 m, n 43°23’54”, e 22°00’36”, 29/09/2016, i. kajevska (leg. n. pavković), m.s.n. 29/09/16 101; gradac resort, above knez selo v. (niš), kalafat mt., fagus forest, on moss rhizoids, 450 m, n 43°23’21”, e 21°00’06”, 18/10/2016, i. kajevska (leg. n. pavković and s. lazarević), m.s.n. 18/10/16 102. neottiela vivida is bryoparasitic species which occurs with stipe base attached to moss rhizoids predominantly on polytrichum spp. important microscopic character of n. vivida which distinguishes it from other neottiella species is its warted spores (senn irlet, 1989; yao & spooner, 1996a; eckstein et al., 2014). distinguishable feature can also be the hair bases with its widened base and median constriction. its occurrence in europe is widespread but it is rarely reported. also, available literature search indicates no published data of this species from serbia. during field studies held in september and october 2016 in the eastern part of serbia, two localities of this species have been recorded (svrljiške planine mt. and gradac resort) and are reported here as new for the country. all findings have been observed at altitude bellow 700 m in fig. 6. microscopic photographs of neottiella vivida, a) ascus with spores and paraphysis in iki; b) spores in iki (x400); c) hymenium (asci, ascospores, and paraphyses) in lactophenol cotton blue solution (lpcb); d) spores in lpcb (x1000); e) ascus base in iki; f) medullary excipulum in lpcb; g) hair in lpcb; h) ectal excipulum in lpcb. photo i. kajevska. biologica nyssana 9 (2) ⚫ december 2018: 77-88 kajevska et al. ⚫ a contribution to the knowledge of ascomycetes... 83 xeric to mesic deciduous forest associations, predominantly quercus spp. *scutellinia legaliae lohmeyer & häffner description: apothecia 2 8 mm in diameter, disc shaped, hymenium dark orange and red, smooth; the outer surface covered with short dark brown thick hairs; margin distinct, covered with thick pointed or tapered hairs; flesh thick and yellowish (fig. 2.a). asci 200 310 x 18 23 μm, inamyloid, cylindrical, operculate, 8 spored, apex obtuse, ascus base with croziers. paraphyses 270 330 x 3 4 μm, cylindrical, septate, filled with orange carotenoid crystals, enlarged tips 6 12 μm. hairs: 350 650 μm, septate, mostly simple and bifurkate at base. ascospores 15.5 17.5 μm, globose, hyaline, surface covered with acuminate spines 2 5 μm long. medullarly excipulum of textura intricata, with hyaline hyphae of 5 15 μm wide. ectal excipulum of textura globulosa with cells of 20 50 μm wide (fig. 7). specimen examined: above gabrovac v., near st. trojica monastery (niš), seličevica mt., quercus cerris forest with carpinus orientalis, on wet clay soil, 450 m, n 43°15’40.6”, e 21°55’25.6”, 04/06/2017, i. kajevska (leg. s. lazarević), m.s.n. 04/06/17 199. according to schumacher (1990), s. legalie is a rarely recorded species in europe. for serbia it is reported for the first time. material examined was found on wet soil along forest pathway in quercus cerris forest on humid and open place unlike s. trechispora which obviously prefers humid but shaded places. although it is referred as more south european species preferring warm climates (schumacher 1990; matočec et al. 1995), hitherto our field studies from the eastern part characterized by warmer climate have resulted in only one data of this species consisting of four small fruiting bodies (two of them in its initial phase). scutellinia trechispora (berk. & broome) lambotte description: apothecia 3 10 mm in diameter, discshaped, hymenium dark orange and red, smooth; the outer surface covered with short dark brown thick hairs; margin distinct, covered with densely thick and sharply pointed hairs; flesh thick and white (fig. 2.c). asci 270 320 x 20 25 μm, inamyloid, cylindrical, operculate, 8 spored, apex obtuse, ascus base with croziers. paraphyses 250 330 x 2 4 μm, cylindrical, few septas, filled with orange carotenoid crystals well vivid in congo red, enlarged tips 6 9 μm. hairs: 500 1700 μm, septate, sharply pointed, mostly bifurkate at base. ascospores 15 18 μm, globose, hyaline, surface covered with truncate spines 2 4 μm high and 1 2 μm wide. medullarly excipulum of textura intricata, with hyaline hyphae of 5 15 μm wide. ectal excipulum of textura globulosa and angularis with cells of 20 50 μm wide (fig. 8). specimen examined: above gadžin han town (niš), suva planina mt., quercus cerris forest, on wet soil, under leaf litter and wood debris, by a stream, 600 m, n 43°12’25”, e 22°03’04”, 01/06/2017, i. kajevska, m.s.n. 01/06/17 213. no published data of this common european species have been reported for serbia up till now. it can be easily delimited from other taxa of the genus by its truncate spores (lohmeyer & häffner, 1983; schumacher, 1990; matočec et al., 1995; yao & spooner 1996b). macro and microscopically, our collection was consistent with published descriptions, except for the length of the hairs which is somewhat shorter that those noted by schumacher (1990). ecologically is also in accordance with the indications given by the aforementioned authors. our specimens consisting of five well-developed fruiting bodies have been found on wet soil in oak forest, barely visible among the humid leaf litter. fig. 7. microscopic photographs of scutellinia legalie, a) hairs (x100) in iki; b) hair tip in lpcb; c) hair base in lpcb; d) ascus apex in lpcb; e) ascus with spores in lpcb; f) spores within ascus (x1000) in lpcb; g) spores (x1000) in lpcb; h) spores (x1000) in iki. photo – i. kajevska. biologica nyssana 9 (2) ⚫ december 2018: 77-88 kajevska et al. ⚫ a contribution to the knowledge of ascomycetes... 84 fam.: sarcosomataceae kobayasi plectania melastoma (sowerby) fuckel description: apothecia 0.3 17 x 0.5 15 mm diam., at first roughly spherical with hollow opening, becoming cup-shaped, concave; hymenium smooth, shining, dark brown or black; excipulum wrinkled, concolorous with the hymenium, covered with orange granules when young especially in the upper part but seldom or missing when mature; flesh tough, gelatinous when wet, gray; margin entire, incurved and covered abundantly with orange granules especially when young. stem 0.2 0.5 mm, if present than central, thick, at base covered with black mycelial threads (fig. 2.f). asci 270 430 x 9 13 μm, cylindrical, long attenuated, 8 spored, uniseriate, thick walled, apex obtuse or truncate but with somewhat eccentric operculum, not staining blue in iodine, ascus base flexuous and tapered. paraphyses 270 400 x 2 μm, filiform, often forked or branched, flexuous, septate. hymenial hairs 260 410 x 2 3 μm, cylindrical, with obtuse apex and one septum at the base. ascospores (20) 21 24(27) x 9 10(12) μm, hyaline, elliptic, fusiform, slightly thick-walled, multiguttulate with small oil drops; smooth or barely visible ornamentation on oil immersion. medullarly excipulum of textura intricata, with hyphae 2 5 μm wide, smooth, hyaline, cylindrical, immersed in a gel. ectal excipulum of textura globulosa-angularis, with cells of 10 13 μm wide, thick walled, brownish. tomentum (basal hyphae) 6 8 μm wide, flexuous, with scarce septa, encrusting pigments, thick walled, dark brown (fig. 9). specimen examined: gabrovac v., near st. trojica monastery (niš), seličevica mt., quercus pubescens and q. cerris forest, on wet fallen oak branches of small diameter, 330 m, n 43°15’16”, e 21°44’75”, 28/02/2015, m. kuštera & i. cvijetan (leg. v. lilić), m.s.n. 28/02/15 – 106; bojanine vode resort, above gornja studena v., suva planina mt., fagus forest, on fallen branches and small sized decaying organic matter, 700 m, n 43°13’18”, e 22°07’20”, 24/04/2015, m. kuštera & i. cvijetan (leg. n. pavković), m.s.n. 24/05/15 107. plectania melastoma is well distinguishable from other species of the genus by the presence of the orange granules on the excipulum surface of the fruiting bodies which may last for centuries as noted by agnelo & carbone (2011). same authors also point out that spores are not quite smooth but has exclusively fine ornamentation (agnelo & carbone, 2011). p. melastoma is widespread species but also reported as rare in many fig. 8. microscopic photographs of scutellinia trechispora. a) ascus with paraphyses in iki; b) hairs (x200) in lpcb; c) hair base in iki; d) spores (x1000) in iki; e) spores (x1000) in lpcb. photo – i. kajevska. fig. 9. microscopic photographs of plectania melastoma, a) hymenium (asci, ascospores, hymenial hairs/seta and paraphyses) in lpcb; b) ascus apex in lpcb; c) ascus base in lpcb; d) paraphysis (branching) in lpcb; e) spores in mlz (x400); f) medullary excipulum in lpcb; g) ectal excipulum in lpcb; h) basal hyphae in mlz; i) spores in iki (x400). photo – i. kajevska. biologica nyssana 9 (2) ⚫ december 2018: 77-88 kajevska et al. ⚫ a contribution to the knowledge of ascomycetes... 85 european countries and listed in many national redlists of fungi (gierczyk et al., 2010). this saprotrophic fungus had not been reported before in scientific literature for serbia. data on p. melastoma published in this paper comes from two localities (gabrovac vill. and bojanine vode resort). investigated specimens were found in the base of a slope where lot of organic matter is accumulated in mixed deciduous forests (carpinus orientalis, quercus cerris, acer campestre, fagus sylvatica, robinia pseudoacacia), humid for a long time. the species was found for the first time in 2015. and all records occur from the southern mountain hills at altitude bellow 700 m. glejdura et al. (2011) states that in slovakia p. melastoma occurs at altitude of 280 – 750 m and its occurrence is confined on small diameter branches, humid places and on localities with no or low sign of human activity and these parameters are in accordance with our findings too. *pseudoplectania nigrella (pers.) fuckel description: apothecia 10 25 x 5 15 mm in diameter, cup shaped to disk-shaped; hymenium shiny black or dark brown, smooth or wrinkled with maturity; the outer surface dark brown to blakish, tomentose with dark brown to blackish, convolute and thick hairs, margin entire or undulate, also with hairs which are light brown when young; flesh thick, white and grayish (fig. 2.e). asci 280 320 x 10 14 μm, inamyloid, cylindrical, operculate, 8 spored, thick walled, apex obtuse or truncate, base attenuated. paraphyses 270 330 x 2 3 μm, filiform, septate, irregularly wry tips, some branched. hymenial hairs: 280 350 x 2 4 μm, narrowly cylindrical, straight, with one septum at base. ascospores (9-)10 12 μm, spherical, smooth, hyaline, thick walled, with gelatinous outer layer, inside contains many small globose oil drops. hairs (outher surface): 4 9 μm in diameter, cylindrical and curved, dark brown, thick-walled, septate. ectal excipulum of textura angularis, with dark brown cells of 18 25 μm wide. medullarly excipulum of textura intricata, with hyaline hyphae of 4 8 μm wide. tomentum (basal hyphae): 4 9 μm wide, cylindrical, curved, dark brown, thick walled, few septas (fig. 10). specimen examined: between gabrovac v. and gornje vlase v. (niš), seličevica mt., pinus sylvestris plantings, on soil, among moss, 398 m, n 43°16’6.34”, e 21°55’7.04”, 24/02/2017, i. kajevska, m.s.n. 24/02/17 158. pseudoplectania nigrella has not been previously reported from serbia and these are new data for the country. it has been found at four different localities, all from the eastern part of the country but exsiccate has been preserved only from the vicinity of gabrovac vill., with two welldeveloped fruiting bodies. macroand microscopically specimens are in accordance with the descriptions given by saver (1913), le gal (1953), paden (1983), breitenbach & kränzlin (1984), korf & zhuang (1991), hansen and knudsen (eds.) (2000), medardi (2006), carbone et al. (2012). data of this species in serbia comes from coniferous forests: 2 collections from fir forest and 2 from pine plantations. ascomata from all collections have been observed at the edge of the forests, about 1-2 m near the conifer trees, on an open area and amongst moss and fallen needles. urnula mediterraneaea (m. carbone, agnello & baglivo) m. carbone, agnello & p. alvarado (syn.: plectania mediterranea m. carbone, agnello & baglivo) description of studied specimen: apothecia 10 50 x 7 30 mm in diameter, cup shaped, hymenium brownish, medium reddish shade of brown, smooth fig. 10. microscopic photographs of pseudoplectania nigrella, a) hymenium (asci, ascospores, hymenial hairs/seta and paraphyses) in iki; b) hymenial hair (in middle) and paraphyses, and ascus with spores in iki; c) ascus apex in cr; d) spores (x1000) in cr; e) hymenial hairs with paraphyses in cr; f) medullary excipulum in iki; g) ectal excipulum in iki; h) basal hyphae in iki. photo – i. kajevska. biologica nyssana 9 (2) ⚫ december 2018: 77-88 kajevska et al. ⚫ a contribution to the knowledge of ascomycetes... 86 at first and wrinkled later; the outer surface is dark brown, rough in touch, covered with densely dark brown and thick hairs; margin crenate; flesh gelatinous, grayish (fig. 2.h). stem 5 20 mm, central, concolorous with the apothecia, with densely dark hairs. asci 450 550 x 12 15 μm, inamyloid, cylindrical, operculate, 8 spored, apex obtuse or truncate, ascus base without croziers. paraphyses 400 570 x 2 4 μm, cylindrical, septate, many with curved tips. hymenial hairs 450 600 x 3 4 μm, cylindrical, straight, apex obtuse. ascospores 22 27(29) x 10 14 μm, globose, hyaline, surface covered with truncate spines 2 4 μm high and 1 2 μm wide. medullarly excipulum of textura intricata, with hyaline hyphae of 3 7 μm wide. ectal excipulum of textura globulosa to angularis with brownish cells of 8 15 μm wide. tomentum (basal hyphae): 4 8 μm wide, cylindrical, curved, dark-brown, thick walled, septate (fig. 11). specimen examined: above gadžn han town (niš), suva planina mt., quercus cerris forest, on wet soil, under leaf litter and wood debris, by a stream, 600 m, n 43°12’25”, e 22°03’04”, 01/06/2017, i. kajevska, m.s.n. 01/06/17 213. urnula mediterranea is a recently described species (carbone et al., 2009) and it is an extremely rare european species. so far it is registered only from: italy, spain, france, serbia and greece (kaounas et al., 2015; milosavljević, 2017). the first record of this mediterranean species in serbia has been surprisingly reported from the central region of the country, a discovery which also represents the first record from the continental europe, and the species was found precisely in 2014, a year followed by heaviest rainfall, floods and temperatures above the average since 1951 (milosavljević, 2017). here we report the second record of u. mediterranea for serbia, located in the eastern part of the country (above gadžin han town, suva planina mt.). specimens on this locality have been registered in 2017 which was normal and dry in a relation of precipitation unlike 2014, but apothecia were found on a very humid place in quercus cerris forest under carpinus orientalis. our collection conforms well to the description given in carbone et al. (2009), although the length of spores has been quite smaller and did not exceed 30 μm but carlo agnello indicates that the dimension of spores is quite variable in this species (pers. comm.). acknowledgements. we are grateful to all members of the current mycological societies from niš for providing the fungal material in further examination. we also want to express our appreciation to prof. mitko karadelev for his valuable comments and advices in the preparation of this manuscript. references agnello, c., carbone, m., 2011: appunti di studio su plectania melastoma. rivista di micologia, 54 (4), 315-337. allard, c., 1998: recontres avec cordyceps gracilis mont. et durieu. le mystére des rendez-vous mycologiques. bulletin trimestriel de la société mycologique de france, 114: 29-33. breitenbach, j. kränzlin, f., 1981: pilze der schweiz band 1. ascomyceten. verlag micologia, luzern, switzerland. 303 p. carbone, μ., agnello, c., baglivo, a., 2009: plectania mediterranea una nuova specie dell’italia mediterranea, con storia e circoscrizionedel genere plectania. rivista di micologia, 52 (3): 245-266. carbone, m., agnello, c., 2012: studio e tipificazione di pseudoplectania nigrella. ascomycete.org, 4 (4): 79-93. dennis, r.w.g., 1960: british cup fungi and their allies. an introduction to the ascomycetes. ray society, london. 280 p. fig. 11. microscopic photographs of urnula mediterranea, a) asci (x400) in cr; b) hymenial hair (x400) in cb; c) hymenial hair tips in cr; d) ascus base in cr; e) spores (x1000) in cr; f) paraphyses tips in cr; g) paraphyses in cr; h) medullary excipulum; i) ectal excipulum (x250) in cr; j) basal hyphae (x250) in cr. photo – i. kajevska. biologica nyssana 9 (2) ⚫ december 2018: 77-88 kajevska et al. ⚫ a contribution to the knowledge of ascomycetes... 87 eckblad, f.e., 1967: the genus cordyceps in norway. nytt magasin for botanik, 14: 68-76. eckstein, j., eckstein, g., vega, m., 2014: noteworthy findings of bryoparasitic pezizales (ascomycota) from germany. boletus, 35 (1): 1725. elliott, m.e., kaufert, m., 1974: peziza badia and peziza badio-confusa. canadian journal of botany, 52 (3): 467-472. gierczyk, b., kujawa, a., wojtowski, m., 2010: plectania melastoma nowe stanowisko w polsce. chrońmy przyrodę ojczystą, 66 (1): 6164. glejdura, s., kunca, v., kučera, v., 2011: plectania melastoma (sarcosomataceae, pezizales) in slovakia. catathelasma, 13: 19-24. hansen, l., knudsen, h., (eds.) 2000: nordic macromycetes vol. 1. ascomycetes. nordsvamp, kobenhavn. 309 p. ivančević, b.n., 2016: prostorna distribucija i ekološke varijacije staništa hipogeičnih makromiceta (mycota) u srbiji. phd thesis, univerzitet u beogradu, biološki fakultet. ivančević, b., beronja, j., 2004: first records of macromycetes from the serbian side of stara planina mts (balkan range). mycologia balcanica, 1 (1), 15-19. kaounas, v., tsampazis, t., agnello, c., 2015: urnula mediterranea (pezizales), a rare species, recorded in greece. ascomycete.org, 7 (2): 97100. karaman, m.a., novaković, m.s., savić, d., matavulj, m.n., 2012: preliminary checklist of myxomycota and ascomycota from fruška gora mountain. zbornik matice srpske za prirodne nauke, 123: 37-49. korf, r.p., zhuang, w.y., 1991: a preliminary discomycete flora of macaronesia: part 11, sarcoscyphineae. mycotaxon, 40: 1-11. le gal, m., 1953: les discomycètes de madagascar. prodrome à flore mycologique de madagascar et dépendances 4: 1-465. lohmeyer, t.r., häffner, j., 1983: beiträge zur taxonomie und verbreitung der höheren ascomyceten in der bundesrepublik deutschland i. einführung in die gattung scutellinia (cooke) lamb. und ihre rundsporigen arten. westfälische pilzbriefe, 10/11: 189-209. mains, e. b., 1951: notes concerning entomogenous fungi. bulletin of the torrey botanical club, 78: 122-133. mains, e. b., 1958: north american entomogenous species of cordyceps. mycologia 50: 169-222. marjanovic, z., grebenc, t., markovic, m., glisic, a., milenkovic, m., 2010: ecological specificity and molecular diversity of truffles (genus tuber) originating from mid west of the balkan peninsula. sydowia, 62 (1), 67-87. matočec, n., antonić, o., mrvoš, d., 1995: the genus scutellinia (pezizales, ascomycotina) in croatia: preliminary part. natura croatica, 4 (1), 1-58. medardi, g., 2006: atlante fotografico degli ascomiceti d’italia. a. m. b. centro studi micologici, trento. 454 p. milosavljević, n., 2016: the genus helvella in central serbia. mycologia montenegrina, 19: 5196. milosavljević, n., 2017: the first record of urnula mediterranea (pezizales) in continental europe. ascomycete.org, 9 (5): 27. moser, m., 1963: ascomyceten (schlauchpilze). kleine kryptogamenflora mitteleuropas. bd. 2a: gustav fischer. stuttgart. 147 p. paden, j.w., 1983: sarcosomataceae (pezizales, sarcoscyphineae). flora neotropica, 37: 1-16. pfister, d.h., 1987: peziza phyllogena an older name for peziza badioconfusa. mycologia, 79 (4): 634. ranojevíc, n., 1910: zweiter beitrag zur pilzflora serbiens. annales mycologici, 6: 347-402. ranojevic, n. v., 1914: dritter beitrag zur pilzflora serbiens. annales mycologici, 12: 393-421. sadiković, d., kuštera, m., 2013: fungal conservation: protected species of fungi in south serbia region. biologica nyssana, 4 (1-2): 35-40. savić, d., 2016: diverzitet gljiva razdela ascomycota na području fruške gore sa posebnim osvrtom na red helotiales. phd thesis, univerzitet u novom sadu, prirodno matematički fakultet. savić, d., karaman, m., 2016: leotiomycetes checklist of mt fruška gora with new records for serbian mycobiota. bulletin of the natural history museum, 7: 31-66. savić, d., grbić, g., bošković, e., hänggi, a., 2016: first records of fungi pathogenic on spiders for the republic of serbia. arachnologische mitteilungen/arachnology letters, 52: 31-34. schumacher, t., 1990: genus scutellinia (pyronemataceae). council for nordic publications in botany. 107 p. seaver, f. j., 1913: the genus pseudoplectania. mycologia, 5 (6), 299-302. senn-irlet, b., 1989: discomyceten aus der alpinen stufe der schweizer alpen und viele weitere interessante beiträge. beiträge zur kenntnis der pilze mitteleuropas, 5: 191-208. skrede, i., carlsen, t., schumacher, t., 2017: a synopsis of the saddle fungi (helvella: ascomycota) in europe–species delimitation, taxonomy and typification. persoonia. molecular biologica nyssana 9 (2) ⚫ december 2018: 77-88 kajevska et al. ⚫ a contribution to the knowledge of ascomycetes... 88 phylogeny and evolution of fungi, 39 (1): 201253. vukojević, j., hadžić, i., knežević, a., stajić, m., milovanović, i., ćilerdžić, j., 2016: diversity of macromycetes in the botanical garden “jevremovac” in belgrade. botanica serbica, 40 (2): 249-259. webster, j., weber, r., 2007: introduction to fungi. cambridge university. press, uk. 841 p. yao, y.j., spooner, b.m., 1996a: notes on british species of octospora. mycological research, 100 (2), 175-178. yao, y.j., spooner, b.m., 1996b: notes on british species of scutellinia. mycological research, 100 (7), 859-865. göse, hacıoğlu doğru, 2019, biologica nyssana 10(2) 10 (2) december 2019: 169-173 doi: 10.5281/zenodo.3600197 antibiofilm activity of verbascum pinnatifidum vahl. ethanolic extract original article mehmet göse department of plant physiology, institute of biology, graduate school of natural and applied sciences, çanakkale onsekiz mart university, çanakkale, turkey m_gose@hotmail.com nurcihan hacıoğlu doğru department of biology, faculty of arts and sciences, çanakkale onsekiz mart university, çanakkale, turkey nurcihan.n@gmail.com (corresponding author) received: july 1, 2019 revised: september 9, 2019 accepted: september 13, 2019 abstract: microbial biofilms pose health risks in clinical environments, food industry and drinking water systems. microorganisms within biofilms are more resistant to antibiotics and chemical agents than planktonic cells in suspension. new alternatives for controlling infections have been proposed focusing on the therapeutic properties of medicinal plants and their antibiofilm activities. here, we investigated in vitro antibiofilm activity of ethanol extract of verbascum pinnatifidum vahl. against escherichia coli nrrl b-3704, pseudomonas aeruginosa atcc 27853, proteus vulgaris atcc 13315, acinetobacter baumanii atcc 19606, bacillus subtilis atcc 6633, staphylococcus aureus atcc 25923, s. haemolyticus atcc 43252 and candida albicans atcc 10231 test microorganisms based on crystal violet binding assay. the highest antibiofilm activity was shown against biofilm formed by b. subtilis atcc 6633 and s. haemolyticus atcc 43252 at the lowest mic values of 2.5 and 10 µg/ml, respectively. the current findings indicated that bacterial biofilm formation can be potentially managed using v. pinnatifidum plant extracts. key words: antibiofilm activity, verbascum pinnatifidum apstract: antibiofilm aktivnost etanolnog ekstrakta verbascum pinnatifidum vahl. biofilmovi mikroorganizama predstavljaju zdravstveni rizik u kliničkim uslovima, prehrambenoj industriji i sistemima pijaće vode. unutar biofilmova, mikroorganizmi su rezistentniji na antibiotike i hemijske agense od planktonskih ćelija u suspenziji. predložene su nove alternative za kontrolisanje infekcija, koje su fokusirane na terapeutske osobine medicinskih biljaka i njihovu antibiofilm aktivnost. ovde je, korišćenjem kristal violet testa, vršeno istraživanje in vitro antibiofilm aktivnosti etanolnog ekstrakta verbascum pinnatifidum vahl. u odnosu na vrste escherichia coli nrrl b-3704, pseudomonas aeruginosa atcc 27853, proteus vulgaris atcc 13315, acinetobacter baumanii atcc 19606, bacillus subtilis atcc 6633, staphylococcus aureus atcc 25923, s. haemolyticus atcc 43252 i candida albicans atcc 10231. najveća antibiofilm aktivnost, sa najnižim mik vrednostima od 2,5 i 10 µg/ml, utvrđena je za biofilmove formirane od strane vrsta b. subtilis atcc 6633 i s. haemolyticus atcc 43252, respektivno. ovi nalazi ukazuju na to da stvaranje bakterijskih biofilmova potencijalno može biti sprečeno korišćenjem ekstrakta v. pinnatifidum. ključne reči: antibiofilm aktivnost, verbacum pinnatifidum introduction microbial biofilms are communities of bacteria, embedded in a self-producing matrix, forming on living and nonliving solid surfaces (vasudevan, 2014). they are considered as an important virulence factor that causes persistent chronic and recurrent infections; they are highly resistant to antibiotics and host immune defenses (grant and hung, 2013). biofilm resistance is due to several reasons, like restricted diffusion of antibiotics into biofilm matrix, expression of multidrug efflux pumps, type iv secretion systems, decreased permeability, and the action of antibiotic-modifying enzymes (alekshun and levy, 2007). the increased biofilm resistance © 2019 göse, hacıoğlu doğru. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work noncommercially under the same license as the original. 169 13th symposium on the flora of southeastern serbia and neighboring regions to conventional treatments enhances the need to develop new control strategies (simões et al., 2007; sanchez et al., 2016). verbascum plants have been used medicinally since ancient times in folk medicine as a remedy for respiratory problems such as bronchitis, dry coughs, whooping cough, tuberculosis, and asthma. the leaves, roots and the flowers have been used also as anodyne, sedative, diuretic, sudorific, expectorant and antidiarrheal agents in traditional world and turkish medicine (baytop, 1999; georgiev et al., 2011). therefore verbascum plant species have been also previously reviewed for their antiviral, antimicrobial, antimalarial, antioxidant, anti-inflammatory, antinociceptive, antitumor, anticancer, cytotoxic, immunomodulatory, anticholinesterase, antiulcerogenic, antihepatotoxic, antihyperlipidemic, anthelminthic, antitussive and antigermination activities (tatli and akdemir, 2006; dulger and hacioglu, 2009; kahraman et al., 2010; kahraman et al., 2011; kozan et al., 2011; ozcan et al., 2011; boğa et al., 2016). however, there is no any literature about verbascum plants antibiofilm activity, except moghaddam et al. (2015), which evaluated the antibiofilm activity of verbascum pinnatifidum vahl. ethanol extract. material and methods plant materials verbascum pinnatifidum was collected from canakkale, kumkale near the lantern d.s. sandy area, (40.008101 n, 26.203127 e) in 2018 and identified with the aid of flora of turkey (davis et al., 1988) by dr. ersin karabacak. voucher specimens were deposited in the biology department at çanakkale onsekiz mart university, çanakkale, turkey. preparation of plant extracts the plant parts were air-dried. dry powdered plant material (10 g) was extracted with 300 ml of 80% ethanol (merck, darmstadt, germany) for 24 h by using soxhlet equipment (khan et al., 1988). the extracts were filtered using whatman filter no.1, and the filtrates were then evaporated under reduced pressure and dried using a rotary evaporator at 55 oc. dried extracts were stored in labelled sterile screwcapped bottles at + 4 oc. test microorganisms gram negative bacteria escherichia coli nrrlb 3704, pseudomonas aeruginosa atcc 27853, proteus vulgaris atcc 13315, acinetobacter baumanii atcc 19606 and gram positive bacteria – bacillus subtilis atcc 6633, staphylococcus aureus atcc 6538, s. haemolyticus atcc 43252 and yeast culture candida albicans atcc 10231 were used as test microorganisms. minimum inhibitory concentration assay to determine the plant extract doses to be used in biofilm inhibition study, minimum inhibitory concentration (mic) values of all samples were determined. mic was investigated as recommended instruction of the clinical and laboratory standards institute (clsi, 2006). the lowest concentration of extract inhibiting the visible growth of each test microorganisms was taken as the mic. the medium, 0.1% (w/v) streptomycin (st), nystatin (nys100) and 10% dmso were used as the nontreated, positive and negative controls, respectively (teanpasian et al., 2017). biofilm inhibition assay microplate biofilm method (merrit et al., 2005) was used to evaluate the inhibition of biofilm formation by v. pinnatifidum plant ethanol extract against test microorganisms. cultures were incubated in 5 ml tryptic soy broth (tsb) medium containing 5% glucose. cultures were diluted 1:100 in tsb and loaded into each well in 4 sterile microplates. different concentrations of plant extract (mic and sub-mic concentrations: 50, 25, 12.5% of mic) were prepared and transferred to each microplate well. after incubation at 37 ± 0.1°c for 48 h planktonic bacteria were removed from the wells and wells were washed twice with distilled water. 200 μl of 0.1% crystal violet solution was added to each well (20 minutes). the crystal violet bounded extracts were poured and washed until the crystal violet removed. the microtiter plates were inverted, and the remaining liquid was drained and dried in room heat. finally, the adhered biofilm bounded crystal violet was eluted in ethanol (95%), and the absorbance was measured at 550 nm by using an automated elisa reader. all experiments were repeated thrice in triplicate. the measurement of the antibiofilm effect of the extract was made by the percentage reduction formulation. % inhibition = (acontrol – asample / acontrol) x 100 acontrol: absorbance of the control (containing 100 µl tbs instead of plant extract) reaction asample: absorbance of the test compound results and discussion ethanol extract of v. pinnatifidum was tested against eight clinical bacterial and fungal strains (tab. 1). ethanol was observed as the best solvent for extracting antimicrobial substances in a previous study (jonathan and fasidi, 2003). results of mic were quite variable between each test microorganisms ranging from 2.5 to 20.0 µg/ 170 biologica nyssana ● 10 (2) december 2019: 196-173 göse, hacıoğlu doğru ● antibiofilm activity of verbascum pinnatifidum vahl. ethanolic extract biofilm is considered to be one of the essential virulence factors that cause persistent chronic and recurrent infections, because of their high resistance to antibiotics and host immune defensive system (grant and hung, 2013). bacteria are more resistant to antibiotics than planktonic cells because they are preserved by exopolysaccharide. this necessitated the screening of new and natural antibiotic sources in the fight against biofilm. new alternatives for controlling infections have been proposed focusing on the therapeutic properties of medicinal plants and their antibiofilm activities. many studies showed that the biofilm formation by pathogens leads to an increase in their virulence (vuong et al., 2004; antunes et al., 2010). therefore, if the biofilm formation is inhibited, the bacterial infection can be prevented (erdönmez et al., 2018). biofilm formation can be controlled by quorum sensing, a bacterial communication system which causes a rapid and coordinated change of expression pattern in the bacterial population in response to population density (pratiwi et al., 2015). the fact that at mic and sub-mic concentrations, v. pinnatifidum extract is capable of disturbing biofilm formation and causes biofilm breakdown, suggests that this disturbance may have been caused by the presence of compounds that inhibit quorum sensing. other plant compounds could attenuate biofilm development by inhibiting bacterial peptidoglycan synthesis (ogunlana et al., 1987), disrupting the permeability barrier of microbial membrane structures, causing the cell to leak out (cox et al., 2000), modify bacterial membrane structure hydrophobicity (türi et al., 1997; das, 2014), or disturbing the extracellular polymeric matrix in the biofilm to release biofilm from the surface of the solid substratum (traba and liang, 2011; pratiwi et al., 2015). further studies need to be performed ml (tab. 1). it was observed that almost all tested microorganisms were sensitive towards the ethanol extract of v. pinnatifidum. there is no data about v. pinnatifidum antimicrobial activity in the literature. our findings about mic ranges confirmed the observations of some other researchers, which stated that some verbascum species have antimicrobial activity against gram positive and negative bacteria and yeast and mold cultures (dulger and hacioglu, 2008;2009; ozcan et al., 2010; morteza-semnani et al., 2012; noori et al., 2012; anil et al., 2016; dulger and dulger, 2018). however, we found that v. pinnatifidum extract was also effective against e. coli nrrlb 3704 and c. albicans atcc 10231, contrary to some studies with the other verbascum species in the literature (dulger and hacioglu, 2008; morteza-semnani et al., 2012; amin et al., 2015). the results of potential inhibition of test microorganism’s biofilm formation by ethanol extract of v. pinnatifidum were shown in tab. 1. the results indicated that v. pinnatifidum extract in 2.5 to 10.0 µg/ml concentrations could inhibit the biofilm formation of all the tested microorganisms except e. coli nrrlb 3704 and c. albicans atcc 10231. throughputs also showed that antibiofilm function of extract was in a dose-dependent manner. the highest antibiofilm activity was noticed against biofilm formed by b. subtilis atcc 6633 and s. haemolyticus atcc 43252 at the lowest mic values of 2.5 µg/ml and 10 µg/ml, respectively. verbascum pinnatifidum has not been investigated in terms of antibiofilm activity. in a study conducted by moghaddam et al. (2015), ethanol extract of v. thapsus had inhibitory effect on biofilm formation of streptococcus mutans, s. sanguinis, and s. salivarius. the current findings indicated that biofilm forming of bacteria could be potentially managed using v. pinnatifidum plant extracts. 171 biologica nyssana ● 10 (2) december 2019: 169-173 göse, hacıoğlu doğru ● antibiofilm activity of verbascum pinnatifidum vahl. ethanolic extract table 1. the percentage of inhibition of biofilm structure of test microorganisms mic (µg/ml) % inhibition of biofilms formation test microorganisms plantextract control st/ ny100 mic mic/2 mic/4 mic/8 e.coli nrrlb 3704 20.0 4.0 7.27±0.33 3.34±0.08 p. aeruginosa atcc 27853 10.0 1.0 74.65±6.23 65.33±5.67 43.56±4.22 34.76±1.76 p. vulgaris atcc 13315 10.0 4.0 70.50±0.54 60.03±1.55 45.54±1.00 40.01±0.23 a. baumanii atcc 19606 10.0 2.0 57.42±0.12 43.12±0.10 b. subtilis atcc 6633 2.5 4.0 90.02±0.01 70.01±0.10 55.78±1.23 43.26±1.43 s. aureus atcc 6538p 10.0 4.0 70.54±0.60 s. haemolyticus atcc 43252 10.0 5.0 76.12±3.11 58.41±3.12 44.45±4.11 23.56±1.56 c.albicans atcc 10231 2.5 2.5 10.45±1.14 5.45±0.99 -: no inhibition of biofilm formation was observed due to the lack of antibiofilm effect. to confirm the actual mode of action of anti-biofilm activity from these extracts. conclusion our investigation was the first report on the antibiofilm property of the v. pinnatifidum ethanol extract. in this research, we found that forming of bacterial biofilm could be potentially being managed using v. pinnatifidum plant extract, especially against gram (+) bacteria. detailed phytochemical investigations are required to determine the types of substances responsible for the biological activities of v. pinnatifidum plant species. we hope that our results will provide useful data for discovering new compounds with better activity than agents currently available. acknowledgments. this investigation is a part of master thesis of mehmet göse. this study was financially supported by the çanakkale onsekiz mart university scientific research projects coordination unit, turkey (fyl-2018-2693). this paper was presented at the symposium on the flora of southeastern serbia and neighboring regions, june, 20-23 2019, serbia. authors would like to thank assoc. prof. dr. ersin karabacak for defining plant species. references alekshun, m.n., levy, s.b. 2007: molecular mechanisms of antibacterial multidrug resistance. cell, 128(6): 1037-1050. amin, j.n, batool, m., abu-hadid, m.m. 2015: screening antibacterial and antifungal activities and evalution of exhaustive extractions yields for verbascum sinuatum l. international research ayurveda pharmacy, 6 : 105-110. anil, s., dosler, s., mericli, a. h. 2016: chemical composition and antimicrobial activity of verbascum caesareum. chemistry of natural compounds, 52(1): 125-126. antunes, l.c.m., ferreira, r.b., buckner, m.m., finlay, b.b. 2010: quorum sensing in bacterial virulence. microbiology, 156(8): 2271-2282. baytop, t. 1999: türkiye’de bitkiler ile tedavi. nobel tıp kitabevleri, i̇stanbul. 480 p. boğa, m., ertaş, a., yılmaz, m.a., kızıl, m., çeken, b., haşimi, n., özden t.y., demirci, s., yener, i̇., deveci, ö. 2016: uhplc-esi-ms/ ms and gc-ms analyses on phenolic, fatty acid and essential oil of verbascum pinetorum with antioxidant, anticholinesterase, antimicrobial and dna damage protection effects. iranian journal of pharmaceutical research, 15(3): 393-405. clsi, 2006. clinical and laboratory standarts 172 institute. methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard-seventh edition. m07a7, villanova, pa, usa. cox, s., mann, c., markham, l., bell, h., gustafson, j. 2000: the mode of action of the essential oil of melaleuca alternifolia (tea tree oil). journal of applied microbiology, 88(1): 170-175. das, m.p. 2014: effect of cell surface hydrophobicity in microbial biofilm formation. european journal of experimental biology, 4(2): 254-266. davis, p.h., mill, r.r., tan, k. (eds.) 1988: flora of turkey and the east aegean islands. 10: 191-193. edinburgh university press, edinburgh. dülger, b., hacioğlu, n. 2008: antimicrobial activity of some endemic verbascum and scrophularia species from turkey. asian journal of chemistry, 20: 3779-3785. dülger, b., hacioğlu, n. 2009: activity of three endemic verbascum species against hospital isolates methicillin-resistant staphylococcus aureus. biotechnology & biotechnological equipment, 23: 760-762. dülger, b., dülger, b. 2018: antibacterial activity of verbascum antinori. konuralp medical journal, 10(3): 395-398. erdönmez, d., kenar, n., türkmen, k.e. 2018: screening for anti-quorum sensing and antibiofilm activity in viscum album l. extracts and its biochemical composition. trakya university journal of natural sciences, 19(2): 175-186. georgiev, m., alipieva, k., orhan, i., abrashev, r., denev, p., angelova, m. 2011: antioxidant and cholinesterases inhibitory activities of verbascum xanthophoeniceum griseb. and its phenylethanoid glycosides. food chemistry, 128: 100-105. grant, s.s., hung, d.t. 2013: persistent bacterial infections, antibiotic tolerance, and the oxidative stress response. virulence, 4(4): 273–283. jonathan, s.g., fasidi i.o. 2003: antimicrobial activities of two nigerian edible macro fungi – lycoperdon pusilum (bat. ex) and lycoperdon giganteus (pers). african journal of biomedical research, 6: 85 –90. kahraman, ç., tatlı, i.i., orhan, i.e., akdemir, z.s. 2010: cholinesterase inhibitory and antioxidant properties of verbascum mucronatum lam. and its secondary metabolites. zeitschrift für naturforschung, 65c: 667– 674. kahraman, ç., ekizoğlu, m., kart, d., akdemir, biologica nyssana ● 10 (2) december 2019: 169-173 göse, hacıoğlu doğru ● antibiofilm activity of verbascum pinnatifidum vahl. ethanolic extract z.s., tatlı, i.i. 2011: antimicrobial activity of some verbascum species growing in turkey. fabad journal of pharmaceutical sciences, 36: 11-15. khan, n.h., kamal, m.s.a., rahman, m. 1988: antibacterial activity of euphorbia thymifolia linn. indian journal of medical research, 87: 395-397. kozan, e., çankaya, i.t., kahraman, ç., akkol, e.k., akdemir, z. 2011: the in vivo anthelmintic efficacy of some verbascum species growing in turkey. experimental parasitology, 129: 211214. merritt, j.h., kadouri, d.e., o’toole, g.a. 2005: growing and analyzing static biofilms. current protocols in microbiology, 1(1b).1-17. moghaddam, h.k., mirzaii, m., khaksari, m., fazli, m., rahimi, f., behzadi, a.a. 2015: antibacterial and anti-adherent activity of great mullein (verbascum thapsus l.) ethanolic extract on in vitro biofilm formation of three oral streptococci. international journal of health studies, 1(2): 34-37. morteza-semnani, k., saeedi, m., akbarzadeh, m. 2012: chemical composition and antimicrobial activity of the essential oil of verbascum thapsus l. journal of essential oil bearing plants, 15(3): 373 – 379. noori, m., malayeri, b., moosaei, m., pakzad, r., piriye, m.h. 2012: effects of heavy metals on the antibacterial properties of verbascum speciosum schard. revista científica udo agrícola, 12(2): 463-471. ogunlana, e., hoeglund, g., onawunmi. o. 1987: effects of lemongrass oil on the morphological characteristics and peptidoglycan synthesis of escherichia coli cells. microbios, 50(202): 43-59. ozcan, b., esen, m., calışkan, m., mothana, r.a., cihan, a.c., yolcu, h. 2011: antimicrobial and antioxidant activities of the various extracts of verbascum pinetorum boiss. o. kuntze (scrophulariaceae). european review for medical and pharmacological sciences, 15: 900-905. pratiwi, s.u.t., lagendijk, e.l., hertiani, t., weert, s., cornellius, a.m.j., vand den hondel, j. 2015: antimicrobial effect of indonesian medicinal plants extracts on planktonic and biofilm growth of pseudomonas aeruginosa and staphylococcus aureus. international journal of pharmacy and pharmaceutical sciences, 7(4): 183-191. sánchez, e., morales, c.r., castillo, s., leosrivas, c., garcía-becerra, l., martínez, d.m.o. 2016: antibacterial and antibiofilm activity of methanolic plant extracts against nosocomial microorganisms. evidence-based complementary and alternative medicine, 2016: 1-8. simões, l.c., simões, m., vieira, m.j. 2007: biofilm interactions between distinct bacterial genera isolated from drinking water. applied and environmental microbiology, 73(19): 6192–6200. tatlı, i.i, akdemir, z.s. 2006: traditional uses and biological activities of verbascum species. fabad journal of pharmaceutical sciences, 31: 85-96. teanpaisan, r., kawsud, p., pahumunto, n., puripattanavong, j. 2017: screening for antibacterial and antibiofilm activity in thai medicinal plant extracts against oral microorganisms. journal of traditional and complementary medicine, 7: 172-177. türi, m., türi, s., koljalg, r. 1997: influence of aqueous extracts of medicinal plants on surface hydrophobicity of escherichia coli strains of different origin. apmis, 105(12): 956-962. vasudevan, r. 2014: biofilms: microbial cities of scientific significance. journal of microbiology & experimentation, 1(3): 1–16. vuong, c., kocianova, s., voyich, j.m., yao, y., fischer, e.r., deleo, f.r., otto, m. 2004: a crucial role for exopolysaccharide modification in bacterial biofilm formation, immune evasion and virulence. journal of biological chemistry, 279(52): 54881-54886. 173 biologica nyssana ● 10 (2) december 2019: 169-173 göse, hacıoğlu doğru ● antibiofilm activity of verbascum pinnatifidum vahl. ethanolic extract anticancer compounds from medicinal plants biologica nyssana 6 (2)  december 2015: 75-80 radojković-kostevski, i. et al.  variations in the headspace volatile profiles… 75 original article received: 11 october 2015 revised: 12 november 2015 accepted: 20 december 2015 variations in the headspace volatile profiles of three different achillea coarctata poir. (asteraceae) populations ivana radojković-kostevski1, goran petrović1, gordana stojanović1, jelena stamenković1, bojan zlatković2 1university of niš, faculty of sciences and mathematics, department of chemistry, višegradska 33, 18000 niš, serbia 2university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia * e-mail: peca@pmf.ni.ac.rs abstract: radojković-kostevski, i., petrović, g., stojanović, g., stamenković, j., zlatković, b.: variations in the headspace volatile profiles of three different achillea coarctata poir. (asteraceae) populations. biologica nyssana, 6 (2), december 2015: 75-80. this study presents a detailed compositional analysis of six achillea coarctata poir. samples obtained by static headspace method and interrelationships based on the volatiles profiles from different plant parts, three different populations and geological substrates, using multivariate statistical analysis. the most dominant components were mutual for aerial vegetative plant parts and inflorescences collected at the same locality. main compounds differed in percentages for two localities (values in parenthesis refer to aerial plant parts and inflorescences, respectively): 1) 1,8-cineole (40.7%; 39.9%), β-pinene (29.6%; 36.4%) and α-pinene (7.2%; 3.3%); 2) 1,8-cineole (51.8%; 53.3%), β-pinene (18.0%; 28.2%) and α-pinene (5.6%; 4.5%). the most abundant constituents identified in third locality were 1,8-cineole (37.2%; 35.6%), β-pinene (18.6%; 11.7%) and ocymene (11.6%; 11,7%). samples collected on different geological substrates are qualitative and quantitative various according to agglomerative hierarchical clustering analysis and can be grouped in two clades and two subclades. key words: achillea coarctata poir., headspace, volatile profiles, statistical analysis apstrakt: radojković-kostevski, i., petrović, g., stojanović, g., stamenković, j., zlatković, b.: razlike headspace profila isparljivih komponenti tri različite populacije achillea coarctata poir. (asteraceae). biologica nyssana, 6 (2), december 2015: 75-80. u ovom radu su predstavljeni rezultati dobijeni ispitivanjem hemijskog sastava lako isparljivih komponenti iz šest uzoraka biljke achillea coarctata poir. headspace statičkom metodom, i ispitana je zavisnost varijacije hemijskih profila od ispitivanih delova biljke i geološke podloge, korišćenjem multivarijantne statističke analize. uzorci nadzemnog dela biljke i cveta prikupljani na jednom staništu sadržali su iste glavne komponente. rezultati su pokazali da je razlika između dva lokaliteta u sadržaju istih glavnih komponenti, čiji su procenti prikazani u zagradama za nadzemni deo biljke i cvet: 1) 1,8-cineol (40.7%; 39.9%), β-pinen (29.6%; 6 (2) • december 2015: 75-80 biologica nyssana 6 (2)  december 2015: 75-80 radojković-kostevski, i. et al.  variations in the headspace volatile profiles… 76 36.4%) i α-pinen (7.2%; 3.3%); 2) 1,8-cineol (51.8%; 53.3%), β-pinen (18.0%; 28.2%) i α-pinen (5.6%; 4.5%). kod trećeg staništa najdominantnije su bile sledeće komponente: 1,8-cineol (37.2%; 35.6%), β-pinen (18.6%; 11.7%) i o-cimen (11.6%; 11.7%), za nadzemni deo biljke odnosno cvet. prema rezultatima ahc analize, uzorci prikupljeni sa različitih podloga se kvalitativno i kvantitativno razlikuju i grupisani su u dva klastera i dva subklastera. key words: achillea coarctata poir., headspace, profili isparljivih komponenti, statistička analiza introduction the genus achillea l. (asteraceae) is comprised of about 115 species found in the northern hemisphere, mostly in the euro-asian continent that are commonly known as yarrows (b e n e d e k et al., 2008; n e me t h & b e r n a t h , 2008; r a d u l o v i ć et al., 2010). the achillea l. species belong to the oldest medicinal plants that are used both for pharmaceutical purposes and in folk medicine. achillea species are diuretic, emmenagogue agents, used for healing wounds, curing stomachache and diarrhea, with antichloristic, antispasmodic, antiseptic and infection preventing properties. they have also been used to reduce sweating and to stop bleeding (a l s o h a i l i & a l f a w w a z , 2014). the achillea genus has a wide distributional range, and the differences in oil composition may be affected by different environmental factors such as plant genetic type, seasonality, and developmental stage, because it is a chemically polymorphic and perennial plant. terpenoids (1,8-cineole, camphor, borneol, pinenes, artemisia ketone, santolina alcohol, farnesane, caryophyllene and its oxides, cubebene, germacrenes, eudesmol, α-bisabolol and oxides, farnesene, γ-gurjunene, γ-muurolene and chamazulene) are the principle components of achillea essential oils (m o t a v a l i z a d e h k a k h k y et al., 2013). a. coarctata is a perennial herb with yellow ligules growing in dry hillsides and sandy oils, with the range restricted to the balcan peninsula, the south ukraine and the asia minor (g a j i ć , 1975; s i m i ć et al., 1999; t z a k o u et al., 2009). as far as we know, there are three reports about chemical composition of a. coarctata essential oil. different components are reported as the main constituents of the oil among these three studies. first known analysis represents caryophyllene oxide, 1,8-cineole and trans-linalool oxide as dominant constituents (s i m i ć et al., 1999). tzakou found 1,8-cineole, camphor and borneol, while toker reported 1,8cineole, camphor and viridiflorol to be major components of the essential oil (t z a k o u et al., 2009; t o k e r et al., 2003). the aims of this study were to perform a detailed compositional analysis of six a. coarctata samples obtained by static headspace method and to establish interrelationships based on the volatiles profiles from different plant parts and different populations using multivariate statistical analysis. material and methods plant material the plant material (flowering stage) was collected at three different locations in serbia (rujan mountain; preševo (mitrovac); pčinja valley (trgovište)), in june 2014. the plant materials were identified by bojan zlatković and the voucher specimens were deposited in the herbarium moesiacum niš (hmn), department of biology and ecology, faculty of science and mathematics, university of niš under the acquisition numbers 9367, 9368 and 9369. types of geological substrates were identified according to basic geological mapp 1:100.000 (k a r a j o v a n o v i ć & h r i s t o v , 1976; b a b o v i ć & c v e t k o v i ć , 1977). sample preparation 300 mg of milled fresh plant material was put into 20 ml hs vial and soaked with 2 ml of distilled water. the sample was heated at 80 °c for 20 minutes with the next mixing program: shaking for 5 seconds, pause for 2 seconds. 500 μl of vapor generated from the aerial parts was drawn out from the vial using a gas-tight syringe (90 °c) and injected directly in the chromatographic column via a transfer line (75 °c). gc and gc/ms analysis the samples were analyzed by a 7890/7000b gc/ms/ms triple quadrupole system in ms1 scan mode (agilent technologies, usa) equipped with a combi pal sampler and headspace for g6501b/g6509b. the fused silica capillary column hp-5ms (5% phenylmethylsiloxane, 30 m x 0.25 mm, film thickness 0.25 μm) was used. the injector and interface operated at 250 and 300 °c, respectively. temperature program: from 50 to 290 °c at a heating rate of 4 °c/min. the carrier gas was helium with a flow of 1.0 ml/min. 500 μl of hs vapor was injected via a transfer line (75 °c). post run: back flash for 1.89 min, at 280 °c, with helium pressure of 50 psi. ms conditions were as follows: biologica nyssana 6 (2)  december 2015: 75-80 radojković-kostevski, i. et al.  variations in the headspace volatile profiles… 77 ionization voltage of 70 ev, acquisition mass range 50-650, scan time 0.32 s. gc analysis was carried out under the same experimental conditions using the same column as described for the gc/ms. the percentage composition of the samples was computed from the gc peak areas without any corrections. identification of volatile compounds hs volatiles were identified by comparison of their linear retention indices (relative to c8-c32 n-alkanes on the hp-5ms column) with literature values and their ms with those of authentic standards, as well as those from wiley 6, nist11, agilent mass hunter workstation b.06.00 software and a homemade ms library with the spectra corresponding to pure substances and components of known essential oils by the application of the amdis software (automated mass spectral deconvolution and identification system, ver. 2.1, dtra/nist, 2011). some components were identified by co-injection of pure substances. multivariate statistical analysis the contents of the components of the a. coarctata headspace volatiles obtained in this study were analyzed by agglomerative hierarchical cluster analysis (ahc). the ahc was performed with euclidean distances as metric and using single linkage method as aggregation criterion using the “statistica, version 8.1” software. results and discussion plant materials were collected at three different locations rujan mountain (samples rn, rc), preševo (mitrovac) (samples pn, pc) and pčinja valley (trgovište) (samples tn, tc). each location has different geological substrate type: 1) serpentine (preševo), 2) silicate (rujan mountain), 3) conglomerates and molasse (pčinja valley). compositions of aerial plant parts and inflorescences headspace volatiles of a. coarctata from three different localities obtained by gc and gc/ms, are presented in tab. 1. number of identified compounds in rn, tn, pn, rc, tc and pc samples were 38, 36, 33, 34, 28 and 32, respectively (representing 98.9% (rn); 98.1% (tn); 98.3% (pn); 98.2% (rc); 98.8% (rc) and 98.6% (tc) of total hs volatiles. the most dominant components were mutual for aerial plant parts and inflorescences collected at the same locality. main compounds found in rn and rc samples were 1,8-cineole (40.7%; 39.9%), βpinene (29.6%; 36.4%) and α-pinene (7.2%; 3.3%). the same components were found as the most dominant in samples tn and tc, with following percentage: 1,8-cineole (51.8%; 53.3%), β-pinene (18.0%; 28.2%) and α-pinene (5.6%; 4.5%), for tn and tc respectively. the most abundant constituents identified in pn and pc samples were 1,8-cineole (37.2%; 35.6%), β-pinene (18.6%; 11.7%) and ocymene (11.6%; 11.7%). as far as we know, there is no any published data on headspace volatiles composition of achillea coarctata. three papers revealed no different compounds as the most abundant in obtained essential oils compositions: (1) caryophyllene oxide, 1,8-cineole and trans-linalool oxide; (2) 1,8-cineole, camphor and borneol and (3) 1,8-cineole, camphor and viridiflorol, respectively (s i m i ć et al., 1999; t z a k o u et al., 2009; t o k e r et al., 2003). content of monoterpenoids, which are more volatile compounds, is higher in headspace volatiles then in essential oil, while sesquiterpenoids, which have relatively high retention times, were only found in traces or not found at all. the results of the ahc analysis are depicted in fig. 1. table 1 lists the identified constituents with their contents of the six a. coarctata headspace volatile samples included in the ahc. the dendrogram depicted in fig. 1, obtained as the result of the ahc, indicates the existence of two statistically different classes of samples (c1–c2). it is obvious that the chemical compositions of the aerial parts and inflorescences of the samples collected at the same locations are almost identical and they are grouped in the same stocks since they have the same main components that differ only in percentages in different parts of plants. samples pc and pn are separated from the rest of the a. coarctata samples and constituted the first clade (c1). the samples tc, tn, rc and rn composed the second clade (c2). there was further subdivision within clade c2, into two more subclades consisted of the samples collected from the same sites. the second clade is more homogeneous than the first one and it is flatter on the dendrogram. samples tc, tn, rc and rn were characterized by high contents of same compounds (1,8-cineole, β-pinene and α-pinene), reason for their strong association (same clade). greater dissimilarity level (1.6) observed in this analysis separated samples pc and pn in clade c1 from the rest of the samples considering that the most abundant constituents identified in these samples were 1,8-cineole, β-pinene and o-cymene. since each population has different geological substrate: serpentine (samples pc and pn, preševo), conglomerates and molasses (samples tc and tn, pčinja valley) and biotite gneiss (samples rc and rn, rujan mountain) it can be concluded that, with respect to the sample origin, different substrates produce various chemotypes. chemical compositions biologica nyssana 6 (2)  december 2015: 75-80 radojković, i. et al.  variations in the headspace volatile profiles… 78 table 1. chemical composition of the a. coarctata volatiles achieved by gc and gc/ms relative amount % riref riexp compound rn tn pn rc tc pc class 801 802 hexanal 0.1 0.3 0.2 tr 0.4 tr o 846 851 2(e)-hexenal 0.2 1.2 0.4 0.3 0.3 tr o 850 854 3(z)-hexenol 0.2 0.7 0.3 o 859 865 2(z)-hexenol 0.1 0.2 0.8 o 863 867 n-hexanol 0.8 1.1 2.1 0.2 0.2 0.3 o 921 923 tricyclene 0.2 tr 0.2 0.2 tr 0.2 m 924 928 α-thujene 0.1 tr 0.3 0.1 0.1 0.3 m 932 935 α-pinene 7.2 5.6 11.5 3.3 4.5 4.7 m 946 951 camphene 3.5 1.6 3.2 3.0 1.7 3.1 m 952 962 benzaldehyde 0.1 0.5 tr 0.2 0.2 0.1 o 969 976 sabinene 0.7 1.0 2.8 1.5 1.5 3.5 m 974 979 β-pinene 29.6 18 18.6 36.4 28.2 25.7 m 988 992 myrcene 0.2 0.3 m 988 994 dehydro-1,8-cineole 0.1 tr 0.1 tr mo 1014 1019 α-terpinene 0.6 0.9 1.3 0.7 0.7 2.8 m 1022 1027 o-cymene 0.5 0.9 11.6 0.2 0.4 11.7 m 1024 1031 limonene 0.2 0.2 0.5 0.2 tr 0.5 m 1026 1034 1,8-cineole 40.7 51.8 37.2 39.9 53.3 35.6 mo 1054 1060 γ-terpinene 1.0 2.3 0.9 1.1 1.2 1.2 m 1065 1069 cis-sabinene hydrate 0.6 0.6 mo 1086 1092 terpinolene 0.2 0.4 0.2 0.2 0.2 0.2 m 1095 1100 linalool 0.3 0.4 0.4 mo 1107* 1108 6-ethenyldihydro-2,2,6trimethyl-2h-pyran-3(4h)-one 3.7 1.9 0.5 3.1 0.9 1.6 o 1122 1128 α-campholenal tr tr mo 1135 1142 trans-pinocarveol 0.1 mo 1141 1148 camphor 3.7 1.5 1.6 1.9 1.1 1.6 mo 1160 1166 pinocarvone tr 0.1 tr tr tr tr mo 1165 1169 borneol 0.2 0.2 0.3 0.3 0.3 0.4 mo 1170 1176 cis-linalool oxide (pyranoid) 0.1 mo 1174 1180 terpinen-4-ol 0.6 1.2 0.4 0.6 0.7 0.6 mo 1186 1193 α-terpineol 0.8 0.9 0.5 0.9 0.8 0.6 mo 1195 1199 myrtenal tr 0.3 0.1 0.1 tr mo 1288 1291 lavandulyl acetate 0.1 0.2 0.1 0.4 mo 1374 1381 α-copaene 0.2 0.9 0.4 0.6 0.2 0.3 s 1417 1427 (e)-caryophyllene 0.4 0.9 1.7 0.4 0.8 1.8 s 1454 1458 (e)-β-farnesene tr 0.1 s 1458 1466 allo-aromadendrene 0.7 0.2 0.8 0.4 s 1474 1482 10-epi-β-acoradiene 0.2 0.2 s 1484 1488 germacrene d 0.7 2.3 0.3 0.6 0.5 0.2 s 1500 1503 bicyclogermacrene 0.1 0.1 tr s 1513 1519 γ-cadinene 0.1 0.1 s 1522 1529 δ-cadinene tr 0.1 0.1 0.1 0.2 s 1582 1592 caryophyllene oxide 0.2 so 1635 1642 cis-cadin-4-en-7-ol 0.6 0.3 so total 98.9 98.1 98.3 98.2 98.8 98.6 monoterpenoids 90.7 87.8 91.3 91.3 95.3 93.5 hydrocarbons(m) 44.0 30.9 51.1 47.2 38.5 53.9 oxygenated(mo) 46.7 56.9 40.2 44.1 56.8 39.6 sesquiterpenoids 3.0 4.4 2.7 3.1 1.5 3.1 hydrocarbons (s) 2.4 4.4 2.7 2.8 1.5 2.9 oxygenated (so) 0.6 0 0 0.3 0 0.2 others (o) 5.2 5.9 4.3 3.8 2.0 2.0 compounds are listed in order of elution from a hp-5 ms column; riref: literature retention indices; riexp: experimental retention indices relative to c8-c32 n-alaknes; (*): identified by nist chemistry webbook retention indices; tr: traces (<0.1%); (-): not detectid. samples rn, tn, pnaerial plant parts (collected at rujan mountain, pčinje valley (trgovište) and preševo (mitrovac), respectively); samples rc,tc, pcinflorescences (collected at rujan mountain, pčinje valley (trgovište) and preševo (mitrovac), respectively). biologica nyssana 6 (2)  december 2015: 75-80 radojković-kostevski, i. et al.  variations in the headspace volatile profiles… 79 fig. 1. dendrogram obtained by agglomerative hierarchical clustering (constituent contents used as cases) of samples collected on biotite gneiss and conglomerates and molasses do not differ to a great extent while the sample from serpentine is slightly different. it has been already noticed that the deficiency of water and indispensable mineral elements result in numerous structural and functional adaptations of plants species that grow on a serpentine substrate (s t e v a n o v i ć et al., 2003). therefore, differences in the chemical composition of the investigated samples can probably be considered as another manifestation of the “serpentine syndrome”. acknowledgements. the authors are grateful to the ministry of education, science and technological development for financial support through the grant within frame of basic research, no 172047. references alsohaili, s. & al-fawwaz, a. 2014: composition and antimicrobial activity of achillea fragrantissima essential oil using food model media. european scientific journal, 10 (30): 156165. babović, m. & cvetković, d. 1977: trgovište sa radomirom, 34-57. in: dimitrijević, m., dragić, d., karamata, s., sikošek, b., veselinović, d. (eds.). osnovna geološka karta 1:100.000, socijalistička federativna republika jugoslavija. savezni geološki zavod, beograd. benedek, b., rothwangl-wiltschnigg, k., rozema, e., gjoncaj, n., reznicek jurenitsch, j., kopp, b., glasl, s. 2008: yarrow (achillea millefolium l. s.l.): pharmaceutical quality of commercial samples. pharmazie, 63 (1): 23-26. gajić, m. 1975: achillea l. in: josifović, m. (ed.). flora sr srbije. 7: 90-110. srpska akademija nauka i umetnosti. beograd. karajovanović, m. & hristov, s. 1976: kumanovo, 34-68. in: dimitrijević, m., karamata, s., sikošek, b., veselinović, d. (eds.). osnovna geološka karta 1:100.000, socijalistička federativna republika jugoslavija. savezni geološki zavod, beograd. motavalizadehkakhky, a., shafaghat, a., zamani, h., akhlaghi, h., mohammadhosseini, m., mehrzad, j., ebrahimi, z. 2013: compositions and the in vitro antimicrobial activities of the essential oils and extracts of two achillea species from iran. journal of medicinal plants research, 7 (19): 1280-1292. nemeth, e. & bernath, j. 2008: biological activities of yarrow species (achillea spp.). current pharmaceutical design, 14 (29): 3151-3167. radulović, n., blagojević, p., skropeta, d., zarubica, a., zlatković, b., palić r. 2010: misidentification of tansy, tanacetum macrophyllum, as yarrow, achillea grandifolia: a health risk or benefit. natural product communication, 5 (1): 121-127. biologica nyssana 6 (2)  december 2015: 75-80 radojković-kostevski, i. et al.  variations in the headspace volatile profiles… 80 simić, n., palić, r., vajs, v., milosavljević, s., djoković, d. 1999: essential oil of achillea coarctata. journal of essential oil research, 11: 700-702. stevanović, v., tan, k., iatrou, g. 2003: distribution of the endemic balkan flora on serpentine i. obligate serpentine endemics. plant systematics and evolution, 242: 149-170. toker, z., özen, h., clery, r., owen, n. 2003: essential oils of two achillea species from turkey. journal of essential oil research. 15 (2): 100-101. tzakou, o., couladis, m., milenković, m., vučićević, d., kovačević, n. 2009: composition and antimicrobial activity of achillea coarctata essential oils from greece. journal of essential oil bearing plant. 12 (5): 541-545. harpke, d., kerndorff, h., raca, i., pasche, e.: a new serbian endemic species of the genus crocus (iridaceae). biologica nyssana, 8 (1), september 2017 biologica nyssana 8 (1)  september 2017: 07-13 harpke, d. et al.  a new serbian endemic species of the genus crocus… 7 original article received: 02 july 2017 revised: 18 july 2017 accepted: 06 september 2017 a new serbian endemic species of the genus crocus (iridaceae) dörte harpke1, helmut kerndorff2, irena raca3, erich pasche4 1 leibniz institute of plant genetics and crop research (ipk), gatersleben, germany 2 casa da eira, são romão, são bras de alportel, portugal 3university of niš, faculty of science and mathematics, department of biology and ecology, višegradska 33, niš, serbia 4 feldstraße, velbert, germany * e-mail: raca.irena@gmail.com abstract: harpke, d., kerndorff, h., raca, i., pasche, e.: a new serbian endemic species of the genus crocus (iridaceae). biologica nyssana, 8 (1), september 2017: 07-13. recent research within the genus crocus (iridaceae) let us doubt that crocus adamii gay from serbia represents the same taxon as c. adamii s. str. of the locus classicus in the caucasian mountains. the latter belongs to a group of crocuses, which is distributed from the anatolian diagonal, a mountain belt in inner anatolia, to iran and the caucasian mountains. to infer (i) if the serbian c. adamii represents a new species and (ii) its taxonomical and phylogenetic affiliation within the genus we combined morphological and molecular investigations. the results show the presence of a morphologically and molecularly differentiated lineages, which both share a close relationship e.g. to c. alexandrii, c. chrysanthus, and c. weldenii but not to c. adamii s. str., which indicates a new species. as a result, we here describe c. randjeloviciorum to honor the serbian botanists novica and vladimir ranđelović. key words: crocus, new species, serbia apstrakt: harpke, d., kerndorff, h., raca, i., pasche, e.: nova endemična vrsta roda crocus (iridaceae) u srbiji. biologica nyssana, 8 (1), septembar 2017: 00-00. skorašnja istraživanja u okviru roda crocus (iridaceae) dovela su u sumnju teoriju da crocus adamii gay iz srbije predstavlja takson istovetan c. adamii s. str. čiji je locus classicus na kavkazu. drugi navedeni takson pripada grupi šafrana, distribuiranih od planinskog pojasa u centralnom delu anatolije, do irana i kavkaskih planina. da bi se zaključilo o tome (i) da li takson c. adamii iz srbije predstavlja novu vrstu, te (ii) sudilo o njegovom taksonomskom i filogenetskom položaju unutar roda, kombinovana su morfološka i molekularna istraživanja. rezultati ukazuju na postojanje morfološki i molekularno diferenciranih linija, srodnim c. alexandrii, c. chrysanthus i c. weldenii, ali ne i c. adamii s. str., što sugeriše da je c. adamii iz srbije nova vrsta za nauku. kao rezultat, u ovom radu biće opisana vrsta c. randjeloviciorum, čiji je naziv dodeljen u čast srpskih botaničara novice i vladimira ranđelovića. ključne reči: crocus, nova vrsta, srbija 8 (1) • september 2017: 07-13 doi: 10.5281/zenodo.962907 biologica nyssana 8 (1)  september 2017: 07-13 harpke, d. et al.  a new serbian endemic species of the genus crocus… 8 introduction crocus l. (iridaceae) comprises currently about 200 species. within the last few years, over 50 new species were described (e r o l et al., 2012; r a n đ e l o v i ć et al., 2012; k e r n d o r f f et al., 2013; s c h n e i d e r , 2014; h a r p k e et al., 2014, 2015; e r o l et al., 2015; r u k s a n s , 2015; m i l j k o v i ć et al., 2016). taxonomically the genus is currently in a complicated situation as many of the newly described taxa are unplaced in the system. moreover, recent phylogenetic analyses proved several units within the genus crocus to be paraor polyphyletic. as a consequence of ongoing revisions of taxonomical groups, with the aim to define monophyletic units, several other taxa are unplaced, too. one of those groups of currently unplaced taxa comprises c. adamii gay and its allies (k e r n d o r f f et al., 2013). it is a very homogenous group and occupies the sister group position to a large clade comprising series aleppici b. mathew, flavi b. mathew, speciosi b. mathew, reticulati b. mathew (h a r p k e et al., 2016). the c. adamii group comprises about 20 taxa (k e r n d o r f f et al., 2013). the taxa of this group are characterized e.g. by toothless basal tunic rings (with the exception of c. aerius herb. which has no rings), silvery bract and bracteole (in some taxa they turn brown with aging), glabrous throat, trifurcate style and vernal flowers. the species of the c. adamii group are inhabitants of the anatolian diagonal, a mountain belt dividing inner anatolia, and occur north-, east-, and south-east of it. considering that despite extensive field studies c. adamii and its allies were never found west of the anatolian diagonal, the presence of c. adamii in serbia became questionable. the serbian plant was mentioned by r a n d j e l o v i ć et al. (1990), located in serbia in the regions of timok, niš, and south morava. from the photograph shown in this work it has obviously some affinity to c. adamii but also to some forms of c. nubigena (h a r p k e et al., 2016). furthermore, its chromosome number (2n) is 18, members of the c. adamii group from the mentioned eastern areas have mostly 2n = 20. material and methods morphological analyses leaf-parameters were measured on fresh material (45 specimens for number, 10 specimens for white stripe, diameter, colour of cataphylls, bract and bracteoles, and ribs underneath). corm and flower parameters were measured on 36 randomly collected and dried specimens from the type-locality hkep 1328. for the investigation of leave anatomy, samples were collected during flowering time (06.03.2016) and preserved in 50% alcohol. anatomical studies were done in the laboratory of plant systematics and ecology, faculty of science and mathematics, university of niš. thirty transverse leaf cross sections were made by manual microtome (g l i g o r i j e v i ć & p e j č i n o v i ć , 1983) and stained with safranin alcian blue. the slides with coverslips were then photographed using a leica dm 1000 microscope. nineteen anatomical features were obtained of leaf cross sections: section height, section length, arm length, white stripe width, lacuna area, adaxial epidermis cells height and width, palisade cells height and width, palisade tissue height, spongy cells height and width, spongy tissue height, abaxial epidermis cells height and width, sclerenchyma area, phloem area, xylem area and number of vascular bundles. measurements of the listed parameters were done in imagej. molecular and phylogenetic analyses molecular analyses were carried out using 3 individuals of one c. cf. adamii populations. extraction of genomic dna and amplification of the nuclear rdna its were conducted according to h a r p k e et al. (2014). both strands of the pcr products were directly sequenced with applied biosystems’ bigdye terminator technology on an abi 3730xl automatic dna sequencer using either the primers from pcr amplifications. forward and reverse sequences were manually checked, edited where necessary, and combined in consensus. the newly obtained sequence was submitted to the embl nucleotide database and is accessible through accession numbers mf766260. sequences were aligned manually. if sequences were found to be identical within the same species or population they were included only once in the analyses. the nuclear data were subjected to phylogenetic analyses using bayesian phylogenetic inference (bi) with mrbayes 3.2 (r o n q u i s t et al., 2012). for bi 2 times 4 chains were run for 2 million generations under the appropriate models of sequence evolution (nuclear data set: gtr+γ+i), sampling a tree every 1000 generations. converging log-likelihoods, potential scale reduction factors for each parameter and inspection of tabulated model parameters in mrbayes suggested that stationary had been reached in all analyses. the first 25% of trees of each run were discarded as burn-in. two independent runs of bi analysis were performed to confirm that separate analyses converged on the same biologica nyssana 8 (1)  september 2017: 07-13 harpke, d. et al.  a new serbian endemic species of the genus crocus… 9 result. in each of the 2 analyses the same topology and similar posterior probabilities (pp) of nodal support resulted. results and discussion affiliation within the genus. crocus cf. adamii from serbia groups within a polytomic clade comprising c. puncatus and its relatives with strong support (pp 1.0; fig. 1). its its sequence differs in at least three positions from its closest relatives c. alexandri (serbia), c. weldenii (ne-italy) and five from c. adamioides (nw-turkey). the seeds (fig. 2d) have more similarity with the latter species than to those of the relatives of the c. adamii-group (k e r n d o r f f et al. 2016). further morphological characters like corm tunics (fig. 2a, fig. 3) also clearly show that it does not belong to the c. adamiigroup. table 1. crocus randjeloviciorum leaf anatomy characters measurements height(µm) width (length)(µm) area(µm2) mean±sd min-max mean±sd min-max mean±sd min-max section 563±75 446-692 3154±311 2644-3668 arm 1393±170 1164-1726 white stripe 434±91 323-599 lacuna 133157±37620 70665-234620 adaxial e. cell 16±2 14-19 16±1 13-18 palisade cell 46±6 34-61 17±1 13-19 palisade tissue 68±11 51-90 spongy cell 18±2 14-22 26±3 21-33 spongy tissue 50±8 37-68 abaxial e. cell 16±2 12-20 18±2 13-22 sclerenchyma 5478±945 3022-7019 phloem 549±123 351-954 xylem 673±128 448-903 mean±sd min-max v. bundles no. 18±3 11-21 sd – standard deviation fig. 1. phylogenetic tree obtained by bayesian phylogenetic inference of the nuclear rdna its regions. numbers along branches give posterior probabilities. sequences obtained by cloning are indicated by a small letter. crocus randjeloviciorum is indicated in bold. biologica nyssana 8 (1)  september 2017: 07-13 harpke, d. et al.  a new serbian endemic species of the genus crocus… 10 description of new species the serbian crocus considered as c. adamii is phylogenetically and morphological quite distinct from c. adamii s. str.. it represents a new species. crocus randjeloviciorum kernd., pasche, harpke & raca sp. nova type: east serbia, tupižnica mt., 950m a.s.l., hkep 1328 (holotype 23042 gat!) diagnosis. crocus randjeloviciorum differs from other species by intermediate outer corm tunic between coriaceous and membranous (e.g. in c. biflorus it is coriaceous, in c. tauri membranous), the 2 ribs in the grooves of the leaves (none in c. biflorus, 2 in c. tauri), no dark spot at the basis of the outer segments, unusually long lobes of the anthers (1.7-3 mm) and very broad connectives. crocus randjeloviciorum has also a very rare property in the genus. the styles are compared to the stamens in more or less the same proportion. this means approximately in 1/3 of the specimens they are shorter, in 1/3 they are equal and in 1/3 they are longer. in the majority of species there is either a tendency to have longer or to have shorter styles compared to the stamens. description. corm sub-globose about 9-11 mm in diameter. outer and inner tunics coriaceous to membranous, the inner ones softer; tunic splits mainly into segments of 2-5 mm rarely into >5 mm, sub-splits absent. neck around 5 mm long consistent of medium-based “triangles”, caps membranous, medium-sized (k e r n d o r f f et al. 2015). rings narrow to normal-sized coriaceous to membranous mostly smoothedged, very rarely with tiny teeth <0.5 mm. basal tunics small plates of 5-7 mm in diameter. cataphylls silvery, skinny, sometimes slightly brownish towards tips. leaves 2 – 3.1 – 4, green, glabrous, 1.5-2.5 mm in diameter with two ribs underneath of both sides of the keel. leaves reach or slightly overtop the flowers at anthesis. white stripe <1/3 to 1/3 of leaf diameter. flowers 1(-2). the outer segments are between 21 and 30 mm but usually 25 mm long, between 6 and 14 mm mostly 9.5 mm wide. the inner segments are between 18 and 28 mm but usually 23 mm long and between 6 and 14 mm in average 10.2 mm wide. segment proportion of length/width of the outer segments is 2.6 (n = 36). the insides of all of segments are light to deep violet blue rarely whitish without markings, the inner ones also at outside with an indistinct brownish-greyish zone or spot near the basis, overlaid by the yellow of the throat which shines through. the outside of the outer segments has either the inside flower colour or can be white or yellowish (buff-coloured) dominated by different numbers of intense brownish-violet stripes, vertically orientated to the tips, occasionally accompanied by many thin ones also upwards orientated but more to the edges than to the tips of the segments, sometimes the whole outside is suffused deep violet. there is no dark blotch or area at the bases of the segments, the violet stripes merge into the perianth tube which is otherwise colourless (white) especially near the ground. prophyll absent. bract and bracteole present, subequal, mostly silverywhite and skinny but sometimes with brownish tips. filaments deep yellow, hairy, 4-5.2-6.5 mm long; anthers yellow 7-11.5-15.2 mm long, having remarkably long lobes of about 1.7-2-3 mm (n = 15), connective very broad and conspicuous, colourless. throat deep yellow, glabrous. pollen yellow. the orange to orange-red red styles are divided into three branches sometimes papillous in the upper part. the branches are 3.6-5.6-8.5 mm long mostly expanded and fringed towards at the apex. the styles are 29% shorter, 37% equal and 34% longer compared to the stamen. capsule small, about 1 cm long, broadly ellipsoid and peaky at top. seeds light brown with an orange tint, main body ellipsoid about 1.6 -2 mm long and 1.3-1.4 mm wide (fig. 2d), caruncle and raphe distinct. chromosome number 2n=18. fig. 2. photographs of corm tunics (a), hairy filaments (b, 9x), leaves with papillae (c, 9x) and seeds (d). biologica nyssana 8 (1)  september 2017: 07-13 harpke, d. et al.  a new serbian endemic species of the genus crocus… 11 distribution and habitat: serbia, in hornbeam-lilac scrub (syringo-carpinetum). etymology: prof. dr. novica ranđelović (1937–) and his son prof. dr. vladimir ranđelović (1965–) are among the most prominent serbian botanists. they extensively studied the flora of the balkan peninsula. the results of their investigations were published in numerous papers. they both are crocus enthusiasts and described several new crocus species and they published a monograph about serbian crocuses (r a n đ e l o v i ć et al., 1990) and authored and co-authored several publications about the genus (r a n đ e l o v i ć et al., 2007, 2011, h a r p k e et al., 2014, 2015, m i l j k o v i ć et al., 2016). phenology: flowering period from february to april. taxonomic relationships: crocus randjeloviciorum belongs to a group of crocuses comprising species e.g. from ne–italy (c. weldenii), the balkan peninsula (c. alexandri) and west turkey (c. adamioides). leaf cross sections leaf blade. leaf cross sections of crocuses are of a unique shape consisting of a central part called the “keel” and two lateral “arms”. the keel is squared, almost rectangular while the arms are re-curved towards the keel (r u d a l l & m a t h e w , 1990). keel corners, length and curving degree of arms vary between different species. the investigated crocus cf. adamii is characterized by four well defined ribs (fig. 4a) on the abaxial side of the keel and arms. v e l e n o v s k y (1907) typified crocus leaf type as bifacial. leaf surface. finger – like papillae can be found on the adaxial side at the end of the arms (fig. 4a). adaxial epidermis cells are square or rectangular, in contrast to the abaxial cells which are more oval. anomocytic stomata occurs in the abaxial epidermis (e r o l et al., 2007). mesophyll. the central part of the keel consists of round parenchyma cells, these cells are mostly broken down forming air space called the lacuna. it can fig. 3. photos of plants from the type population of crocus randjeloviciorum. biologica nyssana 8 (1)  september 2017: 07-13 harpke, d. et al.  a new serbian endemic species of the genus crocus… 12 be noticed as a central white stripe all along the leaf (e r o l et al., 2007; y e t i ş e n et al., 2013) (fig. 4a). the mesophyll of the arm consists of palisade (oriented to the adaxial side) and a spongy tissue below. the palisade parenchyma contains polygonal cells organized in two layers. two or three layers of the oval or irregular shaped cells are present in the spongy parenchyma (fig. 4b). vascular bundles. leaf cross sections have 11-21 collateral vascular bundles positioned in one row all along the mesophyll. four of these bundles are larger. two of them are located in distal parts of the arms and another two in the keel base near corners. the xylem is oriented to the adaxial and phloem to the abaxial side of the leaf (bundles in the end of the arms have opposite orientation of vascular tissues). the fig. 4. anatomic characters of leaves of crocus randjeloviciorum: a-leaf cross sections (5x), b-arm detail (20x), c-big bundle (40x) (ad: adaxial side, ab: abaxial side, la: lacuna area, r: rib, pp: papillae, e: epidermis, pp: palisade paremchyma, sp: spongy parenchyma, st: stomata, sc: sclerenchyma cap, ph: phloem, xy: xylem) table 1. crocus randjeloviciorum leaf anatomy characters measurements height(µm) width (length)(µm) area(µm2) mean±sd min-max mean±sd min-max mean±sd min-max section 563±75 446-692 3154±311 2644-3668 arm 1393±170 1164-1726 white stripe 434±91 323-599 lacuna 133157±37620 70665-234620 adaxial e. cell 16±2 14-19 16±1 13-18 palisade cell 46±6 34-61 17±1 13-19 palisade tissue 68±11 51-90 spongy cell 18±2 14-22 26±3 21-33 spongy tissue 50±8 37-68 abaxial e. cell 16±2 12-20 18±2 13-22 sclerenchyma 5478±945 3022-7019 phloem 549±123 351-954 xylem 673±128 448-903 mean±sd min-max v. bundles no. 18±3 11-21 biologica nyssana 8 (1)  september 2017: 07-13 harpke, d. et al.  a new serbian endemic species of the genus crocus… 13 sclerenchyma tissue is well developed and in a form of a “cap” (fig. 4c). measurements for leaf anatomy are given in tab. 1. acknowledgements. the work was funded by the ministry of education, science and technological development of republic of serbia (project no. 173030). references erol, o., can, l., şık, l. 2012: crocus demirizianus sp. nov. from northwestern turkey. nordic journal of botany, 30: 665-667. erol, o., harpke, d., yıldırım, h. 2015: a new crocus l. (iridaceae) species from se turkey, based on morphological and molecular data. phytotaxa, 239: 223-232. erol, o., küçüker, o. 2007: leaf anatomy of some endemic crocus l. (iridaceae) taxa from western anatolia. international journal of botany, 3 (3): 290-295. gligorijević, s., pejčinović, d. 1983: contribution to the methodology of anatomical sections sreparation. acta biologiae et medicinae experimentalis, 8: 43-45. harpke, d., kerndorff, h., pasche, e., peruzzi, l. 2016: neotypification of the name crocus biflorus mill. (iridaceae) and its consequences in the taxonomy of the genus. phytotaxa, 260: 131143. harpke, d., meng, s., kerndorff, h., rutten, t., blattner, f.r 2013: phylogeny of crocus (iridaceae) based on one chloroplast and two nuclear loci: ancient hybridization and chromosome number evolution. molecular phylogenetic and evolution, 66: 617-627. harpke, d., peruzzi, l., kerndorff, h., karamplianis, t., constantinidis, t., ranđelović, v., ranđelović, n., jušković, m., pasche, e., blattner, f.r. 2014: phylogeny, geographic distribution, and new taxonomic circumscription of the crocus reticulatus species group (iridaceae). turkish journal of botany, 38: 11821198. kerndorff, h., pasche, e. harpke, d. 2015: the genus crocus (liliflorae, iridaceae): life-cycle, morphology, phenotypic characteristics, and taxonomical relevant parameters. stapfia 103: 27–65. kerndorff, h., pasche, e., blattner, f.r., harpke, d. 2013: crocus biflorus miller (liliiflorae, iridaceae) in anatolia-part iv. stapfia, 99: 159186. mathew, b. 1982: the crocus. a revision of the genus crocus (iridaceae). b.t. batsford, london. miljković, m., ranđelović, v., harpke, d. 2016: a new species of crocus (iridaceae) from southern albania (sw balkan peninsula). phytotaxa, 265: 39-49. peruzzi, l., carta, a. 2011: crocus ilvensis sp. nov. (section crocus, iridaceae), endemic to elba island (tuscan archipelago, italy). nordic journal of botany, 29: 6-13. ranđelović, n., ranđelović, v., hill, d.a. 1990: the genus crocus l. in serbia. srpska akademija nauka i umetnosti , beograd. ranđelović. n., ranđelović, v., hristovski, n. 2012: crocus jablanicensis (iridaceae), a new species from the republic of macedonia, balkan peninsula. annales botanici fennici, 49: 99-10. ronquist, f., teslenko, m., van der mark, p., ayres, d.l., darling, a., hohna, s., larget, b., liu, l., suchard, m.a., huelsenbeck, j.p. 2012: mrbayes 3.2: efficient bayesian phylogenetic inference and model choice across a large model space. systematic biology, 61: 539-542. rudall, p., mathew, b. 1990: leaf anatomy in crocus (iridaceae). kew bulletin, 45 (3): 535-544. rukšāns, j. 2015: some new crocus taxa (iridaceae) from western turkey and east aegean islands. international rock gardener, 64: 1-38. schneider, i. 2014: crocus brachyfilus(iridaceae), a new species from southern turkey. willdenowia, 44: 45-50. velenovsky, j. 1907: vergleichende morphologie der pflanzen. teil ii prag yetişen, k., şen, u., yıldırım, t., özdemir, c. 2013: morphological and anatomical study on endemic crocus olivieri gay subsp. istanbulensis mathew subspecies (iridaceae). anadolu university journal of science and technology-c life sciences and biotechnology, 3 (1): 31-37. devetak, jakšić, 2019, biologica nyssana 10(1) 10 (1) september 2019: 35-41 doi: 10.5281/zenodo.3464002 lacewings (insecta: neuropterida: raphidioptera, megaloptera, neuroptera) collected in montenegro with checklist of species original article dušan devetak department of biology, faculty of natural sciences and mathematics, university of maribor, koroška cesta 160, 2000 maribor, slovenia dusan.devetak@guest.arnes.si (corresponding author) predrag jakšić čingrijina 14/25, zvezdara, 11000 beograd, serbia jaksicpredrag@gmail.com received: april 26, 2019 revised: july 1, 2019 accepted: august 13, 2019 abstract: seventeen species of neuropterida (raphidioptera, megaloptera, neuroptera) collected in montenegro during 2017-2018 are reported. an updated checklist of all known species from this country is presented. the list contains 75 species occurring in montenegro. of these, two species were reported for the first time for the country. one was a green lacewing chrysoperla lucasina, which has already been expected for the area. the second species new to montenegro, an owlfly libelloides longicornis, is particularly noteworthy, because its finding in the balkan peninsula was unexpected. key words: lacewings, neuropterida, neuroptera, montenegro, owlfly, libelloides longicornis, checklist apstract: mrežokrilci (insecta: neuropterida: raphidioptera, megaloptera, neuroptera) sakupljeni u crnoj gori, sa ček-listom vrsta dat je prikaz sedamnaest vrsta mrežokrilaca (raphidioptera, megaloptera, neuroptera) utvrđenih u crnoj gori tokom 2017. i 2018. godine. priložena je i inovirana ček-lista svih do sada utvrđenih vrsta u ovoj zemlji. tom listom potvrđeno je prisustvo 75 vrsta mrežokrilaca u crnoj gori. među njima dve vrste su po prvi put prikazane za crnu goru. jedna od njih je vrsta zlatooke chrysoperla lucasina, koja je i očekivana za to područje. druga nova vrsta za crnu goru je libelloides longicornis, čiji je nalaz značajan jer je prisustvo ove vrste na balkanskom poluostrvu neočekivano. ključne reči: mrežokrilci, neuropterida, neuroptera, crna gora, askalafide, libelloides longicornis, ček-lista introduction montenegro is a mountainous mediterranean country in the south-west part of the balkan peninsula, bordering the adriatic sea. despite the fact that montenegro is the second smallest country in the balkan peninsula, the diversity of the landscape, climate and soil created the conditions for high biodiversity. the research has been conducted in pre-selected areas – so-called key biodiversity areas (kbas) – during the field seasons 2017 and 2018 within the frame of the ipa project ‘establishment of natura 2000 network, montenegro’. the oldest known montenegrin record of lacewings (neuropterida: raphidioptera, megaloptera, neuroptera) dates back to 1908, when an owlfly, libelloides lacteus was mentioned in the country (van der weele, 1908). up to the second half of the 20th century, lacewings in the country have been insufficiently investigated, and based mainly on sporadic records (appendix 1). before the wwii, táborský in his studies of owlflies (ascalaphidae) presented the distribution for two ascalaphid species in the balkan countries, also in montenegro (táborský, 1936, 1939). in the sixties, a snakefly raphidia durmitorica was described as a new species (steinmann, 1964), but later this species name was recognized as a synonym of dichrostigma flavipes (aspöck & aspöck, 1966). in the second half of the 20th century, saure (1989) collected twelve lacewing species in montenegro. © 2019 devetak, jakšić. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 35 at the end of the 20th century, in the period from 1985 to 1988, slovenian and serbian entomologists collected lacewings in the durmitor national park, the results of their study is a faunal list that includes data for lacewings of the national park (devetak, 1991). later, a series of papers was published, containing very scattered information on the occurrence of these insects in the country (devetak, 1992, 1995, 1996, 1997; letardi and pantaleoni, 1996; devetak and devetak, 2004, popov, 2004; sziráki, 2014; devetak et al., 2015; devetak et al., 2016; devetak and zeqiri, 2018; appendix 1). until recently, 73 lacewing species were known for montenegro. however, in the preparation for a review of the fauna in selected areas in montenegro, efforts were made to determine whether other species of lacewings might be present in the country. the aim of the paper is to improve the knowledge on the occurrence of lacewings in montenegro. materials and methods lacewings were collected in selected sites during lepidopterological field work of the second author in 2017 and 2018. insects are preserved in the first author’s collection. we followed the nomenclature and taxonomy proposed by aspöck et al. (1980, 2001) and lacewing digital library (oswald, 2017). results in the period from 2017 to 2018, seventeen lacewing species were collected. two species – green lacewing chrysoperla lucasina and owlfly libelloides longicornis are new in montenegro. of these, the finding of the owlfly libelloides longicornis is particularly noteworthy. its finding represents the first documented record for the balkan peninsula. additionally, a checklist of neuropterida (raphidioptera, megaloptera, neuroptera) in montenegro is presented (appendix 1). list of species recorded in montenegro raphidioptera, snakeflies raphidiidae dichrostigma flavipes (stein, 1863) durmitor. sušica, vidikovac; 13/07/2018; p. jakšić leg.; 1 ♀. megaloptera sialidae, alderflies sialis lutaria (linnaeus, 1758) durmitor. zminičko jezero, 1295 m; 43°06’02.9”n, 19°14’54.9”e; 05/06/2017; p. jakšić leg.; 2 ♂ 5 ♀. durmitor. komarnica, nevidio; 05/08/2017; p. jakšić leg.; 1 ♂ 1 ♀. neuroptera osmylidae osmylus fulvicephalus (scopoli, 1763) bijelo polje, bistrica, đalovića klisura; 43°04’14.9”n, 19°54’04.6”e; 01/06/2017; p. jakšić leg.; 1 ♂ 1 ♀. chrysopidae, green lacewings nothochrysa fulviceps (stephens, 1836) durmitor mt., žabljak; 21/07/2017; p. jakšić leg.; 1 ♂ 1 ♀. chrysopa perla (linnaeus, 1758) bijelo polje, đalovića klisura, manastir sv. nikole; 780 m; 43°04’14.1”n, 19°54’12.3”e; 20/07/2014; p. jakšić leg.; 2 ♂. durmitor. bukovica, 999 m; 06/06/2017; p. jakšić leg.; 2 ♂. durmitor. kanjon sušice; 1180 m; 03/06/2017; p. jakšić leg.; 2 ♂ 2 ♀. durmitor. tepca; 880-920 m; 05/06/2017; p. jakšić leg.; 1 ♂. durmitor. zminičko jezero, 1322 m; 06/06/2017; p. jakšić leg.; 1 ♂. ljubišnja planina; 1068 m; 43°21’33”n, 19°06’39”e; p. jakšić leg.; 1 ♀. chrysopa walkeri mclachlan, 1893 komovi. šljivovički vis. 23/06/2018; p. jakšić leg.; 1 ♀. pseudomallada ventralis (curtis, 1834) komovi. šljivovički vis; 23/06/2018; p. jakšić leg.; 1 ♂. chrysoperla cf. carnea (stephens, 1836) bijelo polje, đalovića klisura, manastir sv. nikole; 780 m; 43°04’14.1”n 19°54’12.3”e; 20/07/2014; p. jakšić leg.; 1 ♀. komovi. šljivovički vis; 23/06/2018; p. jakšić leg.; 2 ♀. morača; 42°49’57”n 19°21’14”e; 507 m; 12/ vi/2018; p. jakšić leg.; 1 ♀. prokletije, gusinje: grbaja, 1143 m; 42°31’20”n, 19°47’12”e; 26/06/2018; p. jakšić leg.; 1 ♀. chrysoperla lucasina (lacroix, 1912) durmitor. žabljak; 21/07/2017; p. jakšić leg.; 3 ♀. new species for durmitor! komovi. šljivovički vis; 23/06/2018; p. jakšić leg.; 2 ♀. plužine, bioč. 43°11’02”n, 18°43’51”e; 10/07/2018; p. jakšić leg.; 2 ♀. prokletije, gusinje: grbaja, 1143 m; 42°31’20”n 19°47’12”e; 26/06/2018; p. jakšić leg.; 1 ♀. in the durmitor national park, individuals of a chrysoperla carnea (stephens)-complex of spe36 biologica nyssana ● 10 (1) september 2019: 35-41 devetak, jakšić ● lacewings (insecta: neuropterida: raphidioptera, megaloptera, neuroptera) collected in montenegro cies were found in eighties (devetak, 1991), but at that time it was not possible to separate different species within the complex. today, we are able to distinguish several species in europe (henry et al., 2013), and at least five in the balkan peninsula (e.g. henry et al., 1996, 1999, 2002, 2003; thierry and canard, 2015; devetak and rausch 2016). first record in montenegro! cunctochrysa albolineata (killington, 1935) durmitor. žabljak; 21/07/2017; p. jakšić leg.; 2 ♀. 37 hemerobiidae, brown lacewings megalomus tortricoides rambur, 1842 durmitor. žabljak; 21/07/2017; p. jakšić leg.; 1 ♂ 1 ♀. myrmeleontidae, antlions palpares libelluloides (linnaeus, 1764) ostrog. 10/07/2018; p. jakšić leg.; 1 ♂ (fig. 1-1). myrmecaelurus trigrammus (pallas, 1771) podgorica, cijevna, 38 m; 42°22’50.8”n 19°16’28.6”e; 02/06/2017; p. jakšić leg.; 6 larvae fig. 1. 1) antlion palpares libelluloides (linnaeus, 1764), male, ostrog. forewing length 52 mm. 2) antlion myrmecaelurus trigrammus (pallas, 1771), male. forewing length 35 mm. 3) antlion myrmeleon (myrmeleon) inconspicuus rambur, 1842, female, ulcinj. forewing length 29 mm. 4) antlion creoleon plumbeus (olivier, 1811), female. podgorica. forewing length 30 mm. 5) owlfly libelloides longicornis (linnaeus, 1764), female. đalovića klisura. forewing length 22 mm. 6) owlfly libelloides macaronius (scopoli, 1763), female. kom vasojevićki, trešnjevik. photo: 1-5: d. devetak; 6: p. jakšić. biologica nyssana ● 10 (1) september 2019: 35-41 devetak, jakšić ● lacewings (insecta: neuropterida: raphidioptera, megaloptera, neuroptera) collected in montenegro 38 (l3). adult is shown in fig. 1-2. myrmeleon (myrmeleon) inconspicuus rambur, 1842 podgorica, cijevna, 38 m; 42°22’50.8”n 19°16’28.6”e; 02/06/2017; p. jakšić leg.; 1 larva (l3). an adult from ulcinj is shown in fig. 1-3. creoleon plumbeus (olivier, 1811) podgorica, cijevna; 21/07/2017; p. jakšić leg.; 1 ♀ (fig. 1-4). ascalaphidae, owlflies libelloides longicornis (linnaeus, 1764) bijelo polje, đalovića klisura, manastir sv. nikole; 780 m; 43°04’14.1”n, 19°54’12.3”e; 20/07/2014; p. jakšić leg.; 1 ♀. (fig. 1-5). black spots on the wings of female are typical for l. longicornis (fig. 1-1). it is an extremely interesting finding – the first record in the balkan peninsula! this species occurs in central and western europe and italy (aspöck et al., 1980, 2001) and the finding in montenegro enlarges the knowledge on its occurrence in europe. libelloides macaronius (scopoli, 1763) podgorica, cijevna, 38 m; 42°22’50.8”n 19°16’28.6”e; 09/05/2017; p. jakšić leg.; 1 ♀. observations: bijelo polje, đalovića klisura, manastir sv. nikole; 780 m; 43°04’08.7”n, 19°54’09.4”e; 20/07/2017; 1 ♀ observed by p. jakšić. pivska planina,43°02’23.7”n, 18°48’12.4”e; 1451 m; 12/07/2018; 1 ♂observed by p. jakšić. kom vasojevićki, trešnjevik, 42°44’11.4”n, 19°41’04.2”e; 1303 m, 23/06/2018; 1 ♀ (fig. 1-6), observed and photographed by p. jakšić. discussion the paper presents lacewings collected in selected areas in montenegro during the years 2017 and 2018. with the new findings, the number of known neuropterida in montenegro has now raised to 75. the most spectacular was the finding of an owlfly, libelloides longicornis, which has not yet been recorded in the balkan peninsula. studies in future should confirm whether there is a stable population in montenegro. its known occurrence has ranged from western europe to italy and central europe (aspöck et al., 1980, 2001); in the light of the discovery of this species in montenegro, therefore we can expect more findings in the balkan peninsula in the future. acknowledgements the authors are grateful for helpful suggestions made by two anonymous reviewers on an earlier version of the manuscript. they have greatly helped to improve the manuscript. references aspöck, h., aspöck, u. 1966: studien an europäischen und kleinasiatischen arten des genus raphidia l. (insecta, raphidiodea). mitteilungen der schweizerischen entomologischen gesellschaft, 39: 33-48. aspöck, h, aspöck, u, hölzel, h. (in collaboration with h. rausch) 1980: die neuropteren europas. eine zusammenfassende darstellung der systematik, ökologie und chorologie der neuropteroidea (megaloptera, raphidioptera, planipennia) europas. goecke und evers. krefeld. 2 vols, 495 pp., 355 pp. aspöck, h., hölzel, h., aspöck, u. 2001: kommentierter katalog der neuropterida (insecta: raphidioptera, megaloptera, neuroptera) der westpaläarktis. denisia, 2: 1-606. devetak, d. 1991: neuropteroidea. megaloptera, raphidioptera, planipennia (insecta). fauna durmitora, 4: 135-159. devetak, d. 1992: present knowledge of the megaloptera, raphidioptera and neuroptera of yugoslavia (insecta: neuropteroidea). in: m. canard, h. aspöck, m. w. mansell (eds.): current research in neuropterology, sacco, toulouse, 107-118. devetak, d. 1995: deleproctophylla australis (fabricius, 1787) in istria and quarnero (neuroptera: ascalaphidae). annales, series historia naturalis, 7: 193–198. devetak, d. 1996: palpares libelluloides (linnaeus, 1764) in the northwestern part of the balkan peninsula (neuroptera: myrmeleontidae). annales, annals for istrian and mediterranean studies, 9: 211216. devetak, d. 1997: genus macronemurus costa, 1855 in the northwestern part of the balkan peninsula (neuroptera: myrmeleontidae). annales, annals for istrian and mediterranean studies, 11: 203-208. devetak, d., devetak, p. 2004: neuroleon microstenus (mclachlan, 1898) (neuroptera: myrmeleontidae) in northwestern part of the balkan peninsula. annales, series historia naturalis, 14: 55-58. devetak, d., jakšić, p.n., koren, t., ivajnšič, d. 2015: two sibling green lacewing species, chrysopa pallens and chrysopa gibeauxi (insecta: neuroptera: chrysopidae) in slovenia and western balkan countries. annales, series historia naturalis, 25: 47-54. devetak, d., slavevska-stamenković, v., sivec, i. 2016. alderflies (insecta: megaloptera: sialidae) from serbia and macedonia, with notes on their ocbiologica nyssana ● 10 (1) september 2019: 35-41 devetak, jakšić ● lacewings (insecta: neuropterida: raphidioptera, megaloptera, neuroptera) collected in montenegro 39 currence in the neighbouring balkan countries. acta zoologica bulgarica, 68(1): 39-42. devetak d., rausch h. 2016: checklist of lacewings (neuropterida: raphidioptera, megaloptera, neuroptera) of albania. acta zoologica bulgarica, 68: 457-467. devetak d., zeqiri r. 2018: lacewings (insecta: neuroptera) in the collection of the macedonian museum of natural history in skopje. acta musei macedonici scientiarum naturalium, 21: 113-122. henry c.s., brooks s.j., johnson j.b., duelli p. 1996: chrysoperla lucasina (lacroix): a distinct species of green lacewing, confirmed by acoustical analysis (neuroptera: chrysopidae). systematic entomology, 21: 205-218. henry c.s., brooks, s.j., duelli, p., johnson, j.b. 1999: revising the concept of chrysoperla mediterranea (hölzel), a green lacewing associated with conifers: courtship songs across 2800 kilometres of europe (neuroptera: chrysopidae). systematic entomology, 24: 335-350. henry, c.s., brooks, s.j., duelli, p., johnson, j.b. 2002: discovering the true chrysoperla carnea (insecta: neuroptera: chrysopidae) using song analysis, morphology, and ecology. annals of the entomological society of america, 95: 172-191. henry, c.s., brooks, s.j., duelli, p., johnson, j.b. 2003: a lacewing with the wanderlust: the european song species ‘maltese’, chrysoperla agilis, sp. n., of the carnea group of chrysoperla (neuroptera: chrysopidae). systematic entomology, 28: 131-147. henry, c.s., brooks, s.j., duelli, p., johnson, j.b., wells, m.m., mochizuki, a. 2013: obligatory duetting behaviour in the chrysoperla carneagroup of cryptic species (neuroptera: chrysopidae): its role in shaping evolutionary history. biological reviews, 88: 787-808. letardi, a., pantaleoni, r.a. 1996: i neurotteroidei w-paleartici della collezione del museo di zoologia dell’università di roma (neuropteroidea). fragmenta entomologica, 28: 277-305. oswald, j.d. 2017: lacewing digital library. neuropterida species of the world. version 5.0. http:// lacewing.tamu.edu/speciescatalog/main. accessed on 16 december 2018 popov, a. 2004: the ascalaphidae (neuroptera) of the balkan peninsula. denisia, 13: 229-237. saure, c. 1989: beitrag zur kenntnis der neuropterenfauna jugoslawiens und griechenlands (insecta, planipennia). entomofauna, 10(4): 33-43. steinmann, h. 1964: raphidiopterological studies ii. new raphidia l. and raphidilla nav. species from europe and asia. acta zoologica hungarica, 10: 199-227. sziráki, g. 2014: data to the raphidioptera fauna of the balkan peninsula and crete. folia historiconaturalia musei matraensis, 38: 87-90. táborský, k. 1936: monografické zpracování druhu ascalaphus ottomanus germar. monographische bearbeitung der art ascalaphus ottomanus germar. sborník entomologického oddeleni národního musea v praze, praha, 14: 133-144. táborský, k. 1939: studie druhu ascalaphus macaronius scopoli. časopis národního musea v praze 113: 91-96. thierry, d., canard, m. 2015: contribution to the knowledge of green lacewings of croatia (insecta: neuropterida: chrysopidae). acta entomologica slovenica, 23:21-28. van der weele, h.w. 1908: ascalaphiden monographisch bearbeitet. collections zoologiques du baron edm. de selys longchamps, 8: 1-326. species / taxon literature records comments order r a p h i d i o p t e r a family raphidiidae raphidia (raphidia) ophiopsis ophiopsis linnaeus, 1758 devetak, 1991, 1992 dichrostigma flavipes (stein, 1863) steinmann, 1964; devetak, 1991, 1992; sziráki, 2014 this paper. steinmann (1964) described this species as raphidia durmitorica. phaeostigma (phaeostigma) galloitalicum (h. aspöck & u. aspöck, 1976) devetak, 1991, 1992; sziráki, 2014 appendix 1. checklist of lacewings (neuropterida) in montenegro biologica nyssana ● 10 (1) september 2019: 35-41 devetak, jakšić ● lacewings (insecta: neuropterida: raphidioptera, megaloptera, neuroptera) collected in montenegro 40 species / taxon literature records comment order m e g a l o p t e r a family sialidae sialis lutaria (linnaeus, 1758) devetak, 1991, 1992; devetak et al., 2016; this paper sialis fuliginosa pictet, 1836 devetak, 1991, 1992; devetak et al., 2016 order n e u r o p t e r a family osmylidae osmylus fulvicephalus (scopoli, 1763) saure, 1989; devetak, 1991; this paper family chrysopidae nothochrysa fulviceps (stephens, 1836) devetak, 1991, 1992 this paper nothochrysa capitata (fabricius, 1793) devetak, 1991, 1992 italochrysa italica (rossi, 1790) devetak, 1992 nineta flava (scopoli, 1763) devetak, 1991, 1992 chrysopidia (chrysotropia) ciliata (wesmael, 1841) devetak, 1991, 1992 chrysopa perla (linnaeus, 1758) devetak, 1991, 1992 this paper chrysopa walkeri mclachlan, 1893 devetak, 1991, 1992 this paper chrysopa dorsalis burmeister, 1839 devetak, 1991, 1992 chrysopa formosa brauer, 1851 devetak, 1991, 1992 chrysopa viridana schneider, 1845 devetak, 1991, 1992 chrysopa pallens (rambur, 1838) devetak et al., 2015 pseudomallada flavifrons flavifrons (brauer, 1851) saure, 1989; devetak, 1991, 1992 pseudomallada prasinus (burmeister, 1839) saure, 1989; devetak, 1991, 1992 pseudomallada ventralis (curtis, 1834) devetak, 1991, 1992 this paper pseudomallada clathratus (schneider, 1845) saure, 1989; devetak, 1992 chrysoperla cf. carnea (stephens, 1836) devetak, 1991, 1992 this paper chrysoperla lucasina (lacroix, 1912) this paper. first record in montenegro. cunctochrysa albolineata (killington, 1935) saure, 1989; devetak, 1991, 1992 this paper peyerimhoffina gracilis (schneider, 1851) devetak, 1991, 1992 family hemerobiidae hemerobius humulinus linnaeus, 1758 devetak, 1991, 1992 hemerobius stigma stephens, 1836 devetak, 1991, 1992 hemerobius pini stephens, 1836 devetak, 1991, 1992 hemerobius contumaxtjeder, 1932 devetak, 1991, 1992 hemerobius handschinitjeder, 1957 devetak, 1991, 1992 hemerobius micans olivier, 1793 devetak, 1991, 1992 hemerobius lutescens fabricius, 1793 devetak, 1991, 1992 hemerobius gilvus stein, 1863 devetak, 1991, 1992 hemerobius marginatus stephens, 1836 saure, 1989; devetak, 1991, 1992 wesmaelius concinnus (stephens, 1836) devetak, 1991, 1992 wesmaelius quadrifasciatus (reuter, 1894) devetak, 1991, 1992 wesmaelius nervosus (fabricius, 1793) devetak, 1991, 1992 wesmaelius malladai (navás, 1925) devetak, 1991, 1992 wesmaelius tjederi (kimmins, 1963) devetak, 1991, 1992 wesmaelius subnebulosus (stephens, 1836) devetak, 1991, 1992 sympherobius elegans (stephens, 1836) devetak, 1991, 1992 sympherobius fuscescens (wallengren, 1863) devetak, 1991, 1992 sympherobius pellucidus (walker, 1853) devetak, 1991, 1992 megalomus tortricoides rambur, 1842 devetak, 1991, 1992 this paper drepanepteryx phalaenoides (linnaeus, 1758) devetak, 1991, 1992 micromus variegatus (fabricius, 1793) devetak, 1991, 1992 micromus paganus (linnaeus, 1767) devetak, 1991, 1992 micromus lanosus (zelený, 1962) saure, 1989; devetak, 1991, 1992 family sisyridae sisyra nigra (retzius, 1783) devetak, 1991, 1992 biologica nyssana ● 10 (1) september 2019: 35-41 devetak, jakšić ● lacewings (insecta: neuropterida: raphidioptera, megaloptera, neuroptera) collected in montenegro species / taxon literature records comment family coniopterygidae aleuropteryx juniperi ohm, 1968 devetak, 1991, 1992 helicoconis (ohmopteryx) pseudolutea ohm, 1965 devetak, 1991, 1992 coniopteryx (c.) borealis tjeder, 1930 devetak, 1991, 1992 coniopteryx (c.) pygmaea enderlein, 1906 devetak, 1991, 1992 coniopteryx (c.) tineiformis curtis, 1834 devetak, 1991, 1992 coniopteryx (metaconiopteryx) esbenpeterseni tjeder, 1930 saure, 1989; devetak, 1991, 1992 coniopteryx (metaconiopteryx) arcuata kis, 1965 devetak, 1991, 1992 parasemidalis (parasemidalis) fuscipennis (reuter, 1894) devetak, 1991, 1992 semidalis aleyrodiformis (stephens, 1836) saure, 1989; devetak, 1991, 1992 conwentzia pineticola enderlein, 1905 devetak, 1991, 1992 family dilaridae dilar turcicus hagen, 1858 devetak, 1991, 1992 family mantispidae mantispa styriaca (poda, 1761) devetak, 1991, 1992 family myrmeleontidae palpares libelluloides (linnaeus, 1764) devetak, 1992, 1996 this paper myrmecaelurus trigrammus (pallas, 1771) devetak, 1992 this paper myrmeleon formicarius linnaeus, 1767 devetak, 1991, 1992 myrmeleon inconspicuus rambur, 1842 devetak, 1992 this paper euroleon nostras (geoffroy in fourcroy, 1785) devetak, 1991, 1992 macronemurus bilineatus brauer, 1868 devetak, 1992, 1997 neuroleon (neuroleon) microstenus (mclachlan, 1898) devetak, 1992; devetak and devetak, 2004 distoleon tetragrammicus (fabricius, 1798) devetak, 1991, 1992 creoleon plumbeus (olivier, 1811) devetak, 1992 this paper gymnocnemia variegata (schneider, 1845) letardi and pantaleoni, 1996 family ascalaphidae deleproctophylla australis (fabricius, 1787) devetak, 1992, 1995; popov, 2004 libelloides lacteus (brullé, 1832) van der weele, 1908;táborský, 1936; devetak, 1992; popov, 2004 libelloides longicornis (linnaeus, 1764) this paper. first record in montenegro and in the balkan peninsula. libelloides macaronius (scopoli, 1763) táborský,, 1939; saure, 1989; devetak, 1991, 1992; popov, 2004; devetak and zeqiri, 2018 this paper 41 biologica nyssana ● 10 (1) september 2019: 35-41 devetak, jakšić ● lacewings (insecta: neuropterida: raphidioptera, megaloptera, neuroptera) collected in montenegro 42 stojadinović, d., vidojević, d., crnobrnja-isailović, j.: variation of clutch characteristics in population of eastern hermann’s tortoises (testudo hermanni boettgeri gmelin 1789). biologica nyssana, 8 (1), september 2017 biologica nyssana 8 (1)  september 2017: 123-127 stojadinović, d. et al.  variation of clutch characteristics in population… 123 original article received: 04 january 2017 revised: 23 february 2017 accepted: 11 mart 2017 variation of clutch characteristics in population of eastern hermann’s tortoises (testudo hermanni boettgeri gmelin 1789) dragana stojadinović1, dragana vidojević1, jelka crnobrnja-isailović1,2 1university of niš, faculty of science and mathematics, department of biology and ecology, višegradska 33, niš, serbia 2 institute for biological research „sinišastanković“, university of belgrade, despotastefana 142, 11000 belgrade, serbia * e-mail: draganapenev@yahoo.com abstract: stojadinović, d., vidojević, d., crnobrnja-isailović, j.: variation of clutch characteristics in population of eastern hermann’s tortoises (testudo hermanni boettgeri gmelin 1789). biologica nyssana, 8 (1), september 2017: 123-127. we present here information on some fecundity parameters in local population of eastern hermann’s tortoise situated in the area of kunovica, at the outskirts of the city of niš. the data were collected in the field and in 97% were based on reconstruction of number and dimensions of eggs in destroyed nests what influenced on quality of information. only two of 78 nests recorded from 2010 to 2014 were found intact. other 76 clutches were found destroyed and therefore only some fecundity parameters were measurable, while the others were reconstructed by using formulas proposed in the relevant literature. on the basis of collected data we estimated average clutch size of hermann’s tortoises in kunovica as four eggs what was lower than in populations from greece. however, the most of calculated egg dimensions (e.g. maximal width, volume and mass) were higher than in populations from greece and france. key words: fecundity, clutch size, egg size, testudo hermanni boettgeri apstrakt: stojadinović, d., vidojević, d., crnobrnja-isailović, j.: varijacije karakteristika legla u populaciji istočne podvrste šumske kornjače (testudo hermanni boettgeri gmelin 1789). biologica nyssana, 8 (1), septembar 2017: 123-127. u ovom radu predstavljene su informacije o nekim parametrima fekunditeta lokalne populacije istočne podvrste šumske kornjače sa područja kunovice (okolina grada niša). svi podaci su prikupljeni na terenu i u 97% slučajeva izvršena je rekonstrukcija broja i dimenzija jaja, što utiče na kvalitet informacija. samo dva od 78 pronađenih gnezda u periodu od 2010. do 2014. su bila sa neoštećenim jajima. ostala gnezda su bila uništena, tako da su samo neki parametri mogli da budu izmereni, dok su ostali rekonstruisani pomoću formula datih u odgovarajućoj literaturi. na osnovu prikupljenih podataka, procenili smo da je prosečna veličina legla kod šumske kornjače iz kunovice 4 jaja, što je manje nego kod populacija iz grčke. ipak, većina karakteristika 8 (1) • september 2017: 123-127 doi: 10.5281/zenodo.964581 biologica nyssana 8 (1)  september 2017: 123-127 stojadinović, d. et al.  variation of clutch characteristics in population… 124 jaja ove lokalne populacije (npr. maksimalna širina, zapremina, masa) je bila veća nego kod populacija iz grčke i francuske. ključne reči: fekunditet, veličina legla, veličina jaja, testudo hermanni boettgeri introduction traditional life-history approach (g a d g i l & b o s s e r t , 1970) proposes that reproductive investment increases with age and reaches the peak in the act of terminal investment, where reproduction stops afterwards (w i l l i a m s , 1966; h i r s c h f i e l d & t i n k l e 1975; c l u t t o n b r o c k , 1984). other theory proposes that reproductive investment depends on number of characters subjected to various rates of change, what depends on age of the individual (m c n a m a r a & h o u s t o n , 1996). reptiles in general have greater and more variable fecundity in comparison to endothermous organisms (s h i n e , 2005). in general, female’s fecundity could vary on different levels: within individual female’s life, among females within the same population, among females from conspecific populations or among females of different species. in many reptiles, larger females produce bigger clutches (f i t c h , 1970). reproduction is usually delayed in females until they accumulate enough energy to produce clutch as large as their abdominal cavity is (v i t t & c o n g d o n , 1978; v i t t & p r i c e , 1982). some observations confirmed that decrease of average female body size and average clutch size, caused by environmental change, may decrease survival and growth rates (s h i n e , 2005). egg laying happens from middle may to end of june (c r u c e & r ă d u c a n , 1976; s w i n g l a n d & s t u b b s , 1985; f e r t a r d , 1992; b e r t o l e r o e t al., 2007b) and females repeatedly use the same nesting place (s w i n g l a n d & s t u b b s , 1985). incubation period varies from 90 to 124 days (c r u c e & r ă d u c a n , 1976; c h e y l a n , 1981; n o u g a r è d e , 1998). eggs are of white color, hard shelled and almost ellyptical in shape. incubation temperature varies from 23◦c, to 34◦c (e e n d e b a k , 1995). if incubation temperature keeps constantly at 25◦c or 33-34◦c, mortality of the embrios increases to approximately 50%. on the temperatures lower than 23◦c or higher than 34◦c mortality reaches 100% (e e n d e b a k , 1995). in year 2010 we established regular monitoring of one local population of eastern hermann’s tortoise in the area of village kunovica in vicinity of town of niš in south-eastern serbia. our main goal was to collect data on life history parameters and to record their variation in order to enable population viability analysis in the future. this study presents the most possible projection of female fecundity in population of hermann’s tortoises in kunovica based on data collected during first five years of monitoring. we assumed that average clutch size and egg dimensions could be in range of values reported for other analysed populations of testudo hermanni boettgeri. material and methods field procedures field work was conducted twice per year from 2010 to 2014, always in may and june and seven consecutive days each. detailed description of the studied area could be find in s t o j a d i n o v i ć et al. (2013). during the day researchers were collecting records on different aspects of biology of the species, eggs laying and nests. clutch size was estimated by reconstruction of located destroyed nests only where counting of eggs from shells was possible. direct counting of eggs was possible when females were encountered while laying eggs. in these two cases the eggs from intact nest were carefully taken from the hole, measured for mass and maximal lenght & width by pesola field balance with accuracy of 0.2 g and by digital calliper with precision of 0.01 mm, respectively. the eggs were carefully deposited back in the nest afterwards and covered with soil. we considered egg dimensions as reliable only when length and/or maximal width of egg shells were available to measure and they were included in the analysis. species description testudo hermanni (gmelin 1789) is one of three european tortoise species. two subspecies of hermann’s tortoise are currently recognized: the western subspecies -t. hermanni hermanniinhabits parts of spain, france and italy while eastern subspecies -t. hermanni boettgeriis distributed in croatia, bosnia and herzegovina, montenegro, albania, serbia, fyrm, romania, bulgaria, greece and turkey (fritz et al. 2006). statistical procedures egg volume (ev, mm³) was estimated by applying the equation ev=(π*l*w²)/6000 (longepierre et al., 2003), where π =3.14, l= longer egg axis or egg biologica nyssana 8 (1)  september 2017: 123-127 stojadinović, d. et al.  variation of clutch characteristics in population… 125 lenght, and w = shorter egg axis or egg width (c o l e m a n , 1991). egg mass (em, g) was estimated by applying the equation em=0.435*l+0.0203*w²-14.7 (h a i l e y & l o m b o u r d i s , 1990), where l = egg lenght, and w = egg width. average egg size, shape (l/w), mass and volume were estimated independently for every nest (l o n g e p i e r r e et al., 2003). all statistical analyses were done by software package statistica 7.0 (statsoft inc.). results and discussion only two of 78 hermann’s tortoise clutches recorded during four years of monitoring field site in kunovica were found intact and they contained four and five eggs. females that deposited these clutches were measured for streight carapace length (scl), midbody carapace width (mcw), maximal carapace width (maccw) and weight (w). for female that deposited four eggs measures were: scl-193.58 mm, mcw-141.38 mm, maxcw-143.42 mm, w-1347 g; for female that deposited five eggs measures were: scl184.15 mm, mcw145.93 mm, maxcw 146.13 mm, w-1390 g. average egg lenght in intact clutch was 37.40 mm ± 0.68 mm, average maximal width was 30.25 mm ± 0.39 mm, and average egg mass was 20.0 g ± 0.68 g. the reconstruction of clutch and egg size enabled us to appoximate average clutch size (4 eggs ± 1 egg) in this particular population of eastern hermann’s tortoise. clutch size varied from 1 to 7 eggs. overall number of nests recorded over years was 13 in 2010 (6 in last week of may and 7 in third week of july), 6 in 2011 (4 nests in may and 2in july), 4 in 2012 (4 nests in may), 38 in 2013 (35 in may and 3 in july) and 17 in 2014 (4 nests in may and 13 in july). using h a i l e y and l o m b o u r d i s (1990) formula for estimation of egg mass on the basis of egg dimensions we got hypothetic average egg mass value of 20.8 g ± 4.8 g. observed average egg mass recorded in the field (based on measured intact eggs) was 20.0 g ± 0.68g. additionally, we have also calculated mean values of fecundity parameters for destroyed clutches, and presented their averages in table 1. average egg length per clutch was 37.1 mm, varying from 33.4 mm to 41.3 mm. average value of maximum egg width per clutch was 29.8 mm, varying from 24.0 mm to 35.4 mm. average approximate egg mass per clutch was 19.6 g, varying from 11.8 g to 26.6 g. average egg volume per clutch was 17.5 mm³, varying from 10.3 mm³ to 24.5 mm³. clutch size in testudo hermanni varied from one to seven eggs in western (nominative) subspecies (e s t e b a n , 1987; f e r t a r d , 1992; n o u g a r è d e , 1998; l o n g e p i e r r e et al.,2003; b e r t o l e r o et al., 2007c) and from one to nine eggs in eastern (boettgeri) subspecies (s w i n g l a n d & s t u b b s , 1985; h a i l e y & l o u m b o u r d i s , 1988, 1990) (tab. 1). average clutch size varied from 3.3 in western (e s t e b a n , 1987; f e r t a r d , 1992; n o u g a r è d e , 1998; l o n g e p i e r r e et al., 2003; b e r t o l e r o et al., 2007c) to 4.3 eggs in eastern subspecies (s w i n g l a n d & s t u b b s , 1985; h a i l e y & l o u m b o u r d i s , 1988, 1990). average egg size and weight were 27.2× 34.3 mm and 15.1 g, respectively, in testudo hermanni hermanni (c h e y l a n , 1981; e s t e b a n , 1987; f e r t a r d , 1992; l o n g e p i e r r e et al., 2003; b e r t o l e r o et al., 2007a,c) and 27,9×37,4 mm and 17.1 g, respectively, in testudo hermanni boettgeri table 1. overview of fecundity parameters recorded in hermann’s tortoise populations from different parts of species range (france, greece) and from serbia. n – number of analyzed nests, nav – average clutch size, lav – average egg lenght/clutch, wavaverage maximal egg width/clutch, (l/w)av – average value of egg shape/clutch, evav average value of egg volume/clutch, emav – average value of egg mass/clutch. s.d.-standard devitiation. france 1 (longipierre, 2003) france 2 (longipierre, 2003) greece 1 (hailey, 1988) greece 2 (hailey, 1988) greece 3 (hailey, 1988) serbia (our data, destroyed clutches) serbia (our data, intact clutches) n 76 2 nav ±s.d. 3 ± 1 3 ± 1 5 ± 1 5 ± 1 5 ± 1 4 ± 1 4.5±0.71 lav±s.d. 35.6 37.1± 2.2 37.5 ± 2.2 38.8± 2.7 37.1± 2.3 37.4±0.7 wav±s.d. 27.8 27.0±1.5 28.9 ± 0.9 28.0± 1.7 29.8± 2.9 30.3±0.4 (l/w)av±s.d. 1.28 1.37± 1.5 1.3 ± 1.5 1.38± 1.5 1.25± 0.1 1.2±0.0 emav 16.5 15.8 16.24 18.57 18.09 19.6 20.0 evav 14.4 14.15 16.39 15.92 17.5 17.9 biologica nyssana 8 (1)  september 2017: 123-127 stojadinović, d. et al.  variation of clutch characteristics in population… 126 (c r u c e & r ă d u c a n , 1976; h a i l e y & l o u m b o u r d i s , 1988, 1990;). in general, reproductive traits in female hermann’s tortoises were related to body size and the biggest females have the greatest contribution to overall egg production per reproductive season (c r u c e & r ă d u c a n , 1976; h a i l e y & l o u m b o u r d i s , 1988; f e r t a r d , 1992; l o n g e p i e r r e et al.,2003; b e r t o l e r o et al., 2007c). it was not possible to test this relation in our study as it was almost completely based on data obtained from inspection of broken nests. in fact, only two of 78 recorded tortoise nests were found intact (females were spotted while laying the eggs in may 2010). nonparametric kolmogorov-smirnov two-sample test has shown that there was no significant difference between body size and mass of these two females nor between egg parameters in these two clutches. except 2014, in other years of study the most of the nests was detected in may. it could be explained by the fact that high environmental temperatures starting from july could influence on egg laying time in reptiles with multiple clutches e.g. temperature increase results in earlier nesting time in the summer. studies conducted in france and greece suggested that the first egg laying occursin the midapril, while the last was detected in july. particular flexibility was detected in beginning and the end of the nesting period what could be related to variation in environmental conditions (c r u c e & r a d u c a n , 1976). radiological surveys in those two countries confirmed that the highest number of calcified eggs in oviducts was detected during may and june (b e r t o l e r o et al., 2007b). h a i l e y and l o u m b o u r d i s (1988) were studying reproduction of hermann’s tortoise on three different localities in greece (alyki, litochoron and deskati). average clutch size was 5 eggs (table1). the lowest values of analysed fecundity parameters were recorded on the first locality – dry sandy ground. the other two localities were on fertile soil and similar regading analysed fecundity parameters. localities in france (plaine des mauresand massif des maures) were under the influence of mediterranean climate (l o n g e p i e r r e et al., 2003) what could explain their lower values of fecundity parameters in comparison to greece and serbia (tab. 1). the largest hermann’s tortoise eggs were recorded in serbia (in comparison with data from france and greece, see tab. 1) which should be taken with caution since most of the data were obtained from broken egg shells. egg size could be correlated to body size, but it is not the rule: in some populations larger female tortoises have wider eggs because egg size is constrained by width of the pelvic channel (c o n g d o n & t i n k l e , 1982). however, in some populations smaller females lay more narrow eggs but similar in weight to those of larger females (h a l i l e y & l o u m b o u r d i s , 1988). conclusion reconstruction of number and dimensions of eggs in destroyed nests of hermann’s tortoise from kunovica showed values similar to those obtained from measurement of two intact nests estimated average clutch size of female hermann’s tortoises in kunovica was lower than in populations from greece, but higher than in populations from france. maximal width, volume and mass of eggs were higher than in population in greece and france. acknowledgements. this study represents first results of long-term monitoring on eastern hermann’s tortoises in south-eastern serbia that has been established in 2010 by ds and jci as a part of national project funded by ministry of education, science and technological development of republic of serbia, grant no 173025 „evolution in heterogeneous environments: mechanisms of adaptation, biomonitoring and biodiversity conservation“. our thanks go to a number of students of biology and ecology at the faculty of sciences and mathematics university of niš for participance, especially to j. mijatović for substantal assistance in the field. moreover, logistic support of s. stojadinović and dj. milošević were highly appreciated. field work was approved by permits no 353-011134/2010-03 and no 353-01-29/2011-03 ministry of environment and spatial planning of republic of serbia, no 353-01-505/2012-03 ministry of environment, mining and spatial planning of republic of serbia, no 353-0154/2013-08 and no. 353-01-312/2014-08 ministry of energetics, development and nature protection of republic of serbia. references bertolero, a., cheylan, m., nougarède, j.p., 2007a: accroissement de la fécondité chez la tortue d’hermann testudo hermanni hermanni en condition insulaire: un contre–exemple du syndrome insulaire? revue d’écologie (terre et vie), 62:93–98. bertolero, a., nougarède, j.-p., cheylan, m., 2007b: female reproductive phenology from a population of hermann’s tortoise testudo hermanni hermanni in corsica. herpetological journal, 17:92–96. bertolero, a., nougarède, j.-p., cheylan, m., marín, a., 2007c: breeding traits of hermann’s tortoise testudo hermanni hermanni in two western populations. amphibia-reptilia, 28:77–85. biologica nyssana 8 (1)  september 2017: 123-127 stojadinović, d. et al.  variation of clutch characteristics in population… 127 cheylan, m., 1981: biologie et écologie de la tortue d’hermann testudo hermanni gmelin 1789. contribution de l’espèce a la connaissance des climats quaternaires de la france. montpellier: mémoires et travaux de l’institut de montpellier (e.p.h.e.), vol. 13, 382 pp. clutton-brock, t.h., 1984: reproductive effort and terminal investment in iteroparous animals. american naturalist, 123: 212–229. coleman, m., 1991: measuring parental investment in nonspherical eggs. copeia, 4: 1092-1098. congdon, j.d., tinkle, d.f., 1982: reproductive energetics of the pained turtle (chrysemys picta). herpetologica, 38: 228-237. cruce, m., răducan, i., 1976: reproducerea la broascatestoasă de uscat (testudo hermanni hermanni g.). revue roumaine de biologie, serie biologie animale, 28: 175–180. eendebak, b.t., 1995: incubation period and sex ratio of hermann’s tortoise, testudo hermanni boettgeri. chelonian conservation and biology, 1: 227–231. esteban, i., 1987: estudio de la reproducción de testudo hermanni (gmelin) en cautividad. aquamar, 27:12–20. fertard, b., 1992: etude des caractéristiques radiographiques et chronologiques de la ponte chez testudo hermanni en semi-liberté. in: first international congress of chelonian pathology. gonfaron, france: editions soptom, pp. 190– 199. fitch, h.s., 1970: reproductive cycles in lizards and snakes. miscellaneous publication university of kansas, museum of natural history, 52: 1–247. fritz, u., auer, m., bertolero, a., cheylan, m., fatizzo, t., hundsdörfer, a.k., martín sampayo, m., pretus, j.l., široký, p., wink, m., 2006: a rangewide phylogeography of hermann's tortoise, testudo hermanni (reptilia: testudines: testudinidae): implications for taxonomy. zoologica scripta, 35 (5): 531-543 gadgil, m., bossert, w.h., 1970: life historical consequences of natural selection. american naturalist, 104: 1–24. greer, a.e., 1989: the biology and evolution of australian lizards. chipping norton nsw: surrey beatty & sons. 264 pp. hailey, a., loumbourdis, n.s., 1988: egg size and shape, clutch dynamics, and reproductive effort in european tortoises. canadian journal of zoology, 66: 1527–1536. hailey, a., loumbourdis, n.s., 1990: population ecology and conservation of tortoises: demographic aspects of reproduction in testudo hermanni. herpetological journal, 1:425–434. hirschfield, m.f., tinkle, d.w., 1975: natural selection and the evolution of reproductive effort. proceedings of the national academy of sciences of the united states of america, 72: 2227–2231. longepierre, s., grenot, c., hailey, a., 2003: individual, local and subspecific variation in female hermann’s tortoise (testudo hermanni) reproductive characters. contribution to zoology, 72 (4), http://dpc.uba.uva.nl/ctz/vol72/nr04/ art03. mcnamara, j.m., houston, a.i., 1996: statedependent life histories. nature, 380: 215–221. nougarède, j.p., 1998: principaux traits d’histoire naturelle d’une population de tortue d’hermann (testudo hermanni) dans le sud de la corse. diplôme de l’ecole pratique des hautes etudes, montpellier, france. seigel, r.a., huggins, m.m., ford, n.b., 1987: reduction in locomotor ability as a cost of reproduction in gravid snakes. oecologia, 73 (4): 481-485. shine, r., 2005: life-history evolution in reptiles. annual review of ecology, evolution and systematics, 36: 23-46. shine, r., greer, a.e., 1991. why are clutch sizes more variable in some species than in others? evolution, 45: 1696-1706. stojadinović, d., milošević, đ., crnobrnja-isailović, j., 2013: righting time versus shell size and shape dimorphism in adult hermann’s tortoises: field observations meet theoretical predictions. аnimal biology, 63: 381-396. swingland, i.r., stubbs, d. 1985. the ecology of mediterranean tortoise (testudo hermanni): reproduction. journal of zoology, 205: 595–610. vitt, l.j., congdon, j.d., 1978: body shape, reproductive effort, and relative clutch mass in lizards: resolution of a paradox. american naturalist, 112: 595–608. vitt, l.j., price, h.j., 1982: ecological and evolutionary determinants of relative clutch mass in lizards. herpetologica, 38:237–55. williams, g.c., 1966: adaptation and natural selection. princeton university press. princeton, new jersey. 324p. živković et al., 2019, biologica nyssana 10(1) 10 (1) september 2019: 29-34 doi: 10.5281/zenodo.3464000 susceptibility of different grapevine cultivars to eutypa lata isolate, causing agent of eutypa dieback, originating from serbia original article sanja živković faculty of agriculture, university of niš, kosančićeva 4, 37000 kruševac, serbia gajicsanja43@gmail.com (corresponding author) tanja vasić faculty of agriculture, university of niš, kosančićeva 4, 37000 kruševac, serbia tanjavasic82@gmail.com jordan marković institute for forage crops, 37251 kruševac, serbia jordan.markovic@ikbks.com received: april 25, 2019 revised: may 17, 2019 accepted: august 13, 2019 abstract: the experiment was established in the greenhouse during the two years. the grapevine cultivars used in experiment were riesling blanc, chardonnay, banatski muskat and gamay noir. the susceptibility of grapevine cultivars to the isolate of the fungi eutypa lata, causing agent of eutyposis, was analysed. one isolate el17 was used in the experiment. the highest susceptibility was found in riesling blanc, banatski muskat and gamay noir cultivars, while chardonnay cultivar was less susceptible to the isolates of fungus eutypa lata. key words: grapevine, eutypa lata, cultivar susceptibility apstract: osetljivost različitih sorti vinove loze na izolat eutypa lata, prouzrokovača eutipoze, poreklom iz srbije eksperiment je izveden tokom dve godine u stakleniku. u eksperimentu su korišćene sorte vinove loze rajnski rizling, šardone, banatski muskat i game crni. ispitivana je osetljivost sorti na izolat gljive eutypa lata prouzrokovača odumiranja čokota vinove loze. u ogledu je korišćen jedan izolat el17. najosetljivijom se pokazala sorta rajnski rizling, banatski muskat i game crni, a sorta šardone pokazala se manje osetljivom na izolate gljive eutypa lata. ključne reči: vinova loza, eutypa lata, osetljivost sorti introduction the eutypa dieback is one of the most harmful mycoses on the grapevine (vitis vinifera l.) worldwide caused by fungal pathogen named eutypa lata. this fungus can significantly reduce the yield and quality of the grapevines, but above all, it significantly reduces the longevity of the vine, as it causes partial or complete dying of the trunk (bolay and moller, 1977; carter et al., 1985, 1994; živković et al., 2012a, b). in serbia, during the period from 2010 to 2012, the occurrence the eutypa dieback symptoms on the grapevines were observed. the symptoms first appear in the form of small, chlorotic and necrotic spots along the periphery of the leaves, deformation of the leaves, the appearance of shortened shoots, often with the so-called zig-zag internodes. in time, there is a partial or complete dying of the trunk of the vine (carter at al., 1985; živković et al., 2012a, b). fungus e. lata is a vascular pathogen that infects the grapevine through fresh pruning wounds. the infection occurs when askospores arrive at fresh cross cut sections and in the presence of water droplets penetrate the vascular tissue. the wounds are particularly sensitive immediately after pruning, although infections may occur even after seven weeks following the cut (munkvold, 2001; sosnowski et al., 2007a, b; pitt et al., 2013). phytosanitary and chemical measures are used in the vineyards to suppress the degradation of grapevine. the use of preventive measures is reflected in the selection of the cultivars, the selection of the breeding form of the trunk, the time of the cutting and the removal and burning of diseased parts of the trunk. in a region where there are more hosts of e. lata, control of the disease cannot be done only by sanitary measures. regular cutting and destruction of diseased trunk parts provide control of the inoculum within limits suitable for grapevine. given that © 2019 živković et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 29 there are no resistant grape cultivars to e. lata, planting of vineyards, it is recommended to plant tolerant cultivars, while avoiding sensitive cultivars. as a preventive measure of the fight, it is recommended to select one of the breeding forms, which involves the formation of a lower trunk and mixed cutting (rolshausen et al., 2015; sosnowski, 2016). the main aim of this study was to determine the susceptibility of the grapevine cultivars to isolate of e. lata and finding potential sources of resistance for different grapevine cultivars. for the investigation, the four grapevine cultivars, the one domestic (banatski muskat) and three foreign (riesling blanc, chardonnay, and gamay noir) were selected. a test of the sensitivity of investigated grapevine cultivars to e. lata isolate was carried out by inoculation of unrooted cuttings. materials and methods samples and fungal isolation during the period from 2010 to 2012, symptoms on the grapevine from 14 locations in serbia, it was noticed a plant with dieback. sampling was done in the second half of may and early june in vineyards aged 11 to 22 years. during the collection of samples of diseased plants in the field, fragments of the trunk and cordon of the grapevine, including diseased and healthy tissue, in size from 10 to 20 cm, were taken. symptoms initially appeared as small, chlorotic and necrotic spots along the rim of the leaves, deformation of leaves and the appearance of shortened shoots, often with the so-called zig-zag internodes. over time, partial or complete dying of the vines developed. to isolate the pathogen, diseased parts of grapevine plants were surface-sterilised with 5% sodium hypochlorite for 2 min, followed by three washes with sterile distilled water. surface sterilised tissue was transferred to sterile filter paper and placed on potato dextrose agar (pda) containing streptomycin and incubated at 24 °c in the 24 h uv light for 30 days. single conidia were selected and transferred directly to the pda plate according to the procedures described by choi et al. (1999) and stored on pda in tubes at 4 °c. of the total number of isolates obtained for this study, one isolate el 17 (from cv. cabernet sauvignon) was selected. cultivar test cultivar test was performed according to the methods of peros and berger (1994). unrooted cuttings of four cultivars riesling blanc, chardonnay, banatski muskat and gamay noir wereinoculated with mycelium of one isolate (el17) in a randomised block design with four replication on eight cuttings. the day before the inoculation, 2-node cuttings, with the basal bud eliminated, were prepared and soaked in water overnight. a hole (5 mm in diameter, 5 mm deep) was drilled 4 cm below the upper bud, and a plug (5 mm diameter) of agar and mycelium, cut from a fresh culture on pda medium was inserted into the hole, with the mycelium towards the wood. plugs of pda medium were used for the control. just after inoculation, cuttings were inserted into pieces of rock wool, dimensions 50 x 70 x 30 cm. five and ten weeks after inoculation, the plants were observed using the following scale: 0 (plants without symptoms), 1 (plants with few small and dried leaves), 2 (plants with higher number of small and dried leaves), 3 (plants with barely present leaves) and 4 (plants with dried shoots). after observing, the pieces were put into drained plastic containers, dimensions 12 x 12 x 30 cm, filled with substrate (class 1) (without feeding) and finally placed on benches in a greenhouse. the plants were trimmed in february of the next vegetation cycle. experiments were arranged in a complete randomised design. there were nine replicates of eight cuttings per treatment. five and ten weeks after bud break, plants were observed in the second vegetative growth cycle for each treatment of the cultivar experiment, evaluating the appearance of deformations and necrosis on leaves of inoculated plants. calculation of the intensity of the disease on the shoots was done by direct proportion of the number of diseased leaves on inoculated cuttings and the number of leaves on control cuttings (péros and berger, 1994). statistical analysis statistical analysis was performed to determine the relationship between e. lata isolate and grapevine cultivar. data were analysed by analyse of variance (anova) using the computerised software (proc glm, sas, system, version 8.1; sas institute, cary, nc). to satisfy the assumptions of the anova, the arcsine transformation of the proportion was used (y=2x arcsine √p). homogeneity of groups was assessed using duncan’s test with p=0.05. results by examining the vineyards in 14 locations where grapevine is cultivated in our country in the period from 2010 to 2012, symptoms of eutypa dieback of grapevine have been observed. samples were collected from 14 locations in serbia. during the vegetation, the diseased parts of the vines in vineyards become covered with a new leaf mass, so that the symptoms are more difficult to notice. symptoms on the leaves of diseased plants are ex30 biologica nyssana ● 10 (1) september 2019: 29-34 živković et al. ● susceptibility of different grapevine cultivars to eutypa lata isolate, causing agent of eutypa dieback, originating from serbia) pressed in the form of tiny, chlorotic spots, arranged on the periphery of the leaves. the edges of the leaf are often spiked and bent toward the back, and with stronger infections, the leaf surface is mostly covered with necrotic spots. the central part of the leaf has a wobbly look. over time, the leaves were dried and fall off early. the susceptibility/resistance of different grapevine cultivars, to the selected isolate of e. lata, originating from serbia, under greenhouse conditions was determined. characterisation of morphological and molecular levels had previously been done on the fungi isolate. assessment of susceptibility/resistance was carried out using methods of inoculation of unrooted cuttings of different cultivars of grapevine. this research aimed to determine the susceptibility of different cultivars of grapevines to the isolate based on the appearance of symptoms on leaves and shoots, from the movement of growers on grapevine cuttings. based on the results of this study, the method of inoculation of grape cuttings according to peros and berger (1994) will be used in the future. four grapevine cultivars were selected for testing: riesling blanc, chardonnay, banatski muskat, and gamay noir. tested isolate caused eutyposis symptoms on grapevine cuttings. the reaction of the tested grapevine cuttings, as well as the estimated level of development of the disease after inoculation to selected e. lata isolate, are shown in tab. 1. the foliar expression of eutypa dieback was compared between four cultivars of grapevine following inoculation by e. lata isolate el17 (tables 1). the numbers of cuttings showing foliar symptoms and other abnormalities were recorded five and ten weeks after inoculation in the first vegetative growth cycle and five and ten weeks after bud break in the second cycle vegetative growth cycle in cultivar experiments. all the cultivars inocu31 lated exhibited foliar symptoms of the disease and the other abnormalities. in experiment, the effect of cultivar was highly significant. some of the shoots showing necrotic leaves five weeks after inoculation showed normal growth ten weeks after inoculation and this phenomenon explained the decrease in some percentage. the differences between cultivars were less acute ten weeks after inoculation in first and ten weeks after bud break in second vegetative growth cycle (tab. 1) indicating that the effects of the fungus appeared faster for some cultivars than for others. the foliar symptoms occurred during the vegetative growth cycle on the inoculated unrooted cuttings. the smaller and necrotic leaves were similar to that observed in shoots of infected vines in the vineyard. the cultivars had different responses in the test, indicating that cultivars differ in their susceptibility to fungal toxins and fungal invasion. the high susceptibility of cultivars riesling blanc, banatski muskat and gamay noir was confirmed. cultivars riesling blanc, banatski muskat and gamay was considerably more affected than the chardonnay cultivar, and the inoculation of cuttings caused a faster appearance of abnormal plants for riesling blanc, banatski muskat and gamay noir than for the cultivar chardonnay (tab. 1). the infected plants exhibited the typical symptoms of eutyposis on the shoots and leaves. thus, on the inoculated cuttings of all grapevine cultivars studied, the intensity of the disease was similarly caused by isolate el17 (tab. 1). discussion eutypa lata is one of the most important phytopathogenic fungi that threaten vineyards, causing signifitable 1. effect of grapevine cultivar on percentage of plants showing foliar symptoms and abnormalities after inoculation with e. lata isolate el17 a-c: means in a column followed by the same letter are not significantly different according to duncan’s multiple range test at the p=0.05. biologica nyssana ● 10 (1) september 2019: 29-34 živković et al. ● susceptibility of different grapevine cultivars to eutypa lata isolate, causing agent of eutypa dieback, originating from serbia) i s o l a t e e l 1 7 year n year n+1 5th week 10th week 5th week 10th week cultivar f o l i a r s y m p t o m s ( % ) riesling blanc 82.03 a 76.57 ab 80.21 a 65.63 ab chardonnay 44.53 b 48.44 b 56.25 b 61.46 b banatski muskat 86.72 a 79.69 a 72.92 ab 78.48 a gamay noir 74.22 ab 82.03 a 71.79 ab 77.08 a control 0.00 c 0.00 c 0.00 c 0.00 c 32 cant economic damage (pitt et al., 2013; travadon et al., 2013). according to munkvold et al. (1994) the eutypa dieback is a very dangerous disease of the grapevine because there is no effective chemical protection, and after the advancement of this disease there is a decrease in the yield and dying of parts or whole infected trunks, leading to significant losses in production (rolshausen et al., 2015). in the world, there is a limited number of works on testing the susceptibility of different grapevine cultivars to this disease. earlier studies of the susceptibility of different grapevine cultivars were based on apricot research that was very susceptible to e. lata, and very little was known about the colonisation of grapevine tissue by this fungus (carter et al., 1985; munkvold, 2001). for these reasons, péros and berger (1994) examined the aggressiveness of twelve e. lata isolates and the susceptibility of fifteen cultivars of grapevines in their experiments. sosnowski et al. (2007a) studied the virulence of twenty-eight e. lata isolates isolated from the grapevine, originating from australia, and the susceptibility of three cultivars of grapevines cabernet sauvignon, grenache and merlot. travadon et al. (2013) observed the susceptibility of seven commercial grapevine varieties (cabernet franc, cabernet sauvignon, chardonnay, merlot, riesling, shiraz and thompson seedless) as well as the susceptibility of the interspecific hybrid of vitis labrusca variety concord by inoculation of one-year rooted cuttings during the dormant season. comparison of the obtained symptoms in this study with the symptoms of natural infections in serbia, as well as with the descriptions of this phenomenon in literature, points to their exceptional similarity in the appearance and dynamics of development. the pathogenic isolate of e. lata was used in the sensitivity tests on grapevine cultivars, and it has been demonstrated that this species is capable of contaminating the unrooted cuttings of the grapevine and causing the appearance of symptoms characteristic of the dieback of the grapevine. after the inoculation of unrooted cuttings under greenhouse conditions during the dormant season, after five and ten weeks, in the first vegetation cycle, the symptoms occurred. symptoms were observed on inoculated plants as stunted, chlorotic leaves with curved edges, and chlorotic and necrotic patches along the periphery, which later spread over the entire surface, and subsequently caused drying and falling leaves. the shoots were significantly shortened due to slow growth and have a bright green color, often with the so-called zig-zag internodes. symptoms on shoots which appeared on leaves under greenhouse conditions were similar to symptoms found in grapevines in nature. according to the literature data, leaf necrosis appearing on inoculated cuttings is identical to the necrosis of the leaf-induced fungi culture or to the eutypine toxin obtained by extracting leaves from infected grapevine plants from the vineyard (tey-rulh et al., 1991, loc. cit. péros and berger, 1994; mahoney et al., 2005; jimenez-teja et al., 2006; trouillas and gubler, 2010). according to petzold et al. (1981) and mur (1988), the appearance of leaf symptoms is a consequence of toxic products that move from the inoculation point to the point of growth (travadon et al., 2013). the studied e. lata isolate proved to be the most virulent on grapevine cultivars riesling blanc cultivar, banatski muskat, and gamay noir compared to chardonnay. on the other hand, the chardonnay cultivar exhibits statistically significant resistance to the tested isolate. péros and berger (1994) observed the onset of symptoms five, seven and ten weeks after inoculation in the susceptibility of the cultivars. they concluded that the percentage of symptoms occurring during the fifth to seventh week increased by 70% and remained unchanged in the period from the seventh to the tenth week. cultivars are variously susceptible to the toxin that fungus produce and invasion of fungi (rumbos, 1985; travadon et al., 2013). many inoculated cuttings that exhibited symptoms in the first year did not exhibit symptoms in the second year. this is explained by the fact that the number of toxic products can vary and dilute depending on the development or growth of the shoots (péros and berger, 1994, 1999, 2003). also, this phenomenon can be illustrated by the variation in the intensity of the observed symptoms in some cultivars, the ability to begin to develop regular shoots that overlap deformed shoots after the onset of symptoms. foliar symptoms are initially confined to one span of infected vines, however, as the disease progresses symptoms my spread throughout the entire vine (péros and berger, 1994, 1999, 2003; sosnowsli et al., 2007b; živković et al, 2012a, b; pitt et al., 2013; travadon et al., 2013). the artificial inoculation of the grapevine with isolate of e. lata gave preliminary information on the different susceptibility of four grapevine cultivars. namely, by comparing the reactions of four grapevine cultivars to artificial e. lata infections, it was found that there were statistically very significant differences in the response of inoculated cuttings, which indicates the existence of a difference in the level of susceptibility. péros and berger (1994) in their studies suggest that the tested grapevine cultivars exhibited a different susceptibility to the e. lata toxin fungi and its invasion, which reflects on the intensity of the symptoms on their span. also, by analysing the sensitivity of fifteen grapevine cultivars to e. lata, péros and berger (1994, 1999, 2003) biologica nyssana ● 10 (1) september 2019: 29-34 živković et al. ● susceptibility of different grapevine cultivars to eutypa lata isolate, causing agent of eutypa dieback, originating from serbia) 33 found that the cultivars cabernet sauvignon, ugni blanc, and chasla were the most susceptible. a bit lower degree of susceptibility was revealed by the merlot cultivar. travadon et al. (2013) have found in their experiments that the thompson seedless cultivar has exhibited a high degree of susceptibility to the e. lata isolates examined. likewise, there was no significant difference in the expressed susceptibility between the cultivars of cabernet sauvignon and merlot. unfortunately, currently, there are no developed programs for selection of vines for disease resistance in our country. one of the contributions of this is the examination of the reaction of domestic commercial cultivars, as well as cultivars from the environment, to susceptibility to e. lata under conditions of artificial inoculation for the purpose of preventing the production of vineyard. under experimental conditions, investigated isolate was able to cause typical symptoms on the tested grapevine cultivars. conclusion based on the results during the two-year investigation on susceptibility of four cultivars of the grapevines to the inoculation of e. lata, it can be concluded that the cultivars riesling blanc, banatski muskat and gamay noir manifest high susceptibility compared to cultivar chardonnay. all the cultivars inoculated exhibited foliar symptoms of the disease and the other abnormalities. the differences between cultivars were less acute ten weeks after inoculation in first and ten weeks after bud break in second vegetative growth cycle. references bolay, a. and moller, w.j. 1977: eutypa armeniacae hansf. & carter, agent d’un grave dépérissement de vignes en production. revue suisse de viticulture, arboriculture, horticulture, 9: 241-251. carter, m. v., bolay, a., english, h., rumbos, i. 1985: variation in the pathogenicity of eutypa lata (= e. armeniacae). australian journal of botany, 33(3), 361-366. carter, m.v. 1994: wood and root diseases caused by fungi. eutypa dieback. in: r. c. pearson, and a.c. goheen (ed.), compendium of grape diseases, 3rd ed. aps press. st. paul, minnesota: 32-34. carter, m. v., bolay, a., english, h., rumbos, i. 1985: variation in the pathogenicity of eutypa lata (= e. armeniacae). australian journal of botany, 33(3), 361-366. choi, y.w, hyde, k.d., ho, w.w.h. 1999: single spore isolation of fungi. fungal diversity, 3: 29-38. jimenez-teja, d., fernandez-galan, r., gonzalez collado, i. 2006: metabolites from eutypa species that are pathogen on grapes. natural product report, 23: 108-116. mahoney, n., molyneux, r.j., smith, l.r., schoch, t.k., rolshausen, p.e., gubler, w.d. 2005: dying-arm disease in grapevines: diagnosis of infection with eutypa lata by metabolite analysis. agricultural and food chemistry, 53: 8148-8155. mur, g. 1988: les maladies du bois de la vigne. progrés agricole et viticole, 105: 575-577. munkvold, g.p. 2001: eutypa dieback of grapevine and apricot. online. plant health progress, 2(1): 9. munkvold, g.p., duthie, j.a., marois, j.j. 1994: reductions in yield and vegetative growth of grapevines due to eutypa dieback. phytopathology, 84: 186-192. peros, j.p., berger g. 1994: a rapid method to assess the aggressiveness of e. lata isolates and the susceptibility of grapevine cultivars to eutypa dieback. agronomie, 14: 515-523. peros, j.p., berger, g. 1999: diversity within natural progenies of the grapevine dieback fungus eutypa lata. current genetics, 36, 301-309. peros, j.p., berger g. 2003: genetic structure and variation in aggressivness in european and australian populations of the grapevine dieback fungus, eutypa lata. european journal of plant pathology, 109: 909-919. petzoldt, c.h., moller, w.j., sall, m.a. 1981: eutypa dieback of grapevine: seasonal differences in infection and duration of susceptibility of pruning wounds. phytopathology, 71: 540-543. pitt, w.m., trouillas, f.p., gubler, w.d., savocchia, s., sosnowski, m.r. 2013: pathogenicity of diatrypaceous fungi on grapevine in australia. plant disease, 97: 749-756. rolshausen, p.e., sosnowski, m., trouillas, f.p., gubler, w.d. 2015: diseases caused by fungi and oomycetes. eutypa dieback. in w. f. wilcox, w. d gubler, j. k. uyemoto: compendium of grape diseases, disorders, and pests, 2rd ed. aps press. st. paul, minnesota: 57-61. rumbos, i.c.h. 1985: further pathogenicity studies of eutypa lata (=e. armeniacae) on almond. options mediterraneennes, 85(1): 79-89. sosnowski, m.r., lardner, r., wicks, t.j., scott, e.s. 2007a: the influence of grapevine cultivar and biologica nyssana ● 10 (1) september 2019: 29-34 živković et al. ● susceptibility of different grapevine cultivars to eutypa lata isolate, causing agent of eutypa dieback, originating from serbia) isolate of eutypa lata on wood and foliar symptoms. plant disease, 91: 924-931. sosnowski, m.r., shtienberg, d., creaser, m.l., wicks, t.j., lardner, r., scott, e.s. 2007b: the influence of climate on foliar symptoms of eutypa dieback in grapevines. phytopathology, 97: 12841289. sosnowski, m.r. 2016: best practices management guide. eutypa dieback. available at: http: www. barossa.com/uploads/214/20160621 eutypa – dieback – best – practice – management.guide.pdf [accessed 5 june 2017]. travadon r., rolshausen, p.e., gubler, w.d., cadle-davidson l., baumgartner, k. 2013: susceptibility of cultivated and wild vitis sp. to wood 34 infection by fungal trunk pathogens. plant disease, 97: 1529-1536. trouillas, f.p., gubler, w.d. 2010: host range, biological variation, and phylogenetic diversity of eutypa lata in california. phytopathology, 100 (10): 1048-1056. živković, s., vasić, t., anđelković, s., jevremović, d., trkulja, v. 2012a: identification and characterization of eutypa lata on grapevine in serbia. plant disease, 96 (6): 913. živković, s., vasić, t., trkulja, v., krnjaja, v., and marković, j. 2012b: pathogenicity on grapevine and sporulation of eutypa lata isolates originating from serbia. romanian biotechnological letters, 17(3): 7379-7388. biologica nyssana ● 10 (1) september 2019: 29-34 živković et al. ● susceptibility of different grapevine cultivars to eutypa lata isolate, causing agent of eutypa dieback, originating from serbia) jenačković, d. et al.  contribution to the knowledge of distribution… biologica nyssana 6 (2)  december 2015: 59-65 jenačković, d. et al.  contribution to the knowledge of distribution… 59 original article received: 01 august 2015 revised: 12 october 2015 accepted: 01 december 2015 contribution to the knowledge of distribution of certain macrophytes, invasive and threatened species in serbia dragana jenačković*, milica miljković, dragana mitrović, vladimir ranđelović university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia * e-mail: draganaj@pmf.ni.ac.rs abstract: jenačković, d., miljković, m., mitrović, d., ranđelović, v.: contribution to the knowledge of distribution of certain macrophytes, invasive and threatened species in serbia. biologica nyssana, 6 (2), december 2015: 59-65. this paper contains a short report about new and unpublished chorological data for the following species: hydrocharis morsus-ranae l., bidens cernuus l., najas marina l., potamogeton lucens l., schoenoplectus triqueter (l.) palla, cyperus rotundus l., polypogon monspeliensis (l.) desf. and paspalum distichum l.. despite the fact that this paper provides insight into the distribution of analyzed species, it could be used as a base for invasive species’ paspalum distichum l. expanding patterns research, and as a base for reevaluation of the threatened status of schoenoplectus triqueter (l.) palla and cyperus rotundus l. in serbia. key words: macrophytes, invasive and threatened species, serbia, distribution apstrakt: jenačković, d., miljković, m., mitrović, d., ranđelović, v.: prilog poznavanju rasprostranjenja pojednih makrofitskih, invazivnih i ugroženih vrsta u srbiji. biologica nyssana, 6 (2), december 2015: 59-65. u ovom radu dat je pregled novih i nepublikovanih podataka o rasprostranjenu sledećih vrsta: hydrocharis morsus-ranae l., bidens cernuus l., najas marina l., potamogeton lucens l., schoenoplectus triqueter (l.) palla, cyperus rotundus l., polypogon monspeliensis (l.) desf. i paspalum distichum l.. prikazani horološki podaci pružaju kompletniji uvid u rasprostranjenje analiziranih makrofita, predstavljaju osnovu za detaljnija proučavanja puteva introdukcije i širenja invazivne vrste paspalum distichum l. i od velikog su značaja za procenu statusa ugroženosti vrsta schoenoplectus triqueter (l.) palla i cyperus rotundus l. u srbiji. key words: makrofite, invazivne i ugrožene vrste, srbija, rasprostranjenje introduction in the period of 2011-2014 years, floristic and phytocoenological researching of aquatic and swamp vegetation was performed in the region of the southern serbia. on this occasion, new points of distribution for the following species: hydrocharis morsus-ranae l., bidens cernuus l., najas marina l., potamogeton lucens l., schoenoplectus triqueter (l.) palla, cyperus rotundus l., polypogon monspeliensis (l.) desf. and paspalum distichum l. are recorded (fig. 1). unfortunately, in serbia macrophytes are extensively studied in terms of distribution, ecology and coenotic affiliation on almost entire territory of vojvodina province, while in the other parts of country they are sporadically 6 (2) • december 2015: 59-65 biologica nyssana 6 (2)  december 2015: 59-65 jenačković, d. et al.  contribution to the knowledge of distribution… 60 investigated. significant contribution to the collection of the chorological data for the mentioned plants group on the southern area of the sava and danube rivers are given by j o v a n o v i ć (1965), t o p u z o v i ć et al. (2003), r a n đ e l o v i ć et al. (2007), s t a n k o v i ć et al. (2009), r a n đ e l o v i ć & zlatković (2010). material and methods the data about distribution of analyzed plant species in serbia were collected by reviewing relevant literature sources. further, herbarium specimens deposited in the herbarium of the institute of botany and botanical garden “jevremovac”, university of belgrade (beou) and the herbarium of the faculty of sciences and mathematics, department of biology and ecology, university of niš (hmn) were used as a source of distribution data. the new chorological data were obtained during floristic researching which was carried out at the territory of the se serbia. the distribution of studied species is presented on 10 x 10 km2 utm grid. determination of the collected plant materials was performed using dichotomous identification keys (t u t i n et al. (eds.), 1964-1980; j o s i f o v i ć (ed.), 1970-1980). nomenclature of the studied species is consistent with euro+med plantbase (euro+med, 2006-). the threatened status for all researched species is considered in terms of iucn (2000) criteria and categories. results and discussion 1. bidens cernuus l. (compositae) general distribution: europe, siberia, central asia, southeastern asia, japan, central africa, north america and galapagos. distribution in serbia: published literature data points that this species is presented in the following areas: bačka region (babić, 1971; lazić, 2006), banat region (obradović et al., 1977), šumadija region (topuzović et al., 2003), se serbia (ranđelović & stamenković, 1983; ranđelović & zlatković, 2010) and s serbia (jovanović, 2004) (fig. 2). new and unpublished distribution data: smilovci lake (fn57), caricetum ripariae, 715 m, 07.07.2013., coll./det. d. jenačković. habitat description: this plant prefers wet, muddy habitats which are characterized by significant water level fluctuation during growing season. iucn threatened status in the world: least concern (lc). iucn threatened status in serbia: not threatened. 2. najas marina l. (najadaceae) general distribution: most of europe, most of asia (except west asia), indonesia, australia, mauritius, north africa, south and southeastern africa, most of north america (except alaska and canada), middle america, south america (venezuela, brazil and argentina) and hawaii. accordingly, it could be said that najas marina l. belongs to the group of cosmopolitan species. this species is native to europe, asia and africa. in the last century it was recorded in america where it has the status of alien species. distribution in serbia: the distribution pattern of najas marina l. in serbia could be met according to literature and herbarium data (fig. 2). this species is recorded in banat region (slavnić, 1956; butorac, 1995; polić et al., 2004; radulović, 2005; knežević et al., 2008; polić et al., 2008; ljevnaić-mašić, 2010), bačka region (slavnić, 1956; čanak & dokić 1969; obradović et al., 1977; vučković et al., 1998; lazić et al., 2004; lazić, 2006; stojanović et al., 2007), srem region (obradović et al., 1977; vukov et al., 2012), šumadija region (topuzović et al., 2003), e serbia (stanković et al., 2009), w serbia and vicinity of belgrade (blaženčić, 1997). new and unpublished distribution data: debelica village (fp03), potamo-najadetum, 175 m, 20.07.2011., coll./det. d. jenačković; smilovci lake (fn57), potamo-najadetum, 712 m, 05.08.2012., coll./det. d. jenačković. habitat description: najas marina l. shows preferences to fresh water and brackish water. iucn threatened status in the world: least concern (lc). iucn threatened status in serbia: not threatened. 3. potamogeton lucens l. (potamogetonaceae) general distribution: most of europe, most of asia (except central asia), australia, north africa (morocco), tanzania, madagascar, south america (brazil and argentina), bahamas islands and north america (except alaska and canada). distribution in serbia: potamogeton lucens l. is recorded in banat region (polić et al., 2004; radulović, 2005; knežević et al., 2008; polić et al., 2008; ljevnaić-mašić, 2010), bačka region (slavnić, 1956; stojanović et al., 1994; radulović, biologica nyssana 6 (2)  december 2015: 59-65 jenačković, d. et al.  contribution to the knowledge of distribution… 61 2000; lazić et al., 2004; panjković, 2005; lazić, 2006; stojanović et al., 2007), srem region (panjković, 2005; vukov et al., 2012), e serbia (stanković et al., 2009), se serbia (petković, 1983) and vicinity of belgrade (blaženčić, 1997). the first data about distribution of potamogeton lucens l. in the vicinity of niš is given by sava petrović (petrović, s., 1879, beou) (fig. 2). new and unpublished distribution data: vrtište village (ep60), scirpo-phragmitetum subass. typhetosum angustifoliae, 181 m, 02.06.2013., coll./det. d. jenačković; smilovci lake (fn57), potamo-najadetum, 712 m, 05.08.2012., coll./det. d. jenačković. habitat description: potamogeton lucens l. inhabits relatively depth, clear, alkaline and nutrient poor waters. iucn threatened status in the world: least concern (lc). iucn threatened status in serbia: not threatened. fig. 1. the photographs of studied species 4. schoenoplectus triqueter (l.) palla (cyperaceae) general distribution: central and west europe, most of asia (except north asia), africa (sudan and south africa), north america (the united states). schoenoplectus triqueter (l.) palla is considered an autochthonous species to europe and asia while in the other parts of areal it is introduced. distribution in serbia: there is a little data about distribution of schoenoplectus triqueter (l.) palla in serbia. it has been recorded in bačka region (babić, 1971; panjković et al., 2010) and srem region (slavnić, 1956; stanković, 2011) (fig. 2). only one herbarium data about distribution of this species exists for region positioned outside the boundaries of the vojvodina province. it refers to west serbia (zlatibor mountain). new and unpublished distribution data: se serbia: pukovac village (en67), 190 m, 11.09.2014., coll./det. m. miljković. habitat description: this plant grows on wet and muddy habitats which occur at the banks of rivers, marches and the other types of aquatic habitats. iucn threatened status in the world: least concern (lc). iucn threatened status in serbia: critically endangered vulnerable (data deficient) ((cr vu (dd)). biologica nyssana 6 (2)  december 2015: 59-65 jenačković, d. et al.  contribution to the knowledge of distribution… 62 5. cyperus rotundus l. (cyperaceae) general distribution: europe (except north, central and east europe), asia minor, south, central and southeastern asia, indonesia, australia, africa, north america (except alaska and canada), south america and islands in atlantic, indian and pacific ocean. distribution in serbia: the only literature data about distribution of cyperus rotundus l. in serbia are published in “the red data book of flora of serbia 1” (stevanović (ed.), 1999). according to this literature source, cyperus rotundus l. is recorded on the following locations: the velika morava river near ćuprija (ep26), miljkovo (ep19) and the južna morava river near vrtište (ep60). in hmn is stored exemplar of the mentioned species from the village klisura (en68) while beou contains specimens from the sands around the village vrtište (ep60) (fig. 2). new and unpublished distribution data: se serbia: pukovac village (en67), 190 m, 20.08.2013., coll./det. d. jenačković & d. mitrović. habitat description: cyperus rotundus l. grows at sandy habitats which are positioned on the bank of aquatic bodies. iucn threatened status in the world: least concern (lc). iucn threatened status in serbia: endangered – vulnerable (en b2c vu a1c; b1; e). 6. polypogon monspeliensis (l.) desf. (gramineae) general distribution: europe (except central, north and east europe), asia minor, south, central and east asia, australia, new zealand, africa, north america, middle america, west part of south america, islands in atlantic and pacific ocean. thus, polypogon monspeliensis (l.) desf. could be defined as cosmopolitan species which is native to the area of europe, asia and africa while to the other continent it is introduced. distribution in serbia: distribution of polypogon monspeliensis (l.) desf. in serbia is not known (fig. 2). new and unpublished distribution data: se serbia: pukovac village (en67), 190 m, 20.08.2013., coll./det. d. jenačković & d. mitrović. habitat description: polypogon monspeliensis (l.) desf. shows preference to the humid habitats which are placed around swamps, backwaters and depressions. the specificity of this plant is ability to tolerate the higher level of salinity, so that it often occurs in salt marshes. iucn threatened status in the world: least concern (lc). iucn threatened status in serbia: not threatened. 7. paspalum distichum l. (gramineae) general distribution: europe (except north, central and east europe), asia minor, south asia, east asia, australia, africa (except central and east africa), north america (except alaska and canada), middle america, south america, islands in atlantic and pacific ocean. this plant has a status of alien species for the european area. distribution in serbia: paspalum distichum l. is an invasive, emergent plant. according to the published data (stevanović et al., 2004; polić, 2006), this species massively occurred near the danube river between belgrade and đerdap dam (fig. 2). in the southern part of serbia, it is recorded in the vicinity of niš (ranđelović et al., 2007). the other data about distribution are unpublished and they confirm spreading of paspalum distichum l. in the vicinity of belgrade and banat region. ability to displace the autochthonous vegetation makes it a threat to native biological diversity. new and unpublished distribution data: se serbia: klisura village (en68), 11.10.2014., coll./det. m. miljković. habitat description: paspalum distichum l. grows on humid habitats which are often flooded at the beginning of growing season. iucn threatened status in the world: least concern (lc). iucn threatened status in serbia: not threatened. 8. hydrocharis morsus-ranae l. (hydrocharitaceae) general distribution: most of europe, siberia, caucasus, asia minor, middle asia, north africa (morocco and algeria) and madagascar, north america. hydrocharis morsus-ranae l. is native species to europe, asia and africa while it has been introduced to the territory of north america (catling et al., 2003). distribution in serbia: hydrocharis morsus-ranae l. is common species in aquatic and semi-aquatic vegetation of vojvodina province. according to the literature data, this plant is distributed in the banat biologica nyssana 6 (2)  december 2015: 59-65 jenačković, d. et al.  contribution to the knowledge of distribution… 63 fig.2. the utm grid maps of distribution of the researched species region (slavnić, 1956; butorac, 1995; stojanović & lazić, 1998; polić et al., 2004; radulović, 2005; knežević et al., 2008; ljevnaić-mašić, 2010), bačka region (slavnić, 1956; stojanović et al., 1994; stojanović & lazić, 1998; vučković et al., 1998; radulović, 2000; borišev, 2002; lazić, 2003; lazić et al., 2004; nikolić, 2004; panjković, 2005; lazić, 2006; stojanović et al., 2007), srem region (slavnić, 1956; obradović et al., 1977; igić et al., 2004; stanković, 2011; vukov et al., 2012) and the vicinity of belgrade (jovanović, 2011). in beou a few exemplars of the mentioned species are stored and they refer to localities (mala krsna and obrenovac) which are placed in the south of the sava and danube rivers (fig. 2). the first literature data about presence of this species in central serbia is given by pančić (1874). new and unpublished distribution data: vrtište village (ep60), scirpo-phragmitetum subass. phragmitetosum, 185 m, 15.07.2012., coll./det. d. jenačković. habitat description: hydrocharis morsus-ranae l. grows in shallow, mesotrophic, calcareous waters which are protected from direct wave and wind action. iucn threatened status in the world: least concern (lc). iucn threatened status in serbia: not threatened. conclusion the presented results are important to prepare the new edition of flora serbia and red book of flora serbia. particularly significant are data about distribution of species polypogon monspeliensis (l.) desf. which has been not recorded in serbia until to now. further, presence of species paspalum distichum l. on the banks of the južna morava river is significant for tracking the expanding pattern of this invasive species in our country. information about distribution of threatened species: schoenoplectus triqueter (l.) palla and cyperus rotundus l. are also very important. their significance is reflected in reevaluation of the threatened status and implementation of conservation process. biologica nyssana 6 (2)  december 2015: 59-65 jenačković, d. et al.  contribution to the knowledge of distribution… 64 acknowledgements. this study was supported by the project of ministry of science and technological development of republic of serbia (project no. 173030). references babić, n. 1971: močvarna i livadska vegetacija koviljskog rita. zbornik matice srpske za prirodne nauke, 41: 19-87. blaženčić, j. 1997: florističke karakteristike makrofitske vegetacije savskog jezera kod beograda (srbija, jugoslavija). glasnik instituta za botaniku i botaničke bašte univerziteta u beogradu, 29: 167-173. borišev, m. 2002: akvatične makrofite jegričke (temerin-gospođinci). diplomski rad, prirodnomatematički fakultet, univerzitet u novom sadu. novi sad. butorac, b. 1995: review of aquatic vegetation of the regional park "stari begej". tiszcia, 29: 27-32. čanak, m., dokić, m. 1969: naseljavanje osnovne kanalske mreže hidrosistema dunav-tisa-dunav vodenim makrofitama. letopis naučnih radova poljoprivrednog fakulteta, novi sad, 13: 121132. catling, p. m., mitrow, g., haber, e., posluszny, u., charlton, w. a. 2003: the biology of canadian weeds. 124. hydrocharis morsus-ranae l.. canadian journal of plant science, 83 (4): 10011016. euro+med, 2006(continuously updated): euro+med plantbase the information resource for euro-mediterranean plant diversity. published on http://www.emplantbase.org/home.html igić, r., polić, d., borišev, m., janauer, g. a. 2004: aquatic macrophytes of “krstonošića okno” (obedska bara swamp). limnological reports, proceedings of the 35th iad conference, novi sad, 35: 463-468. iucn world commission on protected areas and europark federation 2000. guidelines for protected area management categories. grafenau, brussels. http://www.iucnredlist.org josifović, m. (ed.) 1970-1980: flora sr srbije i-x. sanu, beograd. jovanović, m. 2004: ruderalna flora vranja. acta herbologica, 13 (1): 83-88. jovanović, m. 2011: herbarium collection by sava petrović in the herbarium of the natural history museum in belgrade (serbia). bulletin of the natural history museum in belgrade, 4: 67-118. jovanović, r. 1965: tipologija, ekologija i dinamika močvarne i livadske vegetacije u dolini velike morave. phd thesis. beograd. knežević, a., ljevnaić, b., nikolić, l., džigurski, d., stojanović, s. 2008: weeds in banatska palanka-novi bečej canal (serbia) in 1997-2007. acta herbologica, 17 (1): 119-128. lazić, d. 2003: florističko-fitocenološka proučavanja biljnog sveta vodotoka jegrička. magistarska teza. poljoprivredni fakultet, univerzitet u novom sadu. novi sad. lazić, d., stojanović, s., boža, p., knežević, a., nikolić, l. 2004: vascular macrophytes of the jegrička watercourse spectra of life forms and floristic elements. acta herbologica, 13 (1): 95100. lazić, d. 2006: vaskularna flora i vegetacija okm hs dtd na području bačke stanje i uticaj na korišćenje i održavanje. phd thesis. prirodnomatematički fakultet, univerzitet u novom sadu. novi sad. ljevnaić-mašić, b. 2010: hidrofite osnovne kanalske mreže hidrosistema dtd na području banata. phd thesis. prirodno-matematički fakultet, univerzitet u novom sadu. novi sad. nikolić, lj. 2004: biljni svet, biomasa i primarna produkcija kao pokazatelji eutrofizacije u jezeru provala. phd thesis. prirodno-matematički fakultet, univerzitet u novom sadu. novi sad. obradović, m., budak v., acević, n. 1977: biljnogeografske karakteristike šire okoline obedske bare u južnom sremu. zbornik radova prirodnomatematičkog fakulteta u novom sadu, 7: 191216. pančić, j. 1874: flora kneževine srbije. državna štamparija, beograd. panjković, b. 2005: akvatična i semiakvatična vegetacija apatinskog i monoštorskog rita. phd thesis. prirodno-matematički fakultet, univerzitet u novom sadu. novi sad. panjković, b., perić, r., stojšić, v. 2010: krčedinska ada značajno područje u podunavlju za očuvanje diverziteta flore i vegetacije. 10th symposium on the flora of southeastern serbia and neighbouring regions. petković, b. 1983: močvarna vegetacija na području tutina. glasnik instituta za botaniku i botaničke bašte univerziteta u beogradu, 17: 61-102. polić, d., igić, r., anačkov, g., janauer, g. a., vukov, d. 2004: aquatic vegetation in dubovačka ada oxbow. limnological reports, proceedings of the 35th iad conference, novi sad, 35: 457-462. polić, d. 2006: florističko-fitocenološko proučavanje labudovog okna. biblioteka academia. zadužbina andrejević, beograd. polić, d., igić, r. s., stojanović, s., lazić, d. 2008: the plant communities of classes hydrocharilemnetea oberd. 1967 and potametea tx. et prsg. 1942 of the labudovo okno locality (serbia). biologica nyssana 6 (2)  december 2015: 59-65 jenačković, d. et al.  contribution to the knowledge of distribution… 65 zbornik matice srpske za prirodne nauke, 115: 101-107. radulović, s. 2000: vodena vegetacija koviljskog rita. magistarska teza. novi sad. radulović, s. 2005: ekologija i disribucija akvatičnih fitocenoza carske bare u gis tematskom modelu. phd thesis. prirodno-matematički fakultet, univerzitet u novom sadu. novi sad. ranđelović, n., stamenković, v. 1983: flora i vegetacija okoline vlasotinca. leskovački zbornik, 23: 315-364. ranđelović, v., matejić, j., zlatković, b. 2007: flora i vegetacija batušinačkih bara kod niša. in: proceedings of 9th symposium on flora of southeastern serbia and neighbouring regions, 19-40. ranđelović, v., zlatković, b. 2010: flora i vegetacija vlasinske visoravni. prirodno-matematički fakultet, univerzitet u nišu. niš. slavnić, ž. 1956: vodena i barska vegetacija vojvodine. zbornik matice srpske za prirodne nauke, 10: 1-72. stanković, m., 2011: rare, threatened and relict species in flora of snr zasavica. biologica nyssana, 2 (1): 77-81. stanković, ž., borišev, m., simić, s., vučković, m., igić, r., vidović, m., miljanović, b. 2009: macrophytes of the grlište reservoir (serbia): fifteen years after its establishment. archives of biological sciences, 61 (2): 267-278. stevanović v. (ed.) 1999: crvena knjiga flore srbije 1 – iščezli i krajnje ugroženi taksoni. ministarstvo za životnu sredinu republike srbije, biološki fakultet univerziteta u beogradu, zavod za zaštitu prirode republike srbije, beograd. stevanović, v., šinžar-sekulić, j., stevanović, b. 2004: expansion of the adventive species paspalum paspaloides (michx.) schribner, echinochloa oryzoides (ard.) fritsch and cyperus strigosus l. in the yugoslav part of the danube reservoir (km 1090-1075). limnological reports, proceedings of the 35th iad conference, novi sad, 35: 399-406. stojanović, s., butorac, b., vučković, m., stanković, ž., žderić, m., kilibarda, p., radak, lj. 1994: biljni svet kanala vrbas-bezdan. prirodnomatematički fakultet, univerzitet u novom sadu. novi sad. stojanović, s., lazić, d. 1998: pregled vaskularne flore tamiša sa flornim elementima i životnim formama. naš tamiš, naučna monografija, prirodno-matematički fakultet, univerzitet u novom sadu, 75-87. stojanović, s., lazić, d., knežević, a., nikolić, l., škorić, m., kilibarda, p., bugarski, r. 2007: flora i vegetacija osnovne kanalske mreže hs dtd u bačkoj. novi sad, poljoprivredni fakultet jvp vode vojvodine. topuzović, m., pavlović, d., ostojić, a. 2003: comparative study of the hydrophilic flora of two reservoirs in the šumadija region (serbia). ekologija, 38 (1-2): 13-24. tutin, t. g., heywood, v. h., burges, n. a., moore, d. m., valentine, d. h., walters, s. m., webb, d. a. (eds.), 1964-1980. flora europaea, i-v. cambridge university press, london. vučković, m., stojanović, s., stanković, ž., žderić, m., radulović, s. 1998: floristical and phytocoenological characteristics of canal network of danube-tisza-danube hydrosystem in bačka. ekologija, 33: 71-76. vukov, d., igić, r., rućando, m., radulović, s. 2012: diversity of vascular hydrophytes in the zasavica river (serbia): changes after thirteen years. archives of biological sciences, 64 (4): 1607-1617. biologica nyssana 6 (2)  december 2015: 59-65 jenačković, d. et al.  contribution to the knowledge of distribution… 66 anticancer compounds from medicinal plants biologica nyssana 5 (1)  september 2014: 37-46 joković, n. et al.  screening of lactic acid bacteria isolated … 37 original article received: 18 june 2014 revised: 25 august 2014 accepted: 10 september 2014 screening of lactic acid bacteria isolated from serbian kajmak for use in starter cultures nataša joković 1 *, jelena rajković 1 , katarina veljović 2 , maja tolinački 2 , ljubiša topisirović 2 1 department of biology and ecology, faculty of science and mathematics, university of nis, visegradska 33, 18000 nis, serbia. 2 institute of molecular genetics and genetic engineering, university of belgrade, vojvode stepe 444a, p.o. box 23, 11010 belgrade, serbia. * e-mail: jole35@open.telekom.rs abstract: joković, n., rajković, j., veljović, k., tolinački, m., topisirović, lj.: screening of lactic acid bacteria isolated from serbian kajmak for use in starter cultures. biologica nyssana, 5 (1), septmeber 2014: 37-46. one hundred and seventy eight isolates of lactic acid bacteria (lab) were isolated by pour plate and enrichment techniques from a sample of milk used for kajmak production and three samples of kajmak from one month ripening period. the identification of isolates was performed by phenotypic characterization followed by molecular identification using (gtg)5-pcr and sequence analysis of 16s rrna gene. isolates belonged to lactococcus lactis and enterococcus faecium were found in milk and kajmak samples while leuconostoc mesenteroides and enterococcus durans were the most frequently isolated species from kajmak samples. streptococcus thermophilus were isolated from milk sample only with enrichment technique. further characterization of lab isolates was done for technological properties which are important for industrial application of lab. strains of lc. lactis and s. thermophilus that showed very good acidification and proteolityc activities and l. mesenteroides strains that metabolized citrate can be used in development of starter cultures for eventual industrial production of kajmak. additionally, producers of antimicrobial compounds belonged to lc. lactis subsp. lactis biovar. diacetylactis can be used for control of undesirable microflora in kajmak production. key words: kajmak, lactic acid bacteria, technological characterization apstract: joković, n., rajković, j., veljović, k., tolinački, m., topisirović, lj.: ispitivanje bakterija mlečne kiseline izolovanih iz srpskog kajmaka za upotrebu u starter kulturama. biologica nyssana, 5 (1), septmeber 2014: 37-46. sto i sedamdeset osam izolata bakterija mlečne kiseline (bmk) izolovano je metodom izlivanja ploča i tehnikom obogaćivanja iz uzorka mleka korišćenog za proizvodnju kajmaka i tri uzorka kajmaka starosti do jednog meseca. preliminarna identifikacija izolata rađena je fenotipskim metodama dok su molekulrne metode identifikacije, pcr sa (gtg)5 prajmrom i sekvenciranje 16s rrnk gena, korišćene za identifikaciju izolata do nivoa vrste. izolati identifikovani kao lactococcus lactis i enterococcus faecium izolovani su iz uzoraka mleka i kajmaka dok su vrste leuconostoc mesenteroides i enterococcus durans najčešće izolovane vrste iz uzoraka kajmaka. streptococcus thermophilus izolati dobijeni su iz uzorka mleka samo tehnikom 5 (1) • september 2014: 37-46 biologica nyssana 5 (1)  september 2014: 37-46 joković, n. et al.  screening of lactic acid bacteria isolated … 38 obogaćivanja. dalja karakterizacija bmk izolata rađena je na osnovu tehnoloških svojstava koja su značajna za industrijsku primenu bmk. sojevi lc. lactis i s. thermophilus koji su imali veoma dobru sposobnost acidifikacije i proteolitičku aktivnost kao i l. mesenteroides sojevi sa sposobnošću metabolizma citrata mogu se upotrebiti za razvoj starter kultura za industrijsku proizvodnju kajmaka. osim toga, izolati identifikovani kao lc. lactis subsp. lactis biovar. diacetilactis koji sintetišu antimikrobna jedinjenja mogu se koristiti za kontrolu nepoželjne mikroflore u proizvodnji kajmaka. ključne reči: kajmak, bakterije mlečne kiseline, tehnološka karakterizacija izolata introduction kajmak is a delicious artisanal dairy product of serbia and neighboring balkan countries. it is made by fermentation of milk fat that rises on the top of cooked milk after slowly cooling. thin layer of aggregated milk fats and proteins is then salted and placed layer by layer in special wooden vessels where ripening takes place. kajmak can be consumed as a fresh up to 7 days of fermentation, or mature kajmak that can be aged for several months. in the most of the dairy products, lactic acid bacteria (lab) are the dominant microbial population which metabolic products play crucial role in formation of the taste, smell, texture and quality of final product (w o u t e r s et al., 2002). in a previous work, we have shown that the most ubiquitous lab isolated from kajmak samples belong to genera leuconostoc, enterococcus, lactobacillus and lactococcus (j o k o v i ć et al., 2008). in serbia, kajmak is made at farmhouse level in central and west part of the country and the sale is mainly limited to the open markets. production of kajmak is always based on non-standardized traditional techniques without addition of starter cultures. therefore, the fermentation process depends entirely on the natural microbial flora that originates from the milk and from the environment. consequently, final products are sensitive to contamination and spoilage and have variable quality, poor hygiene and doubtful safety. on the other hand, some attempts for industrial production of kajmak did not succeed because sensory characteristics of final products were not the same as in original products from households (p u d j a e t a l . , 2008). in modern dairy industry, the main approach to obtain traditional dairy products with improved and reproducible qualities is usage of defined commercial starter cultures made with strains isolated from original products (c h a m m a s et al., 2006). these strains are best adapted on food substrate and their metabolic activities contribute to evolution of typical and unique organoleptic characteristics of traditional products. the searching for strains that can be used in construction of starter cultures includes screening of a large number of isolates selected from naturally occurring processes in term of their technological capabilities that are relevant for obtaining more quality product (a s t e r i et al., 2009). the aim of the present study was screening of lab isolates that were predominant in the early phases of kajmak ripening. for that purpose, isolation and identification of lab from the milk used for manufacturing of kajmak and samples of resulting kajmaks in the first period of ripening were performed. additionally, the technological properties of obtained isolates were characterized. this information could make possible selection of strains that could be eventually used in development of starter cultures for the industrial production of kajmak. material and methods bacterial strains, media, and growth conditions bacterial strains used in this study are presented in table 1. mrs broth (ph 5.7) (merck, gmbh, darmstadt, germany) was used for growth of leuconostoc and lactobacillus strains whereas lactococcus, streptococcus and enterococcus strains were cultured in m17 broth (ph 7.2) (merck, gmbh, darmstadt, germany) supplemented with glucose (0.5%, w/v; gm17 broth). a solid medium was prepared by adding agar (2% w/v; torlak, belgrade, serbia) to each medium. the inoculated media were incubated overnight at appropriate temperatures depending on the strain. manufacture and sampling of kajmak one batch of kajmak was manufactured from cows’ milk in a household in the western part of serbia according to traditional procedures without addition of starter cultures. the household was selected as their kajmak had high quality properties. raw milk was cooked to boiling, with occasional stirring to prevent burnt. then, hot milk was poured into the open wooden shallow vessels that were placed on the shelves in the special room for producing kajmak called “creamery”. the next biologica nyssana 5 (1)  september 2014: 37-46 kamberović, j. et al.  marshland vegetation of the order phragmitetalia … 39 table 1. list of reference strains used bacterial strains lactococcus lactis np45 a  lactococcus lactis ssp. cremoris ns1 a lactococcus lactis subsp. lactis bgmn1-596 a lactococcus lactis subsp. lactis biovar. diacetylactis s50 a enterococcus faecium bggj8-3* enterococcus durans bgzls20-35b* streptococcus thermophilus bgdk1-4a lactobacillus paracasei subsp. paracasei bgbuk2-16/k4 a lactobacillus plantarum a112 a leuconostoc mesenteroides ssp. mesenteroides nrrl b-512 leuconostoc mesenteroides ssp. mesenteroides nrrl b-3470 a -strains used in antimicrobial activity assay *strains identified by molecular methods in the laboratorium voor microbiologie, universitet gent, gent, belgium nrrl – agricultural research service culture collection, peoria, il, usa 12 h milk in vessels was cooling at room temperature with a gradual separation of cream on the surface of the milk. afterwards, the formed layer of fats and proteins was carefully skimmed with perforated spoon, drained and placed in the wooden tub. salt was added between the layers from different vessels. the tub was placed in the creamery where ripening took place for one month. sample of milk immediately after boiling, designated as bgnk1, and the samples of kajmak after 5, 15 and 30 days of ripening (designated as bgnk5, bgnk15 and bgnk30, respectively) were collected for microbiological analyses. the collected samples of milk and kajmak were kept in sterile bags at 4°c until the analyses which were performed within the following 12 h. isolation of lactic acid bacteria (lab) the isolation of lab was performed by pour plate and enrichment techniques. in the pour plate technique, the samples (10 g or 10 ml) were homogenized in 90 ml of sterile 2% (w/v) sodium citrate solution (ph 7.5) preheated to 45°c. decimal dilutions of the homogenates were prepared with 0.85% (w/v) sterile saline and were plated on suitable media for isolation of lab: m17 agar (merck, gmbh, darmstadt, germany) supplemented with glucose (gm17), mrs agar (merck, gmbh, darmstadt, germany) and mayeux, sandine, elliker (mse) agar (mayeux et al., 1962). the plates were incubated at 30°c and 45°c for 3 days. after the incubation period, five single colonies were randomly picked from agar plates and streaked on new agar plates for purification. in the enrichment technique, 0.1 ml of the first dilution was transferred into 10 ml of mrs or gm17 broth as well as into 10 ml of 10% skimmed milk. tubes were incubated at 30 o c and 45 o c for 48 h. after the incubation, loopful of grown culture was streaked on mrs and gm17 agar plates and incubated for additional 48 h at appropriate temperatures and under anaerobic conditions (anaerocult a, gaspakmerck, germany). from each plate, few colonies showing different appearance were randomly picked and streaked on new plates in order to obtain pure colonies. phenotypic identification of isolates gram-positive, catalase-negative isolates were grouped and preliminary identified according to the following phenotypic tests: morphology under microscopic examination; the production of gas from glucose in mrs broth lacking beef extract and containing inverted durham tubes; hydrolysis of arginine; hydrolysis of esculin in esculin broth (torlak, belgrade, serbia); the growth in gm17 or mrs broth with 4% and 6.5% (w/v) nacl for 5 days and the growth in 10% skimmed-milk medium. the growth at different temperatures was determined in mrs broth for bacilli and cocobacilli at 15°c and 45°c or in gm17 broth for cocci at 10°c and 45 for 5 days. bile esculin agar (himedia, mubai, india) was used for the presumptive identification of enterococci. biologica nyssana 5 (1)  september 2014: 37-46 joković, n. et al.  screening of lactic acid bacteria isolated … 40 molecular identification of isolates molecular identification of selected isolates was done by rep-pcr method with (gtg)5-primer and by sequencing of 16s rrna gene. the total dna extraction from pure cultures, pcr amplification with (gtg)5-primer, electrophoresis and statistical similarity among fingerprints of the isolates and the reference strains were performed as previously described (j o k o v i ć et al., 2008). sequencing of the 16s rrna gene was done by using total dna as a template for pcr amplifications with u968 (5΄aacgcgaagaaccttac-3΄) and l1401 (5΄gcgtgtgtacaagaccc-3΄) primers (z o e t e n d a l et al., 1998; r a n d a z z o et al., 2002). the amplified fragments were sequenced by macrogen (sequencing service macrogen, seoul, south korea). the sequences were analysed in the ncbi database using blast, the standard nucleotide-nucleotide homology search (http://www.ncbi.nlm.nih.gov/blast). technological characterization of isolates the acidifying activity of isolates was evaluated by measuring the ph of inoculating 10% (w/v) reconstructed skim milk (rsm). overnight lab cultures grown at appropriate temperatures were centrifuged at 5000 rpm for 10 min, washed with peptone water and inoculated (1% v/v) in 10 ml rsm. ph was measured after 6 and 24 h of incubation at appropriate temperature and values were expressed as ph decrease (ph), in relation to the ph values of non-inoculated control milk. the proteolytic activity of the isolates was detected after growing of isolates for 24 h in rsm as described for acidifying activity. after incubation, the proteins from 0.5 ml of the culture were precipitated with trichloroacetic acid. the proteolytic activity of isolates was determined by the o-pa (o-phthaldialdehyde) method of church and associates (1983). the values were calculated from calibration curve with glycin and expressed in mmglyl -1 . citrate utilization was detected on kempler and mckay agar plates (k e m p l e r & m c k a y , 1981). citrate-positive colonies were blue after 48 h of incubation at appropriate temperature. production of diacetyl in milk was determined after a growing of isolates in rsm for 24 h as described above. qualitative determination of diacetyl was done by addition of 0.5 ml of naphthol (1% w/v) and koh (16% w/v) solution in 1 ml of milk culture. after incubation at 30°c for 10 min results were read. appearance of red ring was considered as positive result (k i n g , 1948). lipolytic activity of lab was identified on tributyrin agar plates (merck, gmbh, darmstadt, germany). the plates were incubated for 7 days at appropriate temperatures and observed daily for clear zones around the colonies. production of exopolysaccharides was detected on reconstructed mrs medium containing 100 g l -1 of sucrose, glucose, lactose, maltose or fructose. the plates were incubated anaerobically for 48 h at appropriate temperature and isolates were tested for slime formation using the inoculated loop method. the lab isolates were screened for antimicrobial activity by the agar-well diffusion method (t a g g & m c g i v e n , 1971). different indicator strains used in this study are listed in table 1. the protein nature of antimicrobial compounds was confirmed by adding a crystal of pronase e (sigma chemie gmbh, deisenhofen, germany) near to the edge of the well containing the strains with a potential production of bacteriocins. the plates were incubated overnight at 30°c. the appearance of a clear zone of inhibition around the well, but not near of pronase e crystal, was considered as positive result for possible bacteriocin production results and discussion identification of isolates sensory properties of dairy products manufactured without addition of starter cultures, as kajmak is, entirely depend on the microflora found in milk and the contaminants from the environment (p o z n a n s k i e t a l ., 2004). in order to determine which part of the lab populations derived directly from the milk, 178 gram-positive and catalase-negative isolates were collected from milk used for kajmak production (bgnk1) and kajmak samples bgnk5, bgnk15 and bgnk30. on the basis of physiological tests that were used for the preliminary identification of isolates to the genus level, all isolates were grouped into eight physiological groups (tab. 2). representatives of various physiological groups, a total of 76, were further selected for molecular identification by (gtg)5-pcr fingerprinting. a dendrogram derived by comparing the similarity of digitally reproduced (gtg)5 fingerprints of isolates and reference strains is presented in fig. 1. biologica nyssana 5 (1)  september 2014: 37-46 kamberović, j. et al.  marshland vegetation of the order phragmitetalia … 41 figure 1. dendrogram based on statistical analysis of the (gtg)5–pcr fingerprints. *indicates the isolates that were subjected to sequencing of 16s rdna the results of physiological tests showed that three groups of coccal isolates can be distinguished (tab. 2). based on a dendrogram derived from (gtg)5-pcr fingerprints these isolates were identified as lactococcus lactis, streptococcus thermophilus, enterococcus faecium and enterococcus durans. the (gtg)5-pcr identification was confirmed by sequencing of 16s rdna for two s. thermophilus, three e. durans and one e. faecium isolates. heterofermentative coccoid isolates showing arginine negative phenotype were supposed to belong to genus leuconostoc (tab. 2). these isolates were identified to species level as leuconostoc mesenteroides (fig. 1) as they had similar (gtg)5-pcr fingerprints with leuconostoc mesenteroides reference strains nrrl b-512 and nrrl b-347. three isolates shaped as very short rods were also heterofermentative with the identical (gtg)5 pcr fingerprints (fig. 1). but these biologica nyssana 5 (1)  september 2014: 37-46 joković, n. et al.  screening of lactic acid bacteria isolated … 42 table 2. differentiation of lab isolated from milk and kajmaks samples based on morphological and physiological characteristics, and the identification of representative strains from each group by (gtg)5-pcr and sequencing of 16s rdna cell morphology and genus 1 p ro d u c ti o n o f c o 2 growth on growth in broth with nacl m il k c lo tt in g hydrolysis of b s a 2 n o i so la te s identified by 45 o c 15 o c 10 o c 4% 6.5% arginine esculin (gtg)5-pcr as sequencing of 16s rdna as cocci lactococcus sp. i nd + + + + + 14 lc. lactis (14) 3 nd enterococcus sp. + nd + + + + + + + 53 e. faecium (5) e. durans (14) e. faecium (1) e. durans (3) streptococcus sp. + nd + + 7 s. thermophilus (4) s. thermophilus (2) coccoid leuconostoc sp. i + + nd + + +v + 53 l. mesenteroides (15) nd leuconostoc sp. ii + + nd + +v + 28 l. mesenteroides (13) nd leuconostoc sp. iii + + nd + + +v 13 l. mesenteroides (5) nd leuconostoc sp. iv + + nd + + 7 l. mesenteroides (3) nd rods (very short) weisella sp. + + nd + + + 3 nd (3) w. cibaria (1) + positive; negative; +v reaction positive for some isolates; nd – not determined 1 -classification based on morphological and physiological characteristics 2 -black colonies on bsa (bile esculin agar) 3 -numbers in parenthesis represent the number of isolates that underwent genetic identification 0 10 20 30 40 50 60 70 80 90 100 bgnk1 bgnk5 bgnk15 bgnk30 samples fr e q u e n c y o f is o la ti o n weissella cibaria leuconostoc mesenteroides enterococcus durans enterococcus faecium lactococcus lactis figure 2. distribution of lab species in milk and kajmak samples based on a pour-plate technique isolates were not identified with rep-pcr method because (gtg)5-pcr fingerprints of these isolates were not related to any band pattern of reference strains from our collection. the 16s rdna sequencing result of one representative isolate from this group showed that it belonged to the species weissella cibaria (table 2). the identification of isolates allowed us to describe the distribution of lab in milk (bgnk1) and kajmak samples (bgnk5, bgnk15 and bgnk30). lab species isolated from milk and kajmak samples were different (fig. 2). e. faecium and lc. lactis were only species isolated from milk sample by a pour plate technique, biologica nyssana 5 (1)  september 2014: 37-46 joković, n. et al.  screening of lactic acid bacteria isolated … 43 while s. thermophilus was isolated from the milk by enrichment technique. lc. lactis, e. faecium, and s. thermophilus are commonly isolated species from milk, while the lactobacilli and leuconostocs are always isolated in lower percentage (f r a n c i o s i et al., 2009; g i a n n i n o et al., 2009). on the other hand, l. mesenteroides was the most dominant species in all analyzed kajmak samples. this species was found in high percentage (56%) in 5-day-old sample of kajmak (bgnk5) and its frequency of isolation increased throughout of the ripening (fig. 2). leuconostocs are ubiquitous in nature due to their ability to survive longer on the surface of different materials or during pasteurization (m a r t l e y & c r o w , 1993; o g i e r et al., 2008). since leuconostocs were not isolated from the milk, they probably got into the kajmak from the environment or they were present in the milk in such small numbers that they could not be detected by used isolation techniques. lc. lactis isolates were isolated by pour plate technique from 5-day-old (bgnk5) and 15-day-old samples of kajmaks (bgnk15) although its frequency of isolation was very low and decreased during fermentation (13% and 9%, respectively) (fig. 2). in the 30-day-old kajmak sample (bgnk30) lactococci were isolated only with enrichment technique. the reduction in the number of lactococci is probably the result of a slight salt content and dry matter increase and ph values decrease during the ripening of kajmak (j o k o v i ć et al., 2008). these conditions inhibited the growth of lactococci, which are much more sensitive to adverse conditions prevailing during the ripening of dairy products compared to leuconostocs and enterococci (l o p e z d i a z et al., 2000). two species of enterococcal isolates were detected in kajmak samples. e. faecium isolates detected in milk, were also isolated from 5-day-old kajmak sample (bgnk5) while e. durans isolates were detected in all samples of kajmak and their number increased slightly during ripening (fig. 2). e. durans isolates were not previously detected in the analyzed samples of kajmak (j o k o v i ć et al., 2008) but they were found commonly in milk and different types of cheeses (o g i e r & s e r r o r , 2008). enterococci have a high tolerance to adverse conditions such as salt and acidity during the ripening of dairy products which explains their dominance in many dairy products (m a r i n o et al., 2003). isolates identified as w. cibaria were found in 5-day-old (bgnk5) and 15-day-old samples of kajmak (bgnk15) in low percentage (7% and 3%, respectively). w. cibaria isolates have been detected in foods of various origins, as well as among some clinical isolates of human and animal origin (b j o r k r o t h et al., 2002; o u a d g h i r i et al., 2009). technological characterization of isolates all isolates were tested for technological characteristics important for use of lab in dairy industry. assays included acidifying, proteolityc and lipolytic activity, ability to metabolize citrate and produce diacetyl and production of eps and inhibitory substances. acidifying and proteolityc activity in all dairy products rapid production of lactic acid by lab at the beginning of fermentation is a crucial step for getting products with good sensory and hygienic properties. fast production of lactic acid by lab strains depends on their proteolytic system, ability to metabolize lactose and bacterial resistance to acid stress (g a l i a et al., 2009). therefore, acidifying and proteolityc activities of the isolates from dairy products are very important for their selection in starter cultures. in our study, streptococci showed the highest acidifying activity in milk (fig. 3) and all strains tested clotted skimmed milk after 24 h of incubation. four of them decreased ph of milk for 6 h of incubation to the value around 5.30. lactococcal strains had lower initial values of ph than streptococci but in latter stage of growing in milk some of them enhanced their acidifying activity reaching ph around 4.40 after 24 h incubation period. lc. lactis and s. thermophilus strains isolated from different dairy products, most often, have better activity in milk than other lab isolates (a y a d et al., 2004; b a d i s et al., 2004). enterococcal strains were weak acid producers and no of the isolate could decrease ph of milk under 5.00 after 24 h of growth in the milk. all strains of l. mesenteroides showed low acidifying ability and 30% could not reduce ph of milk at all while strains of w. cibaria practically could not grow in milk (fig. 3). a limited number of lab strains with good activity in milk was detected by analyzing lab isolates from other artisanal dairy products (a s t e r i et al., 2009; m o h a m m e d et al., 2009). the assay for proteolytic activity using the ophthaldialdehyde method showed that lactococci released the highest amount of amino groups in milk with average value of 1.97 mmgly l -1 (fig. 3). s. thermophilus isolates had lower proteolytic activity followed by e. durans and e. faecium isolates. biologica nyssana 5 (1)  september 2014: 37-46 kamberović, j. et al.  marshland vegetation of the order phragmitetalia … 44 figure 3. acidifying and proteolytic activities of isolates the error bars represent the means of standard deviation isolates identified as leuconostocs and weisella showed the lowest level of proteolytic activity (fig. 3). these results were similar to that described by other authors who showed that lactococci had the best proteolytic activity among lab (g o n z a l e z et al., 2010). five of the fourteen strains of lc. lactis with the highest level of proteolytic activity (2.08-2.28 mmglyl -1 ) were the most rapidly acidifying lactoccocal strains. these strains are good candidates for construction of starter cultures for kajmak production. utilization of citrate and production of diacetyl possibility of diacetyl synthesis from citrate is characteristic of certain lab strains included in the composition of starter cultures. diacetyl is one of the most important compounds that affect the aroma of dairy products giving them flavor similar to that of butter (m a d e r a et al., 2003). in this study, six strains of lc. lactis had blue appearance on kempler and mckey agar plates and produced diacetyl in milk after 24 h of incubation. therefore, these isolates were identified as lactococcus lactis subsp. lactis biovar. diacetylactis. the most of enterococcal isolates had ability of diacetyl production (five e. faecium and twenty-five e. durans isolates) but none of them could use citrate on kempler and mckey agar. these results indicate that enterococci might use other substrates for the synthesis of diacetyl, as have been shown for some lactococci (b a r s & y v o n , 2008). on the other hand, thirty four leuconostoc isolates were able to metabolize citrate on kempler and mckey agar plates but diacetyl synthesis in milk was not detected. since leuconostocs produce diacetyl only in medium with lower ph values (g a r a b a l et al., 2008) that had not been detected in this study due to their poor growth in milk, more detailed investigations on this matter are required. lipolytic activity five e. durans and one e. faecium strains showed lipolytic activity when assayed on tributirin agar. these strains were weakly lipolytic as halos around colonies were very small. in general, lab have poor lypolitic activity, but for certain strains it has been shown to hydrolyze mainly triglycerides containing fatty acids with medium length chains (k a t z et al., 2002). lipolytic strains of the genus enterococcus have increased lipase activity compared to the strains of the genera lactococcus and leuconostoc (a s t e r i et al., 2009). production of eps in the dairy industry, lab strains that synthesize eps have effects on the organoleptic properties of the product and use of these strains in functional starter cultures may be significant (l e r o y & d e v u y s t, 2004). among the tested strains, only sixty-seven l. mesenteroides and three w. cibaria isolates produced eps on the mrs medium with the addition of sucrose. antimicrobial activity the most important antimicrobial substances that are produced by lab are bacteriocins due to their possible application in food biopreservation (s i m o v a et al., 2009). results showed that four lc. lactis subsp. lactis biovar. diacetylactis strains biologica nyssana 5 (1)  september 2014: 37-46 joković, n. et al.  screening of lactic acid bacteria isolated … 45 produced bacteriocins with inhibitory effect on the growth of lactococcal indicator strains lc. lactis ssp. cremoris ns1 and lc. lactis subsp. lactis bgmn1596 used in the experiment. lab strains that produce bacteriocins are a part of starter cultures for improving the microbiological quality and safety of dairy products, while the adjunct starters composed of sensitive strains and bacteriocin producer strains lead to increasing in the degree of autolysis and accelerate the ripening (a y a d et al., 2004). conclusion kajmak is a delicatessen dairy product of balkan countries that is not present in the world market because of not standardized production in households that leads to the variability and poor safety of final products. these problems can be overcome by the industrial production of kajmak with the addition of starter cultures composed of the appropriate strains isolated from kajmak samples and well characterized. the results of the present research indicate that among isolated lab a few interesting strains could be used in construction of starter cultures for industrial production of kajmak. four lactococcal strains that showed fast acidification rate and good proteolityc activity as well as rapid acidifying s. thermophilus strains could be used in starter cultures for rapid production of lactic acid at the beginning of kajmak fermentation. strains of lactococcus lactis subsp. lactis biovar. diacetylactis that produce diacetyl and protein antimicrobial substances may have double role, in getting the specific flavor and control of undesirable microflora. among l. mesenteroides isolates, strains that metabolized citrate, also may have important role in the formation of sensory properties of kajmak. acknowledgments. this work was supported by the ministry of education and science of the republic of serbia grant no.: 173019. references asteri, ia., robertson, n., kagkli, dm., andrewes, p., nychas, g., coolbear, t., holland, r., crow, v. & tsakalidou, e. 2009: technological and flavour potential of cultures isolated from traditional greek cheese-a pool of novel species and starters. international dairy journal, 19:595-604. ayad, ehe., nashat, s., el-sadek, n., metwaly, h. & el-soda, m. 2004: selection of wild lactic acid bacteria isolated from traditional egyptian dairy products according to production and technological criteria. food microbiology, 21:715-725. badis, a., guetarni, d., moussa-boudjemaa, b., henni, d.e., tornadijo, m.e. & kihal, m. 2004: identification of cultivable lactic acid bacteria isolated from algerian raw goat’s milk and evaluation of their technological properties. food microbiology, 21:343-349. bars, d. & yvon, m. 2008: formation of diacetyl and acetoin by lactococcus lactis via aspartate catabolism. journal of applied microbiology, 104:171-177. bjorkroth, kj., schillinger, u., geisen, r., weiss, n., hoste, b., holzapfel, wh., korkeala & hj., vandamme, p. 2002: taxonomic study of weissella confusa and description of weissella cibaria sp. nov., detected in food and clinical samples. international journal of systematic and evolutionary microbiology, 52:141-148. chammas, gi., saliba, r., corrieu, g. & béal, c. 2006: characterisation of lactic acid bacteria isolated from fermented milk "laban". international journal of food microbiology, 110:52-61. church, fc., swaisgood, he., porter, dh & catignani, gl. 1983: spectrophotometric assay using o-phthaldialdehyde for determination of proteolysis in milk and isolated milk proteins. journal of dairy science, 66:1219-1227. franciosi, e., settanni, l., cavazza, a. & poznanski, e. 2009: biodiversity and technological potential of wild lactic acid bacteria from raw cows' milk. international dairy journal, 19:3-11. galia, w., perrin, c., genay, m. & dary, a. 2009: variability and molecular typing of streptococcus thermophilus strains displaying different proteolytic and acidifying properties. international dairy journal, 19:89-95. garabal, ji., rodriguez-alonso, p. & centeno, ja. 2008: characterization of lactic acid bacteria isolated from raw cow’s milk cheeses currently produced in galicia (nw spain). lwt-food science and technology, 41:1452-1458. giannino, lm., marzotto, m., dellaglio, f. & feligini, m. 2009: study of microbial diversity in raw milk and fresh curd used for fontina cheese production by culture-independent methods. international journal of food microbiology, 130:188-195. gonzalez, l., sacristan, n., arenas, r., fresno, jm. & tornadijo, me. 2010: enzymatic activity of lactic acid bacteria (with antimicrobial properties) isolated from a traditional spanish cheese. food microbiology, 27:592-597. biologica nyssana 5 (1)  september 2014: 37-46 joković, n. et al.  screening of lactic acid bacteria isolated … 46 joković, n., nikolić, m., begović, j., jovčić, b., savić, d. & topisirović, lj. 2008: a survey of the lactic acid bacteria isolated from serbian artisanal dairy product kajmak. international journal of food microbiology, 127:305-311. katz, m., medina, r., gonzalez, s. & oliver, g. 2002: esterolytic and lipolytic activities of lactic acid bacteria isolated from ewe's milk and cheese. journal of food protection, 65:1997-2001. kempler, mg. & mckay, ll. 1981: biochemistry and genetics of citrate utilization in streptococcus lactis ssp. diacetylactis. journal of dairy science, 64:1527-1539.king, n. 1948: modification of voges-proskauer test for rapid colorimetric determination of acetyl methyl carbinol plus diacetyl in butter. dairy industry, 13:860-866. leroy, f. & de vuyst, l. 2004: lactic acid bacteria as functional starter cultures for the food fermentation industry. trends in food science and technology, 15: 67-78. lopez-diaz, tm., alonso, c., roman, c., garcialopez, ml. & moreno, b. 2000: lactic acid bacteria isolated from a handmade blue cheese. food microbiology, 17:23-32. madera, c., garcia, p., janzen, t., rodriquez, a. & suarez, je. 2003: characterisation of technologically proficient wild lactococcus lactis strains resistant to phage infection. international journal of food microbiology, 86:213-222. marino, m., maifreni, m. & rondinini, g. 2003: microbiological characterization of artisanal montasio cheese: analysis of its indigenous lactic acid bacteria. fems microbiology letters, 229:133-140. martley, fg. & crow, vl. 1993: interactions between non-starter microorganisms during cheese manufacture and ripening. international dairy journal, 63:461-483. mayeux, jv., sandine, we. & elliker, pr. 1962: a selective medium for detecting leuconostoc organisms in mixed-strain starter cultures. journal of dairy science, 45:655. mohammed, m., abd el-aziz, h., omran, n., anwar, s., awad, s. & el-soda, m. 2009: reppcr characterization and biochemical selection of lactic acid bacteria isolated from the delta area of egypt. international journal of food microbiology, 128:417-423. ogier, jc., casalta, e., farrokh, c. & saihi, a. 2008: safety assessment of dairy microorganisms: the leuconostoc genus. international journal of food microbiology, 126:286-290. ogier, jc. & serror, p. 2008: safety assessment of dairy microorganisms: the enterococcus genus. international journal of food microbiology, 126:291-301. ouadghiri, m., vancanneyt, m., vandamme, p., naser, s., gevers, d., lefebvre, k., swings & j., amar, m. 2009: identification of lactic acid bacteria in moroccan raw milk and traditionally fermented skimmed milk 'lben'. journal of applied microbiology, 106:486-495. poznanski, e., cavazza, a., cappa, f. & cocconcelli, ps. 2004: indigenous raw milk microbiota influences the bacterial development in traditional cheese from alpine natural park. international journal of food microbiology, 92:141-151. pudja, p., djerovski, j. & radovanović, m. 2008: an autochthonous serbian product-kajmak characteristics and production procedures. dairy science and technology, 88:163-172. randazzo, cl., torriani, s., akkermans, adl., de vos, wm. & vaughan, ee. 2002: diversity, dynamics, and activity of bacterial communities during production of an artisanal sicilian cheese as evaluated by 16s rrna analysis. applied and environment microbiology, 68:1882-1892. simova, ed., beshkova, db. & dimitrov, zp. 2009: characterization and antimicrobial spectrum of bacteriocins produced by lactic acid bacteria isolated from traditional bulgarian dairy products. journal of applied microbiology, 106: 692-701. tagg, jr. & mc given, ar. 1971: assay system for bacteriocins. applied microbiology, 21: 943. wouters, jtm., ayad, ehe., hugenholtz, j. & smit, g. 2002: microbes from raw milk for fermented dairy products. international dairy journal, 12:391-109. zoetendal, eg., akkermans, adl. & de vos, wm. 1998: temperature gradient gel electrophoresis analysis of 16s rrna for human fecal samples reveals stable and host-specific communities of active bacteria. applied and environment microbiology, 64:3854-3859. vitkova, a., balabanova, v.: trials on the introduction and cultivation of arnica montana l. (asteraceae) in bulgaria. biologica nyssana, 9 (1). september, 2018: 21-29. biologica nyssana 9 (1) ⚫ september 2018: 21-29 vitkova, balabanova ⚫ trials on the introduction and cultivation of arnica… 21 original article received: 15 mart 2018 revised: 29 june 2018 accepted: 22 august 2018 trials on the introduction and cultivation of arnica montana l. (asteraceae) in bulgaria antonina vitkova1, vessela balabanova2 1institute of biodiversity and ecosystem research, department of plant and fungal diversity and resources, bulgarian academy of science, acad. g. bonchev str., bl. 23, 1113 sofia, bulgaria 2department of pharmacognosy, faculty of pharmacy, medical university sofia, dunav str. 2, 1000 sofia, bulgaria * e-mail: vitkova17@abv.bg abstract: vitkova, a., balabanova, v.: trials on the introduction and cultivation of arnica montana l. (asteraceae) in bulgaria. biologica nyssana, 9 (1). september, 2018: 21-29. the aim of the present study was to consider the possibilities for introduction and sustainable cultivation of arnica montana in bulgaria. the research was conducted on two experimental fields in different floristic regions of the country at 1 400-1 500 m altitude. the experiment was conducted with seeds of two origins: a natural population from the carpathians (ukraine) and the variety "arbo" (germany). comparison between in vivo and in vitro propagated plants under field conditions was carried out. during four vegetation seasons the growth and development of the plants were monitored and the survival rate was evaluated. there were no significant morphological differences between the plants of both studied origins while in vivo and in vitro propagated plants had high parameters variation. the obtained results proved the possibility of sustainable cultivation of a. montana in the high-mountain regions of bulgaria. key words: arnica montana, bulgaria, field cultivation, medicinal plant apstrakt: vitkova, a., balabanova, v.: ispitivanja introdukcije i kultivacije vrste arnica montana l. (asteraceae) u bugarskoj. biologica nyssana, 9 (1). septembar, 2018: 21-29. cilj ovog istraživanja bio je da se razmotre mogućnosti introdukcije i održive kultivacije vrste arnica montana u bugarskoj. istraživanje je bilo izvedeno na dva ogledna polja koja su pripadala različitim florističkim regionima na nadmorskoj visini od 1400-1500 m. eksperiment je izveden sa semenjem čije je poreklo bilo iz dva različita izvora: prirodna populacija sa karpata (ukrajina) i varijetet „arbo“ (nemačka). izvršeno je poređenje in vivo i in vitro propagiranih biljaka u terenskim uslovima. tokom četiri vegetacione sezone, praćeni su rast i razvoj biljaka i procenjena je stopa preživljavanja. nije bilo značajnih morfoloških razlika između biljaka različitog porekla, dok su in vivo i in vitro propagirane biljke imale visoke varijacije parametara. 9 (1) • september 2018: 21-29 doi: 10.5281/zenodo.1470844 biologica nyssana 9 (1) ⚫ september 2018: 21-29 vitkova, balabanova ⚫ trials on the introduction and cultivation of arnica… 22 dobijeni rezultati dokazali su mogućnost održive kultivacije a. montana u visoko planinskim regionima bugarske. ključne reči: arnica montana, bugarska, kultivacija na oglednim poljima, lekovita biljka introduction arnica montana l. (asteraceae) is a valuable medicinal plant widely used in the phytotherapy in the form of numerous preparations (merfort, 2002). the pharmaceutical industry uses the herbal drug arnicae flos (arnica flower heads) (european pharmacopoeia, 2011) that contains sesquiterpene lactones, flavonoids and phenolic acids (lyss et al., 1998; nikolova et al., 2013; todorova et al., 2016) and it is applicable in the production of creams and lotions for muscles and bone injuries as well as in the treatment of hematomas (klaas et al., 2002). the studied species is native to europe. it is distributed from southern norway and latvia, southward to portugal, east across europe to the north apennines in italy and south carpathians in romania (ferguson, 1976; smallfield & douglas, 2008). investigations have been carried out on the influence of environmental factors on plant material quality from natural populations of a. montana (ucenic & mastorakis, 2007), the effect of altitude on the phenolic content in arnica flower heads (spitaler et al., 2008) and also on the presence of mycorrhiza under field conditions (ryszka et al., 2010). the results of the attempts to cultivate mountain arnica in some european countries could help to find appropriate environmental conditions for its sustainable cultivation. such studies have already been made in germany (weyel, 1989; bomme et al., 1995), poland (jurkiewicz et al., 2010; sugier et al., 2013), switzerland (delabays & mangel, 1991), italy (aiello et al., 2012), finland (galambosi, 2004) and serbia (plevljakušić et al., 2014). in bulgaria, experiences on in vivo and in vitro propagation of a. montana have been conducted by balabanova & vitkova (2010), evstatieva et al. (2012) and balabanova et al. (2013). a. montana is not native in the bulgarian flora. however, more than 100 years ago, this species has been found in the subalpine zone in the rila mt. at 1 800 m a.s.l. and a specimen was deposited in the herbarium of sofia university "st. kliment ohridski" (so 86331). the main purposes of the present study were: to establish an approach for cultivation of arnica montana in bulgaria; to find regions in the country with appropriate environmental conditions for its sustainable cultivation; to make a comparison of in vivo and in vitro propagated plants grown in different regions in bulgaria on the basis of their biological features. material and methods our preliminary studies have been conducted on fields in the region sofia (544 m a.s.l.) and the town of pirdop (696 m a.s.l.). the obtained results showed that the climatic conditions at these altitudes are not suitable for successful cultivation of a. montana in bulgaria (tab. 1). after this conclusion we started to look for mountain areas with appropriate table 1. experimental field environmental conditions experimental fields ecological characteristic vitosha mt. sofia city western rhodopes mts. pirdop city gps coordinates n 42o36´ e 23o14´ n 42o41´ e 23o20´ n 41o50´ e 24o07´ n 42°42´ e 24°11´ altitude [m] 1 404 544 1 500 696 exposition sw sw nw s slope [o] 8 1 3 1 soil type brown mountain-forest alluvial-diluvial meadow brown mountain-forest cinnamon forest area [m2] 30 10 30 20 n [%] 0.459 0.147 p2o [mg/100 g] 4.10 19.83 k2o [ mg/100 g] 7.8 16.6 ph 6.1 6.5 humus % 7.02 2.84 biologica nyssana 9 (1) ⚫ september 2018: 21-29 vitkova, balabanova ⚫ trials on the introduction and cultivation of arnica… 23 environmental conditions where the study could be realized. the field experiments were carried out during the period 2009-2013 at two experimental fields of the institute of biodiversity and ecosystem research, bulgarian academy of science: “beglika” locality (western rhodope mts.) at 1500 m a.s.l. and “zlatni mostove” locality (vitosha mt.) at 1400 m a.s.l. the experimental fields are located in the european continental climatic region of bulgaria (subev & stanev, 1963). the experimental fields' environmental characteristics are presented in tab. 1. the soil analysis was carried out at the laboratory of soil analysis at the university of forestry, sofia, bulgaria. the soil type is determined according to ninov (2002). the data for the average temperature and precipitation for the experimental fields were obtained by national hydrometeorological institute, bulgarian academy of science, sofia, bulgaria. the experiment was carried out with seeds (achenes) of two origins: a natural population from the carpathians (ukraine) and a variety "arbo" (germany). comparative study on in vivo and in vitro propagated plants under field conditions was carried out. the field observation on 1-, 2and 3-year old plants was conducted through 2011-2013 growing seasons. in vivo seedlings were produced from seeds of both origins under greenhouse conditions following the smallfield and douglas (2008) methodology. in december 2010, dark and mature achenes were sown in plastic plates with 50 jacks, each 5 cm in diameter, in commercial peat substrate "tera mix" and pine bark (1:1) (fig. 1). thereafter, the soil was slightly pressed in order to improve its close contact with the seeds. the seeds germinated after 10-12 days at 1820 °c. when the plants formed six leaves, they were put into individual pots (d =12 cm) with the above mentioned substrate. we received the in vitro propagated plants bred by assoc. prof. ely zayova from the institute of plant physiology and genetics, bulgarian academy of sciences, sofia, bulgaria. the seedlings were planted in coconut sawdust (noramix group, sri lanka) and their adaptation was conducted under greenhouse conditions. two months later, the seedlings were planted into individual pots (d=12 cm) in peat substrate with natural zeolite (tera mix, bulgaria) and pine bark (1:1). the in vitro plants were included in the study to determine the possibility of plants growing associated with a future selection based on biotechnological breeding approaches. in may 2011, six-month-old in vivo and in vitro plants were transferred to the experimental fields of “beglika” and “zlatni mostove” localities. the seedlings were transplanted in 30 x 40 cm distance, thus ensuring a plant density of 10-12 plant/m2 according to a previous research (sugier et al., 2013). further, the cultivation model should be adjusted for field plantation and the seedlings should be planted in 70 x 30 cm pattern due to the mechanical maintenance. the experimental replication was made up of 46 plants. experimental complete plot consisted of two and three replications for in vitro and in vivo propagated plants, respectively (each replication of 3.8 m2). the areas were kept clean by hand hoeing and a regular irrigation was made. direct seed sowing on the field was conducted in the beginning of april and mid-september. the seeds were sown in shallow furrows in the soil which were at a distance of 30 cm in depth of 4-5 cm. the furrows were filled with a mixture of peat substrate with natural zeolite (tera mix, bulgaria) and pine bark (1:1). after sowing, the soil was treated with a wooden roller and the areas were regularly moistened. the phenological observations were carried out following beydeman (1974). the plant development was monitored throughout 5 phenophases and the duration of each phase was recorded (tab. 2). table 2. duration of phenophases in a. montana development under field conditions experimental fields duration of phenophase (days) vitosha mt. rhodopes mts. vegetative 18 20 flower buds formation 20 16 full flower 30 24 ripe fruit 19 28 end of vegetation 60 55 total duration of vegetation 147 143 in order to establish the plant morphometric variability due to the seed origin, in vivo and in vitro seedlings production and the environmental conditions in the regions of plants cultivation, 8 parameters were examined on 3-year-old plants, as follows: rosette diameter stem height; affiliated rosettes number; leaves number per a rosette; length and width of rosette leaf; flower head diameter; flower heads number per plant. morphometrical measurements were performed on 3-year-old plants during the full flowering stage. the obtained data were statistically calculated by medcalc program, biologica nyssana 9 (1) ⚫ september 2018: 21-29 vitkova, balabanova ⚫ trials on the introduction and cultivation of arnica… 24 expressed by average value (m) and standard deviation (sd), and the differences of the parameters variation were shown (p<0.1%) (tab. 3). results and discussion plants development in greenhouse and adaption to field conditions in vivo propagated plants the present study proved that the seeds from both origins natural population (ukraine) and variety ”arbo” (germany) germinated jointly 10 12 days after sowing under greenhouse conditions and the germination reached a rate up to 70 80%. during the first two months, the seedlings formed roots (5-6 cm) and 4 small leaves (fig.1). six-month-old plants had rosettes with diameter of 10.00 – 12.00 cm consisting of 8-10 leaves. kating & seidel (1967) reported that the nursery stage is critical for arnica cultivation, which prompted us to be careful during this period of plant growing. in vitro propagated plants two-month-old in vitro plants were transferred from the coconut sawdust into a mixture of commercial (tera mix, bulgaria) and pine bark (1:1) substrate. after the transplanting, some of the plants failed to adapt and 15-20% of them died. the surviving plants had a successful adaptation and developed well in the greenhouse conditions during the next 4 months. the 6-month-old plants with well-formed roots and a rosette of leaves with diameter of 10.00-12.00 cm were transferred to the fields. direct seed sowing on the fields the seeds from the two investigated origins did not germinate on the experimental fields of "beglika" and "zlatni mostove" localities. the literary data on cultivated a. montana by direct seed sowing on the field indicated that the seeds germinated jointly, but the juvenile plants were vulnerable to the climate conditions (sugier et al., 2013). our results proved that this approach is not a reliable method for cultivation of mountain arnica in bulgaria. plant adaptation after transferring to the fields in the middle of may 2011, in vivo and in vitro plants were transferred from greenhouse to the mountain experimental fields. plants have successfully adapted to the new conditions. at the end of the studied period table 3. morphometric characteristic of the plants at vitosha mt. and rhodopes mts. parameter [m±sd] sample rosette diameter plant height rosette number leaves number per rosette leaf length leaf width flower head diameter flower heads number амvu1 28.11±4.19 46.33±5.51 3.56±0.92 7.78±1.67 11.80±1.56 3.60±0.76 6.77±0.59 7.94±4.29 амvu2 24.25±6.27 42.94±7.09 5.75±3.20 8.25±1.28 9.60±2.16 3.44±1.10 5.41±0.48 16.63±12.32 амvg1 25.36±5.50 40.90±4.53 3.64±1.69 8.18±1.89 10.62±1.98 3.57±0.79 6.28±0.64 8.09±5.57 амvg2 22.30±2.05 38.50±4.21 4.60±0.89 8.00±1.41 9.20±1.15 3.45±0.72 5.36±0.31 12.40±8.36 амru1 31.87±5.64 33.91±4.12 8.83±2.23 7.65±0.98 11.72±2.51 2.82±0.51 6.57±0.79 23.00±11.87 амru2 29.50±4.04 30.33±3.62 7.67±2.42 7.67±0.82 11.68±1.93 3.77±0.59 6.12±0.80 20.60±10.53 амrg1 33.41±4.10 32.23±5.95 9.71±2.46 8.23±0.89 12.77±2.70 2.61±0.41 6.56±0.80 24.02±17.93 амrg2 25.80±1.92 28.40±6.54 6.00±1.58 7.60±0.89 10.70±1.35 3.10±0.55 5.88±0.25 13.80±3.49 legend: amvu1 a. montana, ukraine, in vivo plants, vitosha mt.; amvu2 a. montana, ukraine, in vitro plants, vitosha mt.; amvg1 a. montana, germany, in vivo plants, vitosha mt.; amvg2 a. montana, germany, in vitro plants, vitosha mt.; amru1 a. montana, ukraine, in vivo plants, rhodopes mts.; amru2 a. montana, ukraine, in vitro plants, rhodopes mts.; amrg1 a. montana, germany, in vivo plants, rhodopes mts.; amrg2 a. montana, germany, in vitro plants, rhodopes mts. fig.1. two-month-old in vivo seedlings bred under greenhouse conditions biologica nyssana 9 (1) ⚫ september 2018: 21-29 vitkova, balabanova ⚫ trials on the introduction and cultivation of arnica… 25 (2013) in the rhodope mts. (“beglika” locality), 80% in vivo and 60% in vitro plants (variety "arbo") and 65% in vivo and 60% in vitro plants (ukraine) have adapted and completed their full development. regarding vitosha mt. (“zlatni mostove” locality), 75% in vivo and 33% in vitro plants (variety "arbo") and 41% in vivo, 25% in vitro plants (ukraine) have been adapted. these results showed that the percentage of successfully adapted in vivo plants at field conditions was higher than in vitro plants and the best results were established in “beglika” locality. our research found a chlorosis in 5% of the plants at “beglika” locality which had resulted on plant death. this is the evidence that the chlorosis correlates with high phosphorus content in the soil (delabays & mange, 1991). the soil analysis has proven that in the locality “beglika” p2o5 content was 19.83 mg/100 g while on the locality “zlatni mostove” it was significantly lower (4.10 mg/100 g). it was found that the plants are very sensitive to drought stress which was also reported by weyel (1989). this conclusion shows that a regular irrigation of the experimental fields is needed, especially during the driest and warmest months july and august. a drought stress was observed in the plants grown on vitosha mt. this is mainly due to the southwest exposure of the experimental area, which leads to intense and prolonged sunshine during the summer months. to the causes of the drought stress on the plants, the soil drying due to the slope of the terrain (8o) can also be added as well as the inability to keep the precipitations falling. due to the meteorological survey, the average monthly air temperatures at “zlatni mostove” locality during april 2009 october 2013 were as follow: at the beginning of the period the temperatures were slightly above the monthly norms (perennial average). during the years 2010, 2011, 2012 and 2013, the temperatures were close to the monthly rates. according to the precipitation values for the above-mentioned period, a significant fluctuation in the amount was recorded. in the spring and late autumn, the rainfalls were generally in the norm, while in summer and early autumn, the received data were close to the minimum. regarding “beglika” experimental field, the average monthly air temperatures during january 2010 october 2013 were generally close to the above-mentioned perennial averages and the temperatures were considerably high in august september. concerning the precipitation values, the data were more variable compared to the perennial average. in the spring and early autumn, rainfalls were close to the average monthly rate, but were below the norm during the period august october. the data obtained in the study showed that the most suitable period for planting and establishing a mountain arnica field plantation in bulgaria is at the end of may or in early june. in this period, the plants assimilate the spring humidity, temperature is appropriate and the plants adapt to the field conditions. if the planting is done in late autumn (november/december), some plants will fail to accommodate to a sharp drop in temperature, freezing of the soil and a large number of them will not survive. if the planting is done at the early october, the plants adaptation will be successful. vegetative propagation it was found that at the beginning of their development, the plants of mountain arnica grown under greenhouse conditions formed offspring of rosettes. at the end of the first vegetation season, 100% of the plants produced up to 2-3 rosettes. two, three and four-year-old arnica plants had about 3-5, 6 10 and up to 11-25 offspring rosettes per plant, respectively (fig. 2). through a division of offspring rosettes, seedlings which can be planted in the field were obtained. the observations showed that 100% of the plants survived and 40% of them blossomed in the same vegetation season. fig. 2. affiliated rosettes of mountain arnica biologica nyssana 9 (1) ⚫ september 2018: 21-29 vitkova, balabanova ⚫ trials on the introduction and cultivation of arnica… 26 phenological observation of plants grown in different floristic regions the phenological observation is important in the process of plant introduction and cultivation into a new geographical area. this study provides information on the duration of the vegetation season and different phenophases. in the presented research it was found that the growing season on “beglika” and “zlatni mostove” experimental fields lasted 143 and 147 days, respectively and that the plant development passed throughout five phenophases (tab. 2). vegetative phase. the growing season of 2-, 3 and 4-year-old plants began in the second half of april in the two experimental fields. regarding vitosha mt., the plants began to form new leaves in the middle of may and the vegetative phase lasted 18 days, while at the rhodopes mts. that happened 5 days later and the vegetative phase lasted 20-22 days. flower buds. this phase started at the end of may in the “zlatni mostove”locality and in the first half of june in “beglika” locality. this phase ended in the second half of june for the plants on both fields. mountain arnica plants began to bloom in the second growing season as 70% of them formed flower buds. in the third growing season, all plants have flower buds and entered into a generative period. it was found that the plants formed single flower buds until the middle of august. full flowering. the plants in “beglika” locality began to bloom two weeks after the flower buds formation and the phase lasted 14 days (fig. 3b). during the period 1-5 july, all the plants were blossoming and unripe seeds in the flower heads were observed. on the same plant, single flower buds, heads in full flowering stage and blooming heads with unripe achenes were present. the full flowering of the plants at “zlatni mostove” locality started in middle of june and ended in middle of july (fig. 3a). these results showed a slightly longer flowering phase for the plants from this locality compared to “beglika” locality. this is probably mainly due to the climatic features of these two plots. the number of flower stems and flower heads increased during the plant growth and development. concerning 4-yearold plants, the flower stem and flower heads number for one plant was 12-20 (25) and 35-50, respectively (fig. 4). ripe fruits. it was found that after the blossoming of the first flower heads, in the middle of july, the seed formation began at both experimental fields. in the beginning of this period, there were a small number of mature seeds in the flower heads. ripe fruit formation was observed in the first half of august, when 80-90% of the seeds were mature. end of vegetation. this phase began in early september when the stems were dying. leaves that have appeared in the same year usually survived the winter. the dried stems fell off at the end of october. variation of plant morphometric characteristics the data on the morphometrical research showed the following results: the rosette diameter was characterized by a very low to medium variability and a large scattering of the sign; stem height was fig. 3. three-year-old plants in flowering phase at “zlatni mostove“ (a) and “beglika“ (b) experimental fields biologica nyssana 9 (1) ⚫ september 2018: 21-29 vitkova, balabanova ⚫ trials on the introduction and cultivation of arnica… 27 characterized by low to medium volatility and a very large sign scattering; rosette number and inflorescence number per plant were characterized by high to very high variability and a large scattering of the parameters; leaves number per rosette was from low to high variability and relatively large sign dissipation; length/width of rosette leaf was characterized by medium to high volatility and a wide scattering of the signs; flower head diameter was characterized by low volatility and low parameter dissipation. plant morphometric variability according to their geographic origin our study found that statistically proven differences exist on plant height, leaves number in the rosette and flower head diameter (p<0.1%) for the plants of both studied origins (tab. 3). the plants from ukraine, grown on both experimental fields, had higher values of the morphometrical characteristics compared to those of variety “arbo” (germany). morphometrical variability of in vitro and in vivo propagated plants statistically proven differences exist on all studied parameters, except stem height and leaves number per rosette (p<0.1%) (tab. 3). morphometrical variability of the plants depending on the region of cultivation significant differences for the plants grown at both experimental fields for the most parameters were proven except leaves number per rosette and leaf length (p<0.1%) (tab. 3). it was found that plants of the two origins grown at “zlatni mostove” locality had higher stems and larger leaves number per a rosette. higher values for number of the flower heads and flower head diameter were established for the plants at “beglika” locality. the least changing parameter was flower head diameter indicating that it is a conservative feature. the flower heads number per plant is an important characteristic for mountain arnica cultivation and its commercial use. the data proved that number of flower heads was significantly larger for the plants cultivated on “beglika” experimental field. arnicae flos yield regarding our previous study, a higher yield was obtained for the plants originating in ukraine. the highest yield was registered for in vivo propagated plants up to 268 kg/ha (balabanova & vitkova, 2016). the harvest of the two studied origins was relatively similar in the experimental plot on vitosha mt. unlike those grown in rhodope mts. the highest quantity of herbal drug arnicae flos was reported for 5-year-old mountain arnica. conclusion a comparative study on the cultivation of a. montana in two floristic regions in bulgaria was carried out for the first time. the obtained results proved good acclimatization of the seedlings to the experimental fields at altitude of 1 400 m (vitosha mt.) and 1 500 m (rhodopes mts.). the in vivo plants were better adapted than those propagated by in vitro methods. the most important factors for a. montana sustainable cultivation were the climate and soil conditions. for the establishment of field plantation should be preferred flat terrain with northern exposure or a northern component. the soil should be well structured and slightly acidic (ph 6.1-6.5). the plants propagated by seeds passed into generative phase during the second year, while those fig.4. four-year-old plants in flowering phase at “beglika” experimental field biologica nyssana 9 (1) ⚫ september 2018: 21-29 vitkova, balabanova ⚫ trials on the introduction and cultivation of arnica… 28 obtained by dividing of offspring rosettes during the first year. the total duration of mountain arnica growing season was 143-147 days and the plants passed throughout five phenological phases. there were no significant morphological differences between the plants of both studied origins while in vivo and in vitro propagated plants had high parameters variation. the most variable was the parameter flower heads number per plant. the present study proves that a. montana could be cultivated successfully in the high-mountainous regions of bulgaria. acknowledgements. the authors are grateful to prof. alexander tashev from the university of forestry in sofia, bulgaria, who collected the mountain arnica seeds from the natural population in the carpathians, ukraine. special thanks also to assoc. prof. ely zayova from the institute of plant physiology and genetics, bulgarian academy of science, sofia, bulgaria, for breeding and providing the in vitro plants. references aiello, n., scartezzini, f., vender, c. 2012: cultivation trial of arnica montana wild accessions – results of second year. acta horticulturae, 955: 253-257. balabanova, v., vitkova, a. 2010: pecularities in ontogenesis of arnica montana l. in bulgaria. comptes rendus de l , academie bulgare des sciences, 2 (63): 1301-1306. balabanova, v., vitkova, a., zeleva-dimitrova, d. 2013: comparative study of germination and seed antioxidant activity of arnica montana l. and a. chamissonis less. (asteraceaea). comptes rendus de l , academie bulgare des sciences, 66: 12611268. balabanova, v., vitkova, a. 2016: flower yield of arnica sp. cultivated in two florestic regions in bulgaria. journal of agriculture and ecology research, 9, (1):10-16 beydeman, i. 1974: methodology for studying the phenology of plants and plant communities. “science”, novosibirsk. p. 153. boome, u., mittermeier, m., regenhardt, i. 1995: results to develop a procedure for fieldcultivation of arnica montana l. 1 and 2. mitteilung. drogenreport, 8(12): 5-10 & (13): 311. delabays, n., mange, n. 1991: the cultivation of arnica montana l .: agronomic and phytosanitary aspects. swiss review of viticulture, arboriculture and horticulture, 23: 313-319. anonimous, 2011: european pharmacopoeia. 7th ed. strasburg, council of europe (coe). european directorate for the quality of medicines (edqm), strasbourg: council of europe. 10531056. evstatieva, l., todorova, m., petrova, m. 2012: cultivation of arnica montana l. in bulgaria. proceedings of the seventh conference on medicinal and aromatic plants of southeast european countries, 27-31 may, subotica, republic of serbia, 263-266. ferguson, i. k., 1976: arnica l. in: tutin, t. g., heywood, v. h. (eds.). flora europaea, vol. 4. cambridge univ. press, london, 189-190. galambosi, b. 2004: introduction of arnica montana l. in finland. arzneiund gewürzpflanzen, 4: 174-179. jurkiewicz, a., ryszka, p., anielska, t., waligorski, p., bialonska, d., goralska, k., tsimilli-michael, m., turnau, k. 2010: optimization of culture conditions of arnica montana l.: effects of mycorrhizal fungi and competing plant. mycorrhiza, 20: 293-306. kating, h., seidel, f. 1967: cultivation experiments with arnica species. planta medica, 15: 148-258 klaas, ch. a., wagner, g., laufer, st., sosa, s., loggia, r. d., bomme, u., pahl, h. l., merfort, i. 2002: studies on the anti-inflammatory activity of phytopharmaceuticals prepared from arnica flowers. planta medica, 68: 385-391. lyss g., schmidt t., merfoft i., pahl., h.1997: helenalin, an anti-inflammatory sesquiterpene lactone from arnica, selectively inhibits transcription factor nf-kb. biological chemistry, 378: 951-61. nikolova, m., petrova, m., zayova, e., vitkova, a., evstatieva, l. 2013: comparative study of in vitro, ex vitro and in vivo grown plants of arnica montana polyphenols and free radical scavenging activity. acta botanica croatica, 72 (1): 13-22. ninov, n. 2002: soils. in: kopralev, i (ed.). geography of bulgaria. sofia, publ. house “farcom”. pljevljakušič, d., janković, t., jelačić, s., novaković, m., menković, n., beatović, d., dajić-stevanović, z. 2014: morphological and chemical characterization of arnica montana l. under different cultivation models. industrial crops and products, 52: 233-244. ryszka, p., blaszkowski, jurkiewicz, a., turnau, k. 2010: arbuscular mycorrhiza of arnica montana under field condition conventional and molecular studies. mycrorrhiza, 20: 551-557. smallfield, b., douglas, m. 2008: arnica montana: a grower , s guide for commercial production in new zealand, christchurch, new zealand. 15 p. spitaler, r., winkler, a., lins, i., yanar, s., stuppner, h., zidorn, ch. 2008: altitudinal biologica nyssana 9 (1) ⚫ september 2018: 21-29 vitkova, balabanova ⚫ trials on the introduction and cultivation of arnica… 29 variation of phenolic contents in flowering heads of arnica montana cv. arbo: 3-year comparison. journal of chemical ecology, 34: 369-375. subev, l., stanev, s. 1963: climatic regions of bulgaria and their climate. state publishing house for agricultural literature. sofia. 178 p. sugier, d., sugier, p., gawlik-dziki, u. 2013: propagation and introduction of arnica montana l. into cultivation: a step to reduce the pressure on endangered and high -valued medicinal plant species. hindawi publishing corporation. the scientific journal, article id 414363, http://dx.doi.org/10.1155/2013/414363 todorova, m., trendafilova, a., vitkova, a., petrova, m., zayova, e., antonova, d. 2016: developmental and environmental effects of sesquiterpene lactones in cultivated arnica montana l. chemistry and biodiversity, 13 (8): 976-981. ucenic, c., mastorakis, n. 2007: the impact to environmental issues in the supply chain for a natural resource: the case study of arnica montana from romania. proceedings of the 2 nd iasme/wseas international conference on energy & enviroment (ee ' 07), portoroz, slovenia, may 15-17. weyel, a. 1989: improvement of the domestication properties of wild plant populations and selected genotype of arnica montana l. phd thesis, univ. giessen, germany. 135 p. stojičić, d., tošić, s., pavlović, j., golubović, a., simonović, j., zlatković, b.: factors influencing axillary bud induction on nodal segments of micromeria pulegium (rochel) benth. biologica nyssana, 8 (1), september 2017 biologica nyssana 8 (1)  september 2017: 93-98 stojičić, d. et al.  factors influencing axillary bud induction on nodal… 93 original article received: 16 june 2017 revised: 29 june 2017 accepted: 15 july 2017 factors influencing axillary bud induction on nodal segments of micromeria pulegium (rochel) benth. dragana stojičić*, svetlana tošić, jovana pavlović, aleksandra golubović, jelena simonović, bojan zlatković university of niš, faculty of science and mathematics, department of biology and ecology, višegradska 33, niš, serbia * e-mail: draganadstojicic@gmail.com abstract: stojičić, d., tošić, s., pavlović, j., golubović, a., simonović, j., zlatković, b.: factors influencing axillary bud induction on nodal segments of micromeria pulegium (rochel) benth. biologica nyssana, 8 (1), september 2017: 93-98. micromeria pulegium (rochel) benth. is an endemic species from family lamiaceae. plants from this family are characterized by presence of secondary metabolites and antioxidant components. micromeria pulegium contains pulegone which is a potential bio-insecticide and a bio-pesticide. natural populations of this species are so small that there is a need for an alternative way of propagate and proliferation of individuals. method of micropropagation was used with the goal of mass production of plants with the chemical composition of essential oils as similar as possible to that in wild-harvested plants. this paper presents the study on influence of concentration of mineral salts, carbon sources (sucrose and maltose) and nitrogen source (casein hydrolysate) on process of in vitro regeneration of plants through induction of axillary buds on the nodal segments of micromeria pulegium. the greatest number of axillary buds was formed in explants grown on ms culture medium with 3% sucrose and 500 mg/l casein hydrolysate. key words: axillary bud induction, shoot culture, biomass production apstrakt: stojičić, d., tošić, s., pavlović, j., golubović, a., simonović, j., zlatković, b.: faktori koji utiču na indukciju aksilarnih pupoljka na nodalnim segmentima vrste micromeria pulegium (rochel) benth.. biologica nyssana, 8 (1), septembar 2017: 93-98. micromeria pulegium (rochel) benth. je endemična vrsta iz familije lamiaceae. ovu familiju karakteriše prisustvo sekundarnih metabolita i antioksidativnih komponenti. micromeria pulegium sadrži pulegon koji je potencijalni bio-insekticid i biopesticid. prirodne populacije ove vrste imaju mali broj jedinki pa se nameće potreba za pronalaženjem načina njihovog gajenja i umnožavanja. u cilju masovne produkcije biljaka čiji će hemijski sastav etarskih ulja biti najsličniji onome koji sadrže biljke iz prirode primenjena je metoda mikropropagacije. u ovom radu ispitan je uticaj jačine mineralnih soli, izvora ugljenika (saharoze i maltoze) i izvora azota (kazein hidrolizata) na proces regeneracije biljaka in vitro putem indukcije aksilarnih pupoljaka na nodalnim segmentima micromeria pulegium. najveći broj aksilarnih pupoljaka je formiran na eksplantatima gajenim na ms hranljivoj podlozi sa 3% saharoze i 500 mg/l kazein hidrolizata. ključne reči: indukcija aksilarnih pupoljaka, kultura izdanaka, produkcija biomase 8 (1) • september 2017: 93-98 doi: 10.5281/zenodo.1003217 biologica nyssana 8 (1)  september 2017: 93-98 stojičić, d. et al.  factors influencing axillary bud induction on nodal… 94 introduction micromeria pulegium (syn. clinopodium pulegium) belongs to section pseudomelissa within the genus micromeria benth. (family lamiaceae). this genus includes four sections: pseudomelissa, micromeria, cymularia and pineolentia (h a r l e y et al., 2004). according to molecular evidence, emphasizing similarity of selected morphological traits, b r ä u c h l e r et al. (2006) have included species of genus micromeria, sect. pseudomelissa, into clinopodium l. m. pulegium is an endemic species of southern carpathians. its range includes romania, serbia and federation of bosnia and herzegovina. in serbia it was recorded in the east, in the area of svrljiški timok gorge, while previously it used to be present at mountain tara (š i l i ć , 1979). gorge of svrljiški timok is situated at low altitude, so summers are very warm and dry, while winters are characterized by strong winds and snow. habitats are rocky and steep, mostly gorges at 1000-1200 m above sea level. micromeria pulegium is a perennial, medium-sized, erect plant (fig. 1), and its leaves are densely covered in small glands, rendering a pleasant aroma to the plant. species of genus micromeria are characterized by presence of secondary metabolites, serving as the foundation of their diverse biological activity (v l a d i m i r k n e ž e v i ć et al., 2000). these species have antimicrobial properties (s a r a c & u g u r , 2007), mostly based on phenol compounds, flavones and flavonoids, terpenoids and alkaloids (c o w a n et al., 1999; c o s e n t i n o et al., 1999). these aromatic plants have been traditionally used as spices and in alternative medicine (a l h a m w i et al., 2011; d u d a i et al., 2001; t e l c i et al., 2007). antioxidant and antimicrobial activities were recorded in both micropropagated plants of m. pulegium and those collected from natural habitats (t o š i ć et al., 2015). chemical composition of m. pulegium essential oils from native and micropropagated plants was also studied (s t o j i č i ć et al., 2016). endemic species with small populations such as those of m. pulegium, situated in vicinity of urban environment, are often under negative anthropogenic influence. in order to preserve biodiversity, various biotechnological methods have been developed, and among them micropropagation of plants in vitro is highly important (p a u n e s c u , 2009; r e e d et al., 2011; s h a r m a & s h a r m a , 2013). through micropropagation it is possible to multiply selected genotypes and chemotypes of different plants, avoiding collection from their natural habitat. in vitro propagation from field-grown plants through multiplication of nodal segments (axillary shoot formation) is a good method of producing a large number of plants without changing the chemical composition (s a n t o s -g o m e s & f e r n a n d e s f e r r e i r a 2003; a f f o n s o et al. 2007). this study was initiated in order to improve the reliable protocol for rapid propagation of m. pulegium through axillary bud induction from nodal explants. material and methods plant material and source of explants aerial parts of m. pulegium plants, at the vegetative stage of development, were collected from natural populations in svrljiški timok gorge, in august 2012 (fig. 1). voucher specimen (nº 6912) was deposited in the herbarium collection of the faculty of science and mathematics, university of niš (hmn). nodal segments (one-node stem segments, 1 cm long and bearing two axillary buds) were surface-sterilized for 30 min with 25% solution of sodium hypochlorite (6% active chlorine) containing two drops of liquid detergent. after three rinsing in sterile distilled water, the explants were treated with 5% solution of nystatin for 24 hours in order to eliminate possible fungal infections. after that nodal segments were rinsed three times with sterile distilled water, and they were placed in different variations of culture medium. every jar closed with polycarbonate cover. fig. 1. wild-growing plant m. pulegium, svrljiški timok george biologica nyssana 8 (1)  september 2017: 93-98 stojičić, d. et al.  factors influencing axillary bud induction on nodal… 95 culture medium and culture conditions isolated nodal segments were placed horizontally on basal murashige and skoog (ms) medium (1962) supplemented with 3% sucrose (w/v) and 0.7% (w/v) agar (torlak, belgrade) in 250-ml glass jars containing 25 ml of the medium, if not stated otherwise. ten explants were placed in each jar. the ph of the media was adjusted to 5.8 prior to autoclaving at 114 °c for 25 min. cultures were maintained at 25 ± 2°c under conditions of a 16 h/8 h photoperiod, with a photon flux density 45 µe m-2 s-1 provided by cool white fluorescent lamps, at 25 ± 2 °c. variations of media medium strength – the effects of five different medium strengths were tested: 0; 0.25; 0.5; 1.0; or 2.0 times those of ms. the other components in all five media were the same as in ms medium. effects of carbohydrates to determine the influence of different carbon sources, ms medium was supplemented with one of two carbohydrates (sucrose or maltose), each at five different concentrations (0; 0.5; 1; 3; or 5%). effect of enzymatic casein hydrolyzate (ch) as a source of organic nitrogen, ch was tested at the following concentrations 0; 125; 250; 375; or 500 mg/l. measured parameters after 4 weeks in culture, the explants formed axillary buds. the explants reacting positively to treatment were recorded and following parameters were measured. the number of explants producing shoots and number of shoots per explant, as well as explant fresh and dry weight were recorded in order to evaluate the effect of nutritive factors on shoot multiplication. the dry weight of shoots was recorded after drying in separate paper containers for 24 h. buds shorter than 1 mm were disregarded. the table 1. effect of medium strength on micromeria pulegium shoot proliferation, number of shoots per explant, shoot length and shoot fresh and dry weight after 28 days of culture ms explants producing shoots (%) number of shoots per explant shoot length (mm) explant fresh weight (g) explant dry weight (g) 0ms 0 0 0 0 0 0.25ms 80.0 ± 0.1a 8.15 ± 0.32a 6.25 ± 0.40a 0.09 ± 0.01a 0.009 ± 0.001a 0.5ms 85.0 ± 0.1b 8.52 ± 0.51a 8.48 ± 0.46b 0.11 ± 0.01b 0.010 ± 0.001a 1ms 93.3 ± 0.2c 12.32 ± 0.71b 9.31 ± 0.80c 0.21 ± 0.02c 0.017 ± 0.002b 2ms 81.7 ± 0.2a 12.12 ± 0.81b 9.88 ± 1.11d 0.22 ± 0.05c 0.019 ± 0.003b values are mean ± se, n = 60. means in the column followed by different letters are different according to lsd multiple range test (p ≤ 0.05) table 2. effect of carbon source on micromeria pulegium shoot proliferation, number of shoots per explant, shoot length and shoot fresh and dry weight after 28 days of culture carbon source (%) explants producing shoots (%) number of shoots per explant shoot length (mm) explant fresh weight (g) explant dry weight (g) 0 51.7 ± 0.1a 2.33 ± 0.93a 4.93 ± 0.33a 0.06 ± 0.02a 0.002 ± 0.001a maltose 0.5 70.0 ± 0.1b 4.84 ± 0.46b 6.41 ± 0.35ab 0.10 ± 0.02ab 0.005 ± 0.001b 1.0 75.0 ± 0.1b 4.94 ± 0.66b 6.43 ± 0.48ab 0.15 ± 0.03bc 0.005 ± 0.001b 3.0 76.7 ± 0.2bc 4.60 ± 0.66b 6.05 ± 0.48ab 0.11 ± 0.03ab 0.006 ± 0.001b 5.0 68.3 ± 0.2b 6.09 ± 0.21c 6.07 ± 0.44ab 0.24 ± 0.01e 0.020 ± 0.001d sucrose 0.5 80.0 ± 0.1c 9.09 ± 0.21d 7.43 ± 0.44c 0.21 ± 0.01d 0.017 ± 0.001c 1.0 85.0 ± 0.1c 12.52 ± 0.21e 7.00 ± 0.44c 0.18 ± 0.01c 0.016 ± 0.001c 3.0 93.3 ± 0.2d 12.32 ± 0.71e 9.31 ± 0.80d 0.21 ± 0.02d 0.017 ± 0.002c 5.0 81.7 ± 0.2c 10.09 ± 0.21de 6.33 ± 0.44ab 0.21 ± 0.01d 0.019 ± 0.001c values are mean ± se, n = 60. means in the column followed by different letters are different according to lsd multiple range test (p ≤ 0.05) biologica nyssana 8 (1)  september 2017: 93-98 stojičić, d. et al.  factors influencing axillary bud induction on nodal… 96 efficiency of different treatments on growth rate was determined by comparing biomass increase and in vitro proliferation rates, using different methods. biomass increase was calculated on both fresh and dry weight basis. proliferation rate was assessed by counting the number of shoots at subculture and following 4-week treatment under previously defined conditions. statistical methods for each treatment, a total of 60 nodal segments (ten explants per jar) were used, divided into two replicates. data collected from experiments were calculated and statistically analyzed, and differences were tested for significance using anova multiple range test at the significance level of p ≤ 0.05. results and discussion micropropagation is a good method for achieving uniform plant material, and at the same time use of nodal segments for plant regeneration with the goal of mass production is considered a reliable method for many lamiaceae species (d o d e et al., 2003). in most species of lamiaceae family, shoot proliferation demands presence of cytokinin in the nutrient medium, with or without auxin (s a h a et al., 2012; b a k h t i a r et al., 2014). however, the nodal explants of m. pulegium cultured on ms medium without plant growth regulators have produced shoots (s t o j i č i ć et al., 2016). these results were used as a foundation for determining the influence of nutritive factors of nutrient substrate on production of axillary buds on nodal segments of m. pulegium. research on the effects of five different medium strengths has shown that medium without salts and vitamins was not sufficient for development of axillary buds, and necrosis of whole explants happened already in the second week (tab. 1). on medium supplemented with salts and vitamins, the percentage of explants developing shoots was greater when concentration of salts increased. the maximum axillary bud proliferation was obtained at 1ms and 2ms, in contrast to results by f a d e l et al. (2010) with mentha spicata l. and m i š i ć et al. (2006) with salvia brachyodon. they achieved the best results by using medium with ms salts reduced to one half (0.5ms). supplementation of the medium with salts and vitamins promoted elongation of the shoot. the greatest average length of axillary buds was developed on explants grown at medium with 2ms. the explants with the greatest average fresh and dry weight were grown on 1ms and 2ms medium. fig. 2. in vitro plantlets cultured on medium with 750 mg/l caseine hydrolysate (left), flowering in vitro plantlets on same medium (right) explants of m. pulegium grown on culture medium ms without a source of carbon were light green, with sporadically developed axillary buds in leaf axils. the percentage of explants with developing axillary buds was the smallest in the whole study (52%). the values of number of buds, length of buds, fresh and dry mass were also significantly smaller than in axillary buds grown in medium supplemented with sugar (tab. 2). sucrose is often the best source of carbon in micropropagation of various species from family lamiaceae (s u j a n a & n a i d u , 2011). after adding maltose or sucrose table 3. effect of casein hydrolyzate on micromeria pulegium shoot proliferation, number of shoots per explant, shoot length and shoot fresh and dry weight after 28 days of culture ch (mg/l) explants producing shoots (%) number of shoots per explant shoot length (mm) explant fresh weight (g) explant dry weight (g) 0 93.3 ± 0.2b 12.32 ± 0.71a 9.31 ± 0.80a 0.21 ± 0.02b 0.017 ± 0.002a 125 80.0 ± 0.1a 12.11 ± 0.45a 9.90 ± 0.70a 0.19 ± 0.02a 0.018 ± 0.002a 250 90.0 ± 0.1b 12.27 ± 0.24a 9.51 ± 0.51a 0.19 ± 0.02a 0.017 ± 0.002a 500 93.3 ± 0.2b 16.93 ± 0.38c 15.36 ± 0.57b 0.25 ± 0.04c 0.023 ± 0.002b 750 81.7 ± 0.2a 15.06 ± 0.42b 15.01 ± 1.10b 0.25 ± 0.02c 0.024 ± 0.002b values are mean ± se, n = 60. means in the column followed by different letters are different according to lsd multiple range test (p ≤ 0.05) biologica nyssana 8 (1)  september 2017: 93-98 stojičić, d. et al.  factors influencing axillary bud induction on nodal… 97 sugars to the nutrient medium, the percentage of m. pulegium explants with axillary buds has significantly increased (68-93%). the explants grown on medium with sucrose were healthy, with good branching, green, with elongated internodes and a large number of developed axillary buds. the results of our study show that sucrose was a better source of carbon for m. pulegium than maltose. in all studied concentrations sucrose caused a greater number of axillary buds than when the medium contained maltose. the maximum number of buds developed on medium with 1% and 3% sucrose, while the greatest length was recorded in axillary buds growing on medium with 3% sucrose. the explants grown on medium with maltose had bushlike form with shorter internodes. therefore the greatest values of average dry and fresh biomass were recorded in explants grown on medium with the greatest concentration of maltose. casein hydrolysate is commonly used in micropropagation as a source of nitrogen. most of the recorded effect was stimulative (s t o j i č i ć et al., 2008). explants of m. pulegium grown on ms medium with or without casein hydrolysate showed no morphological differences. they were elongated, branching explants, dark green in color, with a large number of developed axillary buds (fig. 2). the stimulative effect of casein hydrolysate was manifested in increase of length of axillary buds and their fresh and dry biomass (tab. 3). on medium with the lowest concentrations of casein hydrolysate this increase was not statistically significant. however, use of casein hydrolysate in concentrations of 500 and 750 mg/l is justified as it leads to statistically significant increase in bud length and biomass. the maximum length of axillary buds was recorded in explants grown on medium with 500 and 750 mg/l of casein hydrolysate. some of the explants grown on this medium produced flowers (fig. 2). in some of the explants grown on medium with the greatest concentration of casein hydrolysate adventive roots have formed spontaneously without addition of auxin to the medium. root formation without auxin was observed in a number of lamiaceae species (z u z a r t e et al., 2010; bassolino et al., 2015), where roots were induced on plant growth regulator free medium. however, in m. pulegium shoots rooted spontaneously on the plant growth regulator free medium; hence, the auxin treatment was used to promote rooting (s t o j i č i ć et al., 2016). conclusion micromeria pulegium (rochel) benth. is characterized by relatively high amounts of the essential oils rich in pulegone that potentially may be used as bio-insecticide and bio-pesticide (k o u l et al. 2008). specimens collected in the wild and grown in vitro both produced essential oil of relatively stable composition (s t o j i č i ć et al., 2016). careful selection of the culture conditions may increase accumulation of biomass and production of secondary metabolites, which may be employed to obtain essential oils for commercial use. results indicate that micropropagation of m. pulegium is influenced by composition of nutritive medium. selection of proper mineral salt concentration, carbon source and especially presence of nitrogen source were shown to be important for stimulation of development and growth of axillary buds in this species. the presented protocol may be used as a foundation in further research in order to produce m. pulegium plants with desired characteristics, especially for production of secondary metabolites. acknowledgements. this research was supported by the ministry of education, science and technological development of the republic of serbia (grant 173015, 173030). references affonso, v. r., bizzo, h. r., salguiero lage, c. l., sato, a. 2009: influence of growth regulators in biomass production and volatile profile of in vitro plantlets of thymus vulgaris l. journal of agricultural and food chemistry, 57 (14): 6392– 6395. al-hamwi, m., bakkour, y., abou-ela, m., ellakany, a., tabcheh, m., el-omar, f. 2011: chemical composition and seasonal variation of the essential oil of micromeria fruticosa. journal of natural products, 4: 147-149. bakhtiar, z., mirjalili, m. h., sonboli, a., moridi farimani, m., ayyari, m. 2014: in vitro propagation, genetic and phytochemical assessment of thymus persicus—a medicinally important source of pentacyclic triterpenoids. biologia, 69: 594–603. bassolino, l., giacomelli, e., giovanelli, s., pistelli, l., casetti, a., damonte, g., bisio, a., ruffoni, b. 2015: tissue culture and aromatic profile in salvia dolomitica codd. plant cell, tissue and organ culture (pctoc), 121 (1): 83–95. brauchler, c., meimberg, h., heubl, g. 2006: new names in old world clinopodium—the transfer of the species of micromeria sect. pseudomelissa to clinopodium. taxon, 55: 977–981. cosentino, s., tuberoso, cig., pisano, b., satta, m., mascia, v., arzedi, e. 1999: in vitro antimicrobial activity and chemical composition of sardinian biologica nyssana 8 (1)  september 2017: 93-98 stojičić, d. et al.  factors influencing axillary bud induction on nodal… 98 thymus essential oils. letters in applied microbiology, 29: 130–135. cowan, m. m. 1999: plant products as antimicrobial agents. clinical microbiology reviews, 12 (4): 564-582. dudai, n., larkov, o., ravid, u., putievsky, e., lewinsohn, e. 2001: development control of monoterpene content and composition in micromeria fruticosa (l.) druce. annals of botany, 88 (3): 349-354. fadel, d. kintzios, s., economou, a., moschopoulou, g., constantinidou, h.i. 2010: effect of different strength of medium on organogenesis, phenolic accumulation and antioxidant activity of spearmint (mentha spicata l.). the open horticulture journal, 3: 3135. harley, r. m., atkins, s., budantsev, a. l., cantino, p. d., conn, b. j., grayer, r., harley, m. m., de kok, r., krestovskaja, t., morales, r., paton, a. j., ryding, o., upson, t. 2004: labiatae. in: kadereit, j. w., kubitzki, k. (eds) the families and genera of vascular plants vii. flowering plants. dicotyledons: lamiales (except acanthaceae including avicenniaceae). springer, berlin, pp 167–275. dode, l.b., seixas, f.k., schuch, m.w., braga, e.j.b., bobrowski, v.l., 2003: in vitro clonal propagation of ocimum basilicum l. (lamiaceae). acta scientiarum. biological sciences, 25 (2): 439-441. koul, o., walia, s., dhaliwal, g. s. 2008: essential oils as green pesticides: potential and constraints. biopestic international, 4: 63–84 mišić, d., grubišić, d., konjević, r., 2006: micropropagation of salvia brachyodon through nodal explants. biologia plantarum, 50 (3): 473476. murashige, t., skoog, f., 1962: a revised medium for rapid growth and bioassays with tobacco tissue cultures. plant physiology, 15: 473-497. paunescu, a. 2009: biotechnology for endangered plant conservation: a critical overview. romanian biotechnological letter, 14 (1): 40954103. reed, b.m., sarasan, v., kane, m., bunn, e., pence, v.c., 2011: biodiversity conservation and conservation biotechnology tools. in vitro cellular and developmental biology plant, 47: 1–4. santos-gomes, p.,c., fernandes-ferreira, m. 2003: essential oils produced by in vitro shoots of sage (salvia officinalis). journal of agricultural and food chemistry, 51:2260–2266. saha, s., kader, a., sengupta, c., ghosh, p., 2012: in vitro propagation of ocimum gratissimum l. (lamiaceae) and its evaluation of genetic fidelity using rapd marker. american journal of plant sciences, 3: 64-74. sarac n., ugur a., 2007: antimicrobial activites and usage in folkloric medicine of some lamiaceae species growing in mugla turkey. eurasian journal of biosciences, 4: 28-37. sharma, d.k., sharma, t., 2013: biotechnological approaches for biodiversity conservation. indian journal of scientific research, 4 (1): 183-186. stojičić, d., tošić, s., slavkovska, v., zlatković, b., budimir, s., janošević, d., uzelac, b. 2016: glandular trichomes and essential oil characteristics of in vitro propagated micromeria pulegium (rochel) benth. (lamiaceae). planta, 244: 393-404. sujana, p., naidu, c.v. 2011: impact of different carbohydrates on high frequency plant regeneration from axillary buds of mentha piperita (l.) – an important multipurpose. medicinal plant journal of phytology, 3(5): 1418. šilić, č., 1979: monografija rodova satureja l., calamintha miller, micromeria bentham, acinos miller i clinopodium l. u flori jugoslavije. zemaljski muzej, sarajevo. telci, i., ceylan, m. 2007: essential oil composition of micromeria fruticosa druce from turkyu, chemistry of natural compounds, 43 (5): 629631. tošić, s., stojičić, d., stankov-jovanović, v., mitić, v., mihajilov-krstev, t., zlatković, b., 2015: chemical composition, antioxidant and antimicrobial activities of micropropagated and native micromeria pulegium (lamiaceae) extracts. oxidation communications, 38 (1): 5566. vladimir-knežević, s., blažeković, b., bival štefan, m., alegro, a., köszegy, t., petrik, j., 2011: antioxidant activities and polyphenolic contents of three selected micromeria species from croatia. molecules, 16: 1454-1470. zuzarte, m. r., dinis, a. m., cavaleiro, c., salgueiro, l. r., canhoto, j. m. 2010: trichomes, essential oils and in vitro propagation of lavandula pedunculata (lamiaceae). industrial crops and products, 32:580–587. sarajlić, n., jogan, n.: alien flora of the city of sarajevo (bosnia and herzegovina). biologica nyssana, 8 (2). biologica nyssana 8 (2)  december 2017: 129-136 sarajlić, n., jogan, n.  alien flora of the city of sarajevo… 129 original article received: 09 november 2017 revised: 24 december 2017 accepted: 25 december 2017 alien flora of the city of sarajevo (bosnia and herzegovina) nermina sarajlić1, nejc jogan2 1ornithological society „naše ptice“, semira frašte 6, 71000 sarajevo, bosnia and herzegovina 2university of ljubljana, biotechnical faculty, department of biology, večna pot 111, 1000 ljubljana, slovenia * e-mail: nermina_sarajlic@yahoo.com abstract: sarajlić, n., jogan, n.: alien flora of the city of sarajevo (bosnia and herzegovina). biologica nyssana, 8 (2). december, 2017: 129-136. this paper presents the inventory and brief analysis of 82 alien vascular plant taxa of the urban and suburban area of the city of sarajevo. the checklist is based on field work that has been carried out from summer 2015 to autumn 2017. each taxon is given with information concerning its life-form, time and mode of introduction, geographic origin and previously reported occurrence in the area. most alien plants were the members of the asteraceae (incl. cichoriaceae), therophytes, neophytes, originated from north and south america and deliberately introduced. the values of indicators of anthropogenic changes showed the considerable anthropogenic influence on the total flora. key words: alien plants, urban flora, sarajevo apstrakt: sarajlić, n., jogan, n.: alohtona flora grada sarajeva (bosna i hercegovina). biologica nyssana, 8 (2), decembar, 2017: 129-136. u radu je dat spisak i kratka analiza 82 alohtona taksona vaskularnih biljaka zabeleženih u urbanom i suburbanom delu grada sarajeva od leta 2015. do jeseni 2017. godine. za svaki takson navedeni su podaci o životnoj formi, vremenu i načinu introdukcije, poreklu i najraniji objavljeni podatak o njegovom prisustvu na istraživanom području. izračunati su indikatori antropogene promene flore. najbrojnije alohtone vrste zabeležene tokom ovog istraživanja su pripadnici porodice asteraceae (uključujući cichoriaceae), terofite, neofite poreklom iz severne i južne amerike, koje su u evropu unesene namerno. vrednosti indeksa antropogene promene pokazuju značajan antropogeni uticaj na ukupnu floru grada sarajeva. ključne reči: alohtone biljke, urbana flora, sarajevo 8 (2) • december 2017: 129-136 doi: 10.5281/zenodo.1135953 biologica nyssana 8 (2)  december 2017: 129-136 sarajlić, n., jogan, n.  alien flora of the city of sarajevo… 130 introduction urban habitats are generally characterized by high levels of disturbance, high fertility and heterogeneity of habitats, suitable for development of numerous plant species, including many alien plants. alien (exotic, adventive, introduced, allochthonous, nonindigenous, non-native) plants are those whose presence in a certain area is due to intentional or accidental introduction as a result of human activities (p y š e k , 1995; r i c h a r d s o n et al., 2000; p y š e k et al., 2004). according to their spreading ecology in a certain territory, alien plants can be divided in three groups: casual, naturalized and invasive. according to the time when they were introduced into europe, alien plants can be classified as archaeophytes (introduced before the year 1500) and neophytes (introduced after the year 1500) (p y š e k , 1995, k o w a r i k , 1995; p y š e k et al., 2002; p r e s t o n et al., 2004; l a s o r t e et al., 2007). some of them were introduced deliberately, as ornamental, edible or medicinal plants, and others came to europe accidentally, without the deliberate actions of humans. the city of sarajevo was founded in the 15th century, but expanded, mostly towards the west, only during the post-world war ii era. intense exchange of goods since ancient times and further urbanization, construction of roads and railway enabled the introduction of new plants, some of which slowly become naturalized part of the flora. literature on alien flora of the city of sarajevo is scarce, and the presence of some alien plants is either mentioned in general floristic studies (k o m š a , 1928; p a v l o v i ć , 1987; t o m o v i ć -h a d ž i a v d i ć & š o l j a n , 2006), or studies focused on particular species (s l a v n i ć , 1960; a b a d ž i ć , 1986/87; š o l j a n & m u r a t o v i ć , 2000; š o l j a n , 2011; m e m i š e v i ć -h o d ž i ć et al., 2015; s a r a j l i ć et al., 2016; s u l j i ć et al., 2016). this paper presents a list of hitherto recorded alien species and a brief analysis of the alien flora of the city of sarajevo. the inventory of 82 taxa is provided based on the field work performed in 2015, 2016 and 2017, updating previous floristic knowledge for the investigated area. material and methods study area the city of sarajevo is located in the sarajevo valley, on the banks of miljacka river, at an average altitude between 520 and 750 m a. s. l. (fig. 1). the central parts of the city are situated in the lower parts of fig. 1. geographical position of investigated area (the yellow line limits the area in which the survey was performed) biologica nyssana 8 (2)  december 2017: 129-136 sarajlić, n., jogan, n.  alien flora of the city of sarajevo… 131 sarajevo polje, and the suburbs are scattered on the slopes of surrounding hills and mountains. the area of sarajevo is under influence of mid-european continental climate from the north, and the mediterranean from the south. field investigation the field survey was performed in the urban part of four municipalities (novi grad, novo sarajevo, centar, stari grad) of the city of sarajevo (fig. 1), in an area of 32 km2, from the summer of 2015 to the autumn of 2017. only taxa that had not been deliberately planted were recorded. data analysis the nomenclature follows euro+med plantbase and the international organization for plant information database. asteraceae family was considered in wider sense (incl. cichoriaceae). the taxa are listed in alphabetic order, followed by designations for life form (phanerophyte ph, chamaephyte ch, hemicryptophyte h, geophyte g, hydrophyte hy and therophyte t), period of introduction (archaeophytes arc and neophytes – neo), estimated mode of introduction into the region (deliberate, by planting – del, accidental – acc or in both modes d-a) geographic origin and reported first record for the sarajevo area. life forms were given according to r a u n k i a e r (1934), and time and mode of introduction and geographic origin according to p y š e k et al. (2002), m o s y a k i n & y a v o r s k a (2003) and i g n a t i e v a & k o n e c h n a y a (2004). first records for the area of sarajevo were given according to available literature sources (see references and appendix 1). the indicators of anthropogenic changes in the flora of sarajevo were calculated after j a c k o w i a k (1990, 2006), according to w i t o s ł a w s k i & b o m a n o w s k a (2009) as in tab. 1. results during this research, a total of 82 alien plant taxa from 71 genera and 38 families were recorded. the families with the highest number of alien taxa were asteraceae (21%), poaceae (10%), brassicaceae (7%), fabaceae (6%), amaranthaceae (5%) and solanaceae (5%) (tab. 2). the other families were represented only with one or two taxa. table 2. the most abundant families in the alien flora of the city of sarajevo family no. of taxa % of total alien flora asteraceae 17 20.73 poaceae 8 9.75 brassicaceae 6 7.31 fabaceae 5 6.09 amaranthaceae 4 4.88 solanaceae 4 4.88 analysis of life forms showed the domination of therophytes with 47 taxa (57%), followed by phanerophytes with 14 taxa (17%) geophytes (10 taxa, 12%), hemicryphophytes (10 taxa, 12%), and chamaephytes (1 or 1%). the alien flora of the city of sarajevo consisted of 28 (34%) archaeophytes and 54 (66%) neophytes. analysis of the geographical origin of the alien flora of sarajevo (tab. 3) showed that the majority of species originated from n. and s. america (37 taxa, 45%), followed by those originating from asia (24 taxa, 24%), mediterranean (13 taxa, 16%), eurasia (10 taxa, 12%) and africa (2 taxa, 2%). table 1. indicators of anthropogenic changes (an – total number of alien species, sp – number of native species, mt – number of permanently established alien species (ar + kn df), ar – number of archaeophytes, kn – number of kenophytes (neophytes), df – number of diaphytes (casual aliens)). indicator formula for calculation indicator of total anthropophytization iant = an/(sp+an) ×100% indicator of permanent anthropophytization ianp = mt/(sp+mt) ×100% indicator of total archaeophytization iart = ar/(sp+an) ×100% indicator of permanent archaeophytization iarp = ar/(sp+mt) ×100% indicator of total kenophytization iknt = kn/(sp+an) ×100% indicator of permanent kenophytization iknp = kn/(sp+mt) ×100% indicator of modernization im = kn/mt ×100% indicator of fluctuation changes if = df/(sp+an) ×100% biologica nyssana 8 (2)  december 2017: 129-136 sarajlić, n., jogan, n.  alien flora of the city of sarajevo… 132 table 3. an analysis of the geographical origin of the alien flora of the city of sarajevo region subregion no. of taxa % of total alien flora africa 2 2.44 east (af-e) 1 1.22 north (af-n) 1 1.22 america 37 45.12 south (am-s) 7 8.54 north (am-n) 30 36.58 asia 24 29.27 asia (as) 4 4.88 central (as-c) 1 1.22 east (as-e) 8 9.76 south-west (as-sw) 4 4.88 west (as-w) 3 3.66 eurasia (eu-as) 10 12.19 mediterranean (m) 13 15.85 total 82 100.00 indicators of anthropogenic changes values in the flora of sarajevo are presented in tab. 4. table 4. indicators of anthropogenic changes in the city of sarajevo indicator % iant 12.09 ianp 8.17 iart 4.13 iarp 4.31 iknt 7.96 iknp 8.32 im 65.85 if 4.45 according to our research, which has been taking place since summer 2015, the urban flora of sarajevo consists of 678 taxa. the indicators of anthropophytization (iant = 12.09%; ianp = 8.17%) showed the considerable anthropogenic influence on the total flora of sarajevo, and higher indicator values of kenophytization (iknt = 7.96%; iknp = 8.32%) showed that the flora of sarajevo is more influenced by neophytes than by archaeophytes (iart = 4.13%; iarp = 4.31%). in both archaeophytes and neophytes, the values of total and permanent indicators were similar, showing that alien flora is well established. this was confirmed by the low value of the indicator of fluctuating changes (if = 4.45%). discussion cities usually contain large proportion of alien plants (w i t t i g , 2004), due to developed transport network and diversity of habitats that can be easily colonized by alien species. during this research, we found that the families with the highest number of taxa were asteraceae and poaceae. those two families were also dominant in alien flora of zagreb (h u d i n a et al., 2012), sisak (p r u š a et al., 2013), podgorica (s t e š e v i ć et al., 2014) and mostar (m a s l o , 2015), probably due to high reproduction rate and specialised dispersion structures on the seeds (p y š e k , 1997). in alien flora of sarajevo, therophytes were the dominant life-form. the same was recorded in sisak (croatia) by p r u š a et al. (2013) and in mostar by m a s l o (2015). the domination of therophytes is due to the warm and dry microclimate (the “urban heat-island effect”) that characterize urban environments, providing conditions that allow many alien species with higher temperature requirements and tolerance for arid environments to become established. all mentioned cities have the significant share of phanerophytes in their alien flora, mostly due to woody ornamental aliens escaping from cultivation. during this research, neophytes were more numerous than archaeophytes. more neophytes (55%) were also recorded in alien flora of mostar by m a s l o (2015). according to d e l t r e d i c i (2010), the ratio of neophytes to archaeophytes rises in direct relation to the intensity of human disturbance. this is due to the fact that archaeophytes are typically associated with traditional rural environments or intermediate levels of anthropogenic activities (p r e s t o n et al., 2004), and neophytes are more common in highly disturbed modern anthropogenous habitats covering huge areas with little vegetation coverage and with developed transport facilities and industrial infrastructure contributing to the ‘urban heat-island effect’, which provides distinctive environmental conditions that favor the establishment of species from warmer and drier areas (p y š e k , 1998; p y š e k et al., 2002; g o d e f r o i d & k o e d a m , 2007; l a s o r t e et al., 2007). also, the cultivation of numerous foreign species along roads, in parks and gardens, presents the source for neophyte colonization (w i t t i g , 2004). the analysis of the geographical origin of the alien flora of sarajevo showed that the most plants originated from n. and s. america and asia. the same was found in the city of podgorica (s t e š e v i ć et al., 2014) and mostar (m a s l o , 2015). the alien plants originating from n. and s. america and asia biologica nyssana 8 (2)  december 2017: 129-136 sarajlić, n., jogan, n.  alien flora of the city of sarajevo… 133 also dominate in urban flora of italian cities (c e l e s t i g r a p o w & b l a s i , 1998). this is in a relation with the increased number of neophytes in urban areas, due to the fact that archaeophytes originate primarily from southern europe and the near east, and neophytes from north america and eastern asia (p y š e k et al., 2002; l a s o r t e et al., 2007). the indicators of anthropophytization (iant = 12.09%; ianp = 8.17%) showed considerable anthropogenic influence on the total flora of the city of sarajevo, but were not as high as in savica park area near zagreb (iant = 28.78%; ianp = 26.76%) calculated by a l e g r o et al. (2013) or rzeszów foothills in poland (iant = 21.3%) by jaźwa & s t a d n i c k a -f u t o m a (2015). this is probably due to the fact that some habitats in the city of sarajevo (forests and natural grasslands in the suburban parts of city) had very small number of aliens in comparison to the native plants. according to available literature sources, the first alien plants reported from the area of sarajevo were panicum miliaceum, linum usitatissimum, aesculus hippocastanum, capsella bursa-pastoris, conyza canadensis, helianthus annuus, h. tuberosus, juglans regia, phalaris canariensis, pyrus communis, sinapis arvensis and syringa vulgaris (h o f m a n n , 1882). on the other hand, species recorded only recently before our study were acer negundo, ailanthus altissima, antirrhinum majus, oenothera biennis (t o m o v i ć h a d ž i a v d i ć & š o l j a n , 2006), sedum sarmentosum (š o l j a n , 2011) and reynoutria japonica (s a r a j l i ć et al., 2016). during our research, 37 taxa for which there was no earlier data for the area of sarajevo were found (appendix 1). conclusion the results of 3-years research showed that the alien flora of the city of sarajevo consisted of 82 species from 71 genera and 38 families, of which the asteraceae (21%) were the most numerous, followed by poaceae (10%) and brassicaceae (7%). the analysis of life forms showed the domination of therophytes (57%), followed by phanerophytes (17%). neophytes (66%) were more numerous than archaeophytes (34%). analysis of the mode of introduction showed that most aliens (49%) were introduced to europe deliberately, as horticultural or agricultural plants. most alien plants recorded in the area of the city of sarajevo originated from n. and s. americas (45%) and asia (24%). during this research, 37 taxa for which there was no earlier data for the area of sarajevo were found. the values of indicators of anthropogenic changes showed the considerable anthropogenic influence on the total flora of sarajevo. references abadžić, s. 1986/87: prilog poznavanju horologije i ekologije dviju adventivnih vrsta – echinocystis lobata (michx) torrey et gray i bidens bipinnata l. u flori bosne i hercegovine. glasnik zemaljskog muzeja bosne i hercegovine (prirodne nauke), nova serija, 25-26: 71-77. alegro, a., bogdanović, s., rešetnik, i., boršić, i., cigić, p., nikolić, t. 2013: flora of the seminatural marshland savica, part of the (sub)urban flora of the city of zagreb (croatia). natura croatica, 22 (1): 111–134. beck, g. 1887a: flora von südbosnien und der angrenzenden hercegovina. ii theil. annalen des naturhistorischen museums in wien, 2 (1): 35-76. beck, g. 1887b: flora von südbosnien und der angrenzenden hercegovina. iii theil. annalen des naturhistorischen museums in wien, 2 (2): 81-184. beck, g. 1891: flora von südbosnien und der angrenzenden hercegovina. annalen des naturhistorischen museums in wien, 6: 307-344. beck, g. 1896: flora von südbosnien und der angrenzenden hercegovina viii theil (fortsetzung). annalen des naturhistorischen museums in wien, 11 (1): 39-80. beck, g. 1906: flora bosne, hercegovine i novopazarskog sandžaka ii (3.) dio. glasnik zemaljskog muzeja u bosni i hercegovini, 18 (4): 468-495. beck, g. 1916: flora bosne, hercegovine i novopazarskog sandžaka ii (7.) dio. nastavak. glasnik zemaljskog muzeja u bosni i hercegovini, 28 (1): 41-168. celesti grapow, l., blasi, c. 1998: a comparison of the urban flora of different phytoclimatic regions in italy. global ecology and biogeography letters, 7 (5): 367-378. del tredici, p. 2010: spontaneous urban vegetation reflections of change in a globalized world. nature and culture, 5 (3): 299-315. euro+med (2006-): euro+med plantbase the information resource for euro-mediterranean plant diversity. published on the internet http://ww2.bgbm.org/europlusmed/ [accessed december 2017] formanek, e. 1888: beitrag zur flora von bosnien und der hercegovina. österreichische botanische zeitschrift 38: 271-279. formanek, e. 1889: beiträge zur flora von bosnien und der hercegovina. österreichische botanische zeitschrift, 39: 22-28. biologica nyssana 8 (2)  december 2017: 129-136 sarajlić, n., jogan, n.  alien flora of the city of sarajevo… 134 godefroid, s., koedam, n. 2007: urban plants species patterns are highly driven by density and function of built– up areas. landscape ecology, 22: 1227–1239. hofmann, f. 1882: beitrag zur kenntnis der flora von bosnien. österreichische botanische zeitschrift, 32: 73-81; 111-116; 145-152; 255259. hudina, t., salkić, b., rimac, a., bogdanović, s., nikolić, t.: 2012 contribution to the urban flora of zagreb (croatia). natura croatica, 21 (2): 357–372. ignatieva, m., konechnaya, g. 2004: floristic investigations of historical parks in st. petersburg, russia. urban habitats, 2 (1): 174-216. jackowiak, b. 1990: antropogeniczne przemiany flory roślin naczyniowych poznania. wydawnictwo naukowe uniwersytetu adama mickiewicza w poznaniu, ser. biologia, 42: 1208. jackowiak, b. 2006: methodological proposals for studies on the structure and dynamics of urban flora. polish botanical studies, 22: 251-260. jaźwa, m, stadnicka-futoma, a. 2015: the alien flora of the rzeszów foothills. biodiversity, 38 (1): 25-36. komša, a. 1928: korov sarajevskog polja. rad fitopatološkog zavoda u sarajevu, 38-64. kowarik, i. 1995: on the role of alien species in urban flora and vegetation. in: pyšek, p., prach, k., rejmanek, m., wade m. (eds.), plant invasions general aspects and special problems: 85–103. spb acad. publ., amsterdam. la sorte, f, mckinney, m., pyšek, p. 2007: compositional similarity among urban floras within and across continents: biogeographical consequences of human-mediated biotic interchange. global change biology, 13 (4): 913– 921. maksimović, t. 1990: polenska alergija u dece u odnosu na polenski kalendar sarajevske regije (doktorska disertacija). univerzitet u sarajevu, medicinski fakultet, sarajevo. maly, k. 1904: beiträge zur kenntnis der flora bosniens und der herzegowina. verhandlungen der zoologisch-botanischen gesellschaft in wien, 54: 165-309. maly, k. 1910: prilozi za floru bosne i hercegovine. glasnik zemaljskog muzeja u bosni i hercegovini, 22: 685-694. maly, k. 1912: prilozi za floru bosne i hercegovine. glasnik zemaljskog muzeja u bosni i hercegovini, 24: 587-595. maly, k. 1919: prilozi za floru bosne i hercegovine. glasnik zemaljskog muzeja u bosni i hercegovini, 31: 61-92. maly, k. 1928: prilozi za floru bosne i hercegovine. glasnik zemaljskog muzeja u bosni i hercegovini, 40 (1): 107-172. maly, k. 1933: materialien zu g. beck's flora des ehemaligen bosnien-hercegovina. glasnik zemaljskog muzeja u bosni i hercegovini, 45 (1): 71-142. maslo, s. 2015: alien flora of the city of mostar (bosnia and herzegovina). herbologia, 15 (2): 116. memišević-hodžić, m., mejrić, a., sejdić, a., omerović, s. 2015: cadastre of ragweed`s sites in the sarajevo canton. herbologia, 15 (2): 17-26. mosyakin, s.l., yavorska, o.g. 2003: the nonnative flora of kiev (kyiv) urban area, ukraine: a checklist and brief analysis. urban habitats, 1: 45-65. pavlović, d. 1987: vegetacija voćnjaka okoline sarajeva u uslovima intenzivne obrade. bilten društva ekologa bih, serija a ekološke monografije, 4: 123-133. preston, c.d., pearman, d.a., hall, a.r. 2004: archaeophytes in britain. botanical journal of the linnean society, 145: 257– 294. pruša, m., majić, b., nikolić, t. 2013: invazivna flora grada siska (hrvatska). glasnik hrvatskog botaničkog društva, 1 (3): 4-17. pyšek, p. 1995: on the terminology used in plant invasion studies. in: pyšek, p., prach, k., rejmanek, m., wade, m. (eds.) plant invasions: general aspects and special problems: 71–81. spb academic publishing, amsterdam pyšek, p. 1997: compositae as invadersbetter than the others? preslia, 62: 9–22. pyšek, p. 1998: alien and native species in central european urban floras: a quantitative comparison. journal of biogeography, 25, 155-163. pyšek, p., richardson, d.m., rejmanek, m., webster, g.l., williamson, m., kirschner, j. 2004: alien plants in checklists and floras: towards better communication between taxonomists and ecologists. taxon, 53 (1): 131143. pyšek, p., sáldo, j., mandák, b., 2002: catalogue of alien plants of the czech republic. preslia, 74: 97-186. raunkiaer, c. 1934: the life forms of plants and statical plant geography. clarendon press, oxford. richardson, d. m., pyšek, p., rejmanek, m., barbour, m. g., panetta, f. d., west, c. j. 2000: naturalization and invasion of alien plants: concepts and definitions. diversity & distributions, 6: 93-107. sarajlić, n., đikić, m., gadžo d. 2016: distribution of japanese knotweed (reynoutria japonica biologica nyssana 8 (2)  december 2017: 129-136 sarajlić, n., jogan, n.  alien flora of the city of sarajevo… 135 houtt.) in the city of sarajevo. radovi poljoprivredno-prehrambenog fakulteta univerziteta u sarajevu, 61 (1): 346-349. slavnić, ž. 1960: o useljavanju, širenju i odomaćivanju nekih adventivnih biljaka u bosni i hercegovini. godišnjak biološkog instituta univerziteta u sarajevu, 13 (12): 117-146. stešević d., caković d., jovanović s. 2014: the urban flora of podgorica (montenegro, se europe): annotated checklist, distribution atlas, habitats and life-forms, taxonomic, phytogeographical and ecological analysis. ecologia montenegrina, suppl. 1: 1-171. suljić, n., gadžo, d., karić, n., ðikić, m. 2016: distribution of jerusalem artichoke (helianthus tuberosus l.) in the canton sarajevo area. radovi šumarskog fakulteta univerziteta u sarajevu, 21 (1): 335-341. šoljan, d. 2011: sedum sarmentosum bunge (crassulaceae), an allochthonous species in the flora of bosnia and herzegovina. herbologia, 12 (3): 15-21. šoljan, d., muratović, e., jukić, lj., merdan, a., ravlić, v. 2006: caryophyllaceae, fabaceae, lamiaceae, ranunculaceae i scrophulariaceae u flori kanjona rijeke miljacke od bentbaše do kozije ćuprije. hrvatska misao, 6: 7-23 šoljan, d., muratović, e. 2000: rasprostranjenost vrste ambrosia artemisiifolia l. na području grada sarajeva. herbologia, 1 (1): 41-47. tomović-hadžiavdić v., šoljan, d. 2006: urbana flora sarajeva. glasnik zemaljskog muzeja (prirodne nauke), nova sveska, 32, 121–135. witosławski, p., bomanowska, a. 2009: southern european species in the flora of towns in the central poland. botanica serbica, 33 (2): 115-129. wittig, r. 2004: the origin and development of the urban flora of central europe. urban ecosystems, 7: 323–333. appendix 1. alien flora of the city of sarajevo (lf-life form, pi-period of introduction, mi-mode of introduction, oorigin) species family lf pi mi o 1st record 1. abutilon theophrasti medik. malvaceae t arc acc as-e 2. acer negundo l. aceraceae ph neo del am-n tomović-hadžiavdić & šoljan (2006) 3. aesculus hippocastanum l. hippocastanaceae ph neo del m hofmann (1882) 4. ailanthus altissima (mill.) swingle simaroubaceae ph neo del as-e tomović-hadžiavdić & šoljan (2006) 5. alcea rosea l. malvaceae h neo del m 6. amaranthus albus l. amaranthaceae t neo acc am-n maly (1919) 7. amaranthus caudatus l. amaranthaceae t neo del am-s 8. amaranthus graecizans l. amaranthaceae t neo acc af-e 9. amaranthus retroflexus l. amaranthaceae t arc acc am-n beck (1887a) 10. ambrosia artemisiifolia l. asteraceae t neo acc am-n maksimović (1990) 11. anagallis arvensis l. primulaceae t arc acc m beck (1887b) 12. angelica archangelica l. apiaceae h arc d-a as-e 13. antirrhinum majus l. scrophulariaceae t arc d-a m tomović-hadžiavdić & šoljan (2006) 14. armoracia rusticana gaertn, may & scherb. brassicaceae g arc d-a eu-as beck (1916) 15. artemisia annua l. asteraceae t neo acc as-e beck (1887b) 16. bassia scoparia (l.) a. j. scott chenopodiaceae t neo del as-c maly (1910) 17. bidens frondosus l. asteraceae t neo acc am-n 18. brassica rapa (l.) l. brassicaceae t arc del m maly (1904) 19. calendula officinalis l. asteraceae t arc del m 20. capsella bursa-pastoris (l.) medik. brassicaceae h arc acc eu-as hofmann (1882) 21. catalpa bignonioides walter bignoniaceae ph neo del am-n 22. commelina communis l. commelinaceae g neo del as-e 23. cuscuta campestris yuncker cuscutaceae t neo acc am-n 24. datura stramonium l. solanaceae t neo acc am-n beck (1887b) 25. eleusine indica (l.) gaertn. poaceae t neo acc as 26. erigeron annuus (l.) desf. asteraceae t neo acc am-n maly (1910) 27. erigeron canadensis l. asteraceae t neo acc am-n hofmann (1882) 28. euphorbia prostrata aiton euphorbiaceae t neo acc am-n 29. galinsoga parviflora cav. asteraceae t neo acc am-n maly (1933) 30. galinsoga quadriradiata ruiz & pav. asteraceae t neo acc am-n 31. gleditsia triacanthos l. fabaceae ph neo del am-n šoljan et al. (2006) 32. helianthus annuus l. asteraceae t neo del am-n hofmann (1882) 33. helianthus tuberosus l. asteraceae g neo del am-n hofmann (1882) biologica nyssana 8 (2)  december 2017: 129-136 sarajlić, n., jogan, n.  alien flora of the city of sarajevo… 136 34. hemerocallis fulva (l.) l. xanthorrhoeaceae g neo del as-e maly (1928) 35. hesperis matronalis l. brassicaceae h arc acc eu-as 36. impatiens balfourii hook. f. balsaminaceae t neo del as 37. impatiens glandulifera royle balsaminaceae t neo del as 38. iris germanica l. iridaceae g arc del eu-as 39. juglans regia l. juglandaceae ph arc del as-sw hofmann (1882) 40. juncus tenuis willd. juncaceae h neo acc am-n 41. lepidium virginicum l. brassicaceae t neo acc am-n 42. linum usitatissimum l. linaceae t arc d-a as-w formanek (1889) 43. lonicera nitida e.h. wilson caprifoliaceae ph neo del az-sw 44. lycopersicon esculentum mill. solanaceae t neo del am-s 45. matricaria discoidea dc asteraceae t neo acc am-n maly (1912) 46. medicago arabica (l.) huds. fabaceae t arc acc m beck (1887b) 47. medicago sativa l. fabaceae h arc acc eu-as beck (1896) 48. morus nigra l. moraceae ph arc del as-sw 49. oenothera biennis l. onagraceae h neo del am-n tomović-hadžiavdić & šoljan (2006) 50. oenothera erythrosepala borbas rosaceae h neo del am-n 51. oxalis dillenii jacq. oxalidaceae h neo acc am-n maly (1928) 52. oxalis fontana bunge oxalidaceae h neo acc am-n 53. panicum capillare l. poaceae t neo acc am-n 54. panicum miliaceum l. poaceae t arc d-a as-e formanek (1888) 55. papaver rhoeas l. papaveraceae t arc acc m beck (1916) 56. parthenocissus quinquefolia (l.) planch. vitaceae ph neo del am-n 57. paspalum dilatatum poir. poaceae t neo acc am-s 58. petunia × hybrida (hook.) vilm. solanaceae t neo del am-s 59. persicaria maculosa gray polygonaceae t arc acc eu-as komša (1928) 60. phalaris canariensis l. poaceae t neo acc m hofmann (1882) 61. platycladus orientalis (l.) franco cupressaceae ph arc del eu-as 62. portulaca oleracea l. portulacaceae t arc acc m beck (1906) 63. pyrus communis l. rosaceae ph arc del eu-as hofmann (1882) 64. ranunculus arvensis l. ranunculaceae t arc acc m beck (1891) 65. reynoutria japonica houtt. polygonaceae g neo del as-e sarajlić et al. (2016) 66. rhus typhina l. anacardiaceae ph neo del am-n 67. robinia pseudoacacia l. fabaceae ph neo del am-n maly (1928) 68. sedum sarmentosum bunge crassulaceae ch neo del as šoljan (2011) 69. senecio inaequidens dc. asteraceae t neo acc af-s 70. sinapis arvensis l. brassicaceae t arc d-a m hofmann (1882) 71. solanum tuberosum l. solanaceae g neo del am-s 72. solidago canadensis l. asteraceae g neo del am-n 73. solidago gigantea aiton asteraceae g neo del am-n 74. sorghum halepense (l.) pers. poaceae g arc acc m 75. syringa vulgaris l. oleaceae ph arc del eu-as hofmann (1882) 76. tagetes patula l. asteraceae t neo del am-s 77. tanacetum parthenium (l.) sch.bip. asteraceae t neo del as-w maly (1904) 78. trifolium arvense l. fabaceae t arc del eu-as maly (1904) 79. triticum aestivum l. poaceae t arc del as-sw 80. veronica persica poir. scrophulariaceae t neo acc as-w beck (1887b) 81. xanthium strumarium l. asteraceae t neo acc am-n maly (1912) 82. zea mays l. poaceae t neo del am-s beck (1887a) abbreviations: life forms: ph phanerophyte, ch chamaephyte, h hemicryptophyte, g geophyte, t therophyte; period of introduction: arc archaeophyte, neo – neophyte; mode of introduction: acc accidental, del deliberate, d-a in both ways. anticancer compounds from medicinal plants biologica nyssana 4 (1-2)  december 2013: 1-7 sadakovic, k., kuštera, m..  fungal conservation: protected species… 35 original article fungal conservation: protected species of fungi in south serbia region dušan sadiković , marjan kuštera mycological socety of niš, somborska 81 a/9, 18000 niš, serbia * e-mail: dusan.sadikovic@gmail.com abstract: sadiković, d., kuštera, m.: fungal conservation: protected species of fungi in south serbia region. biologica nyssana, 4 (1-2), december 2013: 35-40. protection and conservation of fungi has only recently became an issue of concern. main motives for increased attention are uncontrolled, mass collecting of edible wild mushrooms and environmental pollution which leads to the rapid decline of their natural habitats, some of which are rich with rare and endangered species. by serbian nature conservation law 2010. there are 38 strictly protected fungal species of which 17 species are recorded in this paper. 11 of those recorded species are on european and/or national red list of endangered fungal species. all investigated territories were in south serbia region. this study is a contribution to conservation of protected and threatened fungi and their respective habitats in serbia. key words: red list, protected species, south serbia, macrofungi. introduction fungi play vital roles in most of the ecosystems on the biosphere. as they are the dominant decomposers of the lignocellulose plant debris and knowing that cellulose is the most abundant organic polymer on the earth (k l e m m , 2005), it is obvious that fungi are integral factors in processes of recycling carbon and reprocessing of nutrients in general. they have established mutualistic symbioses with a wide range of organisms: cyanobacteria and green algae (in lichens), bryophytes, pteridophytes, gymnosperms and angiosperms (in mycorrhizae), and coleopteran, dipteran, homopteran, and isopteran insects (k e n d r i c k , 2011) . also with some hymenopterian insects like fungus-growing ants (attini: formicidae) engage in an obligate mutualism with fungi they cultivate for food (c a l d e r a e t a l ., 2009). in europe more than 15000 species of macroscopic fungi had been identified. this is a conservative estimate because many morphological species concepts are proving to be species complexes. also identifying species of concern has been hampered by the sporadic occurrence of the sexual fruit bodies (sporocarps) of the fungi, the cryptic nature and the lack of morphological characters of the vegetative state, and the difficulty in culturing most species for lab work (l i l l e s k o v e t a l ., 2010). in serbia the official number of recorded species, presented by institute for nature conservation of serbia, is 1300 but it’s estimated that it’s closer to 3000, a figure which would represent about 20% of total european mycodiversity. biogeographical studies on fungi are relatively scarce, due mainly to methodological problems (a r n o l d s , 1997). the awareness that fungi can be threatened came recently, in the early 1980s, when it was noticed that heavy acidification of soil due to acid rains has a receding effect, both qualitatively and quantitatively. acid rain results into acidification of soil, which increases the exchange between 11 th sfses • 13-16 june 2013, vlasina lake 4 (1-2) • december 2013: 35-40 biologica nyssana 4 (1-2)  december 2013: 35-40 sadakovic, k., kuštera, m..  fungal conservation: protected species… 36 hydrogen ion and nutrient cations like potassium (k), magnesium (mg) and calcium (ca) in the soil. these cations are liberated into soil and can be rapidly leached out in soil solution along with sulphate from acid input. acid induced leaching leads to nutrient deficiency in the affected soils and thus negatively affecting microbial processes (s i g h & a g r a w a l l , 2008). also higher levels of nitrogen caused by anthropogenic activities has a reducing effect on mycofloral diversity as it was proven that with increasing the n deposition, ectomycorrhizal root tip abundance and mycelial production can be decreased five and 10-fold. in addition, fungal community can be changed and the species richness decreased (k j ø l l e r e t a l ., 2012). recognizing this problem, national red lists started to emerge, first being in germany in 1982. today more than 35 countries have some form of red list for fungi. evaluations of those red lists in almost every european country, together with many studies regarding fungal conservation, indicate that a large portion, probably in the range of 2000 –3000 species of macrofungi throughout europe are declining and their future is uncertain. european mycologists, through the european council for conservation of fungi (eccf), have prepared list of fungi for possible inclusion in the bern convention. these 33 species represent only a small fraction of all threatened fungal species, but by including them in the convention appendix, the need for conservation of fungi and their habitats would be officially recognized (k j ø l l e r et al., 2012). in serbian legislation, first species of fungi were put under protection in 1991, as “natural rarities threatened by exploitation and trade”, under the decision on amending the decision on putting plant species under protection as natural rarities (1991) but it had inadequate protection measures as well as cardinal taxonomic mistakes. because yugoslavia was in the first half of the last decade the largest exporter of bolets in the, of which the largest quantities were from serbia, the protection only included a few edible species world (for example in 1993. 5 186 100kg of boletus edulis was purchased in serbia) (i v a n c e v i c e t a l ., 2012). currently by the nature conservation law (2009) and regulation on the proclamation and protection of strictly protected and protected wild species of plants, animals and fungi (2010), 38 species of macroscopic fungi are treated as strictly protected. serbian preliminary national red list of endangered species was comprised in 1998 (i v a n c e v i c , 1998) and it contained 96 species. in 2004 the list was corrected and amended to iucn standards. the nprl of endangered species was not fully or not at all considered when making the list of strictly protected species and methods used in comprising this list were unclear. material and methods the areas were investigated between 2011. and 2013. all mycological investigations were conducted in south serbia region (fig. 1.). the fungal specimens were identified on the basis of macroscopic and microscopic morphological characteristics and specific chemical reactions of fruiting bodies according to specific identification keys: (božac 1978; foht 1986; breitenbach & kränzlin 1995; maca et al., 1995; mcknight et al., 1998; jordan 2004; uzelac 2009). author citations for each taxon are abbreviated according to the index fungorum(2013). for the analysis of the legal protection of fungi in serbia and europe, following material was used: guidance for the conservation of mushrooms in europe. convention on the conservation of european wildlife and natural habitats. document prepared by beatrice senn-irlet, jacob heilmannclausen and anders dahlberg for the european council for conservation of fungi (eccf) within the european mycological association (ema). strasbourg, 17 october 2007. одлука о изменама и допунама одлуке о стављању под заштиту биљних врста као природних реткости. службени гласник срс 49, 15. 08. 1991; [decision on amending the decision on putting plant species under protection as natural rarities, 1991] закон о заштити природе. службени гласник републике србије 36, 12.05.2009. i 88/2010; [nature conservation law, 2009 – actual] правилник о проглашењу и заштити строго заштићених и заштићених дивљих врста биљака, животиња и гљива. службени гласник републике србије 5, 05. 02. 2010; [regulation on the proclamation and protection of strictly protected and protected wild species of plants, animals and fungi, 2010 – actual]. the purpose of this study is presenting to the scientific community precise bio geographical data on rare and endangered species of macromycetes in serbia and thus helping in process of making more accurate national red list and national checklist. this is the beginning of more extensive investigations which will revel yet unexplored south serbian mycodiversity. biologica nyssana 4 (1-2)  december 2013: 35-40 sadakovic, k., kuštera, m..  fungal conservation: protected species… 37 figure 1. map of invastigated area jsmt.mali jastrabac, dd-debeli del hill, kf-mt.kalafat, komt.koritnik, jk-jelašnica gorge, se-mt.seličevica, bg-mt.babička gora, gh-mt.suva planina(sw foothill), ds-mt.suva planina, sd-mt.stara planina(sedlar), tdmt.stara planina(arbinje), temt.stara planina(temska area), vs-mt.veliki stol, vp-mt.vlaške planine, mc-mt.mali čemernik, go-mt.goljak, pc-mt.pljačkovica, bk-mt.besna kobila, du-mt.dukat results and discussion of 38 species of macromicetes protected by nature conservation law we located and indentified 17 species, belonging to 13 genera, 7 families and 5 orders. the list of species with number of sightings, location and status is presented in the table (table 1. list of species found with number and localities of findings and status). phylloporus pelletieri (lév.) quél., sarcosphaera coronaria (jacq.) j. schröt. and hericium erinaceus (bull.) pers. are listed on european preliminary red list and are regarded as highly endangered species. p.pelletieri is widespread in europe (very rare in european russia, also in ukraine and moldova), from the lowland up to the montane zone, sometimes subalpine. it is rare in most regions, very local and in low numbers, but more widespread in the alps, e.g. in switzerland. the species is spreading into asia. it is in national lists of endangered fungi in austria, denmark, germany, hungary, moldova, norway, poland, sweden, and the netherlands. in total it has 926 localities in europe and to our knowledge, other than founding in this paper, it has only few unofficial locations in serbia. s.coronaria (syn. s.crassa) is distributed in north europe and mountains of central and south europe up to 1700 m altitude. it is rare in most regions but in some regions more widespread and even numerous in places, for instance in the swiss alps and central italy. it is in national lists of endangered fungi in austria, bulgaria, denmark, estonia (also protected by law), finland, germany, great britain, hungary, norway, poland, and sweden. it has 691 localities in europe with official 2 localities in serbia according to (dahlberg et al., 2006). h.erinaceus is widespread in europe, from the lowland up to the montane zone, north to southern scandinavia; generally rare, for instance in switzerland (5 records between 220 and 930 m) and austria (4 records from the vienna region, altitude 150m). it is locally more common, e.g. in southern england and the central part of the netherlands. it is found on national lists of endangered fungi in armenia, austria, bulgaria, denmark, germany, great britain, greece, macedonia, poland, sweden (also protected by law), switzerland, the biologica nyssana 4 (1-2)  december 2013: 35-40 sadakovic, k., kuštera, m..  fungal conservation: protected species… 38 netherlands (eccf, 2001). it has 435 localities in europe with 3 official in serbia. the rest of the fungal species recorded in this research are also all on other european countries red lists and/or leagaly protected, but because of many lists are still in proces of forming and their data deficiency, we are unable to produce the precise data on the number of their localities and state of populations fully yet. table 1. list of species found with number and localities of findings and status (pnrl-preliminary national red list) taxa total number of recorded localities listed in location pezizales pezizaceae sarcosphaera coronaria (jacq.) j. schröt 1 perl mt. stara planina agaricales hygrophoraceae hygrocybe punicea (fr.) p. kumm 1 pnrl dukat mountain hygropophrous marzuolus (fr.) bres. 6 pnrl mt. suva planina mt.suva planina sw foothill mt.babicka gora mt. seličevica mt. mali jastrebac debeli del hill mt.golemi stol tricholomataceae leucopaxillus giganteus (quel.) singer 1 pnrl mt. stara planina boletales boletaceae boletus impolitus fr. 4 pnrl mt.kalafat mt.seličevica mt.suva planina mt.babicka gora boletus regius kromb. 8 pnrl mt.besna kobila mt. pljackovica mt.selicevica mt.selicevica (gabrovac) mt.mali jastrebac mt.suva planina mt. golemi stol mt.koritnik boletus rhodoxanthus (krombh.) kallenb 2 pnrl mt.vlaška planina debeli del hill boletus satanas lenz pnrl mt. stara planina leccinum crocipodium (letell.) watling 1961 3 mt.seličevica mt.besna kobila jelašnica gorge phylloporus pelletieri (lév.) quél. 1 perl mt.mali čemernik strobilomyces strobilaceus (scop.) berk. 1 pnrl mt.goljak geastrales geastraceae geastrum fornicatum (huds.) hook. 1 mt.dukat geastrum melanocephalum (czern.) v.j. staněk 2 mt.veliki stol debeli del hill phallales phallaceae mutinus caninus (huds.) fr. 1 pnrl mt.veliki stol phallus hadriani vent. 1 pnrl mt. stara planina russulales hericiaceae hericium coralloides (scop.) pers. 1 mt.veliki stol hericium erinaceus (bull.) pers. 1 pnrl perl mt.stara planina (arbinje) biologica nyssana 4 (1-2)  december 2013: 35-40 sadakovic, k., kuštera, m..  fungal conservation: protected species… 39 only 20 species from the red list one found on the strictly protected list and knowing that on the preliminary red list also contained some internationally significant and protected wild species which serbian government had promise to protect (biodiversity strategy of the republic of serbia for the period 2011 – 2018) it is evident that forming of the list of protected species was not based on the preliminary red list data, rather in superficial, subjective and unscientific manner. the fact that in 2007 the author of nprl has presented to ministry of environment and spatial planning project proposition for making the red book of fungi in serbia, which was highly acclaimed by the ministry, was not yet accepted furthermore shows the gap between serbian mycological experts and the government (personal email communication with author). conclusion if a species is rare or endangered in one region or in whole country it doesn’t mean that its state is homogeneous throughout europe and vice versa so biogeographical information on fungi is crucial for understanding biodiversity patterns, evolutionary processes and monitoring. unfortunately these kinds of publications are scarce in serbia, especially for south serbian region due to lack of experts and it’s vastly unexplored, but rich terrains. deficient number of this sort of research may also be contributed to the annual fluctuations of fungal fruiting bodies due to micro and macro climate conditions, uncontrolled wild mushrooms picking, stochastic events and effemeral properties of fungal fruiting bodies. in order to better protect natures rearities, serbia must adopt less anthropocentric and more ecocentric laws. wild mushroom picking itself does not effect the numbers of emerging fruiting bodies but soil trampeling of the mushroom pickers can destroy pre-fruit body primordia formed at the soil surface and thus reduce the frequency of occurrence and reduce their quantity (egli et al., 2006). beside the prevention of picking young and immature specimens and specimens less in size then size allowed for respective species, the state should specify time annually and daily when picking of certain species is allowed. also instead of allowing only picking 2/3 of total number of sporocarps found at the picking site, which is very difficult to assess, government should restrict maximum allowed quantity of collected sporocarps per person and for groups like in some european countries legislations (e.g. in luxembourg one person can pick up to 1kg of sporocarps per day and for a group, the allowed maximum is 3kg). with these measures the disturbance made by trampling of forest floor would be significantly reduced and mushroom picking for sale purposes would be significantly decreased at the same time. in order to bring better nature protecting laws, actual state of serbian mycodiversity must be evaluated. this is only possible trough national and regional checklists, red lists and more extensive biogeographical research. for this to be possible, far better state cooperation with scientific community and experts is imperative. acknowledgments the authors would like to express gratitude to all members of the mycological society of niš for helping in research for this paper. references arnolds, e. 1997: biogeography and conservation. in: wicklow, d., soderstrom, b. (ed.), the mycota iv: environmental and microbial relationships: (2): 115-131, springer-verlag, berlin. biodiversity strategy of the republic of serbia for the period 2011 – 2018. ministry of environment and spatial planning, belgrade 2011. božac, romano. 1978: gljive naših krajeva. grafički zavod hrvatske. breitenbach, j., kränzlin, f. 1995: pilze der schweiz. band 4. verlag mykologia, luzern. 371p. cladera, e., poulsen, e., suen, g., currie, c., 2009: insect symbioses: a case study of past, present, and future fungus-growing ant research. environmental entomology, 38(1):78-92. dahlber, a., croneborg, h., 2006: the 33 threatened fungi in europe. council of europe publishing, belgium. egli, s., peter, m., buser, c., stahel, w., ayer, f., 2006: mushroom picking does not impair future harvests – results of a long-term study in switzerland. biological conservation, 129: 271276. focht, i., 1986: ključ za gljive: ilustrirani uvod u gljivarstvo. naprijed, zagreb. index fungorum. http://www.indexfungorum.org/names/names.asp ivančević, b., 1998: a preliminary red list of the macromycetes of yugoslavia. in: perini, c. (ed.), conservation of fungi in europe: 57-61, università degli studi, siena. ivančević, b., matavulj, m., vukojević, j., karaman, m., 2012: fungi in the legislation of biologica nyssana 4 (1-2)  december 2013: 35-40 sadakovic, k., kuštera, m..  fungal conservation: protected species… 40 the republic of serbia. zbornik matice srpske za prirodne nauke, 123: 51–64 jordan, m., 2004: the encyclopedia of fungi: of britain and europe. frances fincoln ltd., london. kendrick. b. (2011). fungi: ecological importance and impact on humans. wiley online library. kjøller, r ., nilsson, l., hansen, k., schmidt, i., vesterdal, l., gundersen, p., 2012: dramatic changes in ectomycorrhizal community composition, root tip abundance and mycelial production along a stand-scale nitrogen deposition gradient. new phytologist , vol 194(1): 278-286. klemm, d., 2005: cellulose: fascinating biopolymer and sustainable raw material. chemlnform. vol. 36(issue 36), lilleskov, e., hobbie, e., horton, t., 2011: conservation of ectomycorrhizal fungi: exploring the linkages between functional and taxonomic responses to anthropogenic n deposition. fungal ecology, 4: 174-183. mcknight, k., mcknight v., 1998: a field guide to mushrooms: north america. houghton mifflin harcourt, boston. rikardo, m., ileš, m., unković, s., 1995: kako da raspoznate pečurke: jestive gljive i njihovi otrovni dvojnici. evro, beograd. sigh, a., agrawal, m., 2008: acid rain and its ecological consequences. journal of environmental biology, 29 (1): 15-24. the european council for conservation of fungi (eccf). 2001: datasheets of threatened mushrooms of europe, candidates for listing in appendix i of the convention. strasbourg, 13 june 2001. uzelac, b., 2009: gljive srbije i zapadnog balkana. bgv logik, beograd. anticancer compounds from medicinal plants biologica nyssana 6 (1)  september 2015: 41-48 žikić, v. et al.  new data on microgastrinae in serbia and ... 41 original article received: 27 august 2015 revised: 30 august 2015 accepted: 01 september 2015 new data on microgastrinae in serbia and montenegro (hymenoptera: braconidae) and their hosts vladimir žikić, maja lazarević * , saša s. stanković, marijana ilić milošević university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia * e-mail: majalazarević@gmail.com abstract: žikić, v., lazarević, m., stanković, s.s., ilić milošević, m.: new data on microgastrinae in serbia and montenegro (hymenoptera: braconidae) and their hosts. biologica nyssana, 6 (1), september 2015: 4148. the fauna of microgastrinae wasps was investigated on the territory of serbia and montenegro. eighteen species were recorded and most of them emerged from the previously identified hosts. the species belong to seven genera: apanteles (1), cotesia (7), dolichogenidea (2), glyptapanteles (3), microgaster (1), microplitis (2) and pholetesor (2). for the territory of montenegro there is one newly reported species, cotesia tetricus, while for serbia seven species are new: cotesia ofella, dolichogenidea breviventris, glyptapanteles compressiventris, g. porthetriae, microgaster bicolor, microplitis pellucida, m. varipes and pholetesor elpis. apart from microgastrine wasps, some hyperparasitoids were also identified. key words: microgastrinae, parasitoids, lepidoptera, trophic associations apstrakt: žikić, v., lazarević, m., stanković, s.s., ilić milošević, m.: novi podaci o fauni mikrogastrina u srbiji i crnoj gori (hymenoptera: braconidae) i njihovih domaćina. biologica nyssana, 6 (1), septembar 2015: 41-48. istraživana je fauna paraziskih osa iz potfamilije microgastrinae na teritoriji srbije i crne gore. identifikovano je ukupno 18 vrsta, od čega je većina dobijena iz odgajenog i identifikovanog domaćina. registrovane vrste parazitoida pripadaju sledećim rodovima: apanteles (1), cotesia (7), dolichogenidea (2), glyptapanteles (3), microgaster (1), microplitis (2) i pholetesor (2). za teritoriju crne gore registrovana je jedna nova vrsta cotesia tetricus, dok je za teritoriju srbije: cotesia ofella, dolichogenidea breviventris, glyptapanteles compressiventris, g. porthetriae, microgaster bicolor, microplitis pellucida, m. varipes i pholetesor elpis. pored mikrogastrina registrovano je više vrsta hiperparazitoida. key words: microgastrinae, parasitoidi, lepidoptera, trofičke asocijacije introduction members of the subfamily microgastrinae are koinobiont endoparasitoids of larvae lepidoptera and represent one of the most numerous and very important group of braconidae. most of the species are usually small, about 2-5 mm in length, black coloured, sometimes with yellowish parts, 6 (1) • september 2015: 41-48 biologica nyssana 6 (1)  september 2015: 41-48 žikić, v. et al.  new data on microgastrinae in serbia and ... 42 especially on legs. some of the species are solitary parasitoids, in which case only one microgastrine larva develops inside the host body. however, some exhibit different way of parasitism which is gregarious, when several parasitoid individuals develop from only one egg laid by a single female; in which case all parasitoids have the same genotype (s h a ѕ w & h u d d l e s t o n , 1991). beside lepidoptera as hosts for microgastrines larvae, there are several records of other insect groups such as coleopterans, dipterans or hymenopterans (symphyta) (y u et al., 2012). a total of 2000 species of this subfamily have been reported worldwide and of that number about 500 species inhabit the palaearctic (w h i t f i e l d et al., 2002; p é r e z r o d r í g u e z et al., 2013). phylogenetically microgastrinae belongs to the microgastroid complex of subfamilies along with cardiochilinae, cheloninae, ichneutinae, khoikhoiinae, miracinae (w h a r t o n et al., 1992) which is confirmed by later analysis using molecular markers for 18s rrnk, 28s rrnk, protein-coding genes (cad54-405), and the coding gene for acetyl-coenzyme a carboxylase (acc) (s h a r a n o w s k i et al., 2011). classification of microgastrinae is still very confusing because many authors prefer keeping their own systems of identification and separation of some genera and subgenera to new ones. another important fact is that this large subfamily constantly reveals its richness in diversity of species and genera further forcing new reclassifications. the maximum accepted number of genera is 31, which are reported in y u et al. (2012). the biggest problems occur in very large, “traditional genera”, firstly in apanteles förster, cotesia cameron, than in microgaster latreille, microplitis foerster and protapanteles ashmead. the fauna of microgasterinae in serbia and montenegro is poorly investigated. there are few publications that are directly related to the taxonomy of this subfamily: p a p p (1973, 2009), b r a j k o v i ć (1989) and ž i k i ć et al. (2000); whereas most of the cited publications predominantly refer to serbia. sporadically, some records could be found in papers by other authors e.g. (w h i t f i e l d , 1997) but, certainly, the most important are in t o b i a s (1986) and in the global database taxapad by y u et al. (2012). in s a n t o s et al. (2009) there are some data on microgastrinae in citrus orchards. as a result of rather poor and limited research of microgastrinae fauna on the territory of serbia and montenegro, the main aim of the present study was to present some new findings of these wasps through tritrophic associations. material and methods for the purposes of this study the specimens of microgastrinae were collected on the territory of serbia and montenegro during the period of 20012013. material was sampled mainly by collecting caterpillars from plant leaves, both from trees and herbaceous plants. some of the wasp specimens were collected using sweep net and malaise trap. caterpillars were put in specially made cages and they were fed with adequate food plants in the following 10 days. some of the parasitoids emerged directly from caterpillars, while some of them appeared after host pupation (larva/pupal parasitism). most of the collected parasitoids were kept dry and mounted on paper cards and pinned. partly, specimens of each species were conserved in 96% ethanol for further dna analyses. all analysed material has been deposited in the insect collection of the department of biology and ecology, faculty of sciences and mathematics, university of niš, serbia. identification for the identification of feeding plants botanists from the department of biology and ecology, faculty of sciences and mathematics, university of niš, serbia were consulted. larvae of butterflies were identified by boženka hric. parasitoid specimens were identified by dr. jenő papp from hungarian natural history museum, budapest. in this particular study we followed the classification given in the keys proposed by p a p p (1978, 1981, 1982, 1983, 1987). investigated localities all the localities are given the altitude value. all measurements are given in meters above sea level. serbia: bosilegrad, 780 m; deliblatska peščara, 160 m; dukat mt., 1150-1400 m; kruševac, 165 m; lebane, konjino, 275 m; niš, gornji matejevac, 425 m; niš, niška banja 250 m, niš, pantelej, 220 m; sićevačka klisura (gorge), 225 m; tara mt., 1090 m; vlasinsko jezero 1250 m; zasavica, valjevac, 75 m. montenegro: durmitor, žabljak, 1450 m; visitor mt. 1700 m. results the identified species are listed alphabetically. for each species the locality, date, legator’s name and the number of males and females collected were given and those were marked with universal symbols. in cases where there was the host and the plant the information was updated by adding the full names of these members of tritrophic biologica nyssana 6 (1)  september 2015: 41-48 žikić, v. et al.  new data on microgastrinae in serbia and ... 43 associations. additional information and remarks concerning parasitoid biology/strategy and morphology of cocoons are presented in the notes. finally, before the species name new findings for the investigated territories were marked using the following symbols: (*) new to serbia, ( # ) new to montenegro. apanteles lenea nixon, 1986 srb, sićevačka klisura (gorge), 04.06.2011, leg. v. žikić, 1♀; srb, vlasinsko jezero (lake), 05.06.2014, leg. v. žikić, 1♂ on centaurea jacea l. ex larvae verbascum thapsus l. notes: solitary parasitoid. hosts: tortricidae: olethreutes arbutella (l.), oncocera semirubella (scopoli), sparganothis pilleriana (denis & schiffermüller). distribution: austria, bulgaria, czech republic, france, germany, hungary, italy, south korea, romania, russia (chita oblast, primorskye kray, sakhalin oblast), serbia, slovakia, spain, sweden, switzerland, turkey, united kingdom. cotesia jucunda (marshall, 1885) srb, niš, gornji matejevac, 25.05.2014, leg. v. žikić, 25♂, 21♀ on linaria dalmatica subsp. macedonica (griseb.) d. a. sutton ex larvae melitaea didyma (esper). notes: gregarious, separately spun cocoons randomly around dead caterpillar. hosts: geometridae: abraxas grossulariata (l.), alsophila aceraria (denis & schiffermüller), campaea margaritaria (l.), cyclophora linearia (hübner), c. punctaria (l.), c. ruficiliaria (herrichschäffer), epirrita autumnata (borkhausen), erannis defoliaria (clerck), eupithecia dodoneata guenée, operophtera brumata (l.), o. fagata (scharfenberg), phigalia pilosaria (denis & schiffermüller); pieridae: pieris brassicae (l.); pterophoridae: stenoptilia veronicae karvonen; nymphalidae: vanessa atalanta (l.), curculionidae: anthonomus pomorum (l.). distribution: armenia, austria, bulgaria, croatia, czech republic, denmark, estonia, finland, france, germany, greece, hungary, iran, ireland, moldova, mongolia, poland, romania, russia (primorskye kray), serbia, slovakia, spain, sweden, switzerland, turkey, united kingdom. *cotesia ofella (nixon, 1974) srb, kruševac, 03.05.2014, leg. m. lazarević, 65 ♀. notes: gregarious, from unknown yellowish group cocoon associated with chamaecyparis lawsoniana (a. murray) parl. hosts: erebidae: spilosoma lubricipeda (l.); lasiocampidae: euthrix potatoria (l.); noctuidae: acronicta aceris (l.), a. rumicis (l.), simyra dentinosa freyer. distribution: belgium, czech republic, finland, germany, hungary, iran, italy, netherlands, poland, slovakia, spain, switzerland, turkey, ukraine, united kingdom. cotesia sp. 1 srb, bosilegrad, dukat mt., 07.08.2011, leg. v. žikić, 1♂. notes: sweep net. cotesia sp. 2. srb, vlasinsko jezero (lake), 06.06.2014, leg. s. stanković, 3♂, 21♀. notes: gregarious in shared cocoon. hyperparasitized with gelis sp. (ichneumonidae: cryptinae). # cotesia tetricus (reinhard, 1880) mne, durmitor, žabljak, 03.08.2005, leg. v. žikić, 25♀. notes: gregarious from unknown yellowish group cocoon. hyperparasitized by mesochorus sp. (ichneumonidae: mesochorinae). hosts: erebidae: parasemia plantaginis (l.), spilosoma lubricipeda; geometridae: erannis defoliaria, eupithecia exiguata (hübner); nymphalidae: erebia aethiops (esper), lasiommata megera (l.), maniola jurtina; zygaenidae: zygaena filipendulae. distribution: austria, croatia, czech republic, finland, france, germany, greece, hungary, iran, ireland, italy, kazakhstan, latvia, norway, poland, romania, russia (yaroslavl oblast), sweden, switzerland, turkey, united kingdom. cotesia tibialis curtis, 1830 srb, sićevačka klisura (gorge), 13.04.2014, leg. v. žikić, 13♀; srb, vlasinsko jezero (lake), 06.06.2014, leg. s. stanković, 3♂, 21♀. notes: gregarious in shared cocoon. hyperparasitized with gelis sp. (ichneumonidae: cryptinae). hosts: arctiidae: arctia tigrina (villers); bombicidae: bombyx mori l.; cossidae: dyspessa ulula (borkhausen); erebidae: diaphora mendica (clerck), ocnogyna baetica (rambur), o. loewii (zeller), spilosoma lubricipeda (l.); gelechiidae: pexicopia malvella (hübner); geometridae: eupithecia abbreviata stephens, e. innotata (hufnagel), e. tripunctaria herrich-schäffer, phigalia pilosaria (denis & schiffermüller); lasiocampidae: dendrolimus pini (l.), eriogaster lanestris (l.); lycaenidae: plebejus loewii (zeller); biologica nyssana 6 (1)  september 2015: 41-48 žikić, v. et al.  new data on microgastrinae in serbia and ... 44 noctuidae: acronicta aceris, a. euphorbiae (denis & schiffermüller), a. leporina (l.), a. rumicis, agrotis clavis (hufnagel), a. rectangular (denis & schiffermüller), a. segetum (denis & schiffermüller), anarta myrtilli (l.), antoculeora ornatissima walker, autographa gamma (l.), euclidia mi (clerck), catocala nupta (l.), cerastis rubricosa (denis & schiffermüller), cucullia argentea (hufnagel), c. artemisiae (hufnagel), c. asteris (denis & schiffermüller), c. lucifuga (denis & schiffermüller), diachrysia chrystis (l.), discestra trifolii (hufnagel), eupsilia transversa (hufnagel), eurois occulta (l.), euxoa temera (hübner), e. tritici (l.), graphiphora augur (fabricius), lacanobia oleracea (l.), l. pisi (l.), mamestra brassicae (l.), noctua pronuba (l.), orthosia gothica (l.), o. gracilis (denis & schiffermüller), o. incerta (hufnagel), xestia cnigrum (l.), x. exoleta leech, x. stigmatica (hübner), x. triangulum (hufnagel), xylena exsoleta (l.), x. vetusta (hübner); notodontidae: clostera anastomosis (l.), phalera bucephala (l.); nymphalidae: aglais urticae (l.), euphydryas aurinia (rottemburg), aglais io (l.), maniola jurtina (l.), pararge aegeria (l.), pyronia tithonus (l.), vanessa indica herbst; pieridae: aporia crataegi (l.), leptidea sinapis (l.), pieris brassicae; plutellidae: plutella xylostella (l.); pyralidae: loxostege sticticalis (l.), ostrinia nubilalis (hübner), pyrausta cingulata (l.), p. purpuralis (l.), p. sanguinalis (l.); tortricidae: ancylis laetana (fabricius), tortrix viridana l.; yponomeutidae: yponomeuta malinellus zeller; zygaenidae: zygaena filipendulae (l.). distribution: afghanistan, armenia, austria, azerbaijan, belgium, bulgaria, canary islands, china (shanxi, xinjiang), croatia, czech republic, estonia, finland, france, georgia, germany, greece, hungary, iran, israel, italy, japan, kazakhstan, kyrgyzstan, latvia, lithuania, f.y.r. of macedonia, moldova, mongolia, montenegro, netherlands, poland, romania, russia (adygeyskaya, chita oblast, irkutsk oblast, kaliningrad oblast, krasnodor kray, krasnoyarsk kray, moscow oblast, primorskye kray, sankt petersburg, tatarskaya respublika, vladimir oblast, yaroslavl oblast), serbia, slovakia, spain, sweden, switzerland, turkey, turkmenistan, ukraine, united kingdom, uzbekistan. cotesia zygaenarum (marshall, 1885) srb, sićevačka klisura (gorge), 15.05.2012, leg. v. žikić, 155♂ 167♀ ex 5th instar larvae of zygaena filipendulae. notes: gregarious. associated with ulmus sp. host: geometridae: phigalia pilosaria; lycaenidae: polyommatus icarus (rottemburg); noctuidae: diloba caeruleocephala (l.); nymphalidae: euphydryas aurinia; pieridae: colias hyale (l.); zygaenidae: zygaena brizae (esper), z. ephialtes (l.), z. filipendulae, z. laeta (hübner), z. lonicerae (scheven), z. trifolii (esper), z. viciae (denis & schiffermüller). distribution: albania, armenia, austria, azerbaijan, china (hubei), czech republic, finland, france, germany, greece, hungary, iran, italy, japan, korea, f.y.r. of macedonia, moldova, mongolia, poland, romania, russia (dagestanskaya respublika, krasnodar kray, omsk oblast, primorskye kray, ryazan oblast, voronezhskaya oblast), serbia, slovakia, switzerland, turkey, united kingdom. *dolichogenidea breviventris (ratzeburg, 1848): srb, sićevačka klisura (gorge), 13.04.2014, leg. v. žikić, 13 ♀. notes: gregarious. hosts: coleophoridae: coleophora anatipennella (hübner), c. betulella heinemann, c. flavipennella (duponchel), c. gryphipennella (hübner), c. lutipennella (zeller), c. prunifoliae doets, c. serratella (l.); noctuidae: orthosia miniosa (denis & schiffermüller). distribution: canada (newfoundland & labrador), czech republic, egypt, finland, germany, hungary, italy, korea, moldova, netherlands, poland, romania, russia (voronezhskaya oblast), slovakia, sweden, switzerland, turkey, united kingdom. dolichogenidea candidata (haliday, 1834) = d. longicauda (wesmael, 1837) srb, niš, pantelej, 29.05.2014, leg. v. žikić, 1♀ on euonymus europaeus l. ex pupa yponomeuta cagnagella (hübner). notes: solitary parasitoid. hosts: chimabachidae: diurnea lipsiella (denis & schiffermüller); erebidae: euproctis chrysorrhoea (l.); gelechidae: recurvaria leucatella (clerck), r. nanella (denis & schiffermüller); geometridae: abraxas grossulariata; gracillaridae: gracillaria syringella (fabricius); lasiocampidae: malacosoma neustria (l.); tortricidae: hedya nubiferana (haworth), pandemis cerasana (hübner), spilonota ocellana (denis & schiffermüller); yponomeutidae: paraswammerdamia lutarea (goeze), swammerdamia caesiella (hübner). distribution: azerbaijan, bulgaria, cape verde islands, germany, greece (crete), hungary, ireland, f.y.r. of macedonia, mongolia, romania, russia (khabarovsk kray, krasnodar kray, primorskye biologica nyssana 6 (1)  september 2015: 41-48 žikić, v. et al.  new data on microgastrinae in serbia and ... 45 kray, sakhalin oblast), serbia, sweden, tajikistan, turkmenistan, united kingdom, uzbekistan. [d. longicauda (wesmael, 1837)] host: bucculatridae: bucculatrix cristatella (zeller); chimbachidae: diurnea salicella (hübner); choreutidae: choreutis nemorana (hübner), c. pariana (clerck); elachisitdae: agonopterix ocellana (fabricius), blastodacna atra (haworth); erebidae: euproctis chrysorrhoea; gelechiidae: anarsia eleagnella kuznetsov, a. lineatella zeller, dichomeris derasella (denis & schiffermüller), exoteleia dodecella (l.), mirificarma lentiginosella (zeller), recurvaria leucatella, r. nanella, teleiodes paripunctella (thunberg), t. proximella (hübner), t. saltuum (zeller); gracillariidae: acrocercops brongniardella (fabricius), caloptilia populetorum (zeller), gracillaria syringella, phyllonorycter corylifoliella (hübner), p. elmaella doğanlar & mutuura; lasiocampidae: malacosoma neustria; momphidae: mompha subbistrigella (haworth); peleopodidae: carcina quercana (fabricius); plutellidae: plutella xylostella; praydidae: atemelia torquatella (lienig & zeller); psychidae: dahlica lichenella (l.); pyralidae: acrobasis consociella (hübner), a. marmorea (haworth); elegia fallax (staudinger), etiella zinckenella (treitschke), tortricidae: acleris holmiana (l.), a. laterana (fabricius), a. quercinana (zeller), adoxophyes orana (fischer von röslerstamm), archips oporana (l.), a. rosana (l.), choristoneura murinana (hübner), c. rosaceana (harris), ditula angustiorana (haworth), epinotia nigricana (herrich-schäffer), eudemis profundana (denis & schiffermüller), exapate duratella heyden, hedya nubiferana, h. pruniana (hübner), pandemis cerasana, parapandemis chondrillana (herrich-schäffer), rhopobota naevana (hübner), rhyacionia buoliana (denis & schiffermüller), spilonota ocellana, tortricodes alternella (denis & schiffermüller), tortrix viridana, zeiraphera griseana (hübner), z. rufimitrana (herrichschäffer); yponomeutidae: paraswammerdamia albicapitella (scharfenberg), p. lutarea (haworth), swammerdamia caesiella, yponomeuta cagnagella (hübner), zelleria parnassiae braun; ypsolophidae: ypsolopha alpella (denis & schiffermüller); cecidomyiidae: dasineura pyri; tenthredinidae: phyllocolpa benson. distribution: afghanistan, armenia, austria, azerbaijan, belarus, belgium, bulgaria, canada (british colombia), czech republic, estonia, finland, france, georgia, germany, hungary, italy, korea, latvia, lithuania, moldova, mongolia, netherlands, poland, romania, russia (buryatskaya respublika, krasnodar kray, sankt petersburg), serbia, slovakia, slovenia, spain, switzerland, turkey, ukraine, united kingdom. *glyptapanteles compressiventris (muesebeck, 1921) srb, niš, pantelej, 26.04.2014, leg. v. žikić, 42♂, 62♀, ex larva arctia villica (l.). notes: gregarious, separately spun cocoons. hosts: erebidae: amata phegea (l.), diaphora mendica, arctia villica, ocnogyna baetica, o. loewii, o. pierreti (rambur), phragmatobia fuliginosa (l.), spilosoma lubricipeda; noctuidae: amphipyra perflua (fabricius); tortricidae: archips xylosteana (l.). distribution: armenia, azerbaijan, canada (manitoba, northwest territories, nunavut, quebec), croatia, czech republic, finland, germany, hungary, italy, italy (sicily), kazakhstan, lithuania, f.y.r. of macedonia, moldova, netherlands, romania, russia (kamchatka oblast, primorskye kray, sakhalin oblast, sankt petersburg, voronezhskaya oblast), slovakia, spain, switzerland, turkey, usa (new hampshire), united kingdom. glyptapanteles liparidis (bouché, 1834) srb, tara mt., 25.06.2014, leg. v. žikić, 2♀. on ostrya carpinifolia. ex 3rd larval instar lymantria dispar (l.). notes: gregarious. hosts: erebidae: calliteara abietis (denis & schiffermüller), c. pudibunda (l.), arctia villica, euproctis chrysorrhoea, e. taiwana (shiraki), orgyia antiquiodes (hübner), o. australis walker, o. thyellina butler; lasiocampidae: dendrolimus albolineatus matsumura, d. pini, d. punctatus (walker), d. spectabilis (butler), d. superans (butler), eriogaster lanestris, malacosoma neustria; lymantriidae: dasychira pseudabietis butler, ivela auripes butler, lymantria dispar, l. obfuscate walker; noctuidae: acronicta rumicis; notodontidae: clostera anastomosis; tortricidae: rhyacionia buoliana. distribution: austria, belarus, bulgaria, china (beijing, heilongjiang, hunan, jilin, liaoning, nei menggu, shaanxi, taiwan, zhejiang), czech republic, finland, france, germany, hungary, india, iran, italy, japan, kazakhstan, lithuania, moldova, mongolia, poland, romania, russia (chita oblast, irkutsk oblast, kaliningrad oblast, khabarovsk kray, krasnodar kray, novosibirsk oblast, primorskye kray, sakhalin oblast, sankt petersburg, saratov oblast, tomsk oblast, voronezhskaya oblast, yaroslavl oblast), serbia, biologica nyssana 6 (1)  september 2015: 41-48 žikić, v. et al.  new data on microgastrinae in serbia and ... 46 slovakia, spain, sweden, switzerland, ukraine, united kingdom. *glyptapanteles porthetriae (muesebeck, 1928) srb, deliblatska peščara, 17.05.2001, a. ćetković, 1♀. notes: mal. trap. srb, niš, niška banja, 23.05.2014; leg. s. stanković on acer monspssulanum l. and ulmus sp. ex 2nd instar larvae of l. dispar; srb, sićevačka klisura (gorge), 21.05.2014, leg.v. žikić, 2♂, 2♀. notes: solitary, white cocoon underside leaf, on populus tremula l. ex 2nd instar larvae of l. dispar; srb, lebane, konjino, 03.05.2014, leg. s. stanković, 7♂, 10♀ on prunus domestica l. ex 2nd instar larvae of l. dispar; 3♂, 3♀ on cydonia oblonga mill. ex 2nd instar larvae of l. dispar. notes: solitary, white cocoons. hosts: geometridae: alcis repandata (l.), phigalia titea cramer; lymantridae: lymantria dispar, l. obfuscate. distribution: armenia, austria, azerbaijan, bulgaria, china (jilin), croatia, czech republic, finland, france, georgia, germany, greece, hungary, india, iran, italy, sicily, korea, moldova, morocco, poland, portugal, romania, russia (chita oblast, dagestanskaya respublika, moscow oblast, primorskye kray, voronezhskaya oblast, yaroslavl oblast), serbia, slovakia, spain, switzerland, turkey, ukraine, united kingdom. *microgaster bicolor (nees 1834) srb, deliblatska peščara, 17.05.2001, leg. a. ćetković, 1♂, 1♀. notes: mal. trap. hosts: elachistidae: elachista gleichenella (fabricius); erebidae: euproctis similis (fuessly), orgyia thyellina; gelechiidae: chrysoesthia drurella (fabricius); gracillariidae: aspilapteryx tringipennella (zeller), caloptilia semifascia (haworth), gracillaria syringella, parornix fagivora (frey), phyllonorycter acaciella (duponchel), p. acerifoliella (zeller), p. agilella (zeller), p. blancardella (fabricius), p. cavella (zeller), p. celtidella (rondani), p. comparella (duponchel), p. connexella (zeller), p. corylifoliella (hübner), p. cydoniella (denis & schiffermüller), p. emberizaepennella (bouché), p. froelichiella (zeller), p. harrisella (l.), p. heegeriella (zeller), p. junoniella (zeller), p. kleemannella (fabricius), p. lantanella (schrank), p. mespilella (hübner), p. millierella (staudinger), p. nicellii (stainton), p. parisiella (wocke), p. populifoliella (treitschke), p. robiniella (clemens), p. spinicolella (zeller), p. strigulatella (lienig & zeller), p. tenerella (joannis); lyonetiidae: leucoptera malifoliella (o. costa), lyonetia rajella (l.); noctuidae: melanchra persicariae (l.); plutellidae: plutella porrectella (l.); psychidae: acanthopsyche atra (l.); pyralidae: ephestia elutella (hübner); tineidae: psychoides verhuella bruand; tischeriidae: tischeria ekebladella (bjerkander); tortricidae: eucosma aemulana (schläger); cimbicidae: trichiosoma betuleti. distribution: belgium, bulgaria, canary islands, china, china (ningxia), croatia, finland, france, georgia, germany, greece, greece (crete), hungary, iran, ireland, italy, japan, lithuania, moldova, mongolia, new zealand, poland, romania, russia (kamchatka oblast, moscow oblast, sankt petersburg, yaroslavl oblast), serbia, slovakia, spain, switzerland, turkmenistan, ukraine, united kingdom. *microplitis pellucida telenga, 1955 srb, zasavica, valjevac, 19.05.2010, leg. a. ćetković, 5♂. notes: mal. trap; srb, sićevačka klisura, 12.05.2013, leg. v. žikić, 2♂, 3♀. on various plants: populus alba l., ulmus sp., malus domestica borkh. ex lymantria dispar 2nd larval instar. notes: solitary, white cocoon. hosts: no data distribution: denmark, germany, hungary, korea, netherlands, russia (altayskiy kray, primorskye kray). *microplitis varipes (ruthe, 1860): srb, niš, pantelej, 14.09.2014, leg. v. žikić, 1♀. notes: sweep net hosts: crambidae: loxostege sticticalis; noctuidae: cucullia scopariae dorfmeister, hecatera bicolorata (hufnagel), omphalophana antirrhinii (hübner); chrysomelidae: chrysolina didymata. distribution: austria, azerbaijan, china (qinghai, xinjiang), czech republic, finland, germany, hungary, italy, kazakhstan, moldova, mongolia, montenegro, netherlands, poland, russia (chita oblast, krasnodar kray, ryazan oblast, sankt petersburg, yaroslavl oblast), switzerland, turkey, ukraine. *pholetesor elpis (nixon 1973) srb, niš, g. matejevac, 24.05.2014, leg. v. žikić, 30♂, 5♀. notes: gregarious; yellow cocoons distributed in 3 rows, one over another forming kind of cluster. hosts: coleophoridae: coleophora serratella (l.); elachistidae: elachista cingillella herrich-schäffer), e. subnigrella (douglas); gracillariidae: caloptilia rufipennella (hübner), calybites auroguttellus biologica nyssana 6 (1)  september 2015: 41-48 žikić, v. et al.  new data on microgastrinae in serbia and ... 47 (stephens), phyllonorycter blancardella, p. comparella. distribution: austria, azerbaijan, bulgaria, croatia, czech republic, finland, france, greece, hungary, korea, mongolia, netherlands, poland, russia (magadanskaya oblast, primorskye kray, sakhalin oblast), slovakia, ukraine, united kingdom. pholetesor sp. mne, visitor mt, 11.07.2013, leg. v. žikić, 7♀, ex euproctis similis; notes: gregarious, from small white group cocoon. discussion in this study 18 microgastrinae species emerged forms caterpillars from different larval instars or from pupae. the identified species belong to seven genera: apanteles (1), cotesia (7), dolichogenidea (2), glyptapanteles (3), microgaster (1), microplitis (2) and pholetesor (2). although there is a limited number of species reported here, it should be pointed out that eight of the total identified numbers are recorded for the first time on the investigated territories; one for montenegro and seven for the territory of serbia. as a result it is evident that a better knowledge about the diversity of this group of braconidae is needed. concerning the role of microgastrine in ecosystems as a very important factor of biological control, the importance of investigation of this group is even higher. it needs to be added that, beside the primary parasitoids such microgastrinae are, our study revealed various hyperparasitoids which will be considered in the next step of this investigation aiming at connecting them with other members in tetratrophic associations. as for dolichogenidea longicauda the data on hosts and the distribution are presented separately because there are two opinions on its taxonomical status; it is also considered as a different species d. candidate. acknowledgements. we are thankful to dr. jenő papp from hungarian natural history museum, budapest, hungary for his great help in the organisation of this manuscript. we also want to thank to dr. andjeljko petrović for collecting some material for this study. this research was supported by the “ministry of education, science and technological development of the republic of serbia”, no. iii43001. references brajković, m.m. 1989: knowledge of the braconidae (hymenoptera) fauna in yugoslavia. bulletin of the natural history museum, b (43/44): 127-138. [in serbian with english s.]. papp, j. 1973: contributions to the braconid fauna of yugoslavia (hymenoptera, braconidae) i. acta musei macedonici scientiarum naturalium, 14: 1-23. papp, j. 1978: a survey of the european species of apanteles foerster (hym.: braconidae: microgastrinae) ii. the leavigatus group 1.s annales historico-naturales musei nationalis hungarici, 70: 265–300. papp, j. 1981: contributions to the braconid fauna of hungary, ii. microgasterinae (hymenoptera: braconidae). folia entomologica hungarica, 42 (1): 129–141. papp, j. 1982: a survey of the european species of apanteles foerster (hymenoptera: braconidae: microgastrinae) vi. the laspeyresiella, merula, falcatus and validus group. annales historiconaturales musei nationalis hungarici, 74: 255– 267. papp, j. 1983: contributions to the braconid fauna of hungary, iv. microgastrinae (hymenoptera: braconidae). folia entomologica hungarica, 44 (1): 125–138. papp, j. 1987: a survey of the european species of apanteles foerster (hymenoptera: braconidae: microgastrinae). x. the glomeratus group 2. annales historico-naturales musei nationalis hungarici, 79: 207–258. papp, j. 2009: contribution to the braconid fauna of the former yugoslavia, v. ten subfamilies (hymenoptera: braconidae). entomofauna, 30 (1): 1-35. pérez-rodríguez, j., oltra-moscardó, t., perisfelipo, f.j., jiménez-peydró, r. 2013: microgastrinae (hymenoptera: braconidae) in the forest state of artikutza (navarra: spain): diversity and community structure. insects, 4: 493-505. santos, j.p. dos, redaelli, l.r., dal soglio, f.k., foelkel, e., costa, v.a. 2009: variacao sazonal de lepidopteros minadores e seus parasitoides em plantas de crescimento espontaneo em pomar organico de citros em montenegro, rs, brasil. arquivos do instituto biologico (sao paulo), 76 (3): 381-391. sharanowski, b. j., dowling, a. p. g., sharkey, m. j. 2011: molecular phylogenetics of braconidae (hymenoptera: ichneumonoidea), based on multiple nuclear genes, and implications for classification. systematic entomology, 36 (3): 549-572. shaw, m. r. & huddleston, t. 1991: classification and biology of braconid wasps, pp. 80–86. in: handbook for the identification of british biologica nyssana 6 (1)  september 2015: 41-48 žikić, v. et al.  new data on microgastrinae in serbia and ... 48 insects, vol. 7, part 11, royal entomological society, london, england. tobias, v.i. 1986. acaeliinae, cardiochilinae, microgastrinae, miracinae. supplement. pp. 336501. in: medvedev g.s. (ed.) 'opredelitel nasekomych evrospeiskoi tsasti sssr 3, peredpontdatokrylye 4. opr. faune sssr.' 145:1-501. pp. 336-501. [keys to the insects of the european part of ussr. hymenoptera.] wharton, r.a., shaw, s.r., sharkey, m.j., wahl, d.b., woolley, j.b., whitfield, j.b., marsh, p.m., johnson, w. 1992: phylogeny of the subfamilies of the family braconidae (hymenoptera: ichneumonoidea): a reassessment. cladistics, 8 (3): 199–235. whitfield, j.b. 1997: subfamily microgastrinae. in manual of the new world genera of the family braconidae (hymenoptera). the international society of hymenopterists: washington, dc, usa, pp. 339-371. whitfield, j.b., mardulyn, p., austin, a.d., dowton, m. 2002: phylogenetic analysis of relationships among microgastrine braconid wasp genera based on data from the 16s, coi and 28s genes and morphology. systematic entomology, 27: 337-359. yu, d.s., achterberg, c. van, horstmann, k. 2012: taxapad 2012 – world ichneumonoidea 2011. taxonomy, biology, morphology and distribution. on usb flash drive. ottawa, ontario, canada. www.taxapad.com žikić, v., brajković, m., tomanović, ž. 2000: preliminary results of braconid fauna research (hymenoptera: braconidae) found in sićevo gorge, serbia. acta entomologica serbica, 5 (12): 95-110. pepłowska-marczak et al., 2019, biologica nyssana 10(1) 10 (1) september 2019: 63-64 doi: 10.5281/zenodo.3464012 new observations of the woodchat shrike lanius senator linnaeus, 1758 in southeastern serbia short communication danuta pepłowska-marczak kampinos national park, department of science and monitoring, tetmajera street 38, izabelin, poland sowa.ruda@gmail.com dawid marczak university of ecology and management in warsaw, olszewska street 12, warsaw, poland dawid.marczak@gmail.com saša s. stanković university of niš, department of biology and ecology, faculty of sciences and mathematics, višegradska 33, 18000 niš, serbia sasasta@gmail.com (corresponding author) received: december 10, 2018 revised: mart 21, 2019 accepted: august 13, 2019 abstract: during the period from 2013 to 2018 three observations of the woodchat shrike (lanius senator linnaeus, 1758) were made. one pair and two single males were observed in the southeastern serbia on three localities. localities of the three records were marked on the utm map of serbia and utm codes were given. key words: lanius senator, woodchat shirke, birds of serbia apstrakt: nova opažanja crvenoglavog svračka lanius senator linnaeus, 1758 u jugoistočnoj srbiji u periodu od 2013. do 2018. godine zabeležena su tri zapažanja crvenoglavog svračka (lanius senator linnaeus, 1758). jedan par i dva samca su primećeni u jugoistočnoj srbiji na tri lokaliteta. lokaliteti ovih zapisa označeni su na utm mapi srbije i date su utm šifre. ključne reči: lanius senator, crvenoglavi svračak, ptice srbije woodchat shrike lanius senator linnaeus, 1758 is a rare breeding species recorded in several regions of serbia (puzović et al., 2015, šćiban et al., 2015). this species occurs in open areas with shrubs, usually on the hills (šćiban et al., 2015). in serbia the size of population is estimated at 580-790 nesting pairs, out of which in serbia without kosovo i metohija 280-390 nesting pairs respectively. it is estimated that its population is increasing (puzović et al., 2015). woodchat shrike breeds in west palearctic in middle and lower middle latitudes mainly in mediterranean climatic zone (snow & perrins, 1998). the most northern distribution is in northern poland, in gdansk pomerania (neubauer, 2013). in this short note we report three new observations of this species in southeastern serbia, from 2013 to 2018. observations were made using leica 10x50 binoculars, while birdwatching in serbia. during this period, l. senator individuals were spotted on the following localities (fig 1): 1. one pair observed in suitable nesting habitat in breeding season. 17.07.2013. milovanci, near vlasina lake, southeastern serbia. utm grid code: fn12 (42°40’50.54’’ n, 22°21’52.98’’ e) on the map marked with black triangle (fig 1). obs. danuta pepłowska-marczak, dawid marczak. 2. one adult male observed in breeding season in possible nesting habitat. 26.07.2017. čar near the border between serbia and fyr macedonia, southeastern serbia. utm grid code: em57 (42°15’43.27’’ n, 21°42’45.40’’ e) on the map marked with black square (fig 1). obs. danuta pepłowska-marczak, dawid marczak. 3. one adult male observed in breeding season in possible nesting habitat. 10.05. 2018. ivanje, near leskovac, southeastern serbia. utm grid code: en46 (43°00’11.54’’ n, 21°35’07.96’’ e). on the map marked with black circle (fig 1). obs. danuta pepłowska-marczak, dawid marczak, saša stanković. observed individuals occurred in the open area mostly dry meadows covered with individual low bushes. the presence of thorny shrubs, mainly from the family rosaceae such as pyrus spp., crataegus monogyna jacq., prunus spinosa l. and others was notable (fig 2). the males in all cases showed territorial behaviors, with breeding status: possible breeder or probable breeding. © 2019 pepłowska-marczak et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work noncommercially under the same license as the original. 63 biologica nyssana ● 10 (1) september 2019: 63-64 pepłowska-marczak et al. ● new observations of the woodchat shrike lanius senator linnaeus, 1758 in southeastern serbia these observations of l. senator represent a contribution to the distribution and population census of this species, and also the need of protection of its habitat. fig 1. utm map of serbia with tree localities where woodchat shrike specimens were spotted. locality 1: milovanci, near vlasina lake; black triangle. locality 2: čar, serbia and fyr macedonia border; black square. locality 3: ivanje, near leskovac; black circle fig 2. typical habitat of woodchat shrike. ivanje, southeastern serbia. photo s. stanković references neubauer, g. 2013. recent records of lesser grey shrike lanius minor and woodchat shrike l. senator in gdańsk pomerania. ptaki pomorza, 4: 135147. puzović, s., radišić, d., ružić, m., rajković, d., radaković, m., pantović, u., janković, m., stojnić, n., šćiban, m., tucakov, m., gergelj, j., sekulić, g., agošton, a., raković, m. 2015. ptice srbije: procena veličina populacija i trendova gnezdarica 2008-2013. društvo za zaštitu i proučavanje ptica srbije, novi sad. 159 p. snow, d. w., perrins, c. m. 1998. the birds of the western palearctic, concise edition. vol. 2, passerines. oxford university press, oxford, new york. 1697 p. šćiban, m., rajković, d., radišić, d., vasić, v., pantović, u. 2015. ptice srbije kritički spisak vrsta. pokrajinski zavod za zaštitu prirode, društvo za zaštitu i proučavanje ptica srbije, novi sad. 196p. 64 jevtić, a., đekić, v.: influence of growing season on some agronomic characteristics of winter wheat cultivars. biologica nyssana, 9 (2). december, 2018 biologica nyssana 9 (2) ⚫ december 2018: 119-131 jevtić, a., đekić, v. ⚫ influence of growing season on some agronomic... 133 original article received: 24 january 2018 revised: 22 august 2018 accepted: 6 november 2018 influence of growing season on some agronomic characteristics of winter wheat cultivars aleksandra jevtić1, vera đekić2 1high agricultural food school of professional studies, ćirila and metodija 1, 18400 prokuplje, serbia 2small grains research centre, save kovačevića 31, 34000 kragujevac, serbia * e-mail: verarajicic@yahoo.com abstract: jevtić, a., đekić, v.: influence of growing season on some agronomic characteristics of winter wheat cultivars. biologica nyssana, 9 (2). december, 2018: 133-139. the experiment was established in the experimental field of the small grains research centre in kragujevac during the two years. the winter wheat varieties used in the experiment was takovčanka, kruna, toplica and planeta. the following characteristics were analysed: grain yield, plant height, number of plants per m2, 1000 grain weight and test weight. the highest yield of all tested varieties of winter wheat was achieved by takovčanka (4.124 t/ha). the highest plant height for all years was recorded for the cultivar toplica (81.90 cm). the highest 1000 grain weight of investigation on winter wheat takovčanka cultivar (44.05 g). the highest two-year average value of test weight was found in the cultivars kruna and planeta (71.03 kg/hl). analysis of variance was found to have the highly significant effect of years in grain yield, plant height, 1000-grain weight and test weight and significant differences in number of plants per m2 at investigated wheat were found in relation to the year. key words: cultivar, grain yield, winter wheat apstrakt: jevtić, a., đekić, v.: uticaj vegetacije na neke agronomske osobine kod ozime pšenice. biologica nyssana, 9 (2). decembar, 2018: 133-139. eksperiment je izveden na oglednom polju centra za strna žita u kragujevcu tokom dve vegetacione godine. u eksperimentu su korišćene sorte ozime pšenice takovčanka, kruna, toplica i planeta. analizirane su sledeće osobine: prinos zrna, visina biljke, broj biljaka po m2, masa 1000 zrna i hektolitarska masa. najveći prinos kod svih ispitanih sorti ozime pšenice ostvarila je sorta takovčanka (4.124 t/ha). najveću visinu biljaka tokom eksperimenta ostvarila je sorta toplica (81,90 cm). najveću masu 1000 zrna kod ispitivanih sorti ozime pšenice ostvarila je sorta takovčanka (44,05 g). najveću prosečnu dvogodišnju vrednost hektolitarske mase ustanovljena je kod sorti kruna i planeta (71,03 kg/hl). analizom varijanse utvrđen je veoma značajan efekat godine na prinos zrna, visinu biljaka, masu 1000 zrna i hektolitarsku masu, a značajne razlike ustanovljene su kod broja biljaka po m2. ključne reči: ozima pšenica, prinos zrna, sorta 9 (2) • december 2018: 133-139 doi: 10.5281/zenodo.2538606 biologica nyssana 9 (2) ⚫ december 2018: 119-131 jevtić, a., đekić, v. ⚫ influence of growing season on some agronomic... 134 introduction new perspective wheat lines and varieties has more and better filled grain, higher yield, grain mass and farinaceous content, while proteins and lysine were smaller compared with older varieties (đekić et al., 2012, jelić et al., 2013). wheat cultivars that were in production until the end of the eighties were characterized by the lower yields, good technological quality and higher stem sensitive on lodging (zečević et al., 2007). due to lower resistance on lodging, that cultivars were grown at modest soils and therefore they had lower yields (đekić et al., 2014; jelić et al., 2012). agronomic cultivar value depends not only on its genetic potential for yield, but also on its ability to achieve genetic potential under different conditions of production (malešević, 2008). new varieties are characterized by good technological quality, better resistance to lodging and diseases, shorter stem and more efficient assimilates usage (browne et al., 2006; jovanović et al., 2006; malešević et al., 2008; hristov et al., 2011; đekić et al., 2016). several factors are decisive in increasing wheat yields: the cultivar, cultural practices, agroecological conditions, local climatic and soil characteristics, mineral nutrition and adequate protection from plant diseases, pests and weeds. in regard to the elements of mineral nutrition, nitrogen plays a major role in increasing the yield of wheat (kastori et al., 2005; jovanović et al., 2006; malešević, 2008; đekić et al., 2014; jelić et al., 2014). the aim of this study was the determination of the cultivars and ecological environmental factors influence on differences in stability and adaptability of cultivars regard the grain yield, plant height, number of plants per m2, 1000 grain weight and test weight of tested winter wheat cultivars, as well as specificity cultivars exploring in relation to growing seasons conditions. material and methods experimental design and statistical analysis during the 2011/12 and 2012/13 growing seasons, four cultivars of winter wheat (takovčanka, kruna, toplica and planeta), cultivated at the small grains research centre in kragujevac were investigated. experiments have been conducted in randomized block systems, with a plot size of 10 m2 (2 m x 5 m) in five replicates. the usual techniques for wheat production were applied, and it was done in the optimum sowing time in late october. 400 kg/ha of fertilizer npk 15:15:15 was added in the fall on the investigated plots, while during the spring fertilization, 300 kg/ha (kan) was supplemented. the following properties were analyzed: grain yield (t/ha), plant height (cm), number of plants per m2, 1000 grain weight (g) and test weight (kg/hl). on the basis of achieved research results the usual variational statistical indicators were calculated: average values, standard error and standard deviation. statistical analysis was made in the module analyst program sas/stat (sas institute, 2000). meteorological conditions this study was conducted over a three-year period in the šumadija region, central serbia, on a vertisol soil, at kragujevac location, 173-220 m a. s. l. (44° 22′ n, 20°56′ e), in a temperate continental climate having an average annual temperature of 11.5 °c typical of šumadija districts in serbia and a rainfall amount of about 550 mm. kragujevac area is characterized by a moderate continental climate, which general feature is uneven distribution of rainfall by month. the data in tab. 1 for the investigated period (2011-2013) clearly indicate that the years in which the researches were conducted differed from the typical multi-year average of kragujevac region regard the meteorological conditions. table 1. middle monthly air temperature and precipitation amount (kragujevac) year months average x xi xii i ii iii iv v vi mean monthly air temperature (oc) 2011/12 10.4 3.1 4.6 0.7 -3.7 8.1 12.9 16.1 23.0 8.35 2012/13 13.5 9.5 1.7 2.9 4.0 6.5 13.4 18.2 19.9 9.96 average 12.5 6.9 1.9 0.5 2.4 7.1 11.6 16.9 20.0 8.87 the amount of rainfall (mm) 2011/12 33.3 1.3 43.3 117.2 60.1 5.7 74.5 87.3 57.8 480.5 2012/13 56.2 17.7 16.4 62.4 84.3 102.0 41.2 70.8 30.3 481.3 average 57.6 70.4 71.5 58.5 62.7 45.4 48.9 56.6 58.2 529.8 biologica nyssana 9 (2) ⚫ december 2018: 119-131 jevtić, a., đekić, v. ⚫ influence of growing season on some agronomic... 135 the average air temperature in 2011/12 was lower by 0.52 c and 2012/13 was higher by 1.09 c. the sum of rainfall precipitation in 2011/12 was lower by 49.3 mm, where the sum of rainfall in 2012/13 was 48.5 mm lower than the average of many years and with a very uneven distribution of precipitation per months. spring months march and april in 2011/12 were the surplus of precipitation, what affected unfavorable on the crops. during the march in 2012/13 it was 102.0 mm of rainfall, what was 56.6 mm more compared with the perennial average. regarding the high importance of sufficient rainfall amounts during the spring months, particularly may for small grains production, the distribution and amount of rainfall over the growing season 2011/12 were considerably more favorable, resulting with the increment of yields during that year. based on the fact that sufficient amounts of rainfall in these months are very important for the successful production of cereal crops it can be concluded that the years in which the researches were conducted were favorable for the wheat growing. soil and weather conditions before the commencement of the experiment, soil samples were taken from the sample surface and the chemical analysis of soil was performed. on the basis of obtained results, it was revealed that the soil belongs to the smonitza type, with relatively high clay content, and unfavorable physical properties. the humus content in the surface layer of soil was low (2.38-2.64%), and a substitution and total hydrolytic acidity were quite high (ph h2o=5.99, kcl=4.56). the soil was medium provided with total nitrogen (0,11-0,13% n) and easily accessible potassium (10-14 mg/100 g soil k2o), while the available phosphorus content was low (under 10 mg/100 g of soil p2o5). results and discussion average values of grain yield (t/ha), plant height (cm), number of plants per m2, 1000-grain weight (g) and test weight (kg/hl) at investigated kragujevac’s winter wheat cultivars grown at the small grains research centre in kragujevac during two growing seasons, 2011/12 and 2012/13, are presented in tab. 2. during the first year of investigations, cultivar takovčanka achieved the highest grains yield (4.729 t/ha), followed by kruna (4.642 t/ha) and toplica (4.473 t/ha), while the lowest yield was at planeta cultivar (4.395 t/ha). during the second year of investigations (2012/13), the yield of takovčanka cultivar was the highest with 3.518 t/ha, while the slightly lower yield was realized by kruna cultivar (3.276 t/ha). average grains yield observed in the two-year period was the highest at takovčanka variety (4.124 t/ha), while the lowest yield was obtained by kruna cultivar (3.959 t/ha). during the first and second years of investigation, toplica cultivar achieved the highest average plant height (85.80 cm and 78.00 cm) compared with other tested wheat cultivars. in the two-year period, the highest average plant height of winter wheat cultivar investigated was achieved in the toplica cultivar (81.90 cm) and planeta cultivar (78.60 cm). number of plants per m2 of wheat significantly varied across years, from 374 to 454 in 2011/12, from 342 to 368 plants per m2 in 2012/13. during the first year of investigations, the highest average value of number of plants per m2 of wheat achieved the kruna cultivar (454) and planeta (437). during the second year of investigations, the highest average value of number of plants per m2 of wheat achieved the kruna cultivar (368). in the two-year period, the highest average number of plants per m2 of winter wheat cultivar investigated was achieved in the kruna cultivar (411 plants per m2). during the first years of investigation, planeta cultivar achieved the highest average 1000 grain weight (44.90 g) and takovčanka cultivar (44.72 g). during the second year of investigations (2009/10), the highest average value of 1000 grain weight achieved the takovčanka cultivar (43.38 g) compared with other tested wheat cultivars. in the two-year period, the highest average thousand grain weight of winter wheat cultivar investigated was achieved in the takovčanka and planeta cultivars (44.05 g and 43.31 g). the wheat cultivar kruna has achieved the highest test weight in the first years of investigation compared to other tested wheat cultivars (73.05 kg/hl). during the second year of investigations (2012/13), the test weight of planeta cultivar was the highest with 69.81 kg/hl, while the slightly lower test weight was realized by kruna cultivar (69.01 kg/hl). the average two-year value of test weight at kruna and planeta cultivars was 71.03 kg/hl, while the lowest average was at takovčanka and toplica cultivars (70.00 kg/hl and 69.50 kg/hl). table 3 shows the impact of the year, cultivar and interaction of year x cultivar on yield, plant height, number of plants per m2, 1000-grain weight and test weight. analysis of variance was found highly significant effect of year on the grain yield (f=48.911**), plant height (f=26.863**), 1000-grain weight (f=2.616**) and test weight (f=3.634**), and biologica nyssana 9 (2) ⚫ december 2018: 119-131 jevtić, a., đekić, v. ⚫ influence of growing season on some agronomic... 136 significant effect for the number of plants per m2 (f=5.910*). discussion in regard to the tested genotypes winter wheat, variety takovčanka achieved the highest yield of grain (4.124 t/ha). of the tested years, particularly in 2011/12 year, the highest yields were achieved. considerable variation in yield depending on years of research have established biberdžić et al. (2005), jovanović et al. (2006), malešević et al. (2008), hristov et al. (2011), jelić et al. (2013), đekić et al. (2016). perišić et al. (2009), pointing out that among the kg cultivars, very high grain yield demonstrated winter wheat cultivars kruna and vizija. the takovčanka cultivar had stable and high grain yields, while cv. toplica demonstrated good yield followed by high quality. wheat productivity and grain quality in central serbia are governed by a range of factors, notably climate, soil, genetics and crop nutrition. soil acidity in wheat fields in central serbia has become a severe problem that leads to a significant decline in grain yield and quality of wheat (đekić et al., 2013, jelić et al., 2015). the yield per unit area is the result of the action of factors of variety in interaction with environmental factors. therefore, the yield a relative term and is determined by the variety, environmental conditions and the level of applied technology. yield is largely dependent on the genetic potential, which could be defined the yield of variety which was grown in conditions on which it had been adapted, with adequately amounts of water and nutrients and efficient control of pests, diseases, weeds and other stresses (đekić et al., 2016). yields considerably vary primarily as a result of agro-ecological conditions during the growing season (zečević et al., 2010; hristov et al., 2011; đekić et al., 2015). analysis of variance showed highly significant effect of year on the grain yield (p<0.01). considerable variation in yield depending on years of research has been established by glamočlija et al. (2010), milovanovic et al. (2011), đekić et al. (2012; 2014) and jelic et al. (2015). table 2. average values of investigated wheat cultivars characteristics varieties 2011/12 2012/13 average x s sx x s sx x s sx grain yield, t/ha takovčanka 4.729 0.567 0.253 3.518 0.566 0.253 4.124 0.832 0.263 kruna 4.642 1.014 0.454 3.276 0.278 0.124 3.959 1.005 0.318 toplica 4.473 0.258 0.115 3.125 0.692 0.309 3.799 0.864 0.273 planeta 4.395 0.757 0.338 3.160 0.402 0.180 3.778 0.866 0.274 plant height, cm takovčanka 83.60 9.317 4.166 71.20 2.588 1.158 77.40 9.180 2.903 kruna 83.40 4.722 2.112 72.20 3.365 1.505 77.80 7.056 2.231 toplica 85.80 9.418 4.212 78.00 3.021 1.351 81.90 7.770 2.457 planeta 81.80 6.140 2.746 75.40 2.631 1.177 78.60 5.587 1.767 number of plants per m2 takovčanka 374 73.245 32.756 362 38.220 17.093 368 55.44 17.532 kruna 454 66.969 29.949 368 88.136 39.416 411 86.493 27.351 toplica 375 71.688 32.060 342 46.785 20.923 359 59.630 18.857 planeta 437 82.384 36.843 362 48.711 21.784 399 75.113 23.753 1000 grain weight, g takovčanka 44.72 1.994 0.892 43.38 1.240 0.554 44.05 1.717 0.543 kruna 43.86 2.317 1.036 41.82 1.215 0.543 42.84 2.049 0.648 toplica 44.24 1.212 0.542 41.20 2.051 0.917 42.72 2.256 0.713 planeta 44.90 1.044 0.467 41.92 1.305 0.583 43.41 1.925 0.609 test weight, kg/hl takovčanka 72.92 0.784 0.351 67.08 1.994 0.892 70.00 3.393 1.073 kruna 73.05 1.789 0.800 69.01 2.128 0.952 71.03 2.823 0.893 toplica 71.37 1.647 0.736 67.63 2.706 1.210 69.50 2.889 0.913 planeta 72.25 1.661 0.743 69.81 1.152 0.515 71.03 1.863 0.589 biologica nyssana 9 (2) ⚫ december 2018: 119-131 jevtić, a., đekić, v. ⚫ influence of growing season on some agronomic... 137 milovanovic et al. (2011), states that in serbia higher yields are achieved by varieties with shorter growing season because they manage to form the largest part of the yield before the occurence of high temperatures. in this study, in both years, the wheat was not exposed to extremely high temperatures so early growth did not come into its own. the 1000-grain weight of winter wheat significantly varied across cultivars, from 42.72 g in cultivar toplica to 44.05 g in cultivar takovčanka. highly significant influence of the year on 1000-grain weight was established at investigated winter wheat cultivars by variance analysis. a number of authors (jelić et al. 2013; đekić et al. 2014, 2016), underline that 1000-grain weight is a cultivar-specific trait, with considerably higher variations being observed among genotypes than among treatments or environmental factors. jelic et al. (2015) have presented the 1000grain weight of three winter wheat cultivars (pobeda, planeta and nora). the 1000-grain weight of wheat variety was 42.62 g in control to 47.06 g while wheat treatment in the combined treatment with lime, manure and mineral npk fertilizer at the rates of caco3 of 5 t/ha and phosphorus of 100 kg/ha. a number of authors underlined that 1000-grain weight is a cultivar-specific trait, with considerably higher variations being observed among genotypes than among treatments or environmental factors (malešević et al., 2008; hristov et al., 2011; đekić et al., 2012, 2013; jelic et al., 2015). the genotype kruna had an extreme value (73.05 kg/hl) in the first years (2011/12) were recorded. the average two-year value of test weight at kruna and planeta cultivars was 71.03 kg/hl. particularly in the second year, genotypes had significantly lower test weight compared to first year. based on the analysis of variance, it can be concluded that there are significant differences in test weight in regard to the year of investigation, which is in accordance with the results obtained by đekić et al. (2015). the test weight of winter wheat ranged from 80 (takovčanka) to 82 kg/hl in cultivar toplica (protić et al., 2013). jelic et al. (2015) have examined the average test weight of winter wheat, which ranged from 68.66 kg/hl to 70.91 kg/hl. a number of authors (browne et al., 2006; đekić et al., 2013, 2016; jelić et al., 2013, 2014) underline that grain of investigated wheat cultivars was characterized by good physical characteristics; especially regarding the test weight and 1000 grain weight. determined average values of these characteristics in the study were slightly higher than the values obtained by jelić et al. (2013) and đekić et al. (2015). table 3. analysis of variance of the tested parameters (anova) effect of year on the traits analyzed traits mean sqr effect mean sqr error f(df1,2) 1, 38 p-level grain yield (t/ha) 16.64 0.340 48.91138 0.000000 plant height (cm) 893.03 33.243 26.86321 0.000007 number of plants per m2 26419.60 4470.631 5.90959 0.019886 1000-grain weight (g) 55.22 2.616 21.10920 0.000047 test weight (kg/hl) 161.20 3.634 44.35568 0.000000 effect of cultivar on the traits analyzed traits mean sqr effect mean sqr error f(df1,2) 3, 36 p-level grain yield (t/ha) 0.259 0.800 0.324121 0.807880 plant height (cm) 41.825 56.411 0.741432 0.534361 number of plants per m2 6177.467 4938.089 1.250983 0.305709 1000-grain weight (g) 3.715 3.986 0.932025 0.435230 test weight (kg/hl) 5.878 7.824 0.751257 0.528824 effect of the year x cultivar interaction traits mean sqr effect mean sqr error f(df1,2) 3, 32 p-level grain yield (t/ha) 0.015 0.378 0.040450 0.988943 plant height (cm) 19.825 33.697 0.588333 0.627103 number of plants per m2 3028.667 4445.800 0.681242 0.570020 1000-grain weight (g) 1.658 2.603 0.636829 0.596804 test weight (kg/hl) 4.906 3.305 1.484619 0.237321 * statisticaly significant difference (p<0.05) ** statisticaly high significant difference (p<0.01) biologica nyssana 9 (2) ⚫ december 2018: 119-131 jevtić, a., đekić, v. ⚫ influence of growing season on some agronomic... 138 in the growing season 2011/12 which were more favourable for wheat production in terms of agro-meteorological conditions, a significantly higher grain yield was obtained in all winter wheat cultivars. highly significant impact of the year on grain yield, plant height, 1000-grain weight and test weight were determined in investigated winter wheat cultivars by using the variance analysis, while the influence of year on number of plants per m2 was statistically significant. there are significant phenotypic differences among wheat cultivars with regard to grain quality. these differences result from the action of different genes that control characters for wheat quality (perišić et al., 2009). nonetheless, environmental factors play a major role in the expression of genotype characteristics (misic & mladenov, 1998; glamočlija et al., 2010; milovanović et al., 2011; đekić et al., 2013; jelić et al., 2013). their impact, however, is rarely optimal; one or more of them will always limit the yield and quality of the product. for this reason, it is very important to determine the variation of environmental factors and their effects on the processes that determine wheat quality. the present results confirm the opinion of many authors that the traits analyzed are genetically determined but are strongly modified by the nutrient status of the environment and weather conditions (jelić et al., 2014; đekić et al., 2016). due to the global change of climate, drought is a frequent occurrence both in southern parts of central europe and in the balkan. owing to such climatic conditions, southern parts of central europe and southern europe are considered suitable for the production of bread wheat (misic and mladenov, 1998; glamočlija et al., 2010; milovanović et al., 2011). conclusion based on the results during the two-year investigation on four kragujevac’s winter wheat cultivars, it can be concluded that the grain yield of wheat ranged from 3.778 t/ha (planeta) to 4.124 t/ha (takovčanka). the highest average plant height of winter wheat cultivar investigated was achieved in the toplica cultivar (81.90 cm). during the two-year the 1000 grain weight of investigation on winter wheat cultivars ranged from 42.72 g (toplica) to 44.05 g (takovčanka). winter wheat cultivars had test weight greater than 70 kg/hl, except of cultivar toplica (69.50 kg/hl). during 2011/12, statistically significantly higher grain yield per area unit, as well as 1000 grain weight was achieved, compared with 2012/13 year. highly significant differences in yield, plant height, 1000-grain weight and test weight and significant differences in number of plants per m2 at investigated wheat were found in relation to the year. the interaction of grain yield, plant height, number of plants per m2, 1000-grain weight and test weight and year x cultivar had no significant differences at investigated wheat cultivars. rainy weather in the spring month during 2013 caused water lodging, the occurrence of plant lodging and plant diseases, which influenced poorer quality and lower average yield of wheat in the 2013/14 season compared to the 2011/12 season. acknowledgements. investigations necessary for this paper are part of the project tp 31054 "development of new cereals cultivation technologies on acid soils by usage of modern biotechnology", financed by the ministry of education, science and technology development of republic of serbia. references biberdžić, m., đorđević, m., barać, s., deletić, n., & stojković, s. 2005: productivity of some winter wheat genotypes. genetika, 37(2), 131-136. browne, r.a., white, e.m., burke, j.i. 2006: responses of developmental yield formation processes in oats to variety, nitrogen, seed rate and plant growth regulator and their relationship to quality. journal agriculturae science, 144: 533545. đekić, v., milovanović, m., staletić, m., stevanović, v., milivojević, j. 2012: influence of growing season on some agronomic characteristics of six winter wheat cultivars grown in acidic soil. proceedings. 47rd croatian and 7rd international symposium on agriculture, 13.-17. februar, opatija, croatia, 478-482. đekić, v., staletić, m., jelić, m., popović, v., branković, s. 2013: the stability properties of wheat production on acid soil. proceedings, 4 th international symposium "agrosym 2013", 03-06. oktober, jahorina, 84-89. đekić, v., milovanović, m., popović, v., milivojević, j., staletić, m., jelić, m., perišić, v. 2014: effects of fertilization on yield and grain quality in winter triticale. romanian agricultural research, 31: 175-183. đekić, v., milovanović, m., milivojević, j., staletić, m., popović, v., simić, d., mitrović, m. 2015: impact of year and grain quality of winter wheat. proceedings of research papers pkb agroekonomik, belgarde, 21(1-2): 79-86. đekić, v., milivojević, j., jelić, m., popović, v., branković, s., staletić, m., terzić, d. 2016: the influence of mineral nutrition on winter wheat yield. proceedings, vii international scientific biologica nyssana 9 (2) ⚫ december 2018: 119-131 jevtić, a., đekić, v. ⚫ influence of growing season on some agronomic... 139 sympozium "agrosym jahorina 2016", 06-09. october, jahorina, 392-398. glamočlija, đ., staletić, m., ikanović, j., spasić, m., đekić, v., davidović, m. 2010: possibilities alternative grain production in the highlands area of central serbia. economics of agriculturemultifunctional agriculture and rural development (v), ep 2010 (57) si-2, 71-77, belgrade. hristov, n, mladenov, n, kondić-špika, ankica, marjanović-jelonmela, ana, jocković, b, jaćimović, g. 2011: effect of enviromental and genetic factors on the correlation and stability of grain yield components in wheat. genetics, 43(1): 141-152. jelić, m., milivojević, j., paunović, a., biberdžić, m., nikolić, o., madić, m., đekić, v. 2012: response of wheat genotypes to liming and fertilization on pseudogley soil. proceedings. 47rd croatian and 7rd international symposium on agriculture, 13.-17. februar, opatija, croatia, 488-491. jelić, m., dugalic, g., milivojevic, j., djikic, s., đekić, v., savic, n., gudzic, n. 2013: influence of perennial application of fertilizers and ameliorative measures on changes of pseudogley soil properties and grain yield of winter wheat. 1 st international congress soil-water-plant, 23-26 september, belgrade, 195-207. jelić, m., milivojević, j., đekić, v., paunović, a., tmušić, n. 2014: impact of liming and fertilization on grain yield and utilization of nitrogen and phosphorus in wheat plant grown on soil type pseudogley. proceedings of research papers pkb agroekonomik, belgrade, 20(1-4): 49-56. jelic, m., milivojevic, j., nikolic, o., đekić, v., stamenkovic, s. 2015: effect of long-term fertilization and soil amendments on yield, grain quality and nutrition optimization in winter wheat on an acidic pseudogley. romanian agricultural research, 32: 165-174. jovanović, z., djalović, i., komljenović i., kovacević v., cvijović, m. 2006: influences of liming on vertisol properties and yields of the field crops. cereal research communications, 34(1): 517-520. kastori, r. et al. 2005: nitrogen: agrochemical, cultural practice, physiological and ecological aspects, monografija, urednik r. kastori, institute of field and vegetable crops, novi sad, 1-419. malešević, m. 2008: mineral nutrition of small grains in integrated crop management system. proceedings institute of field and vegetable crops, 45(1): 179-193. malešević, m., starčević, lj., jaćimović, g., đurić, v., šeremešić, s., milošev, d. 2008: the yield of winter wheat depending on the conditions and the level of nitrogen fertilization. proceedings, xiii counseling on biotechnology, čačak, 28-29. march, 2008, faculty of agronomy, 13(14): 135141. milovanović, m., staletić, m., đekić, v., nikolić, o., luković, k. 2011: seed production and contribution of kg varieties to biodiversity of small grains in the period 2006-2010. beograd, economics of agriculture, belgrade, book ii, (58), cб/si-1, str. 103-111. misic, t., and mladenov, n. 1998: results of winter wheat breeding at the novi sad institute. proceeding of 2 nd balkan symposium of field crops. s. stamenkovic, ed. inst. field and vegetable crops: novi sad, vol. 1, 15-22. perišić, v., milovanović, м., đulaković, v., janković, s., staletić, m. 2009: actual cultivars of winter wheat, barley and spring oat created in kragujevac [serbia]. poljoprivredne aktivnosti. protic, r., todorovic , g., protic, n., djordjevic, r., vicentijevic, d., delic, d., kopanja, m., prodanovic, r. 2013: effect of genotype x environment interaction on grain yield of winter wheat varieties. bulgarian journal of agricultural science, 19(4), 697-700. sas/stat (2000). user's guide, version 9.1.3. sas institute inc. zečević, v., knežević, d., mićanović, d. 2007: variability of technological quality components in winter wheat. genetics, belgrade, 39: 365-374. zečević, v., bošković, j., knežević, d., mićanović, d., madić, m. 2010: ecological and genetic variability of wheat quality components. kragujevac journal science, 32: 89-94. savić, a., đorđević, m., jušković, m., pešić, v.: ecological analysis of macroinvertebrate communities based on functional feeding types: a case study in southeastern serbia. biologica nyssana, 8 (2). biologica nyssana 8 (2)  december 2017: 159-166 savić, a. et al.  ecological analysis of macroinvertebrate communities… 159 original article received: 22 november 2017 revised: 30 november 2017 accepted: 17 december 2017 ecological analysis of macroinvertebrate communities based on functional feeding groups: a case study in southeastern serbia savić ana1*, đorđević miodrag2 , jušković marina1, vladimir pešić3 1department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, 18000 nis, serbia 2department of mathematics, faculty of sciences and mathematics, university of niš, višegradska 33, 18000 nis, serbia 3department of biology, university of montenegro, podgorica, montenegro * e-mail: anka@pmf.ni.ac.rs abstract: savić, a., đorđević, m., jušković, m., pešić, v.: ecological analysis of macroinvertebrate communities based on functional feeding groups: a case study in southeastern serbia. biologica nyssana, 8 (2). december, 2017: 159-166. to examine if the macroinvertebrate community corresponds to the rcc (river continuum concept), which describes longitudinal patterns in allochthonous and autochthonous energy input and the associated feeding categories of macroinvertebrates along the lotic continuum, it was necessary to determine the functional feeding groups – ffgs. the goals of the research were: to examine whether a macroinvertebrate community matches the rcc; to use the functional feeding groups to determine the attributes of the ecosystem; to test if the itc index is appropriate for use in this part of the world and to determine water quality along the river course of the nišava river. macroinvertebrate samples and physicochemical data were analyzed for 10 localities along the 151 km long stretch of the nišava river in southeastern serbia, over a one-year period. out of all the collected specimens (9837 individuals) 49.6% belong to shredders, 31.77% to scrapers, 12.25% to collectors and 6.4% to predators. on the annual level only locality nine belongs to autotrophic type; relation p/r=1.44. locality four has the most heterotrophic character, the lowest channel stability and the most disturbed predator-prey relationships. the results of the research reveal that the trends found in relative functional group abundance do not correspond with the tendencies predicted by the rcc. the ffg ratios surrogate are consistent with the observations of the properties of the ecosystem at the sampling localities. according to the index of trophic completeness, most of the localities included in the research belong to good water quality (seven out of ten), while other localities belong to moderate water quality (three out of ten). key words: macroinvertebrates, functional feeding groups, index of trophic completeness apstrakt: savić, a., đorđević, m., jušković, m., pešić, v.: ekološka analiza zajednice makroinvertebrata na osnovu funkcionalnih grupa zasnovanih na tipu ishrane: primer iz jugoistočne srbije. biologica nyssana, 8 (2), decembar, 2017: 159-166. rcc (river continuum concept) opisuje longitudinalne šablone u inputu autohtone i alohtone energije, i njihovu povezanost sa kategorijama ishrane makroinvertebrata duž rečnog kontinuuma. da bi se utvrdilo da li 8 (2) • december 2017: 159-166 doi: 10.5281/zenodo.1135973 biologica nyssana 8 (2)  december 2017: 159-166 savić, a. et al.  ecological analysis of macroinvertebrate communities… 160 zajednica makroinvertebrata prati rcc neophodno je odrediti funkcionalne grupe na osnovu tipa ishrane (ffg). tokom jedne godine makroinvertebratska zajednica i fizičko hemijski podaci su analizirani na 12 lokaliteta duž 151 km rečnog toka nišave, koja se nalazi na jugoistoku srbije. od ukupnog broja individua (10519) 48.99% pripada sekačima, 31.49% grebačima, 13.02% sakupljačima i 6.5% predatorima. na osnovu ffg odredjeni su sledeći ekosistemski atributi: autotrofnost/heterotrofnost lokaliteta, stabilnost rečnog kanala, odnos predator-plen i odnos cpom/fpom (krupne čestice organske materije/sitne čestice organske materije). na godišnjem nivou, samo lokalitet 9 ima autotrofnu prirodu (p/r=1.44). lokalitet 4 je sa najizraženijim heterotrofnim karakterom, sa najmanjom stabilnošću rečnog kanala i sa najnarušenijim odnosom predator-plen. odnos cpom/fpom pokazuje da reka nišava ima regularnu asambleju sekača (na svim lokalitetima odnos je >0.25) što je povezano sa uslovima u riparijalnoj zoni. ključne reči: makroinvertebrate, funkcionalne grupe na osnovu tipa ishrane, indeks of trophic completeness introduction of all the freshwater organisms that have been considered for use in biological monitoring, benthic macroinvertebrates are recommended most often (c a r t e r et al., 2007). macroinvertebrates have been used to evaluate the effects of anthropogenic stressors at all levels of biological organization, from the molecular to the ecosystem (r o s e n b e r g & r e s h , 1993). functional feeding group (ffg) approach, described more than 40 years ago (c u m m i n s , 1973), has been modified in some details since then (e.g., c u m m i n s & k l u g , 1979, w a l l a c e & m e r r i t t , 1980, c u m m i n s & w i l z b a c h , 1985, m e r r i t t & c u m m i n s 1996, m e r r i t t et al., 1999, 2002), but the basis of ffg relationships remains quite simple. ffgs are based on a direct correspondence between the categories of nutritional resources present in the environment and the populations of freshwater invertebrates that are adapted to efficiently harvest a given food resource (m e r r i t t & c u m m i n s , 2007). the analysis of the trophic structure of benthic macroinvertebrate communities can be used in biological assessments of the condition of river ecosystems. using the trophic or functional approach, the index of trophic completeness (itc) was developed (p a v l u k et al., 2000). also, ffg ratios can be used as surrogates for these aquatic ecosystem attributes and serve as a useful assessment of the ecological condition freshwater ecosystems (m e r r i t t & c u m m i n s , 2007). in this study, we wanted to examine whether the macroinvertebrate community matched the rcc (river continuum concept) (v a n o t e et al., 1980) in the river, or its trophic structure (distribution pattern of ffg) was more influenced by pollution. the goal was to use functional feeding groups to determine the ecosystem attributes: indication of autotrophic/heterotrophic type; ratio between fpom fig. 1. the map of the studied area with localities biologica nyssana 8 (2)  december 2017: 159-166 savić, a. et al.  ecological analysis of macroinvertebrate communities… 161 in transport and fpom in sediment; stability of channels (river channel resistance capacity to the detachment of bed and bank materials); and predator control. another goal of the research was to test is itc index appropriate for use in this part of the world, because there was no evidence about its use in the region of the balkan peninsula. in addition, the study set out to determine water quality along the river course of the nišava river based on these two types of analysis. material and methods the nišava river (fig. 1) belongs to the black sea drainage basin. it originates in western bulgaria, on the stara planina mt., and flows in a southeastnorthwest direction. it is 218 km long, of which 67 km flows through bulgaria, and 151 km through serbia. ten localities were chosen along the entire course of the nišava river in serbia. the odd numbered localities are positioned upstream of the settlements, and even numbered downstream of the settlements. sampling was performed each month, from may 2006 to april 2007. all localities were sampled on a single day during each field trip. biochemical oxygen demand (bod5) was estimated using the standard methodology recommended by apha (1999). dissolved oxygen, ph and conductivity were measured using a wtw®multi 340i probe. the concentration of total nitrogen (tn) and phosphorus (tp) were determined in the field, using a photometer – system pc multi direct lovibond ® meter. the percentage of substrate was observed visually; the classification of mineral substrates by particle sizes according to w e n t w o r t h (1922) and v e r d o n s c h o t (1999) was used. since larger substrates require greater stream power for movement, they are physically more stable (g u r t z & w a l l a c e 1984); thus, pebbles, cobbles and boulders consolidated a stable substrate. water turbidity was measured with a lovibond® checkit device. stream order was determined according to s t r a h l e r (1952). macrozoobenthos were sampled at each locality over a 50 m river stretch with a square frame kick net (35 × 35 cm, mesh size 300 μm). three 3minute samples were taken during each visit to include different substrates (boulder, cobble, pebble, sand, silt, and detritus) and flow regime zones at each location. the net was held perpendicular to the flow and the substrate was vigorously disturbed in front of the net. as the substrate was disturbed, sampling moved progressively upstream. the three samples were then pooled, representing a single monthly sample for each site. this sampling procedure was previously evaluated by preliminary test sampling, and three replicates proved to be sufficient to capture the maximum number of taxa. all samples were elutriated in the field and the organisms were fixed in 4% formaldehyde solution and returned to the laboratory for sorting. the material was identified using identification keys: for oligochaeta b r i n k h u r s t & j a m i e s o n (1971) and h r a b e (1981); for hirudinea m a n n & w a t s o n (1964); for freshwater snails m a c a n & c o o p e r (1994) and p f l e g e r (1990, 2000); for odonata n i l s o o n (1997) and b e š o v s k i (1994); for ephemeroptera b e l f i o r e (1983) and e l l i o t et al. (1988), for trichoptera w a l a c e et al. (1990), e d i n g t o n & h i l d r e w (1995) and p e s c a d o r et al. (1995), for plecoptera h y n e s (1967) and z w i c k (2004); for diptera n i l s s o n (1997), v a l l e n d u u k & p i l l o t (2007) and p i l l o t (2009). for each taxon the functional feeding group was determined based on: m o o g (1995), g r a f et al. (2006) and p a v l u k et al. (2000). the ecosystem attributes where calculated according to m e r r i t t & c u m m i n s (2007) and m e r r i t t et al. (2002). itc index and water quality class are determined by de v a a t e & p a v l u k (2004). quality class score is calculated by the formula:    n i itot cc 1 , where ctot is the total score, n is the number of trophic guilds present in the data-set, and ci is the ln transformed indication value of trophic guild i. the relation between ctot and the quality classes is given in de v a a t e & p a v l u k (2004). results the community of macroinvertebrates was collected at ten localities along the entire course of the nišava river. the river is a 4th to 7th stream order (according to s t r a h l e r , 1952) (tab. 1). table 1 shows that the concentrations of total nitrogen and total phosphorus are the highest at localities 4 and 10. these localities are under the highest anthropogenic pressure. the 4th locality is under the impact of industrial wastewater from tire factory ‘tigar’. the 10th locality is downstream from the biggest city along the entire length of the river – niš. during one-year research 9837 individuals of macroinvertebrates were collected. out of the total number, shredders were the most present 4877 individuals. they were followed by scrapers (3125 individuals), collectors (1205 individuals) and finally predators (630 individuals) (fig. 2). biologica nyssana 8 (2)  december 2017: 159-166 savić, a. et al.  ecological analysis of macroinvertebrate communities… 162 table 2 shows that the highest number of predators were detected at localities 4 and 10, which have already been mentioned as localities with the highest concentrations of nutrients. the greatest percentage of predators are present at locality 4 (fig. 3). fig. 2. percentage of ffg on annual level according to the ratio p/r (tab. 3) at annual level, nine localities on the nišava river were characterized as heterotrophic (the exception is locality 9). according to the ratio tfpom/bfpom, fpom in transport is greater than normal particulate loading in suspension on locality 7. at other localities, there is more fpom in sediments (benthos) than in suspension. the stability of the river channel is the lowest at locality 4. also, it is low at locality 10. the ratio cpom/fpom indicates that the river nišava has normal shedder association linked to functioning riparian system along the whole river stretch. the predator-prey relationship is the most disturbed at locality 4. according to d e v a a t e & p a v l u k (2004), localities 2, 3, 5 and 7 belong to class ii (good quality), subclass i; locality 8 belongs to class ii (good quality), subclass ii; localities 6 and 9 belong to class ii (good quality), subclass iii. on the other hand, localities 1, 4 and 10 belong to class iii (moderate), subclass ii (tab. 4). discussion the trends found in relative functional group abundance in the research do not correspond with the tendencies predicted by the rcc. similar results were obtained in the research conducted by s k a f f (2010). the rcc assumes that the highest proportion of shredders will be present in lowstreams order (v a n n o t e et al. 1980). the data obtained in this study illustrate that this is not the case more shredders (in %) were found at locality 10 (the 7th stream order) than at locality 1 (the 4th stream order). one possible explanation for the disparity between the observed and the expected feeding group proportions may be relatively small ranges of physical stream characteristics found between stream orders. the 6th order (locality 3) has average depth 89.5 cm, while the 7th order (the locality 10) has average depth 96.5 cm (s a v i ć , 2012). similar results were obtained by s k a f f (2010). the use of stream order to characterize a stream is sometimes deemed to be a misrepresentation of the true size. some researchers consider drainage area and discharge to be better indicators (a l l e n & h o e k s t a , 1992). another explanation could be simply a general lack of riparian cover to supply litter inputs (c u m m i n s et al., 2005) in low stream order parts of the river stretch. according to the p/r ratio, nine sites (out of ten) were characterized as heterotrophic (tab. 3). it means that the dominant base food chain for the invertebrate communities, at most localities, was judged to be allochthonous detritus, largely from the riparian zone. locality 4 was the most heterotrophic. 12% 6% 32% 50% collectors predators scrapers shredders table 1. average annual values of environmental parameters at each locality (loc) studied along the nišava river. so-stream order; tp-total phosphorus in mg/l; tn-total nitrogen in mg/l; o-oxygen in mg/l; bod5biochemical oxygen demand in mg/l; tu-turbidity in ntu; con-conductivity in s/cm; ss-stable substrate in %. loc 1 loc 2 loc3 loc 4 loc 5 loc 6 loc 7 loc 8 loc 9 loc 10 so 4 5 6 6 7 7 7 7 7 7 tp 0.02 0.08 0.04 0.10 0.07 0.07 0.07 0.06 0.07 0.11 tn 0.27 0.28 0.08 0.34 0.10 0.12 0.12 0.08 0.09 0.21 o 6.72 7.28 8.04 7.09 7.85 8.34 8.37 7.98 7.56 6.50 bod5 1.68 3.04 2.74 4.16 3.08 3.37 3.61 3.08 3.17 2.99 ph 7.56 6.54 6.17 6.59 6.98 6.56 6.30 7.34 6.15 6.54 tu 26.60 35.92 12.33 18.20 21.98 20.72 5.28 12.90 6.94 3.75 con 496.00 536.42 460.08 459.42 395.75 403.83 414.17 411.08 413.58 575.67 ss 67.50 79.67 78.33 46.50 76.67 52.58 83.75 89.17 66.67 70.42 biologica nyssana 8 (2)  december 2017: 159-166 savić, a. et al.  ecological analysis of macroinvertebrate communities… 163 this is in accordance with the fact that the highest value of bod5 and the lowest value of oxygen concentration were found there (tab. 1). the tfpom/bfpom ratio revealed significant amounts of fpom in transport at locality 7 (tab. 3) which was an indicator of high quality of water (c u m m i n s et al., 2005). this is in accordance with the fact that the highest concentration of oxygen was detected at locality 7 (tab. 1). the stability of the stream channel is the key attribute of a stream/river ecosystem (m e r r i t t & c u m m i n s , 2007) as reflected in the relative permanence (stability) of various bottom materials. if the bottom is stable, invertebrates that cling to surfaces of stones or large wood, while feeding on attached algae will be more abundant (c u m m i n s et al., 2005). the ffg surrogate ratio for channel stability revealed that localities 1, 2, 4 and 10 were below the threshold of 0.50. localities 3 and 5 were on the borderline. the lowest values were at locality 4. this is also in accordance with the fact that the lowest percentage of stable substrates were found at locality 4 (tab. 1). according to m e r r i t t et al. (2002) for the ffg surrogate ratio for predator-prey balance the locality 4 is (tab. 3) the locality with the most deviation from typical values of this ratio (<0.15). in other words, only localities 4 and 10 are not with ‘normal’ predator-prey balance, or top-down control. table 2. number of specimens of ffg at each locality localities 1 2 3 4 5 6 7 8 9 10 collectors 34 228 301 16 115 91 187 124 68 41 predators 24 60 62 170 45 34 80 27 29 99 scrapers 38 197 186 3 834 329 407 371 731 29 shredders 99 416 247 90 1687 573 563 379 432 391 fig. 3. percentage of ffg at ten localities table 3. ecosystem attributes calculated using ffg according to m e r r i t t and c u m m i n s (2007). localities p/r tfpom/bfpom stability of the river channel top-down control 1 0.29 0.12 0.32 0.14 2 0.31 0.37 0.43 0.07 3 0.34 0.18 0.47 0.09 4 0.02 0 0.03 1.56 5 0.45 0.07 0.47 0.02 6 0.49 0.38 0.55 0.02 7 0.54 0.62 0.71 0.07 8 0.74 0.1 0.79 0.03 9 1.44 0.23 1.51 0.01 10 0.07 0.02 0.07 0.2 biologica nyssana 8 (2)  december 2017: 159-166 savić, a. et al.  ecological analysis of macroinvertebrate communities… 164 this is in accordance with the fact that proportion of some ffg could be changed under some pollutants (p a v l u k et al., 2000). the index of trophic completeness (itc) at locality 3 was with high value (itc=27.4), at locality 4 (downstream of water discharge point from tire factory ‘tigar’) it dropped to itc=17.7, then after a distance of 34.9 km, at locality 5, it increased again to itc=26.5; at locality 6 it dropped to itc=22.3 and 8.1 km further downstream, at locality 7, it increased to itc=26.9 (s a v i ć , 2012). on the other hand, the situation was different between localities 8 and 9, where instead of the expected increase of itc, its value decreased from itc=24.1 to itc=21.9. the distance between these two localities is only 4.8 km, which suggest that the minimal distance for the river self-purification processes to become effective is at least 8 km (s a v i ć et al., 2013). the odd-numbered localities are positioned upstream, and evennumbered downstream of the settlements. a clear table 4. values for itc on investigated localities. n number of species in each guild; a – relative number of species in each trophic guild; c=100/a. guild loc 1 2 3 4 5 6 7 8 9 10 11 12 ctot 1 n 6 3 3 5 0 9 11 3 1 0 0 0 a 14.6 7.32 7.32 12.2 0 21.9 26.8 7.32 2.44 0 0 0 c 6.83 13.7 13.7 8.19 0 4.57 3.73 13.7 41 0 0 0 ln c 1.92 2.61 2.61 2.1 0 1.52 1.32 2.61 3.71 0 0 0 18.4 2 n 5 4 7 3 0 16 17 5 1 2 0 2 a 8.05 6.45 11.3 4.84 0 25.7 27.4 8.05 1.61 3.22 0 3.22 c 12.4 15.5 8.86 20.7 0 3.88 3.65 12.4 62.1 31.1 0 31.1 ln c 2.52 2.74 2.17 3.03 0 1.35 1.28 2.52 4.13 3.43 0 3.43 26.6 3 n 7 6 8 5 0 15 22 3 1 2 0 1 a 10 8.56 11.4 7.13 0 21.4 31.4 4.28 1.43 2.86 0 1.43 c 10 11.7 8.75 14 0 4.66 3.17 23.4 69.9 35 0 69.9 ln c 2.3 2.46 2.17 2.63 0 1.54 1.14 3.15 4.25 3.55 0 4.25 27.4 4 n 2 1 0 2 0 4 5 1 3 3 0 0 a 9.52 4.75 0 9.52 0 19.1 23.8 4.75 14.3 14.3 0 0 c 10.5 21.1 0 10.5 0 5.25 4.19 21.1 7 7 0 0 ln c 2.34 3.04 0 2.34 0 1.65 1.42 3.04 1.94 1.94 0 0 17.7 5 n 4 2 3 3 1 12 12 2 5 1 0 0 a 8.88 4.44 6.66 6.66 2.22 26.7 26.7 4.44 11.1 2.22 0 0 c 11.3 22.5 15 15 45 3.75 3.75 22.5 9 45 0 0 ln c 2.42 3.11 2.71 2.71 3.81 1.32 1.32 3.11 2.19 3.81 0 0 26.5 6 n 6 3 4 3 0 12 9 0 4 1 0 1 a 13.9 6.97 9.3 6.97 0 27.9 20.9 0 9.3 2.32 0 2.32 c 7.17 14.3 10.8 14.3 0 3.57 4.77 0 10.8 43.1 0 43.1 ln c 1.96 2.66 2.36 2.66 0 1.26 1.55 0 2.36 3.75 0 3.75 22.3 7 n 7 4 7 6 0 23 14 2 4 2 0 1 a 10 5.71 10 8.56 0 32.9 20 2.86 5.71 2.86 0 1.43 c 10 17.5 10 11.7 0 3.04 5 35 17.5 35 0 69.9 ln c 2.3 2.86 2.3 2.46 0 1.11 1.61 3.55 2.86 3.55 0 4.25 26.9 8 n 5 3 3 3 0 20 15 1 3 1 0 0 a 9.25 5.55 5.55 5.55 0 37 27.8 1.85 5.55 1.85 0 0 c 10.8 18 18 18 0 2.7 3.6 54.1 18 54.1 0 0 ln c 2.37 2.88 2.88 2.88 0 0.98 1.27 3.98 2.88 3.98 0 0 24.1 9 n 4 4 4 4 0 12 14 2 3 2 0 0 a 8.15 8.15 8.15 8.15 0 24.5 28.6 4.07 6.12 4.07 0 0 c 12.3 12.3 12.3 12.3 0 4.07 3.5 24.6 16.3 24.6 0 0 ln c 2.51 2.51 2.51 2.51 0 1.4 1.25 3.2 2.79 3.2 0 0 21.9 10 n 1 2 0 3 0 12 10 0 1 3 0 0 a 3.12 6.25 0 9.37 0 37.5 31.3 0 3.12 9.37 0 0 c 32.1 16 0 10.7 0 2.66 3.2 0 32.1 10.7 0 0 ln c 3.46 2.77 0 2.36 0 0.98 1.15 0 3.46 2.36 0 0 16.5 biologica nyssana 8 (2)  december 2017: 159-166 savić, a. et al.  ecological analysis of macroinvertebrate communities… 165 pattern can be seen: values of ctot are higher at oddnumbered localities, except when it comes to localities 1 and 2. this could be explained by facts that all other pairs of localities (3 and 4; 5 and 6; 7 and 8; 9 and 10) belong to the same stream order (tab. 1). locality 1 belongs to the 4th stream order, while locality 2 belongs to the 5th stream order. according to that, the index of trophic completeness could be sensitive on river section which is opposite to conclusion to some investigation in different parts of the world. conclusion the trends found in relative functional group abundance in this study do not correspond with the tendencies predicted by the rcc. a possible explanation for the disparity between the observed and expected feeding group proportions may be the relatively small ranges of physical stream characteristics found between stream orders. another explanation could be disturbance of riparian vegetation. surrogate measures for ecosystem attributes were used for the very first time in this region. the ffg ratios surrogate are consistent with the observations of the properties of the ecosystem at the sampling localities. the results show that the use of surrogates of ecosystem attributes is quite adequate for this purpose since the results obtained in this way match the results obtained by direct measurements of the attributes. however, direct measurements are often more difficult and require additional time and money. therefore, this principle should be used more often. the index of trophic completeness is considered to be a promising tool for determination of water quality in our region. according to this index, most of the localities included in the research belong to good water quality (seven out of ten) while other localities belong to moderate water quality (three out of ten). references allen, t.f.h., hoeksta, t.w. 1992: toward a unified ecology. columbia university press, new york, 384 p. apha, 1999. standard methods for the examination of water, wastewater. port city press, baltimore, maryland. belfiore, c. 1983: efemerotteri (ephemeroptera). in: ruffo s. (ed.) guide per ilriconoscimentodelle specie animalidelleacque interne italiane, 24. consiglio nazionale delle ricerche, roma. brinkhurst, r. o., jamieson, b. g. m. 1971: aquatic oligochaeta of the world. 1st ed. university of toronto press, toronto, 860 p. carter, j.l., resh, v.h., hannaford, m.j., myers, m.j. 2007: macroinvertebrates as biotic indicators of environmental quality. in: hauer, f.r., lamberti, g.a (eds.), methods in stream ecology: 805-833, elsevier academic press, burlington. cummins, k. w., 1973: trophic relations of aquatic insects. annual review of entomology, 18: 183– 206. cummins, k.w., klug, m.j. 1979: feeding ecology of stream invertebrates. annual review of ecology and systematics, 10:147–172. cummins, k.w., merrit, r.w., andrade, p.c.n. 2005: the use of invertebrate functional groups to characterize ecosystem attributes in selected streams and rivers in south brasil. studies on neotropical fauna and environment, 40 (1): 6989. cummins, k.w., wilzbach, m.a. 1985: field procedures for the analysis of functional feeding groups in stream ecosystems. appalachian environmental laboratory, contribution no. 1611, university of maryland, frostburg, md. de vaate, b.a., pavluk, t. i. 2004: practicability of the index of trophic completeness for running waters. hydrobiologia, 519: 49-60. edington, j. m., hildrew, a. g. 1995: a revised key to the caseless caddis larvae of the british isles (with notes on their ecology). freshwater biological association, scientific publication, 53, ambleside, 173 p. elliot j.m., humpesch u.h., macan t.t. 1988: larvae of the british ephemeroptera: a key with ecological notes. freshwater biological association, scientific publication, 49, ambleside, 145 p. graf w., murphy, j., dahl, j., zamora-muñoz, c., lópez-rodríguez m.j., schmidt-kloiber., a. 2006: trichoptera indicator database. eurolimpacs project, workpackage 7 – indicators of ecosystem health, task 4, www.freshwaterecology.info, version 5.0. gurtz m.e., wallace j.b. 1984: substrate-mediated response of stream invertebrates to disturbance. ecology, 65: 1556–1569. hrabě, s. 1981: vodnímáloštětinatci (oligochaeta) československa. acta universitatis carolinae – biologica, praha, 167 p. hynes, h.b. 1967: a key to the adults and nymphs of the britush stoneflies (plecoptera). freshwater biological association, scientific publication, 17, ambleside, 91 p. biologica nyssana 8 (2)  december 2017: 159-166 savić, a. et al.  ecological analysis of macroinvertebrate communities… 166 macan, t.t., douglas cooper, r. 1994: a key to the british freshand brackishwater gastropods with notes on their ecology. freshwater biological association, scientific publication, 13, reprinted fourth edition, ambleside, 46 p. mann, k.h., watson, e.v. 1964: a key to the british freshwater leeches. freshwater biological association scientific publication, 14, second edition, ambleside, 50 p. merritt, r.w., cummins, k.w. 1996: an introduction to the aquatic insects of north america, 3rd ed. kendall/hunt, dubuque, ia. merritt, r.w., cummins, k.w. 2007: trophic relationships of macroinvertebrates. in: hauer, f.r., lamberti, g.a (eds.), methods in stream ecology: 585-611, elsevier academic press, burlington. merritt, r.w., cummins, k.w., berg, m.b., novak, j.a., higgins, m.j., wessell, k.j., lessard, j.l. 2002: development and application of a macroinvertebrate functional-group approach in the bioassesment of remnant river oxbows in southwest florida. journal of the north american benthological society, 21: 290–310. merritt, r.w., cummins, k.w., berg, m.b., novak, j.a., higgins, m.j., wessell, k.j., lessard, j.l. 2002: development and application of a macroinvertebrate functional group approach in the bioassessment of remnant river oxbows in southwest florida. journal of american benthological society, 21 (2): 290-310. merritt, r.w., higgins, m.j., cummins, k.w., vandeneeden, b. 1999: the kissimmee riverriparian marsh ecosystem, florida: seasonal differences in invertebrate functional feeding group relationships. in: batzer, d., rader, r. b., wissinger, s.a (eds.), invertebrates in freshwater wetlands of north america: 55– 79,wiley and sons, new york, ny. moog, o. 1995: fauna aquatica austriaca. wasserwirtschaftskataster, bundesministerium für landund fortswirtschaft. wien, loseblattsammlung. nilsson, a., 1997: aquatic insects of north europe. a taxonomic handbook, vol. 2. apollo books, steenstrup, 440 p. pavluk, t.i., de vaate, b.a., leslie, h.a. 2000: development of an index of trophic completeness for benthic macroinvertebrate communities in flowing waters. hydrobiologija, 427: 135-141 pescador, m. l., rasmussen, a. k., harris, s. c. 1995: identification manual for the caddisfly (trichoptera) larvae of florida. state of florida, department of environmental protection, division of water facilities, tallahassee, 132 p. pfleger, v. 1990: molluscs. blitz, leicester, 216 p. pfleger, v. 2000: a field guide in colour to molluscs. silverdale books. prague. 216 p. pillot, h. k. m. m., 2009: chironomidae larvae of the netherlands, adjacent lowlands: biology, ecology of the chironomini. knnv publishing, zeist, 144 p. rosenberg, d. m., resh, v. h. 1993: freshwater biomonitoring and benthic macroinvertebrates. chapman & hall, new york, ny. 488 p. savić, a., 2012: ekološka analiza zajednice makrozoobentosa reke nišave. phd thesis. biološki fakultet, univeryitet u beogradu. savić, a., ranđelović, v., đorđević, m., karadžić, b., đokić, m., krpo-ćetković, j. 2013: the influence of environmental factors on the structure caddisfly (trichoptera) assemblage in the nišava river (central balkan peninsula). knowledge and management of aquatic ecosystems, 409: 03. skaff, n. 2010: the applicability of the river continuum concept to the upper reaches of a neotropical lower montane stream. american journal of undergraduate research, 9 (1): 9-18. strahler a.n., 1952: hypsometric (area-altitude) analysis of erosional topology. geological society of american bulletin, 63: 1117–1142. vallenduuk, h. j., pillot, h. k. m. m. 2007: chironomidae larvae of the netherlands, adjacent lowlands: general ecology, tanypodinae. knnv publishing, zeist, 270 p. vannote r.l., minshall g.w., cummins k.w., sedell j.r., cushing c.e., 1980: the river continuum concept. canadian journal of fisheries and aquatic sciences, 37: 130–137. verdonschot p.f.m., 1999. micro-distribution of oligochaetes in a soft-bottomed lowland stream (elsbeek; the netherlands). hydrobiologia, 406: 149–163. wallace, i.d., wallace, b., philipson, g. n. 1990: a key to the case-bearing caddis larvae of britain and ireland. freshwater biological association, scientific publication 51, ambleside, 237 p. wallace, j. b., merritt, r. w. 1980: filter-feeding ecology of aquatic insects. annual review of entomology, 25:103–132. wentworth c.k., 1922: a scale of grade and class terms for clastic sediments. the journal of geology, 30: 377–392. zwick, p. 2004: key to the west paleartic genera of stoneflies (plecoptera) in larval stage. limnologica, 34: 315-348. бешовски, в. л. 1994. фауна на българия, 23: insecta, odonata. издателствона бан. софия, 372 p. petrović, g., stamenković, j., stojanović, g., zlatković, b., jovanović, o.: essential oil analysis of different plant parts of geocaryum cynapioides (guss.) l. engstrand. biologica nyssana, 9 (1). september, 2018: 31-35. biologica nyssana 9 (1) ⚫ september 2018: 31-35 petrović et al. ⚫ essential oil analysis of different plant parts of geocaryum… 31 original article received: 29 january 2018 revised: 29 mart 2018 accepted: 27 june 2018 essential oil analysis of different plant parts of geocaryum cynapioides (guss.) l. engstrand goran petrović1, jelena stamenković1, gordana stojanović1, bojan zlatković2, olga jovanović1 1university of niš, faculty of science and mathematics, department of chemistry, višegradska 33, 18000 niš, serbia 2university of niš, faculty of science and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia * e-mail: jelena.stamenkovic@pmf.edu.rs abstract: petrović, g., stamenković, j., stojanović, g., zlatković, b., jovanović, o.: essential oil analysis of different plant parts of geocaryum cynapioides (guss.) l. engstrand. biologica nyssana, 9 (1). september, 2018: 31-35. phytochemical analysis by gc and gc/ms of the essential oil samples, obtained from fresh aerial parts, shoots and inflorescences of geocaryum cynapioides (guss.) l. engstrand, allowed the identification of 55 components in total, comprising 99.5%, 99.7% and 99.0% of the oils compositions, respectively. regarding the aerial parts essential oil, the major of 52 identified volatile compounds were (e)-β-farnesene (66.6%), 7epi-cis-sesquisabinene hydrate (17.4%) and (e,e)-α-farnesene (3.7%). the same compounds were dominant among the 48 components detected in the shoots and 42 volatiles in inflorescences oil, but in different proportions. hydrocarbon sesquiterpenes had the highest contribution in all investigated geocaryum cynapioides essential oils, with a share of about three quarters of all, followed by one quarter of oxygenated sesquiterpenes, while the monoterpenoid and nonterpenoid compounds were detected only in trace amounts. key words: geocaryum cynapioides, essential oil, phytochemical analysis, gc/ms apstrakt: petrović, g., stamenković, j., stojanović, g., zlatković, b., jovanović, o.: analiza etarskih ulja iz različitih biljnih delova vrste geocaryum cynapioides (guss.) l. engstrand. biologica nyssana, 9 (1). septembar, 2018: 31-35. fitohemijskom analizom uzoraka etarskih ulja izolovanih iz svežeg biljnog materijala različitih delova (nadzemnog dela, stabljike i cvasti) biljne vrste geocaryum cynapioides (guss.) l. engstrand, pomoću gc i gc/ms metode, identifikovano je ukupno 55 komponenti, što predstavlja 99,5% nadzemnog dela, 99,7% stabljike i 99,0% cvasti. u etarskom ulju izolovanom iz svežeg nadzemnog dela biljke identifikovane su ukupno 52 komponente među kojima su (e)-β-farnezen (66,6%), 7-epi-cis-seskvisabinen hidrat (17,4%) i (e,e)-α-farnezen (3,7%) bile najzastupljenije. iste te komponente su bile dominantne i u etarskim uljima 9 (1) • september 2018: 31-35 doi: 10.5281/zenodo.1470846 biologica nyssana 9 (1) ⚫ september 2018: 31-35 petrović et al. ⚫ essential oil analysis of different plant parts of geocaryum… 32 izolovanim iz stabljike i cvasti ali u različitim procentnim udelima. u svim ispitivanim uzorcima tri četvrtine ukupnog sastava etarskog ulja činili su ugljovodonični seskviterpeni, zatim oksigenovani seskviterpeni koji su činili jednu četvrtinu, dok su monoterpenoidi i neterpenoidne komponente detektovani samo u tragovima. ključne reči: geocaryum cynapioides, etarsko ulje, fitohemijska analiza, gc/ms introduction the genus geocaryum l. (apiaceae) is small genus, comprised of only 8 accepted species (the plant list, 2012). it belongs to the tribe scandiceae, subtribe scandicinae (spalik & downie, 2001) with 11 another larger genera like chaerophyllum, anthriscus, osmorhiza etc. geocaryum cynapioides (guss.) l. engstrand is endemic species which inhabits pastures and meadows of the high mountains in southern parts of the balkan peninsula and central and southern parts of italy (ball, 1968). it is a perennial herb, 45 cm tall, with flexuous or curved internodes and undivided uppermost cauline leaves, linear or filiform lamina up to 20 mm, flowering from may to july. according to the plant list database, g. cynapioides has as many as 18 synonymous names classifying it in other genera such as bunium, carum or even chaerophyllum. downie (downie et al., 2010) finally hemotaxonomically defined its position within the tribe scandiceae on the basis of his investigation of nuclear ribosomal dna. but, it is very unusual that there are no any previous data about the chemical composition of essential oils, as well as about the extracts of species belonging to this genus, and their biological activities. as far as we know, there is only one report on chemical composition of g. cynapioides essential oil (radulović et al., 2008) which relates to the plant as a whole, including underground tubers, for which it is literally known that they might be completely different in chemical composition. the aim of this study was to perform a detailed phytochemical analysis of geocaryum cynapioides (guss.) l. engstrand essential oils obtained from different plant parts. material and methods plant material the plant material (flowering stage) was collected at vlasina lake plateau, serbia, in june 2017 and was identified by one of the authors bojan zlatković. the voucher specimen was deposited in the herbarium moesiacum niš (hmn), department of biology and ecology, faculty of science and mathematics, university of niš under the acquisition number 13317. sample preparation the essential oils samples were prepared by hydrodistillation of fresh chopped whole aerial plant parts (194 g), shoots (158 g) and inflorescences (54 g), for 2.5 hours using clevenger type apparatus, according to the method recommended in british pharmacopoeia (british pharmacopoeia, 1988). the essential oils were extracted with n-hexane, dried over anhydrous sodium sulfate and evaporated. the yields of the oils were 0.06%, 0.05% and 0.08%, based on the weight of fresh plant, respectively. samples were re-dissolved in n-hexane to obtain the desired optimal concentrations and stored at -20 °c in the dark until analyzed. hexane analytical reagent grade, fisher chemical, uk; na2so4 pro analysis grade, sigma-aldrich, germany. gc and gc/ms analysis the sample of essential oil (20 mg/ml) was analyzed by a 7890/7000b gc/ms/ms triple quadrupole system in ms1 scan mode (agilent technologies, usa) equipped with a combi pal sampler. the fused silica capillary column hp-5 ms (5% phenylmethylsiloxane, 30 m x 0.25 mm, film thickness 0.25 μm) was used. the injector and interface operated at 250 and 300 °c, respectively. temperature program: from 50 to 290 °c at a heating rate of 4 °c min-1. the carrier gas was helium with a flow of 1.0 ml min-1. one microliter of the oil solution in hexane was injected (1:100, split ratio 40:1). post run: back flash for 1.89 minutes, at 280 °c, with helium pressure of 50 psi. ms conditions were as follows: ionization voltage of 70 ev, acquisition mass range 40-440, scan time 0.32 seconds. gc-fid analysis was carried out under the same experimental conditions using the same column as described for the gc/ms. the percentage composition of the sample was computed as an average of the gc peak areas obtained in triplicate, without any corrections. identification of volatile compounds eo constituents were identified by comparison of their linear retention indices relative to c8-c32 alkanes on the hp-5 ms column (van den dool & kratz, 1963), with literature values and their ms with biologica nyssana 9 (1) ⚫ september 2018: 31-35 petrović et al. ⚫ essential oil analysis of different plant parts of geocaryum… 33 table 1. chemical compositions of the g. cynapioides essential oils obtained by gc/ms relative amount % riexp riref compound so io ao class 998 988 myrcene tr tr tr m 1034 1024 limonene tr tr tr m 1042 1032 (z)-β-ocimene 1.5 0.2 1.0 m 1052 1044 (e)-β-ocimene 0.6 0.1 0.4 m 1083 1088* 1-nonen-3-ol 0.1 tr o 1091 1086 terpinolene 0.2 0.1 m 1101 1095 linalool tr tr mo 1100 1100 undecane tr o 1106 1100 n-nonanal tr 0.1 tr o 1147 1144 2(z)-nonen-1-al 0.1 tr o 1161 1157 2(e)-nonen-1-al tr tr o 1287 1283 isobornyl acetate tr tr mo 1291 1288 lavandulyl acetate tr tr mo 1299 1300 tridecane tr tr o 1278 1374 α-copaene tr tr tr s 1383 1379 geranyl acetate 0.1 tr mo 1388 1387 β-bourbonene 0.1 tr s 1394 1389 β-elemene 0.1 tr s 1406 1405 sesquithujene 0.1 0.1 tr s 1416 1410 α-cedrene tr tr tr s 1423 1417 (e)-caryophyllene 0.8 1.6 1.0 s 1429 1429* himachala-2,4-diene 0.2 0.1 0.1 s 1432 1430 β-copaene tr . tr s 1437 1432 α-trans-bergamotene tr tr tr s 1444 1440 (z)-β-farnesene tr tr tr s 1461 1454 (e)-β-farnesene 61.0 72.0 66.6 s 1481 1481 γ-curcumene 0.1 0.1 s 1485 1484 germacrene d 2.7 3.0 2.8 s 1494 1491* (z,e)-α-farnesene 1.8 1.2 1.5 s 1500 1500 bicyclogermacrene 0.6 0.2 0.4 s 1503 1506 (z)-α-bisabolene 0.1 tr 0.1 s 1508 1505 (e,e)-α-farnesene 4.4 2.6 3.7 s 1513 1514 β-curcumene 0.5 0.2 0.4 s 1525 1521 β-sesquiphellandrene tr tr tr s 1525 1522 δ-cadinene 0.2 tr 0.1 s 1533 1529 (e)-γ-bisabolene tr tr tr s 1555 1542 cis-sesquisabinene hydrate 2.1 1.4 1.7 so 1564 1561 (e)-nerolidol 0.4 0.3 0.3 so 1579 1574 germacrene d-4-ol 0.1 tr so 1583 1577 trans-sesquisabinene hydrate 0.4 0.1 0.1 so 1593 1586* 7-epi-cis-sesquisabinene hydrate 19.3 13.1 17.4 so 1611 1611 tetradecanal tr tr o 1634 1632 α-acorenol 0.6 0.3 0.4 so 1651 1636 β-acorenol 0.2 0.1 0.1 so 1672 1670 epi-β-bisabolol 0.7 0.4 0.5 so 1685 1683 epi-α-bisabolol 0.2 0.1 so 1698 1699 β-sinensal tr tr tr so 1900 1900 nonadecane 0.1 tr o 2000 2000 eicosane tr tr o 2100 2100 heneicosane 0.1 tr o 2200 2200 docosane tr o biologica nyssana 9 (1) ⚫ september 2018: 31-35 petrović et al. ⚫ essential oil analysis of different plant parts of geocaryum… 34 those of authentic standards, as well as those from adams (adams, 2007), wiley 6, nist11, agilent mass hunter workstation b.06.00 software and a homemade ms library with the spectra corresponding to pure substances and components of known eos by the application of the amdis software (automated mass spectral deconvolution and identification system, ver. 2.1, dtra/nist, 2011). results and discussion chemical compositions of the aerial plant parts, shoots and inflorescences essential oils of geocaryum cynapioides, obtained by gc and gc/ms analyses, are presented in tab. 1. number of identified compounds in aerial parts (ao), inflorescences (io) and shoots (so) samples were 52, 42 and 48 respectively, representing 99.5%, 99.0% and 99.7% of total volatiles. the composition of the essential oils of the shoots and the oil obtained from inflorescences was found to be quite comparable. hydrocarbon sesquiterpenes had the highest contribution in all investigated g. cynapioides essential oils, with a share of about three quarters of all, followed by one quarter of oxygenated sesquiterpenes. the shoot essential oil had a little bit lower content of hydrocarbon sesquiterpenes compared to the inflorescences oil, 72.6% and 81.1%, respectively. in both essential oils this class of compounds predominated over the oxygenated sesquiterpenes. a reverse distribution was observed for the oxygenated sesquiterpenes which are prevalent in shoots essential oil over the inflorescences oil (24.0% vs. 15.7%, respectively). hydrocarbon monoterpenes were present in negligible amounts in both oils, while the oxygenated monterpenes were not detected at all. obtained results indicate that the composition of the g. cynapioides aerial parts essential oil represents the average value of the contents of essential oils derived from inflorescences and shoots essential oils. phytochemical analysis by gc and gc/ms of the essential oil samples, obtained from fresh aerial parts, shoots and inflorescences of g. cynapioides, allowed the identification of 55 components in total. regarding the aerial parts essential oil, the major of 52 identified volatile compounds were (e)-βfamesene (66.6%), 7-epi-cis-sesquisabinene hydrate (17.4%) and (e,e)-α-farnesene (3.7%). the same compounds were dominant among the 48 components detected in the shoots and 42 volatiles in inflorescences oil, but in different proportions. as we already shown, chemical composition of inflorescences could be pretty similar (kostevski et al., 2016) or essentially various (petrović et al., 2018; stamenković et al., 2015) in comparison to the composition of the shoots essential oil. on the other hand, the composition of the volatile components obtained from all the above-ground parts of the plant and root is always completely different (petrović et al., 2017; stamenković et al. 2015). compared with the previously published results of g. cynapioides essential oil (radulović et al., 2008), certain differences in chemical compositions of aerial parts essential oils can be observed. that can be explained by the fact that sample a consisted of fresh aerial parts (inflorescences, stems and leaves) and underground tubers, while sample b was composed of stems, leaves and umbels full of ripe fruit (fruiting stage). sample b differs significantly both by the number of identified components and by the relative content of compound classes, especially in relation to monoterpenoids content (hydrocarbon monoterpenes relative amount % riexp riref compound so io ao class 2300 2300 tricosane 0.1 0.6 0.2 o 2500 2500 pentacosane tr 0.4 0.1 o 2700 2700 heptacosane 0.1 0.2 0.1 o 2900 2900 nonacosane 0.3 0.4 0.2 o total 99.7 99.0 99.5 monoterpene hydrocarbons m 2.3 0.3 1.5 monoterpene oxygenated mo 0.1 sesquiterpene hydrocarbons s 72.6 81.1 76.8 sesquiterpene oxygenated so 24.0 15.7 20.6 others o 0.7 1.9 0.6 compounds are listed in order of elution from a hp-5 ms column; riref: literature retention indices; riexp: experimental retention indices relative to c8-c32 n-alkanes; (*): identified by nist chemistry webbook retention indices; tr: traces (<0.1%); (-): not detected. essential oils: so shoots; io inflorescences; ao aerial plant parts. biologica nyssana 9 (1) ⚫ september 2018: 31-35 petrović et al. ⚫ essential oil analysis of different plant parts of geocaryum… 35 6.5% and oxygenated monterpenes 4.9%). however, the main difference that can be noticed is that one of the main components identified in our investigation, 7-epi-cis-sesquisabinene hydrate, was not found at all. the probable reason is the similarity of the mass spectra and the proximity of the retention indices of this compound and trans-sesquisabinene hydrate, which was found in a significant amount, which led to a misidentification. according to the results of this study, the essential oil of g. cynapioides consisted mainly of sesquiterpene compounds, implying that there are great differences in composition of investigated essential oil compared to other reports about the scandicinae species` essential oils. considering the fact that geocaryum genus is not investigated at all, more studies are needed regarding essential oil constituents of its species. obtained data could be helpful in future research to clarify the chemotaxonomic relationships between the genera within the tribe scandiceae and subtribe scandicinae. acknowledgements. the authors are grateful to the ministry of education, science and technological development for financial support through the grant within frame of basic research, no 172047. references adams, r. p. 2007: identification of essential oil components by gas chromatography and mass spectrometry (4th ed.). carol stream: allured publishing corporation. ball, p. w. 1968: huetia p. w. boiss. in: t. g. tutin, v. h. heywood, n. a. burges, d. m. moore, d. h., valentine, s. m. walters, d. a. webb (eds.), flora europaea 2 (pp. 330). cambridge: cambridge university press. downie, s. r., katz-downie, d. s., spalik, k. 2000: a phylogeny of apiaceae tribe scandiceae: evidence from nuclear ribosomal dna internal transcribed spacer sequences. american journal of botany, 87: 76-95. kostevski, i., petrović, g., stojanović, g., stamenković, j., zlatković, b. 2016: chemical composition of achillea coarctata essential oil and headspace volatiles. natural product communications, 11 (4): 543-545. petrović, g., ilić, m., stankov-jovanović, v., stojanović, g., jovanović, s. 2018: phytochemical analysis of saponaria officinalis l. shoots and flowers essential oils. natural product research, 32 (3): 331-334. petrović, g., stamenković, j., stojanović, g., mitić, v., zlatković, b. 2017: chemical profile of essential oils and headspace volatiles of chaerophyllum hirsutum from serbia. natural product communications, 12 (9): 1513-1515. radulović, n., đorđević, n., zlatković, b., palić, r. 2008: composition of the essential oil of geocaryum cynapioides (guss.) l. engstrand. chemical papers, 62 (6): 603-607. spalik, k. & downie, s. 2001: the utility of morphological characters for inferring phylogeny in scandiceae subtribe scandicinae (apiaceae). annals of the missouri botanical garden, 88 (2): 270-301. stamenković, j., stojanović, g., radojković, i., petrović, g., zlatković, b. 2015: chemical composition of the essential oil from chaerophyllum temulum (apiaceae). natural product communications, 10 (8): 439-441. the plant list [internet]. royal botanic gardens, kew and missouri botanical garden; [version 1.1, september 2013]. available from: http:// http://www.theplantlist.org/ van den dool, h. & kratz, p. d. 1963: a generalization of the retention index system including linear temperature programmed gasliquid partition chromatography. journal of chromatography, 11, 463-471. plants and insects in interactions: multitrophic associations 75 reviev article received: 31 october 2016 revised: 12 november 2016 accepted: 24 november 2016 plants and insects in interactions: multitrophic associations vladimir žikić, marijana ilić milošević, maja lazarević*, saša s. stanković university of niš, faculty of science and mathematics, department of biology and ecology, niš, serbia * e-mail: majalazarevic9@gmail.com abstract: žikić, v., ilić milošević, m., lazarević, m., stanković, s.s.: plants and insects in interactions: multitrophic associations. biologica nyssana, 7 (2), december 2016: 75-82. studying a biological species, we are very often obliged to examine many other species which are directly or indirectly related to the particular species in order to have broader perspective of its being. interactions between organisms can be perceived on various levels concerning biological study areas, such as ecology, physiology, morphology etc. in this place, the authors have given some peculiar examples concerning parasitism. this specific interaction is very common in the animal kingdom, having in mind that about a half of all known organisms are parasitic. the importance of this phenomenon is reflected on many aspects of life, propelling evolution and speciation of each member in the relation. key words: parasitism, parasitoids, hosts, defending strategies apstrakt: žikić, v., ilić milošević, m., lazarević, m., stanković, s.s.: interakcije biljaka i insekata: multitrofičke asocijacije. biologica nyssana, 7 (2), decembar 2016: 75-82. proučavajući određene biološke vrste često smo primorani da izučavamo i mnoge druge koje su direktno ili indirektno povezane sa njom u cilju šireg sagledavanja njene celokupne biologije. interakcije između organizama mogu biti proučavane na različitim biološkim nivoima, kao što su ekologija, fiziologija, morfologija itd. u ovom slučaju, autori su prezentovali neobične primere parazitizma. ova specifična interakcija je veoma česta u životinjskom carstvu, imajući u vidu da skoro polovina poznatih organizama pripada parazitima. značaj ovog fenomena se ogleda u mnogim aspektima života, podstičući evoluciju i specijaciju svakog člana posmatrane interakcije. ključne reči: parazitizam, parazitoidi, domaćini, odbrambene strategije introduction the vast majority of plants have evolved along with insects. a lot of insect species directly depend on plants which represent a resource for the whole, or at least a part of insects’ life cycle. in this trophic association plants can offer roots, bulbs, trunks, branches, leaves, flowers, fruits and seed. even dead plants or their parts can be of use for decomposing insects. other connections between plants and insects imply symbiosis or mutualism. a specific relationship between plants and most insects is pollination; this particular act can be performed by hymenopterans, butterflies and moths, 7 (2) • december 2016: 75-82 12th sfses • 16-19 june 2016, kopaonik mt doi: 10.5281/zenodo.200402 biologica nyssana 7 (2)  december 2016: 75-82 žikić, v. et al.  plants and insects in interactions… 76 horned beetles, many flies etc., of which a great number represent very specialized forms as far as their morphology or behaviour is concerned (b r o n s t e i n et al., 2006). for example, moths from the family sphingidae are associated with plants whose flowers have deep calyx forming tubes (f a e g r i & v a n d e r p i j l , 1979; h a b e r & f r a n k i e , 1989). hovering around the plants these moths suck nectar through a very elongated proboscis. this type of feeding is not unique in the animal world; convergent evolution has appeared four times in non-related nectar feeders. in mammals, bats are a very good example, while among birds there are hummingbirds, and also two groups of insects: hoverflies (diptera: syrphidae) and hawk moths (k i t c h i n g , 2002). the list of attended plants is huge, ranging from those with small flowers with short calyx such as verbena l., lantana l., lobelia l. and buddleja l., over petunia juss. or nicotiana l., and furthermore to plants with very large flowers with deep calyx such as various species of datura l. or darwin orchid, angraecum sesquipedale thouars with a calyx which is 30 cm deep. otherwise, the group of solitary bees (megachilidae), e.g., eucera longicornis (l.), some digger wasps (crabronidae) and some mining bees (andrenidae) specialize in the pollination of various orchids imitating the females; in the first place from the genus ophrys l. (w i l l m e r , 2011). for many snapdragon species the only solution of being pollinated was to establish an intimate relation with some bumblebees which are capable to open the “lips” of the flowers using their strong hind legs (e.g., o d e l l et al., 1999). myrmecochory is a special kind of ant-plant interaction where ants are engaged in seed dispersal. for this purpose myrmecochorous plants shaped their seeds to carry elaiosomes, various external appendages rich of nutrients such as lipids, amino acid, or other attractive substances to ants (g i l a d i , 2006). some stick insects (phasmida) have taken advantage of this ant activity by producing seed-like eggs which are carried by ants into their nests where the conditions are favourable for their development (c o m p t o n & w a r e , 1991; s t a n t o n et al., 2015). in central america there is an example of true symbiosis between ants and plants. the acacia ant – pseudomyrmex ferruginea f. smith, lives in small colonies in the base of the horns of the bullhorn acacia – vachellia cornigera (l.) finding a shelter there, but at the same time offering protection to its host (j a n z e n , 1967; p i p e r , 2007) as ants attack all approaching animals and plants by stinging them. a specific way of insect parasitism takes place in galls. galls or cecidia are transformations of plant parts (external tissues) such as leaves, branches, roots, petals or flowers, caused by insect or mite activities. plant tissues show abnormal outgrowths like tumours in animals. the most important gallinducing insects are gall wasps, cynipidae (hymenoptera), gall midges cecidomyiidae (diptera), gall aphids aphididae (hemiptera) and some species from the group of leaf-miner flies, agromyzidae (diptera) (e.g., s p e n c e r , 1973; s t o n e et al., 2002; y u k a w a & r o h f r i t s c h , 2005; f l o a t e , 2010). galls and cecidia arise after females inject cecidogenous fluid, along with egg, in the plant tissue, e.g. tenthredinidae (hymenoptera) (k o p e l k e , 1998). afterwards, parasite larvae force the neighbouring plant cells into gall to start the division process of meristems. parasites use the galls as their habitat and shelter; also exploiting the sugars accumulated in it, they also use the galls as a food source (l a r s o n & w h i t h a m , 1991). a very bizarre relationship between plants and insects is found in carnivorous plants such as drosera l., nepenthes l., sarracenia l. or dionaea muscipula ellis that “consume” insects. leaves of those mainly bog inhabiting plants form traps, usually pitfalls, filled with water and enzymes (phytotelmata). all kind of traps in carnivorous plants are supplied with attractive colour, odour and specialized nectary glands. those glands produce sweet smelling nectar to attract and catch insects later digesting them, using externally excreted enzymes. in this way, plants obtain minerals from insect’s body, since the boggy soil is poor. once those plants have to be pollinated, they need insects again. then, carnivorous plants suspend all the attractants in their traps, focusing on their flower equipment in order to attract pollinators. this convergent behaviour evolved independently in both dicotyle and monocotyle plant lineages, terrestrial and underwater plants (a l b e r t et al., 1992). plants, hosts and parasitoids predators, parasites and parasitoids have an indirect connection with plants via their herbivorous pray or host which is essential for their life cycles (p r i c e et al., 1980; s t o t z et al., 1999). in the case of insect parasitoids, the host is usually required during the larval stage. the plant role in this play is to unintentionally help the host to become less attractive or even poisonous for its enemies. the six-spot burnet, zygaena filipendulae l. is capable of sequestering alkaloids from the food plant lotus corniculatus l. in this cases the cyanogenic glucoside linamarin, also lotaustralin are biosynthesized in poison thus decreasing predation of larvae and adults (d a v i s & n a h r s t e d t , 1979; z a g r o b e l n y et al., 2007). this poison protects a biologica nyssana 7 (2)  december 2016: 75-82 žikić, v. et al.  plants and insects in interactions… 77 very high percentage of adult moths while larvae still remain exposed for various parasitoids such as various hymenopterans and tachinids (tachinidae: diptera) (ž i k i ć et al., 2013). the great majority of parasitoid insects belong to the hymenoptera insect order. with some exceptions, they come from the division parasitica. parasitoid members of the other division aculeata, are in minority and their kind of parasitism is a little different (e.g., chrysididae, sphecidae, etc.) (w c i s l o et al., 1985; w i n t e r h a g e n , 2015). the second important group of parasitoids are tachinid flies (feener & brown, 1997); also, there are some sporadic cases in coleoptera, lepidoptera, trichoptera, neuroptera and strepsiptera (p i e r c e , 1995; h e r a t y , 2009). a simple association between a plant (primary food producer), an herbivorous host (primary food consumer) and a parasitoid (secondary food consumer) is being considered through trophic chains; this threesome is usually referred to as a tritrophic association. higher trophic levels with more members include more than one parasitoid species and also hyperparasitoids primary food consumer forming a multitrophic association. parasitoid larvae are carnivorous, feeding outside or inside the host body. conversely, adults are free living organisms feeding on flowers (nectar) or honeydew produced by green aphids. the final effect of host-parasitoid interaction is death of the host, which makes these organisms very powerful biological weapon. nowadays, several companies have developed technologies for commercial production of a few dozen parasitoid species on artificial nutrient medium. those species are applicable both in open fields as well as in greenhouses (o l k o w s k i et al., 2003; h a l e & h e n s l e y , 2010). there are various classifications of parasitoids based on different criteria concerning their behaviour, life cycle or number of eggs laid per single host. on the basis of oviposition location parasitoids can be ectoor endoparasitoids; whether or not parasitoid larvae alter hosts behaviour they could be idiobionts or koinobionts accordingly; according to the number of eggs laid per host there are two cases, solitary (a single egg per host) or gregarious (usually few to more than a hundred eggs laid per host) (g o d f r a y , 1994). ectoparasitoids lay their eggs on the host body surface. if the female causes a permanent paralysis of the host and parasitoid larvae feed outside the host body, those species represent idiobionts (many ichneumonidae and some braconidae). on the contrary, if the host continues its life after the larvae have been hatched on its body, those parasitoids are considered as koinobionts. those are parasitoids which only temporarily paralyse the host, and they are capable of regulating its behaviour and life cycle until they reach maturity (d e s n e u x et al., 2009). usually, parasitoid larva kills the host at the end of its larval stage. an example of ectoparasitic koinobionts is an ichneumonid wasp acrodactyla quadrisculpta (gravenhorst) which parasitizes a spider, tetragnatha montana simon (m i l l e r et al., 2013). the majority of koinobionts represent endoparasitoids, which develop inside the host body. in some cases, such as the species macrocentrus curtis (braconidae), the female lays only one egg in the host body which undertakes the process of polyembryony and ultimately gives dozens of individuals which are genetically the same (clones) and thus increasing its parasitic capacity (h o w a r d , 1906; c u s h m a n , 1913; p a r k e r , 1931; q u i c k e , 2014). another strategy of increasing parasitic capacity can be seen in gregarious parasitoids (e.g., eulophidae, encyrtidae, braconidae) where females lay a couple of eggs per host, but their offspring is not genetically the same and both sexes are developed (q u i c k e , 2014). multitrophic associations usually include hyperparasitoids, the species that parasitize primary parasitoids. however, the hyperparasitism does not end here; on the highest level of trophic chains we are talking about hyperparasitoids of the second and the third level. clearly, in the trophic chain with a maximum number of members in a raw is consisted of: 1) primary food producer (plant), 2) primary food consumer (host), 3) secondary food consumer (primary parasitoid), 4) tertiary food consumer (secondary parasitoid = primary hyperparasitoid), 5) quaternary food consumer (tertiary parasitoid = secondary hyperparasitoid) and 6) quinary food consumer (quaternary parasitoid = tertiary hyperparasitoid). h a r v e y et al. (2009) explained this phenomenon working on the wasp cotesia glomerata (l.) (braconidae) as a primary parasitoid which is hyperparasitized by lysibia nana gravenhorst (ichneumonidae) which is a secondary parasitoid. furthermore, the species l. nana was hyperparasitized by another ichneumonid wasp gelis agilis (fabricius), appearing as a tertiary parasitoid. finally, there are some other species from the same genus, gelis attack g. agilis. interspecific parasitism is considered in more details in s u l l i v a n (1987). a kind of parasitism which is known as kleptoparasitism is the interaction where one animal takes food from another. in parasitoids kleptoparasitism occurs in cuckoo wasps (chrysididae). the cuckoo wasp omalus panzer is the parasitoid of the larvae of another hymenopteran family, crabronidae (w i n t e r h a g e n , 2015). this species does not oviposit directly in crabronid larva, but in aphids, even though an aphid is many times biologica nyssana 7 (2)  december 2016: 75-82 žikić, v. et al.  plants and insects in interactions… 78 smaller than the cuckoo wasp larva. crabronid females collect the aphids and carry them in a previously made nest to serve as food for their larvae. accidentally, a crabronid female picks up a parasitized aphid with the egg of a cuckoo wasp which has been laid inside its body. in this way a crabronid wasp brings the parasitoid in its own nest. ultimately, when the cuckoo wasp larva emerges from its egg and then from the aphid body, it starts looking for food devouring the crabronid larva, and also the collected aphids. exceptions exist in almost every group of sibling organisms. well know example of exception is panda – being herbivore among the mammal’s order carnivora. similarly, among the large groups of parasitica, there are some exceptions as well; many species of the family agaonidae (fig wasps) have developed a unique mutualistic association with various ficus l. trees, and more specifically with their flowers. the odour released from the fig flowers (syconium) attracts a fertilized female wasp to enter the syconium through a tiny opening in the centre. in this way, the wasp is carrying pollen from the flower from which it emerged. however, the main reason of its presence is to lay eggs in fig’s female flowers turning a syconium to a fig fruit (k j e l l b e r g et al., 2005). plant defending strategies and plant communications there is a secret language in which plants communicate with each other in many ways (e.g., allelopathy) (b a l u š k a & n i n k o v i ć , 2010). modern definition of allelopathy is “chemical interactions that involve toxic allelochemicals and should be distinguished from plant chemical communication” (s c h e n k et al., 1999; s c h e n k & s e a b l o o m , 2010). a way of emanating evaporative chemical signals via air is the one that is best investigated (v e t & d i c k e , 1992; b r u c e et al., 2005). f o w l e r & l a w t o n (1985) found that the chemicals are a blend of organic molecules such as alcohols, aldehydes, ketones and esters which are known as volatile organic compounds (vocs) also called kairomones (more recent, semiochemicals). recent studies reveal that plants as the lowest trophic level, when attacked by herbivores such as aphids, scale insects or caterpillars, emit any substance produced by an individual of one species that benefits both the producer and the recipient which is of a different species synomones which attract natural enemies i.e. parasitoids and predators in order to defend themselves (v e t & d i c k e , 1992; t u r l i n g s et al., 1995). this group of authors also revealed that the signalling role probably evolved secondarily as a result of plant responses that produce toxins and repellents against pests and also antibiotics against pathogens. studying olfactory physiology and behaviour of a predaceous beetle thanasimus formicarius (l.) (coleoptera: cleridae), the authors z h a n g & s c h l y t e r (2010) showed that the specific chemical substances from other nontarget hosts and plants, which are not even a part of the particular trophic association, can inhibit the attraction of chemical signals of attacked host/prey. in other words, neighbouring target plants resort to mimicry, thus stopping to smell out, becoming less attractive for pests. the other way of plant communication is through soil. they can do that directly by roots, or most often using an “internet” made of fungi. by linking to the fungal network, plants exchange information, nutrients and toxic chemicals with their neighbours (s o n g et al., 2010). mutually-beneficial relationships between plants and fungi, known as mycorrhiza can be found in a great majority of plants. host-parasitoid coevolution under the pressure of parasitoids, hosts develop various defending strategies (k r a a i j e v e l d et al., 1998). a simple type of protection that females use for their offspring is laying eggs in hidden places. for example, some siricidae (hymenoptera: symphyta) have very long and strong ovipositors with which they drill wood and lay their eggs deep inside it, thus minimizing the chance of their larvae to be parasitized. some other species, such as sawflies (hymenoptera: argidae and tenthredinidae) saw plant stems where they will oviposit. in symphyta there are also the species causing leaf mines or galls as it was previously explained (h a w k i n s , 1988; h e r i n g , 2013). the immune system can protect the host against parasitoids to varying degrees (k r a a i j e v e l d & g o d f r a y , 1999; c a r t o n et al., 2008). in a process of encapsulation hosts can eliminate parasitoids, forming a multi-layered cyst made of hemocytes. the hemocytes found in insect’s hemolymph are cells with the phagocyte function (g o d f r a y , 1994). endosybionts are present in many living beings, irrespective of whether they are algae, plants, fungi or animals. these are organisms that live within the body or cells of other organisms. the most famous examples of endosymbiosis are nitrogenfixing bacteria associated with legume roots (franche et al., 2009), single-cell algae living in reef-building corals (r o w a n , 1998) and in some turbellaria (b a r n e a h et al., 2007), or some protists and prokaryotes which inhabit the intestine of termites biologica nyssana 7 (2)  december 2016: 75-82 žikić, v. et al.  plants and insects in interactions… 79 (o h k u m a , 2008) and other wood-consuming insects. there are also bacterial endosymbionts that protect host insects, e.g., aphids against parasitoids. bacteria from the genus buchnera overproduce tryptophan (plasmid borne enzyme) and other amino acids that minimize the exposure of aphids to parasitoids (r o u h b a k h s h et al., 1997). hamiltonella defensa is a species of bacteria, that lives as an endosymbiont of aphids (most studied is the pea aphid – acyrthosiphon pisum harris) providing its host protection against parasitoids. mechanism of protection implies that h. defensa is infected with temperate bacteriophages called acyrthosiphon pisum secondary endosymbionts (apses) (v a n d e r w i l k et al., 1999; v o r b u r g e r , 2014). those bacteriophages encode different toxin genes killing the parasitoid eggs or larvae thus protecting the aphid host (d e g n a n & m o r a n , 2008). endosymbiotic male-killing bacteria from the genera wolbachia and rickettsia eliminate only the male sex in parasitoid embryos defined by genetics. it is not yet clear why males are more sensitive to wolbachia; it might be due to the fact that they have only one x chromosome or because they lack the whole garniture of chromosomes (haploids) (e.g., h u r s t & j i g g i n s , 2000; k o n d o et al., 2002; b e l s h a w & q u i c k e , 2003; w e r r e n at al., 2008). parasitoids also have evolved a variety of strategies to overcome host immune responses, but on the other hand they developed strategies to defend themselves against hyperparasitoids. there are several strategies to accomplish host defending mechanisms: by avoiding oviposition in body parts accessible to host hemocytes or by disrupting the host immune system injecting specific chemicals into the host at the time of oviposition; for example: apanteles (hymenoptera: braconidae) injects baculovirus-like nucleocapsids in the caterpillar body (s t o l t z & v i n s o n , 1977). also, remnants of polydnaviruses found in larvae of some parasitoid taxa disrupt the capsule around the parasitoid which is made of host hemocytes (b é z i e r et al., 2009). polydnaviruses destroy those hemocytes or alter their ability to aggregate and form a strong layer around parasitoid larvae (v i n s o n , 1990). these endosymbionts played one of the main roles in evolution of host range and in defining parasitoidhost compatibility (s c h l e n k e et al., 2007). conclusion although this overview represents only a glimpse of a fantastic array of interactions among insects themselves and plants, it gives an interesting insight and perspective of these phenomena. beside the fact that many details are concealed or maybe beyond our reach, the accumulated knowledge and permanent investigation of this topic has provided us with many benefits. the most useful is pest management and using biocontrol agents to combat pest insects. this has some serious repercussions in healthier food production and sustainable agriculture. “let it be borne in mind how infinitely complex and close-fitting are the mutual relations of all organic beings to each other and to their physical conditions of life” (d a r w i n , 1872). acknowledgements. we would like to thank to the organisation board of the 12th symposium on the flora of south-eastern serbia and neighbouring regions, kopaonik mt. review of english language was done by antonis mylonopoulos, komotiní, greece. we also thank to dr. andjeljko petrović for the selection of references to write this manuscript. the authors participate in the project of the ministry of education, science and technological development of the republic of serbia, grant no. iii43001.this study is partly supported by eu project biocomes (fp7-kbbe-2013-7). references albert, v.a., williams, s.e., chase, m.w. 1992: carnivorous plants: phylogeny and structural evolution. american association for the advancement of science, 257 (5076): 1491-1495. baluška, f., ninković, v. 2010: plant communication from an ecological perspective. springer science & business media. 252pp barneah, o., brickner, i., hooge, m., weis, v.m., lajeunesse, t.c., benayahu, y. 2007: three party symbiosis: acoelomorph worms, corals and unicellular algal symbionts in eilat (red sea). marine biology, 151 (4): 1215-1223. belshaw, r., quicke, d.l. 2003: the cytogenetics of thelytoky in a predominantly asexual parasitoid wasp with covert sex. genome, 46 (1): 170-173. bézier, a., annaheim, m., herbinière, j., wetterwald, c., gyapay, g., bernard-samain, s., pfister-wilhem, r. 2009: polydnaviruses of braconid wasps derive from an ancestral nudivirus. science, 323 (5916): 926-930. bronstein, j.l., alarcón, r., geber, m. 2006: the evolution of plant–insect mutualisms. new phytologist, 172 (3): 412-428. bruce, t.j., wadhams, l.j., woodcock, c.m. 2005: insect host location: a volatile situation. trends in plant science, 10 (6): 269-274. carton, y., poirié, m., nappi, a.j. 2008: insect immune resistance to parasitoids. insect science, 15 (1): 67-87. biologica nyssana 7 (2)  december 2016: 75-82 žikić, v. et al.  plants and insects in interactions… 80 compton, s., ware, a.b. 1991: ants disperse the elaisosome-bearing eggs of an african stick insect. psyche, 98 (2-3): 207-214. cushman, r.a. 1913: biological notes on a few rare or little known parasitic hymenoptera. proceedings of the entomological society of washington, 15: 153-155. darwin, c. 1872: on the origin of species by means of natural selection, or the preservation of favoured races in the struggle for life. john murray. london. 502 p. lulu. com. davis, r.h., nahrstedt, a. 1979: linamarin and lotaustralin as the source of cyanide in zygaena filipendulae. comparative biochemistry and physiology, 64b: 395-397. degnan, p.h., moran, n.a. 2008: diverse phageencoded toxins in a protective insect endosymbiont. applied and environmental microbiology, 74 (21): 6782-6791. desneux, n., barta, r.j., delebecque, c.j., heimpel, g.e. 2009: transient host paralysis as a means of reducing self-superparasitism in koinobiont endoparasitoids. journal of insect physiology, 55 (4): 321-327. faegri, k., van der pijl, l. 1979: principles of pollination ecology. pergamon press, oxford. 244p feener jr, d.h., brown, b.v. 1997: diptera as parasitoids. annual review of entomology, 42 (1): 73-97. floate, k.d. 2010. gall-inducing aphids and mites associated with the hybrid complex of cottonwoods, populus spp. (salicaceae), on canada’s grasslands. in shorthouse, j.d. and floate, k.d. (eds.) – arthropods of canadian grasslands. vol. 1: ecology and interactions in grassland habitats, biological survey of canada (bsc), chapter 13, 281-300 p. fowler, s.v., lawton, j.h. 1985: rapidly induced defenses and talking trees: the devil's advocate position. american naturalist, 126(2): 181-195. franche, c., lindström, k., elmerich, c. 2009: nitrogen-fixing bacteria associated with leguminous and non-leguminous plants. plant and soil, 321 (1-2): 35-59. giladi, i. 2006: choosing benefits or partners: a review of the evidence for the evolution of myrmecochory. oikos, 112 (3): 481-492. godfray, h.c.j. 1994: parasitoids: behavioral and evolutionary ecology. princeton university press. princeton, new jersey. 488p haber, w.a., frankie, g.w. 1989: a tropical hawkmoth community: costa rican dry forest sphingidae. biotropica, 21 (2): 155-172. hale, f.a., hensley, d. 2010: sp290-z-commercial sources of predators, parasitoids and pathogens. the university of tennessee, agricultural extension service, htp://trace.tennessee.edu/utk_agexcomhort/33. harvey, j.a., wagenaar, r.,bezemer, t.m. 2009: interactions to the fifth trophic level: secondary and tertiary parasitoid wasps show extraordinary efficiency in utilizing host resources. journal of animal ecology, 78 (3): 686-692. hawkins, b.a. 1988: do galls protect endophytic herbivores from parasitoids? a comparison of galling and non‐galling diptera. ecological entomology, 13 (4): 473-477. heraty, j. 2009. parasitoid biodiversity and insect pest management. in: foottit, r.g. and adler, p.h. (eds.) insect biodiversity: science and society. chapter 19: 445-462. john wiley & sons. hering, e.m. 2013: biology of the leaf miners. dr. w. junk. berlin. 420p. howard, l.o. 1906: polyembryony and the fixing of sex. science, 24: 810-818. hurst, g.d., jiggins, f.m. 2000: male-killing bacteria in insects: mechanisms, incidence, and implications. emerging infectious diseases, 6 (4): 329-336. janzen, d.h. 1967: interaction of the bull's-horn acacia (acacia cornigera l.) with an ant inhabitant (pseudomyrmex ferruginea f. smith) in eastern mexico. the university of kansas science bulletin, 47: 315-558. kitching, i.j. 2002: the phylogenetic relationships of morgan's sphinx, xanthopan morganii (walker), the tribe acherontiini, and allied long‐tongued hawkmoths (lepidoptera: sphingidae, sphinginae). zoological journal of the linnean society, 135 (4): 471-527. kjellberg, f., jousselin, e., hossaert-mckey, m., rasplus, j.y. 2005. biology, ecology, and evolution of fig-pollinating wasps (chalcidoidea, agaonidae). in:raman, a., schaefer, c.w., withers, t.m. (eds.), biology, ecology and evolution of gall-inducing arthropods: 539-572, enfield, nh: sciences publisher, inc. kondo, n., nikoh, n., ijichi, n., shimada, m., fukatsu, t. 2002: genome fragment of wolbachia endosymbiont transferred to x chromosome of host insect. proceedings of the national academy of sciences, 99 (22): 1428014285. kopelke, j.p. 1998: oviposition strategies of gallmaking species of the sawfly genera pontania, euura and phyllocolpa (hymenoptera, tenthredinidae: nematinae). entomologia generalis, 22 (3): 251-275. kraaijeveld, a.r., van alphen, j.j.m., godfray, h.c.j. 1998: the coevolution of host resistance biologica nyssana 7 (2)  december 2016: 75-82 žikić, v. et al.  plants and insects in interactions… 81 and parasitoid virulence. parasitology, 116 (s1): s29-s45. kraaijeveld, a.r., godfray, h.c.j. 1999: geographic patterns in the evolution of resistance and virulence in drosophila and its parasitoids. the american naturalist, 153 (s5): s61-s74. larson, k.c., whitham, t.g. 1991: manipulation of food resources by a gall-forming aphid: the physiology of sink-source interactions. oecologia, 88 (1): 15-21. metcalf, r.l. 1985. plant kairomones and insect pest control. in: godfrey, g.l., bouseman, j.k., edwards, w.r., robertson, k., zewadski, r.m. (eds.) 125 years of biological research 18581983: a symposium. illinois natural history survey bulletin, 033: 03. miller, j., belgers, j.d., beentjes, k., zwakhals, k., van helsdingen, p. 2013: spider hosts (arachnida, araneae) and wasp parasitoids (insecta, hymenoptera, ichneumonidae: ephialtini) matched using dna barcodes. biodiversity data journal, 1: e992. odell, e., raguso, r.a., jones, k.n. 1999: bumblebee foraging responses to variation in floral scent and color in snapdragons (antirrhinum: scrophulariaceae). the american midland naturalist, 142 (2): 257-265. ohkuma, m. 2008: symbioses of flagellates and prokaryotes in the gut of lower termites. trends in microbiology, 16 (7): 345-352. olkowski, w., dietrick, e., olkowski, h., quarles, w. 2003: commercially available biological control agents. the ipm practitioner: the newsletter of integrated pest management. parker, h.l. 1931: macrocentrus gifuensis ashmead: a polyembryonic braconid parasite in the european corn borer. technical bulletin no. 230, us department of agriculture, washington d.c, 62p. pierce, n.e. 1995: predatory and parasitic lepidoptera: carnivores living on plants. journal of the lepidopterists society, 49 (4): 412-453. piper, r. 2007: extraordinary animals: an encyclopedia of curious and unusual animals. greenwood publishing group. london. 321 p. price, p.w., bouton, c.e., gross, p., mcpheron, b.a., thompson, j.n., weis, a.e. 1980: interactions among three trophic levels: influence of plants on interactions between insect herbivores and natural enemies. annual review of ecology and systematics, 11: 41-65. quicke, d.l. 2014: the braconid and ichneumonid parasitoid wasps: biology, systematics, evolution and ecology. john wiley & sons. new jersey, usa 704p. rouhbakhsh, d., clark, m.a., baumann, l., moran, n.a., baumann, p. 1997: evolution of the tryptophan biosynthetic pathway in buchnera (aphid endosymbionts): studies of plasmidassociated trpeg within the genus uroleucon. molecular phylogenetics and evolution, 8 (2): 167-176. rowan, r. 1998: diversity and ecology of zooxanthellae on coral reefs. journal of phycology, 34 (3): 407-417. schenk, h.j., callaway, r.m., mahall, b.e. 1999: spatial root segregation: are plants territorial? advances in ecological research: 28: 146-180 schenk, h.j., seabloom, e.w. 2010. evolutionary ecology of plant signals and toxins: a conceptual framework. in baluška, f., ninković, v. (eds.), plant communication from an ecological perspective: 1-19, springer. berlin, germany. schlenke, t.a., morales, j., govind, s., clark, a.g. 2007: contrasting infection strategies in generalist and specialist wasp parasitoids of drosophila melanogaster. plos pathogens, 3(10): e158. song, y.y., zeng, r.s., xu, j.f., li, j., shen, x., yihdego, w.g. 2010: interplant communication of tomato plants through underground common mycorrhizal networks. plos one, 5 (10): e13324. spencer, k.a. 1973: agromyzidae (diptera) of economic importance (vol. 9). springer science & business media. berlin. 418 p. stanton, a.o., dias, d.a., o’hanlon, j.c. 2015: egg dispersal in the phasmatodea: convergence in chemical signalling strategies between plants and animals? journal of chemical ecology, 41 (8): 689-695. stoltz, d.b., vinson, s.b. 1977: baculovirus-like particles in the reproductive tracts of female parasitoid wasps ii: the genus apanteles. canadian journal of microbiology, 23 (1), 28-37. stone, g.n., schönrogge, k., atkinson, r.j., bellido, d., pujade-villar, j. 2002: the population biology of oak gall wasps (hymenoptera: cynipidae). annual review of entomology, 47 (1): 633-668. stotz, h.u., kroymann, j., mitchell-olds, t. 1999: plant-insect interactions. current opinion in plant biology, 2 (4): 268-272. sullivan, d.j. 1987: insect hyperparasitism. annual review of entomology, 32 (1): 49-70. turlings, t.c., loughrin, j.h., mccall, p.j., röse, u.s., lewis, w.j., tumlinson, j.h. 1995: how caterpillar-damaged plants protect themselves by attracting parasitic wasps. proceedings of the national academy of sciences, 92 (10), 41694174. van der wilk, f., dullemans, a.m., verbeek, m., van den heuvel, j.f. 1999: isolation and biologica nyssana 7 (2)  december 2016: 75-82 žikić, v. et al.  plants and insects in interactions… 82 characterization of apse-1, a bacteriophage infecting the secondary endosymbiont of acyrthosiphon pisum. virology, 262 (1): 104-113. vet, l.e., dicke, m. 1992: ecology of infochemical use by natural enemies in a tritrophic context. annual review of entomology, 37 (1): 141-172. vinson, s.b. 1990: how parasitoids deal with the immune system of their host: an overview. archives of insect biochemistry and physiology, 13 (1‐2): 3-27. vorburger, c. 2014: the evolutionary ecology of symbiont‐conferred resistance to parasitoids in aphids. insect science, 21 (3): 251-264. wcislo, w.t., low, b.s., karr, c.j. 1985: parasite pressure and repeated burrow use by different individuals of crabro (hymenoptera: sphecidae; diptera: sarcophagidae). sociobiology, 11 (2): 115-126. werren, j.h., baldo, l., clark, m.e. 2008: wolbachia: master manipulators of invertebrate biology. nature reviews microbiology, 6 (10): 741-751. willmer p.g. 2011: pollination and floral ecology. princeton university press. princeton, usa. 792p. winterhagen, p. 2015: strategy for sneaking into a host's home: the cuckoo wasp omalusbiaccinctus (hymenoptera: chrysididae) inserts its eggs into living aphids that are the prey of its host. european journal of entomology, 112 (3): 557– 559. yukawa, j., rohfritsch, o. 2005: biology and ecology of gall-inducing cecidomyiidae (diptera). in: a., schaefer, c.w., withers, t.m. (eds.), biology, ecology, and evolution of gallinducing arthropods 1: 273-304, enfield, nh: sciences publisher, inc. zagrobelny, m., bak, s., ekstrom. c.t., olsen, c.e., moller, b.l. 2007: the cyanogenic glucoside composition of zygaena filipendulae (lepidoptera: zygaenidae) as effected by feeding on wild-type and transgenic lotus populations with variable cyanogenic glucoside profiles. insect biochemistry and molecular biology, 37 (1): 10-18. zhang, q.h., schlyter, f. 2010: inhibition of predator attraction to kairomones by non-host plant volatiles for herbivores: a bypass-trophic signal. plosone, 5 (6): e11063. žikić, v., stanković, s.s., petrović, a., ilićmilošević, m., van achterberg, c. 2013: parasitoid complex of zygaena filipendulae l. (lepidoptera: zygaenidae). archives of biological sciences, 65 (3): 1027-1035. coumarin structure as a lead scaffold for antibacterial agents molecular docking study biologica nyssana 7 (2)  december 2016: 167-170 veselinović, j.b. et al.  coumarin structure as a lead scaffold… 167 original article received: 30 october 2016 revised: 14 november 2016 accepted: 24 november 2016 coumarin structure as a lead scaffold for antibacterial agents molecular docking study jovana b. veselinović1*, jelena s. matejić2, aleksandar m. veselinović2, dušan sokolović2 1health institution filly farm, pharmacy filly 63, cara dušana 31, 18000 niš, serbia 2faculty of medicine, university of niš, bulevar dr zorana ðinđića 81, 18000 niš, serbia * e-mail: milosavljevic.jovana@hotmail.com abstract: veselinović, j.b., matejić, j.s., veselinović, a.m., sokolović, d.: coumarin structure as a lead scaffold for antibacterial agents molecular docking study. biologica nyssana, 7 (2), december 2016: 167-170. coumarins owe their class name to “coumarou”, the vernacular name of the tonka bean (dipteryx odorata willd, fabaceae), from which coumarin was isolated in 1820. many molecules based on the coumarin structure have been synthesized utilizing innovative synthetic techniques. various synthetic routes have led to interesting derivatives including the furanocoumarins, pyranocoumarins and coumarinsulfamates which have been found to be useful in photochemotherapy, antitumor and anti-hiv therapy, as stimulants for central nervous system, antiinflammatory therapy, as anti-coagulants, etc. one of important pharmacological activity of coumarin molecules is their potential as antibacterial agents since they show inhibitory activity toward isoleucyl-transfer rna (trna) synthetase. in the presented research molecular docking studies of selected coumarin compounds inside isoleucyltransfer rna (trna) synthetase active site were performed. molecular docking scores of all studied compounds were obtained through score functions. presented results indicate that from all studied coumarin compounds the strongest interactions with studied enzyme has 7,8-dihydroxy-4-phenyl coumarin followed by 5,7-dihydroxy-4-phenyl coumarin. presented results are in accordance with in vitro obtained results for their antibacterial activity. presented findings suggest that 4-phenyl hydroxycoumarins may be considered as good molecular templates for potential antibacterial agents and can be used for further chemical modifications for improving their antibacterial activity. key words: verbascum davidoffii, endemic species, genetic diversity, conservation apstrakt: veselinović, j.b., matejić, j.s., veselinović, a.m., sokolović, d.: coumarin structure as a lead scaffold for antibacterial agents molecular docking study. biologica nyssana, 7 (2), decembar 2016: 167-170. kumarini su dobili ime od reči "coumarou", narodnom nazivu za tonku (dipterix odorata willd, fabaceae) iz koje je kumarin izolovan 1820. godine. mnogi molekuli bazirani na kumarinskoj strukturi su sintetisani primenom inovativnih hemijskih tehnika. razičiti putevi sinteze su doveli do interesantnih derivata koji uključuju furanokumarine, piranokumarine i kumarinsulfamate koji imaju primenu u fitohemoterapiji, antitumorskoj i anti-hiv terapiji, kao stimulanti za centralni nervni sistem, kao antinflamatorni i antikoagulativni agensi, itd. jedna od važnih farmakoloških aktivnosti kumarinskih jedinjenja je njihov potencijal kao antibakterijskih agenasa, jer pokazuju inhibitornu aktivnost prema izoleucil-transfer rnk sintetazi (trnk). u predstavljenom istraživanju studija molekularnog dokinga unutar aktivnog mesta izoleucil7 (2) • december 2016: 167-170 12th sfses • 16-19 june 2016, kopaonik mt doi: 10.5281/zenodo.200417 biologica nyssana 7 (2)  december 2016: 167-170 veselinović, j.b. et al.  coumarin structure as a lead scaffold… 168 transfer rnk sintetaze (trnk) je primenjena za odabrana kumarinska jedinjena. vrednosti molekularnog dokinga za sva ispitivana jedinjenja su dobijene primenom odgovarajućih skoring funkcija. predstavljeni rezultati ukazuju da od svih ispitivanih kumarinskih jedinjenja najviše interakcija sa ispitivanim enzimom ima 7,8-dihidroksi-4-fenil kumarin, a zatim 5,7-dihidroksi-4-fenil kumarin. predstavljeni rezultati su u skladu sa in vitro dobijenim rezultima za njihovu antibakterijsku aktivnost. prikazani rezultati ukazuju da se 4-fenil hidroksikumarini mogu smatrati dobrim molekuskim osnovama za potencijalne antibakterijske agense i da se mogu koristiti za dalje hemijske modifikacije u cilju poboljšanja antibakterijske aktivnosti. key words: 4-fenil hidroksikumarini, antibakterijska aktivnost, molekularni doking introduction great hopes that the discovery and the application of penicillin and other antibiotics would forever solve the problem of bacterial infections lasted for a relatively short time period. seven decades after antibiotics were introduced, infectious diseases as a cause of mortality are in third place in europe. in the european union (eu) around 2 million people are hospitalized and around 200,000 die from some infectious diseases annually. as a gloom example in south asia in every 2 minutes one infant dies due to resistance to existing antibiotics. it is considered that the main reason for this "failure" of the treatment is a large percentage of drug-resistant and multidrugresistant bacteria. today, antibacterial resistance is a growing public health threat of major concern around the world. a post-antibiotic era (in which common infections and minor injuries can kill), far from being an apocalyptic fantasy, is instead a very real possibility for the 21st century (who, 2014). bacterial resistance is a natural biological phenomenon that can be associated with their fight for survival. mechanisms of bacterial resistance to antibiotics are very different and complex and depend both on the bacteria and the structure of antibiotics. also, it involves a range of resistance mechanisms acting an ever-widening range of bacteria, most of which can cause a wide spectrum of diseases in humans and animals. factors that favor the development of bacterial resistance are: overusing prescribing antibiotics, improper use the compliance, inadvertent application of antibiotics and slow development of new effective antibiotics. of all identified bacterial strains resistant to antibiotics of particular concern are methicillinresistant staphylococcus aureus (mrsa), penicillinresistant streptococcus pneumoniae, vancomycinresistant enterococcus, and mycobacterium tuberculosis, since many of these organisms are resistant to several classes of established antibiotics and they can cause infections which are very difficult to treat. above stated facts are forcing the search for novel antibacterial agents which affect targets that are essential to bacteria (p a y n e et al., 2007; b o l l e n b a c h , 2015). in this regard, the aminoacyltrna synthetase (aars) enzymes have been a focus of recent antibacterial drug research. inhibition of these enzymes halts protein biosynthesis which in turn results in the attenuation of bacterial growth under both in vitro and infectious conditions and for this reason bacterial aarss present a vast number of opportunities for the identification of novel inhibitors that can be considered as candidates for development as antibiotics (o c h s n e r et al., 2007). today resistant bacterial strains occur significantly faster than the synthesis and the release of new effective antibiotics. it is well known fact that the development of new drugs in recent decades has become extremely expensive and time-consuming process. for this reason novel computational methods like molecular docking are used to speed up the process of drug design and development. the main aim of molecular docking is the determination of a suitable geometry and binding affinity of the tested molecule (ligand) to the active site of the target macromolecules (receptors) with the application of the “scoring” functions (k r o e m e r , 2007). coumarins are a group of molecules found extensively in plants and they show various biological and pharmacological activities including anticoagulant, estrogenic, vasodilator, hypothermic, anthelmintic, sedative, analgesic, anti-inflammatory and antiulcer. further, coumarin derivates have a wide range of structural modifications (b o r g e s et al., 2005) and they can serve as molecular templates for new drugs. coumarin derivates are also considered as potential antibacterial agents (v e s e l i n o v i ć et al., 2015). main goal of presented research was to establish the influence of 4-phenyl group addition and the number and position of hydroxyl groups within main coumarine structure on the interaction with isoleucyl-transfer rna (trna) synthetase using molecular docking study. studied interactions are considered as main influence on enzyme inhibition which can lead to antibacterial activity. material and methods molecular docking. in order to gain insight into the plausible mechanism of antibacterial action docking simulations were performed. the crystal structure of isoleucyl-transfer rna (trna) synthetase (ilers) biologica nyssana 7 (2)  december 2016: 167-170 veselinović, j.b. et al.  coumarin structure as a lead scaffold… 169 was obtained from the brookhaven protein data bank http://www.rcsb.org/pdb (pdb entry: 1qu3). the compounds were docked into enzyme binding sites using the molegrovirtual docker (mvd) and published methodology [7]. three dimensional structures of compounds (7-hydroxy-4-phenyl coumarin (7c), b) 5,7-dihydroxy-4-phenyl coumarin (57c) and c) 7,8-dihydroxy-4-phenyl coumarin (78c)) used for docking simulation were constructed using marvinsketch 6.1.0, 2013, chemaxon (http://www. chemaxon.com). geometry optimization was carried out by employing mmff94 molecular force field. fig. 1. above: chemical structures of investigated compounds: a) 7-hydroxy-4-phenyl coumarin (7c), b) 5,7-dihydroxy-4-phenyl coumarin (57c) and c) 7,8-dihydroxy-4-phenyl coumarin (78c). bellow: the structure of ilers with best docking poses for all investigated coumarins inside enzyme binding pocket. results and discussion chemical structures of investigated coumarines are presented in fig. 1. the least energy binding mode of compounds has been studied and best docking poses for all investigated coumarins inside enzyme binding pocket is presented in fig. 1. binding affinity of a ligand to the active site of the enzyme can be estimated based on the score values obtained using scoring functions form the molecular docking method. table 1 presents score values for all studied compounds. presented results indicate that of all studied compounds the highest interactions with enzyme has 78c followed by 57c. according to presented results the difference between their interaction energies is not as big as difference between their interaction energies and 7c interaction energy. it is of great importance that presented results are in accordance with in vitro obtained results for antibacterial activity. interesting result was obtained with molecular docking study – that 7c don’t form any hydrogen bond with the active site of studied enzyme. this can be one of the reasons why 7c show the lowest antibacterial activity, because number, bond length, and bond energy of hydrogen bonds formed between ligand and enzyme have an important role in ligand effect on investigated activity. two dimensional representations of formed hydrogen bonds for 57c and 78c are presented in fig. 2. it was observed from in silico studies that 57c forms one hydrogen bond oxygen atom from carbonyl group from the lacton part of molecule with lys-71 (2.80 å). 78c forms four hydrogen bonds. oxygen atom from carbonyl group from the lacton part of molecule forms one hydrogen bond with lys71 (2.79 å). hydroxyl group at position 7 forms fig. 2. two dimensional representations of the best docking pose for a) 5,7-dihydroxy-4-phenyl coumarin, and b) 7,8-dihydroxy-4-phenyl coumarin inside isoleucyl-transfer rna (trna) synthetase active site. biologica nyssana 7 (2)  december 2016: 167-170 veselinović, j.b. et al.  coumarin structure as a lead scaffold… 170 one hydrogen bond with his585 (3.23 å). hydroxyl group at position 8 forms two hydrogen bonds with his585 (3.19 å) and gly66 (2.60 å). conclusion molecular docking simulations against ilers were performed and correlation between the observed inhibitory activity and the in silico molecular docking scores of the compounds was obtained through hydrogen bonding interactions. these findings suggest that 4-phenyl hydroxycoumarins may be considered as good molecular templates for potential antibacterial agents and can be used for further chemical modifications for improving antibacterial activity what will be the main goal of our further research. acknowledgements. this work has been financially supported by ministry of education and science, republic of serbia, under project number 43012.. references bollenbach, t. 2015: antimicrobial interactions: mechanisms and implications for drug discovery and resistance evolution. current opinion in microbiology, 27: 1-9. borges, p., roleira, f., milhazes, n., santana, l., uriarte, e. 2005: simple coumarins and analogues in medicinal chemistry: occurrence, synthesis and biological activity. current medicinal chemistry, 12 (8): 887-916. kroemer, r.t. 2007: structure-based drug design: docking and scoring. current protein and peptide science, 8 (4): 312-328. ochsner, u.a., sun, x., jarvis, t., critchley, i., janjic, n. 2007: aminoacyl-trna synthetases: essential and still promising targets for new antiinfective agents. expert opinion on investigational drugs, 16 (5): 573-593. payne, d.j., gwynn, m.n., holmes, d.j., pompliano, d.l. 2007: drugs for bad bugs: confronting the challenges of antibacterial discovery. nature reviews. drug discovery, 6 (1): 29-40. veselinović, j.b, veselinović, a.m., nikolić, g.m., pešić, s.z., stojanović, d.b., matejić, j.s., mihajilov-krstev, t.m. 2015: antibacterial potential of selected 4-phenyl hydroxycoumarins integrated in vitro and molecular docking studies. medicinal chemistry research, 24 (4): 16261634. who. 2014: antimicrobial resistance: global report on surveillance. world health organization who library cataloguing-in-publication data. table 1. score values (kcal/mol) for all studied coumarin compounds. moldock rerank interaction inter. hbond docking score 78c -125.476 -108.328 -138.994 13.517 -6.0007 -127.625 57c -118.820 -101.209 -130.653 11.832 -3.8847 -124.704 7c -106.467 -88.612 -116.639 10.172 0 -104.336 anticancer compounds from medicinal plants biologica nyssana 4 (1-2)  december 2013: 15-17 ballian, d., mujagić-pašić, a..  morphological variability of the fruit… 15 original article morphological variability of the fruit and seed of wild cherry (prunus avium l.) in a part of its natural distribution in bosnia and herzegovina dalibor ballian 1 , aida mujagić-pašić 2 1 faculty of forestry, university of sarajevo, zagrebačka 21, 71000 sarajevo, bosnia and herzegovina 2 botanical faculty, university of bihać, luke marjanovića bb, 77000 bihać, bosnia and herzegovina * e-mail: balliand@bih.net.ba abstract: ballian, d., mujagić-pašić, a.: morphological variability of the fruit and seed of wild cherry (prunus avium l.) in a part of its natural distribution in bosnia and herzegovina . biologica nyssana, 4 (1-2), december 2013: 15-17. the paper discusses morphological variability of the fruit and seed of wild cherry (prunus avium l.) which have been collected in 12 localities of its natural distribution in bosnia and herzegovina. a total of 2,926 fruits have been collected, and the following properties have been studied: fruit length, fruit width, fruit thickness, seed length, seed width and seed thickness. a discriminant analysis by groups formed according to classes of altitude and ecological and vegetation zoning has shown that there are no separations within the studied populations, which implies the possibility to use seed and planting material from different altitudes and ecological and vegetation zones within the studied area. key words: bosnia and herzegovina, variability of the fruit and seed, wild chery introduction the wild cherry (prunus avium l.) has great and multiple importance. the fruit of the wild cherry is used for several purposes (as food for people, birds and other animals, as well as in phytotherapy). literature data about the morphological variability of the wild cherry are scarce and only general information for particular characteristics can be found, usually for seed and less for flower characteristics (b a l l i a n , 2000, 2002), as well as some data about fruit size which serve for the taxonomic determination of the wild cherry (h e r m a n , 1971). there are several different pieces of information available about the origin of the wild cherry. it is generally assumed that it originated in west asia and caucasus (v a v i l o v , 1935), while others believe its origin is in the mediterranean (ž u k o v s k y , 1965). however, some authors (m i š i ć , 1987) state that the wild cherry originated in asia minor, as well as south and mid europe, whereas others point out the pontic area (n i n k o v s k i , 1998). material and methods the plant material was collected from 12 natural populations in bosnia and herzegovina. the fruit and seed were collected from marginal or solitary trees, usually from the south-facing, outer sun-exposed parts of the tree crown. the following characteristics were measured: fruit length (fl), 11 th sfses • 13-16 june 2013, vlasina lake 4 (1-2) • december 2013: 15-17 biologica nyssana 4 (1-2)  december 2013: 15-17 ballian, d., mujagić-pašić, a..  morphological variability of the fruit… 16 fruit width (fw), fruit thickness (ft), seed length (sl), seed width (sw) and seed thickness (st). (figure 1. wild cherry seed). all statistical analyses of the data were made using the spss 15.0 package for windows. fig. 1. wild cherry seed results and discussion the results obtained show the presence of a high level of intrapopulational, as well as interpopulational, morphological variability in the natural populations of the wild cherry which have been investigated. analyses of population differentiation have not confirmed our expectations. a discriminant analysis by groups formed according to classes of altitude (fig. 1) and ecological and vegetation zoning (fig. 3) has shown that there are no separations within the studied populations, which implies the possibility to use seed and planting material from different altitudes and ecological and vegetation zones within the studied area. conclusion the analyses of 6 morphological characteristics in 12 natural populations of the wild cherry in bosnia and herzegovina showed statistically significant differences between investigated populations. differentiation in natural populations of the wild cherry was very low and identified only in fruit dimension characteristics. fig. 2. discriminant analysis by groups according to classes of altitude fig. 3. discriminant analysis by groups according to ecological and vegetation zoning biologica nyssana 4 (1-2)  december 2013: 15-17 ballian, d., mujagić-pašić, a..  morphological variability of the fruit… 17 references ballian, d. 2000: početna istraživanja varijabilnosti morfoloških svojstava sjemena divlje trešnje (prunus avium l.). šumarski list, 124 (5– 6): 271–278. ballian, d. 2002: variability of characteristics of the wild cherry blossom (prunus avium l.) in the region of central bosnia. ann sci for, 25(2): 19. herman, j. 1971: šumarska dendrologija. stanbiro. zagreb, 466 p. ninkovski, i. 1998: trešnja. nauka i praksa, beograd, 151 p. vavilov, n. i. 1935: teoretičerskie osnovi selekcii rastenij. gos izdat kolhoznoj i sovhoznoj literaturi, moskva-leningrad. žukovsky, p. m. 1965: main gene centers of cultivated plants and their wild relation within the territory of the ussr. euphytica, p 14. dill (anethum graveolens l.) seeds essential oil as a potential biologica nyssana 7 (1)  september 2016: 31-39 stanojević, lj.p. et al.  dill (anetum graveolens l.) seeds essential oil… 31 original article received: 18 january 2015 revised: 19 february 2016 accepted: 01 mart 2016 dill (anethum graveolens l.) seeds essential oil as a potential natural antioxidant and antimicrobial agent ljiljana p. stanojević*, mihajlo z. stanković, dragan j. cvetković, bojana r. danilović, jelena s. stanojević university of niš, faculty of technology, bulevar oslobođenja 124, 16000 leskovac, serbia * e-mail: stanojevic@tf.ni.ac.rs abstract: stanojević, lj.p., stanković, m.z., cvetković, d.j., danilović, b.r., stanojević, j.s.: dill (anethum graveolens l.) seeds essential oil as a potential natural antioxidant and antimicrobial agent. biologica nyssana, 7 (1), september 2016: 31-39. synthetic antioxidants and antimicrobial agents can induce many undesired side effects, which attracts interest of food producers and consumers in finding ingredients of natural origin. the antioxidative and antimicrobial activity of essential oil from dill (anethum graveolens l.) seeds was investigated in terms of its possible application as natural antioxidant and antimicrobial agent. dpph test and frap method have been used for the investigation of antioxidative activity of essential oil. disc-diffusion method has been used for investigation of oil antimicrobial activity on following microorganisms: staphylococcus aureus, listeria monocytogenes, bacillus subtilis, escherichia coli, salmonella enteritidis and candida albicans. essential oil, in concentration of 29 mg/ml, incubated for 60 minutes has shown the highest degree of dpph radicals’ neutralization (79.62%). frap activity of oil was 40.63 μmol fe2+/g of essential oil. essential oil showed the best antimicrobial activity on staphylococcus aureus. furthermore, there was a significant antimicrobial activity on all investigated microorganisms. key words: anethum graveolens l., dill seeds, essential oil, antioxidant activity, antimicrobial activity apstrakt: stanojević, lj.p., stanković, m.z., cvetković, d.j., danilović, b.r., stanojević, j.s.: etarsko ulje semena mirođije (anethum graveolens l.) kao potencijalni prirodni antioksidans i antimikrobni agens. biologica nyssana, 7 (1), septembar 2016: 31-39. sintetski antioksidansi i antimikrobni agensi mogu dovesti do brojnih neželjenih efekata, pa je zato sve veće interesovanje proizvođača i potrošača hrane za sastojcima prirodnog porekla. proučavana je antioksidativna i antimikrobna aktivnost etarskog ulja semena mirođije (anethum graveolens l.) u cilju moguće primene kao prirodnog antioksidansa i antimikrobnog agensa. antioksidativna aktivnost etarskog ulja je određena primenom dpph-testa i frap metode.antimikrobna aktivnost ulja je određena disk-difuzionom metodom, na sledeće mikroorganizme: staphylococus aureus, listeria monocytogenes, bacilus subtilis, escherichia coli, salmonela enteritidis i candida albicans. najveći stepen neutralisanja dpph radikala (79,62%) pokazuje ulje inkubirano 60 minuta, u koncentraciji 29 mg/ml. frap vrednost etarskog ulja iznosi 40,63 μmol fe2+/g etarskog ulja. ulje pokazuje najbolje antimikrobno dejstvo na staphylococcus aureus. takođe, postoji značajna antimikrobna aktivnost na sve ispitivane mikroorganizme. key words: anethum graveolens l., seme mirođije, etarsko ulje, antioksidativna aktivnost, antimikrobna aktivnost 7 (1) • september 2016: 31-39 doi: 10.5281/zenodo.159101 biologica nyssana 7 (1)  september 2016: 31-39 stanojević, lj.p. et al.  dill (anetum graveolens l.) seeds essential oil… 32 introduction there are an increasing number of scientific investigations to find natural products that exhibit different biological activities, and antioxidant, antimicrobial and anti-inflammatory activities are the most commonly studied (t e p e et al., 2004; b a k k a l i et al., 2008; m i š i ć et al., 2008). one of the most important trends in food industry is discovery of natural antioxidants from plant material (d e l a q u i s , 2002). one of the most efficient ways of lipid peroxidation inhibition is the addition of synthetic antioxidants to the oils and foods, such as ascorbyl palmitate (ap), tert-butyl-4hydroxyanisole (bha), tert-butyl-4-hydroxytoluene (bht), propyl gallate (pg), butyl gallate (bg), octyl gallate (og), dodecyl gallate (dg). but, synthetic antioxidants have some undesirable side effects wherefore natural antioxidants are increasingly used (m a e s t r i et al., 2006). plants have long been used for various infectious diseases treatment and some of these traditional medicines are still involved in the treatment of various diseases (m e n d o n ç a f i l h o , 2006). essential oils and herbal extracts, as sources of natural products, have become interesting in recent decades. they represent an alternative to synthetic antioxidants and antimicrobial agents in food industry (t e p e et al., 2004; h i n n e b u r g et al., 2006) as well as in pharmaceutical industry, alternative medicine and natural therapy (b u r t , 2004; t e p e et al., 2004; m i š i ć et al., 2008). anethum graveolens l., commonly known as dill, is an annual and sometimes biennial medicinal plant from the family apiaceae (umbelliferae). dill is one of the most significant spices in food industry (o r h a n et al., 2013; l e u n g & f o s t e r , 2003). dill is native plant to mediterranean region, southeastern europe and central southern asia (k a u r & a r o r a , 2010). dill herb and dill seeds have been used as flavoring agent in food industry for sauces, salads and seafood (p i n o et al., 1995; k a u r & a r o r a , 2010). the food industry often uses essential oil instead of dill leaves and seeds (p i n o et al., 1995) due to its characteristic aroma and flavor (j i r o v e t z et al., 2003). it has been reported that dill has antimicrobial, antihyperlipidemic, diuretic, hypotensive, antispasmodic, antiemetic, laxative effect (k o p p u l a & c h o i , 2011; h o s s e i n n z a d e h et al., 2002; t u c a k o v , 1997) and anticancer activity (p e e r a k a m et al., 2014). bioactive components of dill are: essential oil, fatty oil, proteins, carbohydrates, fiber, mineral elements (potassium, calcium, magnesium, phosphorous, sodium), vitamin a and niacin (k a u r & a r o r a , 2010). essential oil is present in all parts of plant, but its content is the highest in the seeds (2-5%) (l e u n g & f o s t e r , 2003). the major component in dill seeds essential oil is carvone (20-60%) (l e u n g & f o s t e r , 2003; r a d u l e s c u et al., 2010, d e l a q u i s et al., 2002). besides carvone, there are also present: limonene, phelandrene, -pinene, -terpinene, apiole, dill apiole, 1,8-cineole, dihydro carvone and p-cymene (l e u n g & f o s t e r , 2003; p i n o et al., 1995). in their study, s t a n o j e v i ć et al. (2015) and coworkers have found a high content of carvone (about 90%) in dill seeds essential oil from the territory of southeast serbia. since dill seeds are one of the most commonly used spices in serbian traditional cuisine and food industry, and, at the same time, dill is a plant with many medicinal properties, the aim of this study was to investigate antioxidant activity of essential oil from dill seeds by two antioxidant assay: dpph and frap as well as antimicrobial activity against some intestinal pathogens. material and methods plant material the commercial sample of non-disintegrated dill seeds (anethi fructus) was purchased („planta mell“, svrljig, southeast serbia) and used for investigations. chemicals and reagents ethanol, 96% (centrochem, zemun, serbia), 2,4,6tris (2-pyridyl)-1,3,5-triazine (tptz reagent), 1,1diphenyl-2-picrylhydrazyl (dpph radical), butylated hydroxy toluene (bht), iron (iii) chloride hexahydrate, iron (ii) sulfate heptahydrate (sigma chemical company, st. louis, usa), dimethyl sulfoxide (dmso; bdh, milan, italy). all other chemicals were analytical-grade. isolation of essential oil essential oil from dill seeds was isolated by classic clevenger-type hydrodistillation (cohobation) according to ph. jug. v (2000). dill seeds (15 g) were immersed in 300 ml of water in round bottom flask, and the oil was isolated using a clevenger-type apparatus for 3 h. the obtained essential oil was dried over anhydrous sodium sulfate and used for analysis (s t a n o j e v i ć et al., 2015). antioxidant activity dpph assay antioxidant activity of essential oil was deterimined by the use dpph test (a q u i n o et al., 2002; c h o i et al., 2002; s a n c h e z -m o r e n o , 2002). biologica nyssana 7 (1)  september 2016: 31-39 stanojević, lj.p. et al.  dill (anetum graveolens l.) seeds essential oil… 33 the essential oil was dissolved in ethanol (96%) and a series of different concentration solutions were prepared (0.23 to 29 mg/ml). the ethanol solution of dpph radical (1 ml, 3×10-4 mol/l) was added to 2.5 ml of each essential oil solutions. absorbance of one sample was immediately measured at 517 nm, while the other samples were incubated at room temperature in the dark, for 20, 30, 45 and 60 minutes, and the absorbance was also measured at 517 nm (au). the absorbance at 517 nm was measured for pure ethanol solution of dpph radical prepared as described above – 1 ml of the dpph radical (3×10-4 mol/l) diluted with 2.5 ml of ethanol, ak), as well as for the essential oil before treatment with dpph radical (2.5 ml of essential oil diluted with 1 ml of ethanol, ab). free radical scavenging capacity was calculated by the following equation (s t a n o j e v i ć et al., 2015a): dpph radicals scavenging capacity (%) =          k bu a aa 100 100 (1) essential oil concentration needed for the neutralization of 50% of the initial dpph radical concentration is called ec50 value. this value was determined by interpolation from the linear regression analysis in the concentration range between 0.23 and 29 mg/ml of essential oil added to the reaction mixture. bht was used as the reference compound (ec50 = 0.021 mg/ml). frap assay the antioxidant activity of essential oil by frap assay is determined using benzie and strain method with some modifications (b e n z i e & s t r a i n , 1996). frap reagent was prepared from acetate buffer (300 mmol/l, ph = 3.6), tptz reagent (10 mmol/l in 40 mmol/l hcl) and fecl3×6 h2o (20 mmol/l) in 10:1:1 ratio. ethanol solution of essential oil (0.1 ml, concentration 9.25 mg/ml) and 3 ml of frap reagent were added in a test tube. absorbance was measured at 593 nm after 30 minutes of incubation at 37 °c against blank control. the calibration curve for frap values determination was obtained by measuring the absorbance of fe2+ (0.2 to 1 mmol/l fe2so4×7h2o) standard solution, which was treated in the same way as the essential oil samples. frap value was expressed as mol fe2+/g of the essential oil. antimicrobial activity microorganisms and substrates. six microorganisms were selected to determine the antimicrobial activity: staphylococcus aureus (atcc 25923), bacillus subtilis (atcc 6633), escherichia coli (atcc 25922), listeria monocytogenes (atcc 19166), salmonella enteritidis (atcc 13076) and candida albicans (atcc 10259). mediums used for the growth of the microorganisms: antibiotic agar no. 1 for microbiology (merck, darmstadt, germany) for bacteria and sabouraud dextrose agar (torlak, belgrade) for fungi. microorganisms are from the collection of the microbiological laboratory of the faculty of technology, leskovac. disc-diffusion method. the agar disc-diffusion method was used for testing antimicrobial activity of dill seeds essential oil (k i e h l b a u c h et al., 2000). the mediums were sterilized for 15 minutes in an autoclave at 121oc. the suspension was prepared with overnight culture and adjusted to 0.5 mcfarland standard. the inoculum of 0.1 ml of suspension was added to 10 ml of medium and poured into the petri dishes. for screening, sterilized filter paper disks (12.7 mm dia., schleicher & schuell) were placed on the surface of inoculated mediums and impregnated with 60 l of essential oil. plates were incubated for 24 hours at 37 °c for bacteria, and 48 hours at 25 °c for yeast. antimicrobial activity was expressed as the diameter of inhibition zones (mm) obtained by investigated sample. standardized discs of amoxicillin (30 μg/disc, hemofarm, a.d. vršac), cephalexin (30 μg/disc, panfarma, beograd), amracin (30 μg/disc, galenika, a.d. zemun) and nystatin (100 u/disc, bioanalyse) served as positive controls. all experiments were carried out in three replications. data were expressed as mean ± standard deviation. the obtained data were analyzed by microsoft excel 2007 and origin 7 trial. results and discussion essential oil composition the moisture content and initial oil content in dill seeds were 7.33% and 4.0 ml/100 g of dry plant material, respectively. the yield of essential oil was 2.80 ml/100 g dry plant material (s t a n o j e v i ć et al., 2015). in the essential oil twenty nine components have been identified (99.9% of all components). these results are presented in our previous studies where the influence of the technique on the yield, composition and kinetics of essential oil hydrodistillation from dill seeds have been investigated (s t a n o j e v i ć et al., 2015). it has been found by gc-ms analysis of essential oil that carvone has the highest content (85.9%). carvone content in dill essential oil is usually 20 to 60% (l e u n g & f o s t e r , 2003; d e biologica nyssana 7 (1)  september 2016: 31-39 stanojević, lj.p. et al.  dill (anetum graveolens l.) seeds essential oil… 34 c a r v a l h o et al., 2006). besides carvone, limonene (5.1%), cis-dihydrocarvone (3.0%), transdihydrocarvone (2.7%), cis-carveol (1.8%) and trans-carveol (1.4%), all other components of oil were identified in much lower concentrations (s t a n o j e v i ć et al., 2015). the higher content of carvone in oil from serbia compared to its content in oils from other areas (bulgaria, canada, india and romania) (d e l a q u i s et al., 2002; j i r o v e t z et al., 2003; s i n g h et al., 2005) is probably due to different climatic conditions, as well as genetic characteristics of seeds (s t a n o j e v i ć et al., 2015). antioxidant activity antioxidant activity of essential oil investigated by dpph test (ability of oil in different concentrations to scavenge free dpph radical) is shown on fig. 1 (results for not-incubated and 20, 30, 45 and 60 min incubated samples are represented). fig. 1. dpph radicals scavenging activity by dill (anethum graveolens l.) seeds essential oil it can be noticed that the degree of dpph neutralization depends on incubation time, for all investigated concentrations of oil. the highest degree of dpph radicals’ neutralization is for 60 minutes incubation, in concentration of 29 mg/ml (79.62%). ec50 values of essential oil are shown in tab. 1. non-incubated samples of essential oil have not achieved ec50 value in the investigated range of concentrations. the tested synthetic antioxidant has showed better antioxidant activity compared to the essential oil. ec50 values of essential oils are lower than the ec50 values of synthetic antioxidant, bht. these synthetic antioxidants are used in food industry despite their documented undesirable side effects (t e p e et al., 2005). based on these results (fig. 1 and tab. 2) it can be concluded that the incubation time has effect on dpph radicals’ neutralization. dill seeds essential oil from thailand has shown lower extent of dpph neutralization (ec50 = 128.49 mg/ml) than oil obtained in our investigation (n a n a s o m b a t & w i m u t t i g o s o l , 2011). table 1. ec50 values of dill (anethum graveolens l.) seeds essential oil incubation time, min ec50, mg/ml* 20 20.27 ± 0.811 30 18.20 ± 1.019 45 13.45 ± 0.807 60 12.20 ± 0.464 *all data represent the mean of tree replications ± standard deviation (mean  sd). removal of free radicals is very important in food and food products preservation (h i n n e b u r g et al., 2006; t e p e et al., 2004). essential oil of dill seeds can be an alternative to dill as spice in the form of powdered plant (whole or some parts). oil is also a potential source of natural antioxidants, as a possible alternative to synthetic antioxidants. but, for this potential application of dill oil it is necessary to perform in vivo tests, which will be the aim of our further investigation. essential oil of dill seeds has lower dpph neutralization activity compared to acetone extracts of dill seeds (s i n g h et al., 2005). better activity of extract compared to unstable oil is probably due to presence of nonvolatile phenol compounds. in addition, some of the compounds with a different polarity, which are present in very small amounts in the extract, are also able to contribute to better antioxidative activity of extract. some compounds can originate in extract during hydrolysis or other processes of decomposition. some chemical reactions initiated by heating can also drive up to activities changes of complex extract, composed of a number of compounds with different chemical and physical properties (s i n g h et al., 2005). s i n t i m et al. (2015) reported on the significant effect of the antioxidant capacity of the dill seed essential oil using the oxygen radical absorbance capacity (orac) method. in addition, it was found that dill essential oil from the aerial parts of the plant had antioxidant activity. kazemi reported that dill oil exhibited a high activity in each antioxidant system with a special attention for β-carotene bleaching test and reducing power (k a z e m i , 2015). frap value, as a measure of essential oil antioxidant activity, was 40.63 μmol fe2+/g of essential oil. l a d o et al. (2004) determined the antioxidant properties of commercially purchased essential oils using the frap assay (cumin, biologica nyssana 7 (1)  september 2016: 31-39 stanojević, lj.p. et al.  dill (anetum graveolens l.) seeds essential oil… 35 coriander, dill, chamomile, hyssop, lavender, parsley, rosemary, sage and yarrow). frap value of the investigated dill essential oil was 42.64 μmol/g. the essential oil, investigated in our work, was incubated for 30 min at 37 c with frap reagent, while l a d o et al. (2004) have been incubated the oil only for 5 minutes. due to the long incubation time our oil should express greater frap value. however, lower frap value of our essential oil is probably the result of different chemical composition compared to the oil used by lado and coworkers. different origin of plant material can be also the reason of such differences in obtained results. tab. 2 shows the frap values of dill essential oil as well as literature frap values of some volatile components of oil (carvone, limonene and linalool). table 2. frap value of dill (anethum graveolens l.) seeds essential oil as well as literature frap values of some volatile oil components essential oil or volatile component frap value, μmol fe2+/g essential oil dill seeds essential oil 40.63 ± 3.23* dill seeds essential oila 42.64a carvonea 15.77a linaloola 25.73a limonenea 37.44a a l a d o et al. (2004) *all data represent the mean of tree replications ± standard deviation (mean  sd). the dill essential oil has the highest frap value (tab. 2), but the individual components identified in the essential oil also have the reductive capacities (l a d o et al., 2004). frap value obtained in our study is most likely the result of reducing ability of components present in the oil, especially carvone and limonene, which are present in the highest content. such activity of oil is due to the presence of minor component too, such as dihydrocarvone, cisand trans-carveol and linalool. so, frap value of oil is a result of synergistic effect of all present components since reducing ability of oil is higher than reducing ability of individual components (oil shows the highest frap activity). a significant number of natural products investigations suggest that essential oils have antioxidant activity. it is believed that antioxidants are directly responsible for antimutagenic and anticarcinogenic activity due to their radical scavenging properties. essential oil with antioxidant activity could be beneficial for human health (b a k k a l i et al., 2008). antimicrobial activity antimicrobial activity of dill essential oil as well as activity of reference antibiotics are shown in tab. 3. essential oil of dill has effect on all tested microorganisms. the highest effect was observed on s. aureus. tested antibiotics showed much less activity on this bacterium. it has also a significant effect on b. subtilis and e. coli. staphylococcus aureus is a gram-positive bacterium that is, among many other harmful effects to humans, one of the most frequent mastitis agents in herds of dairy cows (s i n g & p r a k a s h , 2008; m i l a n o v et al., 2010). milk contaminated with s. aureus, as well as products from such milk can cause a variety of infections, by bacteria itself and by their enterotoxins (s a m a r ž i j a et al., 2007). the relatively high effect of dill essential oil upon these bacteria suggests the potential use of oil as a natural antimicrobial agent in milk products. however, in order to use the obtained oil in such way, detailed studies are necessary to establish its minimum inhibitory concentration as well as its acute toxicity in particular concentration which would be the goals of our further studies. it is significant that investigated essential oil has a higher effect on b. subtilis compared to widely used cephalexin and amoxicillin antibiotics. bacillus species most likely cause alimentary toxic infections in humans. toxic infections are consequence of various food products consumption, in which starch and proteins are dominating, such as rice, meat and meat products, desserts, and other canned food. they are very often present as contaminants in food of animal and vegetable origin since they can survive various physical and chemical conditions because of resistant spores. in addition to alimentary infection, bacillus causes a number of other diseases: septic meningitis, cellulitis, gangrene, and many eye infections (k o t i r o n t a et al., 2000). based on this results it can be concluded that dill seeds essential oil can be used for natural antimicrobial formulation production. essential oil from dill seeds shows higher antimicrobial activity on e. coli (zone diameter 38 mm) compared to cephalexin and amoxicillin antibiotics. e. coli is considered as a dominant bacterium species in the digestive tract. the presence of this bacterium in water and food is a reliable indicator of fecal contamination. this bacterium commonly contaminates meat and dairy products, as well as fruit and vegetables (m a r k o v et al., 2009). presence of enteropathogenic e. coli in the food products can cause vomiting and diarrhea in infants and young children (s i n g h & p r a k a s h , 2008). http://www.ncbi.nlm.nih.gov/pubmed/10742691 biologica nyssana 7 (1)  september 2016: 31-39 stanojević, lj.p. et al.  dill (anetum graveolens l.) seeds essential oil… 36 the good antimicrobial activity of essential oil against e. coli probably mainly originates from carvone, which is present in the amount of 86% in the oil. this result is in accordance with studies of naigre and coworkers who determined the effect of carvone on enterococcus faecium, escherichia coli and aspergillus niger (n a i g r e et al., 1996). salmonella are gram-negative bacteria from enterobacteriaceae family and they are the most common causes of food poisoning. salmonella are natural inhabitants of animals’ gastrointestinal tract; they are widespread in soil, water and plants. all representatives of salmonella genus are potential human pathogens. they cause three types of disease in humans enteric fever, sepsis and gastroenteritis. about 95% of salmonella are ingested through food and the most common sources of infection are milk and dairy products, eggs, meat and meat products (c o x , 2000; m a r k o v et al., 2009). there are scientific papers on antimicrobial activity of commercial dill seeds essential oil on salmonella typhimurium (d e l a q u i s et al., 2002). there is no data about the effect of dill seeds essential oil from the southeast serbia region on salmonella species. the investigated dill seeds oil had a significant effect against s. enteritidis which is in the range with the effect of the commercial antibiotics. l. monocytogenes is a pathogenic bacterium that leads to listeriosis disease after consumption of food. this is a particularly dangerous pathogen since it can survive at low temperatures (foods that are kept in the fridge). listeriosis is one of the most common diseases with a fatal outcome (30%) (m a r k o v et al., 2009). there are studies of essential oils effects on l. monocytogenes (d e l a q u i s et al., 2002). dill seeds essential oil has less effect on bacteria compared to fractions of oils that are rich in carvone and limonene (d e l a q u i s et al., 2002). carvone, as a major component of the oil isolated in our study exhibited antimicrobial activity against l. monocytogenes (d e c a r v a l h o et al., 2006), so it is probably the most responsible for the effect of oil on these bacteria. isolated essential oil showed antifungal activity against c. albicans, which is, again, most probably due to the high content of carvone in the oil. carvone and limonene are the main components of caraway essential oil which showed strong antifungal activity against c. albicans. limonene is a carvone precursor and it is mainly present in mentha species being toxic to most microorganisms. carvone is widely used in the manufacture of aromas and fragrances and it has antifungal activity (p i n a et al., 2012). commercial dill seeds essential oil, with content of limonene and carvone of 46.3% and 49.5% respectively, shows antimicrobial activity against pseudomonas fragi, escherichia coli, salmonella typhimurium, listeria monocytogenes, staphylococcus aureus and saccharomyces cerevisiae. the fractions of essential oils with high content of limonene (more than 90%), and fractions with high content of carvone (67-99%) have shown better activity against gram-positive and gramnegative bacteria. thereby, the fractions rich in carvone have showed weaker activity in some extents compared to fractions rich in limonene (d e l a q u i s et al., 2002). essential oils are complex mixtures and their biological properties are result of synergistic effects of all components or major compounds. in most cases, biological activity of main components only, like thymol, carvacrol, linalool, terpineol, eugenol, carvone, geraniol, citronellol, nerol, safrole, eucalyptol, limonene, cinnamaldehyde, were analyzed. generally, the main components of table 3. antimicrobial activity of dill (anethum graveolens l.) seeds essential oil microorganism essentail oil antibiotic cephalexin amracin amoxicillin nystatin inhibition zone diameter, mm escherichia coli 38.0 ± 1.52 26.0 ± 1.04 n.t. 28.0 ± 0.84 n.t. staphylococcus aureus 80.0 ± 2.16 26.0 ± 0.78 n.t. 27.0 ± 0.97 n.t. salmonela enteritidis 28.0 ± 0.97 30.0 ± 1.08 25.0 31.0 ± 0.90 n.t. listeria monocytogenes 20.0 ± 0.83 34.0 ± 0.88 n.t. 36.0 ± 0.72 n.t. bacillus subtilis 52.0 ± 1.38 48.0 ± 0.96 52.0 ± 0.99 37.0 ± 0.59 n.t. candida albicans 18.0 ± 0.57 n.t. n.t. n.t. 17.0 ± 0.32 n.t.-not treated; all data represent the mean of tree replications ± standard deviation (mean  sd). biologica nyssana 7 (1)  september 2016: 31-39 stanojević, lj.p. et al.  dill (anetum graveolens l.) seeds essential oil… 37 essential oils are the ones on which biophysical and biological properties of oils depend (b a k k a l i et al., 2008). antimicrobial activity of dill seeds essential oil in our study probably originates from carvone, having in mind literature data about antimicrobial effect of carvone on a large number of bacteria and fungi (s i n g h et al., 2005). there are also data of limonene antimicrobial activity, a component that is represented with about 5% in isolated oil in this study (d e l a q u i s et al., 2002; s i n g h et al., 2005). other components of the oil probably also contribute to this activity. the high content of carvone, monotherpenic ketone, with numerous biological properties, indicates the possible application of dill seeds essential oil for medical purposes beside food industry, as a bioactive product of natural origin. antimicrobial activity of dill seeds essential oil is significant for human and animal pathogens as well as for food protection (b a k k a l i et al., 2008). conclusion essential oils are the source of natural products with different pharmacological activities. these oils represent the complex mixture of number components what is the reason for difficult explanation of their pharmacological activities. the presented data on antimicrobial and antioxidant activities of dill seeds essential oil showed that the isolated oil (southeast serbia, svrljig), with a high content of carvone is a potential source of natural antioxidants and antimicrobial agents. the results indicate possible application of essential oils in food and pharmaceutical industry as a safer alternative to synthetic antioxidants and antimicrobial agents. bearing in mind the results obtained in this work, dill seeds essential oil should be considered for further investigation with practical applications in different food and pharmaceutical systems. acknowledgements. this research is a part of project tr34012 which is supported by ministry of education, science and technological development of the republic of serbia. references aquino, r., morelli, s., tomaino, a., pellegrino, m., saija, a., grumetto, l., puglia, c., ventura d., bonina, f., grumetto, l. 2002: antioxidant and photoprotective activity of a crude extract of culcitium reflexum h. b. k. leaves and their major flavonoids. journal of ethnopharmacology, 79: 183–191. bakkali, f., averbeck, s., averbeck, d., idaomar, m. 2008: biological effects of essential oils a review. food and chemical toxicology, 46 (2): 446–475. benzie, i.f.f., strain, j.j. 1996: the ferric reducing ability of plasma (frap) as a measure of ‘‘antioxidant power’’: the frap assay. analytical biochemistry, 239 (1): 70–76. burt, s. 2004: essential oils: their antibacterial properties and potential application in foods – a review. international journal of food microbiology, 94 (3): 223–253. choi, w.c., kim, c.s., hwang, s.s., choi, k.b., ahn, j.h., lee, y.m., park. h.s., kim, k.s., lee, y.m. 2002: antioxidant activity and free radical scavenging capacity between korean medicinal plants and flavonoids by assay-guided comparison. plant science, 163: 1161–1168. cox, j. 2000. salmonella. in: robinson, r.k., batt, c.a., patel, p.d. (eds.), encyclopedia of food microbiology: 1928–1937. academic press, london. de carvalho, c.c.c.r., da fonseca, m.m.r. 2006: carvone: why and how should one bother to produce this terpene. food chemistry, 95 (3): 413–422. delaquis, p.j., stanich, k., girard, b., mazza, g. 2002: antimicrobial activity of individual and mixed fractions of dill, cilantro, coriander and eucalyptus essential oils. international journal of food microbiology, 74 (1-2): 101–109. hinneburg, i., dorman, h.j.d., hiltunen, r. 2006: antioxidant activities of extracts from selected culinary herbs and spices. food chemistry, 97 (1): 122–129. hosseinnzadeh, h., karimi, g.r., ameri m. 2002: effects of anethum graveolens l. seed extrats on experimental gastric irritation models in mice. bmc pharmacology, 2 (21): 1471–2210. jirovetz, l., buchbauer, g., stoyanova, a.s., georgiev, e.v., damianova, s.t. 2003: composition, quality control, and antimicrobial ativity of the essential oil of long-time stored dill (anethum graveolens l.) seeds from bulgaria. journal of agricultural and food chemistry, 51 (13): 3854–3857. kaur, g.j., arora, d.s. 2010: bioactive potential of anethum graveolens, foeniculum vulgare and trachyspermum ammi belonging to the familiy umbelliferae current status. journal of medicinal plants research, 4 (2): 087–094. kazemi, m. 2015: phenolic profile, antioxidant capacity and anti-inflammatory activity of anethum graveolens l. essential oil. natural products research, 29 (6): 551–553. biologica nyssana 7 (1)  september 2016: 31-39 stanojević, lj.p. et al.  dill (anetum graveolens l.) seeds essential oil… 38 kiehlbauch, j.a., hannett, g.e., salfinger, m., archinal, w., monserrat, c., carlin, c. 2000: use of the national committee for clinical laboratory standards guidelines for disk diffusion susceptibility testing in new york state laboratories. journal of clinical microbiology, 38 (9): 3341–3348. koppula, s., choi, d.k. 2011: anethum graveolens linn (umbelliferae), extract attenuates stressinduced urinary biochemical changes and improves cognition in scopolamine induced amnesic rats. tropical journal of pharmaceutical research, 10 (1): 47–54. kotironta, a., lounatma, k., haapasolo, m. 2000: epidemology and pathogenesis of bacillus cereus infections. microbes and infection, 2 (2): 189– 198. lado, c., then, m., varga, i., szoke, e., szentmihalyi, k. 2004: antioxidant property of volatile oils determined by the ferric reducing ability. zeitschrift naturforschung c, 59 (5-6): 354–358. leung, a.y., foster, s. 2003: encyclopedia of common natural ingredients (used in food, drugs, and cosmetics), second edition, a john wiley & sons, inc., hoboken, new jersey. 649 p. lu, li.-c., chen, y.-w.c., chou, c.-c. 2003: antibacterial and dpph free radical-scavenging activities of the ethanol extract of propolis collected in taiwan. journal of food and drug analysis, 11 (4): 277–282. maestri, d.m., nepote, v., lamarque, a.l., zygadlo, j.a. 2006. natural products as antioxidants. in: imperato f. (ed.), phytochemistry: advances in research, research signopost: 105–135, kerala, india. markov, k., frece, j., čvek, d., delaš, f. 2009: listeria monocytogenes i drugi kontaminanti u svježem siru i vrhnju domaće proizvodnje s područja grada zagreba. mljekarstvo, 59 (3): 225– 231. mendonça-filho, r.r. 2006. bioactive phytocompounds: new approaches in the phytosciences, in: ahmad, i., aqil, f., owais m. (eds.), modern phytomedicine, turning medicinal plants into drugs: 1-24, wiley-vch verlag gmbh & co. kgaa, weinheim. milanov, d., lazić s.,vidić b., petronijević j., bugarski d., šugeljev z. 2010: slime production and biofilm forming ability by staphylococcus aureus bovine mastitis isolates. acta veterinaria (beograd), 60 (2-3): 217–226. mišić, d., žižović i., stamenić m., ašanin, r., ristić, m., petrović, s.d., skala, d. 2008: antimicrobial activity of celery fruit isolates and sfe proces modeling. biochemical engineering journal, 42 (2): 148–152. naigre, r., kalck, p., rogues, c., roux, i., michel, g. 1996 : comparison of antimicrobial properties of monoterpenes and their carbonylated products. planta medica, 62 (3): 275–277. nanasombat, s., wimuttigosol p. 2011: antimicrobial and antioxidant activity of spice essential oils. food science and biotechnology, 20 (1): 45–53. orhan, e.i., senol, f.z., ozturk, n., celik, s.a., pulur a., kan, y. 2013: phytochemical contents and enzime inhibitory and antioxidant properties of anethum graveolens l. (dill) samples cultivated under organic and conventional agricultural conditions. food and chemical toxicology, 59: 96–103. peerakam, n., wattanathorn, j., punjaisee, s., buamongkol, s., sirisa-ard , p. , chansakaow s. 2014: chemical profiling of essential oil composition and biological evaluation of anethum graveolens l. (seed) grown in thailand. journal of natural sciences research, 4 (16): 34– 41. pharmacopoeia jugoslavica v, 2000: savremena administracija, beograd (in serbian), vol. 1: 118. pina, e.s., coppede, j. da s., sartoratto, a., fachin, a.l., bertoni, b.w., suzelei de castro frança, s. de c., pereira, a.m. 2012: antimicrobial activity and chemical composition of essential oils from aloysia polystachya (griseb.) moldenke grown in brazil. journal of medicinal plants research, 6(41): 5412–5416. pino, j.a., rosado, a., goire, i., roncal, e. 1995: evaluation of flavor characteristic compounds in dill herb essential oil by sensory analysis and gas chromatography. journal of agricultural and food chemistry, 43 (5): 1307–1309. rădulescu, v., popescu, m.l. ilieş, d.-c. 2010: chemical composition of the volatile oil from different plant parts of anethum graveolens l. (umbelliferae) cultivated in romania. farmacia, 58 (5): 594–600. samaržija, d., damjanović s., pogačić t. 2007: staphylococcus aureus u siru. mljekarstvo, 57 (1): 31–48. sanchez-moreno, c. 2002: methods used to evaluate the free radical scavenging activity in foods and biological systems. food science and technology international, 8 (3), 121–137. singh, g., maurya s., de lampasona, m.p., catalan, c. 2005: chemical constituents, antimicrobial investigations, and antioxidative potentials of anethum graveolens l. essential oil and acetone extract: part 52. journal of food science, 70 (4): 208–215. http://www.google.rs/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&cad=rja&uact=8&ved=0ahukewipnuclxlpkahug3iwkhqababgqfggamaa&url=http%3a%2f%2fwww.tjpr.org%2f&usg=afqjcnhssdshe9uc_xlk0jlrebpodgyjea&bvm=bv.112064104,d.bgg http://www.google.rs/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&cad=rja&uact=8&ved=0ahukewipnuclxlpkahug3iwkhqababgqfggamaa&url=http%3a%2f%2fwww.tjpr.org%2f&usg=afqjcnhssdshe9uc_xlk0jlrebpodgyjea&bvm=bv.112064104,d.bgg http://www.ncbi.nlm.nih.gov/pubmed/10742691 http://www.ncbi.nlm.nih.gov/pubmed/10742691 biologica nyssana 7 (1)  september 2016: 31-39 stanojević, lj.p. et al.  dill (anetum graveolens l.) seeds essential oil… 39 singh, p., prakash, a. 2008: isolation of escherichia coli, staphylococcus aureus and listeria monocytogenes from milk products sold under market conditions at agra region. acta agriculturae slovenica, 92 (1): 83–88. sintim, h.y., burkhardt, a., gawde, a., cantrell, l.c., astatkie, t., obour, a.e., zheljazkov, v.d., schlegel, v. 2015: hydrodistillation time affects dill seed essential oil yield, composition, and bioactivity. industrial crops and products, 63: 190–196. stanojević, l.p., radulović, n.s., djokić, t.m., stanković, b.m., ilić, d.p., cakić, m.d., nikolić, v.d., 2015: the yield, composition and hydrodistillation kinetics of the essential oil of dill seeds (anethii fructus) obtained by different hydrodistillation techniques. industrial crops and products, 65: 429–436. stanojević, j.s., stanojević, lj.p., cvetković, d.j., danilović, b.r., 2015a: chemical composition, antioxidant and antimicrobial activity of the turmeric essential oil (curcuma longa l.). advanced technologies, 4 (2): 19–25. tepe, b., donmez, e., unlu m., candan, f., daferera, d., vardar-unlu, g., polissiou, m., sokmen, a. 2004: antimicrobial and antioxidative activities of the essential oils and methanol extracts of salvia cryptantha (montbret et aucher ex benth.) and salvia multicaulis (vahl). food chemistry, 84 (4): 519–525. tepe, b., sokmen, m., akpulat h.a., daferera, d., polissiou m., sokmen, a. 2005: antioxidative activity of the essential oils of thymus sipyleus subsp. sipyleus var. sipyleus and thymus sipyleus subsp. sipyleus var. rosulans. journal of food engineering, 66 (4): 447–454. tucakov, j. 1997: lečenje biljem, rad-beograd. beograd (in serbian). 717 p. anticancer compounds from medicinal plants biologica nyssana 4 (1-2)  december 2013: 19-33 jotić et al.  the vascular flora of the tepoš… 19 original article the vascular flora of the tepoš plateau around pirot city (eastern serbia) branko jotić, milica miljković, marija marković, bojan zlatković, vladimir ranđelović university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia * e-mail: marijam@pmf.ni.ac.rs abstract: jotić, b., miljković, m., marković, m., zlatković, b., ranđelović, v.: the vascular flora of the tepoš plateau around pirot city. biologica nyssana, 4 (1-2), december 2013: 19-33. the flora of the karst plateau tepoš, which is the part of vidlič mountain (eastern serbia) was studied. based on the analysis of the collected material and data from the literature, it was noted that the flora of tepoš plateau is represented by 486 species, distributed in 284 genera and 68 families of vascular plants. the biological spectra was shown. the hemicriptophytic-terophytic character was established by analyzing the presence of plant life forms. by phytogeographical analysis the presence of 25 area types was established. the euroasian florystic chorion with 258 species of eurasian area type is the most abundant, followed by mediterranean-submediterranean (58 species), central-european (47 species), cosmopolitan (27 species) and pontian area type (24 species). in the flora of tepoš plateau the 9 balkan endemic taxa were recorded, which is 3,14% of total balkan endemic taxa in flora of serbia. a small number of endemic taxa is expected, considering the low diversity of habitats. balkan endemic taxa recorded in this area are: acer hyrcanum subsp. intermedium, bupleurum flavicans, crucianella graeca, eryngium palmatum, pastinaca hirsuta, hypericum rumeliacum subsp. rumeliacum, thymus praecox subsp. jankae, linaria rubioides subsp. nissana and viola tricolor subsp. macedonica. key words: flora, pirot city, tepoš plateau introduction karst plateau tepoš (650-690m) is situated in eastern serbia, 14 km se of pirot city, above the krupac village (fig. 1). this is the part of mt. vidlič, westernmost part of the mountain system stara planina. the karst plateau is elongated in the southeastern-northwestern direction. in contrast to other regions of mountain system stara planina, mt. vidlič are represented by unique geological structure and climate conditions. the geological structure of the most part of mt. vidlič is represented by limestone, while other serbian parts of stara planina are built of silicate (v i d a n o v i ć , ed., 1960). the pedological substrate is consisted of reddish soil tiller and mountain soil. the climate is transitional humid continental, between dry climate of pirot region and wet climate on mt. stara planina (v i d a n o v i ć , ed., 1960). although the flora of this part of mt. stara planina was investigated by pančić (1884) and adamović (1911), it has not been well known. the tepoš plateau is not investigated up to now. material and methods the flora of tepoš plateau were studies in the period between 2009 and 2011. herbarium specimens are deposited at the herbarium moesiacum, university of niš (hmn). determination of plant material was carried out using the keys from the regional flora reference 11 th sfses • 13-16 june 2013, vlasina lake 4 (1-2) • december 2013: 19-33 biologica nyssana 4 (1-2)  december 2013: 19-33 jotić et al.  the vascular flora of the tepoš… 20 (j o s i f o v i ć , ed. 1970-1977; в е л ч е в , ed. 19821989). the nomanclature folows med-checklist (greuter et al., 1984-1989) and flora europaea (tutin et al., eds., 1964-1980). for the area types classification meusel et al. (1965, 1978), meusel and jäger (1992) and stevanović (1992) were used. life forms were determined according to mueller-dombois and ellenberg (1974) and s t e v a n o v i ć (1992a). results as a results of the floristic investigations of tepoš plateau in eastern serbia, 486 plant taxa belonging to 284 genera and 68 families were recorded (tab. 1). figure 1. geographical position of investigated area localities: l1 – road from village krupac to village visočki odorovci (500 – 750 m), l2 – ″kragujevac″ (810 m), l3 – ″kurjasi vrh″ (934 m), l4 – ″kočino″ (857 m), l5 – ″strči krak″ (861 m), l6 – ″begova rudina″ (651 m) table 1. list of vascular flora with life forms and area-types taxa life form area type locality polypodiopsida aspleniaceae asplenium ruta-muraria l semp ch herb caesp hol l5 asplenium trichomanes l. semp ch herb semiros csm l5 woodsiaceae cystopteris fragilis (l.) bernh. fo dec ch herb semiros csm l2 magnoliopsida acanthaceae acanthus balcanicus heywood & i.b.k.richardson a meg-alt h scap semiros eas l2 biologica nyssana 4 (1-2)  december 2013: 19-33 jotić et al.  the vascular flora of the tepoš… 21 taxa life form area type locality aceraceae acer campestre l. fo dec mes p scap eas l1 acer hyrcanum fischer & c. a. meyer subsp. intermedium (pančić) bornm. fo dec mes p scap me l1 acer monspessulanum l. fo dec mi-mes p scap med l1 acer platanoides l. fo dec mes p scap me l3 acer pseudoplatanus l. fo dec mes p scap me l3 acer tataricum l. fo dec mi-mes p scap eas l2 amaranthaceae amaranthus retroflexus l. a mes-alt t scap adv l1 anacardiaceae cotinus coggygria scop. fo dec mi p caesp eas l6 apiaceae aegopodium podagraria l. v-a mes-meg h scap/g rhiz eas l1 bifora radians m. bieb. a meg t scap eas l1 bupleurum commutatum boiss. & bal. a mac-meg t scap p l1 bupleurum flavicans boiss. & heldr. a mi-mes t scap med l6 bupleurum praealtum l. v-a mi-mes t scap med l1 carum rigidulum (viv.) koch ex dc. a mes h scap eamt l1 caucalis platycarpos l. v-a mes t scap eas l1 chaerophyllum temulentum l. a mes t scap/h bienn eas l1 daucus carota l. a meg t/h scap hol l1 eryngium campestre l. v-a mes-meg h semiros eas l6 eryngium palmatum pančić et vis. a mes-meg h scap semiros med l5 ferulago sylvatica (besser) reichenb. a meg-alt h scap eamt l2 orlaya grandiflora (l.) hoffm. a mes t scap eas l1 pastinaca hirsuta pančić a mes-meg h scap/t scap bienn eamt l1 peucedanum alsaticum l. a-aut mes-alt h scap eas l6 physocaulis nodosus (l.) koch a mes-meg t scap med l1 seseli peucedanoides (bieb.) kos. pol. a mes-meg h scap eas l2 smyrnium perfoliatum l. a mes-meg h scap med l2 tordylium maximum l. a meg t scap med l1 torilis leptophylla (l.) reichenb. a mes t scap eas l1 apocynaceae vinca herbacea waldst. et kit. v-a mi-mes ch suff rept p l6 araliaceae hedera helix l. aut fo semp alt s lig me l5 aristolochiaceae aristolochia pallida willd. v-a mes g rad med l1 asarum europaeum l. fo semp ch herb rept eas l5 asclepiadaceae vincetoxicum hirudinaria medicus a mes-alt h scap eas l2 asteraceae achillea clypeolata sibth. & sm. a mes-meg h scap med l2 achillea crithmifolia waldst. et kit. a mes-meg h scap msm l5 achillea millefolium l. a meg h scap hol l1 anthemis arvensis l. a mes-mac t scap med l1 arctium lappa l. a mes-alt h scap bienn eas l1 arctium tomentosum miller a-aut meg-alt h scap bienn eas l6 artemisia alba turra aut meg fo dec ch suffr caesp eamt l5 artemisia vulgaris l. aut meg-alt h scap hol l1 bellis perennis l. v-a mi-mes h ros eas l1 carduus acanthoides l. a mac h scap me l1 biologica nyssana 4 (1-2)  december 2013: 19-33 jotić et al.  the vascular flora of the tepoš… 22 taxa life form area type locality carduus candicans waldst. et kit. subsp. globifer (velen.) kazmi a mes-meg h scap msm l6 carduus nutans l. a mes-meg h scap me l1 carthamus lanatus l. a mes-mac t scap eas l6 centaurea biebersteinii dc. a mes-meg h scap eas l1 centaurea calcitrapa l. a mes-meg h caesp eas l1 centaurea phrygia l. a mes-meg h scap me l3 centaurea salonitana vis. a mac h scap med l2 centaurea scabiosa l. a meg h scap eas l2 centaurea solsticialis l. a alt t scap msm l1 chamomilla recutita (l.) rauschert a mes-meg t scap csm l1 chondrilla juncea l. a mes-meg t scap bienn med l6 cichorium intybus l. a mac h scap csm l1 cirsium eriophorum (l.) scop. a mes-alt t/h scap bienn eamt l1 cirsium vulgare (savi) ten. a mes-alt t/h scap bienn eas l1 conyza canadensis (l.) cronquist a-aut mes-alt t scap adv l1 crepis biennis l. v-a mac h scap bienn me l1 crepis foetida l. a mes-meg t ros eas l6 crepis sancta (l.) babcock v mi-mes t scap eas l6 doronicum columnae ten. v mes-meg h scap/g rhiz eamt l2 echinops ritro l. subsp. ruthenicus (bieb.) nyman a-aut mac-meg h scap eas l1 erigeron annuus (l.) pers. v-a mes-meg t scap adv l1 hieracium pilosella l. a mi-mes h ros eas l6 hieracium praealtum vill. ex gochnat subsp. bauhinii (besser) petunnikov a meg-alt h scap me l6 inula britannica l. a mes-meg h scap eas l1 inula conyza dc. a mes-meg h scap eas l1 inula oculus-christi l. a mes-meg h scap p l3 lapsana communis l. a meg-alt t scap eas l1 leontodon crispus l. a mi-mes h ros med l3 leontodon hispidus l. a mi-mac h ros eas l2 leucanthemum vulgare lam. a mes-meg h scap eas l2 logfia minima (sm.) dumort. a mi-mes t scap eas l6 onopordum acanthium l. a meg-alt h scap bienn eas l1 ptilostemon afer (jacq.) w. greuter a mac-meg h scap bienn med l1 pulicaria dysenterica (l.) bernh. a mes-mac h scap eas l1 scorzonera hispanica l. a mac-meg h scap p l2 senecio vernalis waldst. et kit. v mes t scap eas l1 sonchus asper (l.) hill a-aut meg-alt t scap bienn eas l1 tanacetum corymbosum (l.) schultz bip. a meg-alt h scap eas l5 taraxacum officinale weber v-aut mes h ros eas l1 tragopogon balcanus velen. a mes-meg h scap bienn med l1 tragopogon pratensis l. a mes-meg h scap eas l1 xanthium spinosum l. a mes-meg t scap adv l1 xanthium strumarium l. a meg-alt t scap csm l1 xeranthemum annuum l. a mes-meg t scap med l6 boraginaceae anchusa barrelieri (all.) vitman a mac-alt h scap bienn eas l6 anchusa officinalis l. a alt h scap bienn eas l1 buglossoides arvensis (l.) i. m. johnston v-a mes t scap eas l1 buglossoides purpurocaerulea (l.) i. m. johnston v-a mac h scap eas l1 cerinthe minor l. a mi-meg h scap bienn eas l1 echium italicum l. v-a mac-meg h scap bienn med l1 biologica nyssana 4 (1-2)  december 2013: 19-33 jotić et al.  the vascular flora of the tepoš… 23 taxa life form area type locality echium vulgare l. a mac h scap eas l1 heliotropium europaeum l. a mes-meg t scap eas l6 myosotis arvensis (l.) hill v-a mi-mes t scap hol l6 nonea pulla (l.) dc. v-a mes-mac h scap bien eas l3 pulmonaria officinalis l. v mes-mac h scap me l2 symphytum tuberosum l. v mes-mac h scap/g tub msm l2 brassicaceae alliaria petiolata (bieb.) cav. & grande v-a mes-meg t/h scap eas l1 alyssum alyssoides (l.) l. v mi-mes t scap eas l1 alyssum montanum l. v mi-mes fo dec ch suffr rept eas l1 alyssum repens baumg. v mi-mes fo dec ch suffr p l1 arabis hirsuta (l.) scop. a mes-meg h ros bienn csm l1 arabis recta vill. v meg h scap bienn eas l1 arabis sagittata (bertol.) dc. a mes-meg h ros bienn eas l1 arabis turrita l. v-a meg h scap semiros eas l1 berteroa incana (l.) dc. a mes-mac t/h scap eas l1 calepina irregularis (asso) thell. a mes-meg t scap eas l1 capsella bursa – pastoris (l.) medicus v-aut mi-meg t/h ros bienn csm l1 cardamine bulbifera (l.) crantz v-a mes-mac g rhiz eas l2 cardamine graeca l. v mes t scap ros med l1 cardamine hirsuta l. a mi-mes t scap ros eas l1 draba muralis l. v mi-meg t scap csm l1 erophila verna (l.) besser v mi-mes t ros eas l2 erysimum cuspidatum (bieb.) dc. a mac t/h ros bienn p l1 erysimum diffusum ehrh. a mes-meg t/h scap eas l1 lepidium campestre (l.) r. br. a meg t/h scap bienn eas l1 rorippa pyrenaica (lam.) reichenb. a mes-mac h ros eas l1 sinapis arvensis l. v-a mes-mac t scap eas l1 sisymbrium officinale (l.) scop. a mes-mec t scap eas l1 sysimbrium orientale l. a meg t scap bienn eas l1 syrenia cana (piller & mitterp.) neilr. a mes t scap ros/h scap ros p l1 thlaspi alliaceum l. v-a mes-meg t semiros med l1 thlaspi goesingense halascy a mes t scap eas l1 thlaspi kovatsii heuff. a mes-alt h scap p l1 thlaspi perfoliatum l. v mi-mes t scap semiros eas l1 campanulaceae asyneuma anthericoides (janka) bornm. a mes-meg h scap me l2 asyneuma canescens (w. et k.) gris. et sch. a mes-meg h scap eas l2 campanula bononiensis l. a mes-meg h scap eas l5 campanula cervicaria l. a mes-meg h scap eas l3 campanula patula l. subsp. abietina (griseb.) simonkai a mes-meg h scap me l3 campanula persicifolia l. a mes-meg h scap me l2 campanula rapunculus l. a mes-meg h scap eas l5 campanula sparsa friv. a mes-mac t scap me l5 caprifoliaceae lonicera xylosteum l. fo dec mi p caesp eas l2 sambucus ebulus l. a alt g rad/h scap eas l1 sambucus nigra l. fo dec mi p scap eas l1 viburnum lantana l. fo dec n-mi p caesp eas l1 caryophyllaceae agrostemma githago l. a meg t scap eas l6 arenaria serpyllifolia l. v-a mi-mes t scap eas l1 cerastium glomeratum thuill. v-a mi-mes t scap csm l1 biologica nyssana 4 (1-2)  december 2013: 19-33 jotić et al.  the vascular flora of the tepoš… 24 taxa life form area type locality cerastium pumilum curt. a mes h scap eas l1 dianthus diutinus kit. a mes-meg h scap msm l1 herniaria incana lam. a mi h caesp rept eas l6 holosteum umbellatum l. a mi t scap eas l1 lychnis coronaria (l.) desr. a mes-meg h scap eas l2 minuartia verna (l.) hiern. a mi-mes h caesp hol l1 moenchia mantica (l.) bartl. a mes t scap eas l5 petrorhagia prolifera (l.) p. w. ball & heywood a mes t scap eas l6 petrorhagia saxifraga (l.) link a mes-mac ch caesp eas l6 silene italica (l.) pers. a mes-meg h scap ros med l2 silene viridiflora l. a mes-meg h scap eas l2 silene vulgaris (moench.) garcke a mes-meg h scap/g rad eas l1 stellaria graminea l. a mes-mac h scap eas l2 stellaria holostea l. a mes-mac h scap eas l1 stellaria media (l.) vill. a mes-meg h scap rept csm l1 celastraceae evonymus europaeus l. fo dec n-mi p caesp eas l2 evonymus latifolius (l.) miller fo dec n-mi p caesp eas l2 chenopodiaceae atriplex patula l. a-aut mes-meg t scap hol l1 chenopodium album l. a mes-meg t scap csm l1 cistaceae fumana procumbens (dunal) gren. & gordon v-a mi-mes ch frut eas l1 helianthemum nummularium (l.) miller a mes-meg fo dec ch suffr caesp eas l3 helianthemum salicifolium (l.) miller v mi-mes t scap med l5 convolvulaceae convolvulus arvensis l. a mes-alt s herb eas l1 convolvulus canthabrica l. a mes-meg h scap eas l6 cornaceae cornus mas l. fo dec mi p caesp eas l2 cornus sanguinea l. fo dec mi-mes p caesp me l2 corylaceae carpinus betulus l. fo dec meg-alt p caesp scap eas l3 carpinus orientalis miller fo dec mi-mes p caesp med l2 corylus avellana l. fo dec meg-alt p caesp eas l1 corylus colurna l. fo dec mes p scap med l1 crassulaceae sedum acre l. a mi-mes ch herb scap succ me l1 sedum album l. a mi-mes ch herb scap succ med l1 sedum annuum l. a n-mi ch herb scap succ ab l1 sedum hispanicum l. a mi t/h succ eas l1 sedum ochroleucum chaix. a mes ch herb succ med l1 sedum sexangulare l. a mi-mes ch herb succ me l1 dipsacaceae dipsacus laciniatus l. a mes-alt t scap bienn eas l1 knautia integrifolia (l.) bertol a mes-meg t scap med l2 knautia macedonica griseb. a mes-meg h scap msm l2 scabiosa argentea l. a mes-meg h scap p l2 euphorbiaceae euphorbia amygdaloides l. a mes-mac t scap eas l2 euphorbia cyparissias l. v-a mes-mac h scap eas l1 euphorbia esula l. a meg-alt h scap me l1 euphorbia esula l. subsp. tommasiniana a mes-meg h scap eas l6 biologica nyssana 4 (1-2)  december 2013: 19-33 jotić et al.  the vascular flora of the tepoš… 25 taxa life form area type locality (bertol.) nyma euphorbia helioscopia l. a mi-meg t scap eas l1 euphorbia seguierana necker subsp. niciciana (borbás ex novák) rech. fil. a mes-meg h scap eas l1 fabaceae anthyllis vulneraria l. a mes-meg h scap eas l1 astragalus glycyphyllos l. a mes-meg h scap rept eas l2 astragalus onobrychis l. a mes fo ch suffrut rept eas l1 chamaecytisus glaber (l. fil.) rothm. v-a mes-meg fo dec ch frut caesp eamt l1 chamaecytisus rochelii (wierzb.) rothm. v-a mes-meg fo dec ch frut caesp msm l2 chamaespartium sagittale (l.) p. gibbs a mes fo dec ch suffrut caesp eas l1 colutea arborescens l. fo dec mi p caesp eas l2 coronilla scorpioides (l.) koch v mi-mes t scap med l2 coronilla varia l. a mes-meg h scap eas l2 cytisus procumbens (waldst. & kit. ex willd.) sprengel a mes fo dec ch suffrut caesp med l2 dorycnium pentaphyllum scop. subsp. germanicum (gremli) gams a mes fo dec ch suffrut caesp msm l2 genista tinctoria l. a mes-mac fo dec ch suffrut caesp eas l2 gleditsia triacanthos l. fo dec mes p scap adv l1 lathyrus aphaca l. a mes t/s herb eas l1 lathyrus niger (l.) bernh. a mes-meg h scap me l2 lathyrus nissolia l. a mes-meg t scap eas l1 lathyrus pratensis l. a mes-meg h scap eas l5 lathyrus tuberosus l. a meg h scap/g tub eas l1 lathyrus venetus (mill.) wohlf. a mes-meg h scap p l3 lotus corniculatus l. a mes h scap hol l6 medicago lupulina l. a mes t/h scap hol l6 medicago minima (l.) bartal v mi-mes t scap eas l6 medicago rigidula (l.) all. v mi-mes t scap eas l1 medicago sativa l. a mes-meg h scap adv l1 medicago sativa l. subsp. falcata (l.) arcangeli a mes-meg h scap eas l6 melilotus officinalis (l.) pallas a meg t/h scap bienn eas l1 onobrychis alba (waldst. et kit.) desv. a mes-meg h scap med l1 onоnis pusilla l. a-aut mi-mes fo dec ch suffrut caesp eas l1 ononis spinosa l. a mes-meg fo dec ch suffrut caesp eas l1 robinia pseudoacacia l. fo dec mes p scap adv l2 trifolium alpestre l. a mes-mac h scap eas l2 trifolium arvense l. a mes t/h scap bienn eas l3 trifolium badium schreber a mes-meg h scap eamt l3 trifolium campestre schreber a mes-mac t scap eas l2 trifolium incarnatum l. a mes-meg t scap eas l2 trifolium medium l. v-a mes-mac h scap eas l2 trifolium montanum l. a mes h scap eas l3 trifolium pratense l. a mes h scap eas l1 trifolium repens l. a mi h rept hol l1 trifolium scabrum l. a mi-mes t scap med l5 vicia cracca l. a meg-alt h/s scap herb eas l1 vicia grandiflora scop. v-a mes-mac h scap/sh herb eas l1 vicia hirsuta (l.) s. f. gray a mes-meg t/s scap herb eas l5 biologica nyssana 4 (1-2)  december 2013: 19-33 jotić et al.  the vascular flora of the tepoš… 26 taxa life form area type locality vicia lathyroides l. v-a mes-meg t scap eas l2 vicia pannonica crantz v-a mes t/s scap herb p l1 vicia sativa l. subsp. cordata wulfen ex hoppe v-a mes-meg t scap bienn eas l1 fagaceae fagus moesiaca (k. maly) czecz. fo dec meg-alt p scap me l2 quercus cerris l. fo dec mes p scap med l2 quercus petraea (mattuschka) liebl. fo dec meg-alt p scap me l2 quercus pubescens willd. fo dec mes p scap med l2 fumariaceae corydalis solida l. v mes g tub me l2 gentianaceae gentiana cruciata l. a mes-meg g rad scap eas l3 geraniaceae erodium ciconium (l.) l’her. v-a mi-mes t scap msm l1 geranium dissectum l. a mi-meg t scap eas l1 geranium lucidum l. a mi-meg t scap eas l1 geranium molle l. v mes t scap eas l1 geranium purpureum vill. a mi-meg t scap med l5 geranium pyrenaicum burm. fil. a mes-meg h scap eas l2 geranium sanguineum l. v-a mes h semiros eas l1 hypericaceae hypericum hirsutum l. a mes-meg h scap eas l1 hypericum perforatum l. a mes-meg h scap eas l1 hypericum richeri vill. a mes-meg h scap eamt l2 hypericum rumelicum boiss. a mi-mes h scap med l1 lamiaceae acinos alpinus (l.) moench a mes t scap/g rhiz eas l1 acinos arvensis (lam.) dandy a mi-mes t scap/g rhiz eas l6 ajuga chamaepytus (l.) shreber subsp. chia (schreber) arcangeli a mi-mes t scap med l5 ajuga genevensis l. v-a mes-mac h scap ros eas l1 ajuga laxmannii (l.) bentham v-a mes h scap/g rhiz p l2 ballota nigra l. a meg h scap eas l1 clinopodium vulgare l. a mes-meg h scap hol l1 glechoma hirsuta waldst. et kit. a mes h rept eas l3 lamiastrum galeobdolon (l.) ehrend. & polatschek v-a mes-mac h scap me l2 lamium amplexicaule l. v mi-mes t scap hol l1 lamium maculatum l. v mes-mac h scap eas l1 lamium purpureum l. v mi-mes t scap eas l1 marrubium peregrinum l. a mes-meg h scap eas l1 marrubium vulgare l. a meg h scap eas l2 mentha longifolia (l.) hudson a mes-meg h scap hol l1 nepeta nuda l. a mac-meg h scap eas l1 origanum vulgare l. a mes-meg h scap eas l2 prunella laciniata (l.) l. a mes-mac h scap eas l3 prunella vulgaris l. a mi-mes h scap semiros eas l1 salvia aethiopis l. a mes-mac h scap p l1 salvia austriaca jacq. a mes-mac h scap p l1 salvia nemorosa l. a mes-meg h scap eas l1 salvia pratensis l. a mes-meg h scap msm l2 salvia sclarea l. a mes-mac h scap med l1 salvia verticillata l. a mes-mac h scap eas l1 satureja montana l. subsp. kitaibelii (wierzb.) p.w. ball a mes ch suffrut caesp eamt l1 biologica nyssana 4 (1-2)  december 2013: 19-33 jotić et al.  the vascular flora of the tepoš… 27 taxa life form area type locality scutellaria columnae all. a mes-mac h scap med l1 scutellaria galericulata l. a mes-meg h scap bor l2 sideritis montana l. a mi-mes t scap eas l6 stachys germanica l. a meg h scap msm l6 stachys officinalis (l.) trevisan a mes-meg h scap eas l3 stachys recta l. a mes-meg h scap eas l6 stachys sylvatica l. a mes-meg h scap eas l5 teucrium chamaedrys l. a mes ch suffrut caesp eas l1 teucrium montanum l. a mes ch suffrut caesp eamt l1 thymus glabrescens willd. a mi-mac ch herb rept eas l1 thymus pannonicus all. a mi-mac ch rept me l1 thymus praecox opiz subsp. jankae (čelak.) jalas a mi ch herb rept pulv med l1 linaceae linum catharticum l. a mi-mes t scap eas l2 linum hologynum reichenb. a mes-mac h caesp med l2 malvaceae althaea officinalis l. a meg h scap bienn eas l5 hibiscus trionum l. a mes t scap bien hol l1 lavatera thuringiaca l. a meg-alt h scap eas l3 malva sylvestris l. v-a mes-alt h scap eas l1 oleaceae fraxinus excelsior l. fo dec mes p scap eas l3 fraxinus ornus l. fo dec mi-mes p scap eas l2 ligustrum vulgare l. fo dec mi p caesp eas l1 syringa vulgaris l. fo dec mi p caesp med l3 papaveraceae papaver dubium l. a meg t scap eas l1 papaver rhoeas l. a mes-meg t scap eas l1 plantaginaceae plantago argentea chaix a mi-meg h ros eamt l6 plantago lanceolata l. a mi-meg h ros eas l1 plantago major l. a mes-meg h ros csm l1 plantago media l. a mes-meg h ros eas l1 polygalaceae polygala comosa schkuhr a mi-mes h scap eas l1 polygonaceae rumex acetosa l. a meg h scap hol l1 rumex arifolius all. a meg h scap p l2 rumex conglomeratus murray a meg h scap csm l1 rumex sanguineus l. a meg h scap hol l1 primulaceae lysimachia nummularia l. a n-mes ch herb rept me l1 lysimachia vulgaris l. a mes-meg h scap eas l3 primula veris l. v mi-mes h ros eas l1 ranunculaceae adonis aestivalis l. a mes-mac t scap eas l1 adonis vernalis l. v mes-meg g rhiz p l1 anemone ranunculoides l. v mi-mes g rhiz me l5 clematis integrifolia l. v mes-meg g rhiz scap p l2 clematis vitalba l. a dec s lig eas l1 consolida regalis s. f. gray a mes-meg t scap eas l1 delphinium fissum waldst. et kit. a meg-alt g tub med l3 helleborus odorus waldts. & kit. v meg g rhiz me l1 isopyrum thalictroides l. v mes g rhiz eas l2 biologica nyssana 4 (1-2)  december 2013: 19-33 jotić et al.  the vascular flora of the tepoš… 28 taxa life form area type locality nigella arvensis l. a-aut mes t scap eas l1 ranunculus bulbosus l. a mes-meg h scap eas l2 ranunculus ficaria l. v mi-mes g tub me l1 ranunculus millefoliatus wahl. v-a mes h scap semiros med l2 thlalictrum minus l. a meg-alt h scap eas l5 resedaceae reseda lutea l. a mes-meg t/h scap bienn eas l1 rosaceae agrimonia eupatoria ledeb. a meg h scap eas l1 aremonia agrimonioides (l.) neck. v mi-mes h scap ros eamt l2 crataegus monogyna jacq. fo dec mi p caesp eas l1 cydonia oblonga mill. fo dec np caesp adv l1 filipendula vulgaris moench a mes-meg h scap eas l1 fragaria vesca l. a mes h rept csm l1 fragaria viridis duchesne a mes h rept eas l1 geum urbanum l. a mes-meg h scap hol l1 malus pumila miller fo dec mi-mes p scap p l1 potentilla argentea l. a mes-meg h scap me l1 potentilla cinerea chaix ex vill. v mi-mes h caesp rept eas l1 potentilla micrantha ramond ex d.c. v mi-mes h scap med l2 potentilla recta l. a mes-meg h scap eas l2 prunus avium l. fo dec mes p scap eas l2 prunus spinosa l. fo dec n p caesp eas l1 pyrus amygdaliformis vill. fo dec n p caesp med l1 pyrus pyraster burgsd. fo dec mes p scap eas l1 rosa canina l. fo dec n p caesp eas l1 rosa micrantha borrer ex sm. fo dec n p caesp eas l1 rubus caesius l. fo dec np rept eas l1 rubus canescens dc. fo dec np rept eas l1 sanguisorba minor scop. a mes-meg h scap eas l1 sorbus aria (l.) crantz fo dec mes p scap me l3 sorbus torminalis (l.) crantz fo dec mes p scap eas l2 rubiaceae asperula cynanchica l. v-a mes-mac h scap eas l1 asperula taurina l. v-a mes-mac h scap eas l5 crucianella angustifolia l. a mi-mes t scap med l1 crucianella graeca boiss. a mi-mes t scap med l1 cruciata glabra (l.) ehrend. v-a mi-mes h scap eas l1 cruciata laevipes opiz v-a mes-mac h scap eas l1 galium album miller a meg-alt h/s scap herb me l1 galium aparine l. v-a mes-meg t/s herb csm l1 galium sylvaticum l. a mes-mac h scap/g rhiz scap me l2 galium verticillatum danth. v-a m-mes t scap med l1 galium verum l. a mes-meg h scap eas l1 sherardia arvensis l. v-a mi-mes t scap med l1 rutaceae dictamnus albus l. v-a mes-alt ch suffrut caesp eas l2 salicaceae populus tremula l. fo dec mes p scap eas l1 santalaceae thesium alpinum l. a mes-mac h scap eamt l6 saxifragaceae saxifraga tridactylites l. v-a mi t ros me l1 scrophullariaceae biologica nyssana 4 (1-2)  december 2013: 19-33 jotić et al.  the vascular flora of the tepoš… 29 taxa life form area type locality digitalis grandiflora miller a mac-alt h scap eas l5 digitalis lanata ehrh. a mac-alt h scap me l6 euphrasia pectinata ten. a mi-mes t scap/ep semipar eas l1 euphrasia salisburgensis funck a mi-mes t scap/ep semipar eamt l1 kickxia elatine (l.) dumort. a mes t rept eas l1 lathraea squamarria l. a mes-meg g par eas l2 linaria rubioides vis. & pančić subsp. nissana (petrović) niketić & tomović a mes h scap med l5 linaria vulgaris l. a mes-mac h scap eas l1 melampyrum arvense l. a mes-mac t scap/ep semipar eas l2 melampyrum cristatum l. a mes-meg t scap/ep semipar eas l2 rhinanthus angustifolius c. c. gmelin a mi-mac t scap/ep semipar me l2 rhinanthus rumelicus velen. a mi-mac t scap/ep semipar eas l2 verbascum densiflorum bertol. a mac-alt t scap ros bienn eas l6 verbascum lychnitis l. a mac-alt t/h scap ros bienn eas l1 verbascum phlomoides l. v-a mes-alt h ros eas l6 verbascum phoeniceum l. v-a mac-meg h ros eas l6 veronica arvensis l. a mes-mac t scap eas l1 veronica austriaca l. a mes-mac h scap eas l2 veronica beccabunga l. a mes-mac h scap eas l1 veronica chamaedrys l. v-a mi-mes h scap eas l2 veronica hederifolia l. v mi-mes t scap eas l1 simaroubiaceae ailanthus altissima (miller) svingle fo dec mes p scap adv l6 solanaceae datura stramonium l. a-aut meg-alt t scap hol l1 hyascyamus niger l. v-a mes-meg t scap bienn eas l1 lycium barbarum l. fo dec np caesp med l1 solanum dulcamara l. a meg-alt s lig eas l1 solanum nigrum l. a-aut mes-meg t scap hol l1 staphyleaceae staphylea pinnata l. fo dec mi p caesp/mi p scap me l1 tiliaceae tilia tomentosa moench fo dec mes p scap med l3 urticaceae urtica dioica l. a mes-meg t/h scap hol l1 valerianaceae valeriana officinalis l. a meg-alt h scap eas l2 valerianella locusta (l.) laterrade v mes t scap csm l1 valerianella rimosa bast. v mes t scap eas l1 verbenaceae verbena officinalis l. a mes-meg h scap eas l1 violaceae viola alba besser v mi-mes h rept semiros eas l2 viola arvensis murray v-aut mi-mac t scap csm l1 viola jordanii hanry v mes h scap ros p l1 viola kitaibeliana schultes v n-mi t scap eas l6 viola odorata l. v mi-mes h rept ros eas l1 viola tricolor l. subsp. macedonica (boiss. & heldr.) a. schmidt v mi-mes t/h scap med l1 liliopsida araceae arum maculatum l. v mes-meg g rhiz me l6 biologica nyssana 4 (1-2)  december 2013: 19-33 jotić et al.  the vascular flora of the tepoš… 30 taxa life form area type locality cyperaceae carex caryophyllea latourr. a mes-meg h caesp eas l1 eleocharis palustris (l.) roem. & schult. a mes-meg emer hyd g rhiz csm l1 dioscoreaceae tamus communis l. v alt s/g herb rhiz med l3 iridaceae gladiolus illyricus w. d. j. koch a mes-meg g tub scap med l5 iris graminea l. v-a mes-mac g rhiz eas l1 juncaceae juncus effusus l. a mes-meg g rhiz caesp csm l1 luzula campestris (l.) dc. v mes-meg h caesp csm l1 liliaceae allium albidum fischer ex bieb. a mes g bulb p l6 allium flavum l. a mes-mac g bulb msm l1 allium scorodoprasum (l.) subsp. rotundum (l.) stearn a mes-meg g bulb eas l6 anthericum ramosum l. a mes-meg g rhiz eas l2 asparagus officinalis l. a meg-alt g rhiz p l6 asparagus tenuifolius lam. a meg-alt g rhiz eas l3 colchicum autumnale l. aut mes g bulb eas l6 erythronium dens – canis l. v mes g bulb eas l2 lilium martagon l. a meg-alt g bulb me l2 muscari comosum (l.) miller v mes g bulb eas l2 muscari neglectum guss. ex ten. v mes g bulb eas l1 muscari tenuiflorum tausch v mes g bulb p l1 ornithogalum ortophyllum ten. subsp. kochii (parl.) zahar. v mi-mes g bulb eas l1 ornithogalum pyramidale l. v mi-mes g bulb eas l1 polygonatum odoratum (miller) druce a mes-meg g rhiz eas l2 veratrum nigrum l. a meg-alt g rhiz eas l5 orchidaceae cephalanthera damasonium (miller) druce a mes-mac g tub scap eas l1 epipactis atrorubens (hoffm.) besser a mes g tub scap p l5 epipactis helleborine (l.) crantz a mes g tub scap eas l2 epipactis microphylla (ehrh.) swartz a mes g rhiz scap p l2 himantoglossum hircinum (l.) sprengel v-a mac-meg g tub med l1 limodorum abortivum (l.) swartz a mes-meg g rhiz scap p l2 neottia nidus-avis (l.) l. c. m. richard a mes-mac sapr g rhiz eas l2 ophrys apifera hudson a mes g tub scap eas l6 ophrys cornuta steven a mes g tub scap eas l6 orchis morio l. v-a mes g tub scap eas l6 orchis purpurea hudson v-a mac-meg g tub med l6 platanthera chlorantha (custer) reichenb. a mes g tub scap eas l1 poaceae aegilops geniculata roth. a mi-mes t caesp med l1 alopecurus pratensis l. a mes-meg h caesp eas l1 antoxanthum odoratum l. a mes-meg h caesp eas l1 arrhenatherum elatius (l.) beauv. ex j. & c. presl a mes-alt h caesp eas l1 avena versicolor vill. a meg h scap eamt l6 brachypodium pinnatum (l.) beauv. a mac-alt h caesp eas l1 brachypodium sylvaticum (hudson) beauv. a mac-alt h caesp eas l2 briza media l. a mes-meg h caesp me l1 bromus racemosus l. a mes-meg t caesp me l6 bromus sterilis l. a mes-meg t caesp eas l6 biologica nyssana 4 (1-2)  december 2013: 19-33 jotić et al.  the vascular flora of the tepoš… 31 taxa life form area type locality chrysopogon gryllus (l.) trin. a alt h caesp eas l1 dactylis glomerata l. a meg h caesp eas l1 danthonia alpina vest a mes-meg h caesp eamt l6 dasypyrum villosum r. br. ex fresen. a mes-meg t caesp med l1 dichanthium ischaemum (l.)roberty a mes-meg h caesp eas l6 elymus repens (l.) gould a mes-meg g rhiz caesp csm l1 festuca valesiaca schleicher ex gaudin a mes-mac h caesp eas l6 holcus lanatus l. a mes-mac h caesp eas l6 hordeum murinum l. subsp. leporinum (link) arcangeli a mes t caesp med l1 koeleria glaucovirens domin a mes-meg h caesp med l6 koeleria nitidula velen. a mes-meg h caesp med l6 melica ciliata l. a mes-meg h caesp eas l6 melica transsilvanica schur a mes-meg h caesp p l6 melica uniflora retz. a mes-mac h caesp me l2 phleum pratense l. a mes-alt h caesp me l1 poa annua l. a-aut mi-mes t/h caesp csm l1 poa bulbosa l. a mes-meg h caesp eas l1 poa compressa l a mes-mac h caesp csm l1 poa nemoralis l. a mes-meg h caesp hol l2 poa pratensis l. a mes-meg h scap eas l1 setaria italica (l.) beauv. a mes-meg t scap hol l1 setaria pumila (poiret) roem. & schultes a mes-meg t caesp csm l1 setaria viridis (l.) beauv. a mes-mac h caesp eas l1 stipa capillata l. a mac-meg h caesp eas l1 trisetum flavescens (l.) beauv. a mes-meg h caesp eamt l1 potamogetonaceae potamogeton obtusifolius mert. & koch a rad sbm hyd t hol l1 typhaceae typha latifolia l. a alt emer hyd g rhiz csm disscusion taxonomic analysis divisio pteridophyta was represented by single class polypodiopsida with only 3 species. divisio magnoliophyta was represented by 481 species, from which 408 are classified as dicotyledons (magnoliopsida) and 73 as monocotyledons (liliopsida). in taxonomic spectrum of the flora of investigated area the most numerous in genera were asteraceae (34), poaceae (24), lamiaceae (19), fabaceae (18), apiaceae (17), brassicaceae (16). families with the most number of species were asteraceae (55), fabaceae (46), lamiaceae (38) and poaceae (35). the analysis of the particular genera occurrence in flora of the tepoš plateau has shown domination of representatives of genera trifolium (10), following by the genuses acer, campanula, centaurea, euphorbia, geranium, lathyrus, salvia, sedum, vicia and viola with 6 species in each genus. phytogeographical analisys for the phytogeographical analysis, all plant taxa were classified into 25 area types (fig. 3) according to the classification by s t e v a n o v i ć (1992). eurasian area type (eas) was the most dominant in the flora of tepoš plateau with 258 taxa (56%). the second one was mediterraneansubmediterranean area type (med) with 58 species (12 %). there were 9 balcan endemic taxa in the flora of the tepoš plateau. these represents 3,14% of the total number of balkan endemics in serbian flora, which includes 287 taxa in rank of species and subspecies (s t e v a n o v i ć e t a l , 1999). small number of endemics are expected due to uniformity of habitats in the wider area of tepoš plateau. the recorded balkan endemics are: acanthus balcanicus, acer hyrcanum subsp. intermedium, bupleurum flavicans, eryngium palmatum, pastinaca hirsuta, hypericum rumeliacum, thymus biologica nyssana 4 (1-2)  december 2013: 19-33 jotić et al.  the vascular flora of the tepoš… 32 praecox subsp. jankae, linaria rubioides subsp. nissana, viola tricolor subsp. macedonica. me 10% med 12% p 5% hol 5% csm 6% adv 2% eam 3% eas 57% msm 4% ab 0% figure 2. area spectrum of the flora of vučje hill: csm – cosmopolitan, hol – holarctic, ab – arctoalpian + boreal, me – middle-european, eas – euroasian, eamt – euroasian mountain, p – pontic, msm – merridional-submerridional, med – mediterranean-submediterranean, adv adventitious biological spectrum the analysis of plant life forms (fig. 2) showed that flora of investigated area was of hemicryptophyta type, as well as the flora of serbia (diklić, 1984) and balkan penninsula (turill, 1929). the hemicryptophytes were represented with 45%; therophytes followed with 27% and geophytes with 11%. the climatics characteristics of this area caused the high percentage of hemicryptophyta. h 45% g 11% hyd 1% t 27% p 9% ch 7% figure 3. biological spectrum of the flora of tepoš plateau: p – phanerophyta, ch – chamaephyta, h – hemycryptophyta, g – geophyta, t – terophyta, hydhydrophyta conclusion as a results of the floristic investigations of the tepoš plateau between 2009 and 2011, 486 plant taxa belonging to 284 genera and 68 plant families were recorded. the flora of tepoš plateau has similar taxonomic characters as the serbian and european flora. phytegeographical analysis showed that the species of eurasian area type are the most abundant ones. there were 9 endemic taxa in the flora of investigated area. the hemicriptophytic character of flora with significant contribution of therophytes and geophytes, is established by analyzing the presence of plant life forms. although the flora of this region is studied in detail, the expected number of taxons of karst plateau tepoš must be higher, presenting a base for a future studies in this area. acknowledgements. the authors are grateful to ministry of education and science of republic of serbia for partial support (projects number 173030 and 171025). references adamović, l., 1911: flora jugoistočne srbije. jazu. zagreb. diklić, n., 1984: životne forme biqnih vrsta i biološki spektar flore sr srbije. in: sarić, m., ed: vegetacija sr srbije, i: 291-316. sanu, posebna izdanja. beograd. greuter, w., burdet, h.m., long, g., eds., 19841989: med-checklist, 1,3,4. gèneve. josifović, m., ed.,1970-1976: flora sr srbije, i-ix. sanu. beograd. meussel, h., jäger, e., weinert, e., 1965: vergleinchende chorologie der zentraleuropaischen flora. veb. gustav fischer verlag, 1. jena. meussel, h., jäger, e., raischert, s., weinert, e., 1978: vergleinchende chorologie der zentraleuropaischen flora. veb. gustav fischer verlag, 2. jena. meussel, h., jäger, e., 1992: vergleinchende chorologie der zentraleuropaischen flora. veb. gustav fischer verlag, 3. jena. mueller – dombois, d., ellenberg, h. (1974.): aims and methods of vegetation ecology. john wiley and sons. new york. pančić, j., 1884.: dodatak flori kneževine srbije. kraljevska srpska državna štamparija, beograd. stevanović, v. ed., 1999: crvena knjiga flore srbije. 1, iščezli i krajnje ugroženi taksoni. ministarstvo za životnu sredinu republike srbije, zavod za zaštitu prirode srbije. beograd. stevanović, v., 1992: floristička podela teritorije srbije sa pregledom viših horiona i odgovarajućih flornih elemenata. in sarić, m. biologica nyssana 4 (1-2)  december 2013: 19-33 jotić et al.  the vascular flora of the tepoš… 33 (ed.): flora srbije, i. (drugo izdanje). sanu. beograd. 49 – 65. stevanović, v., 1992a: klasifikacija životnih formi flore srbije. in sarić, m. (ed.): flora srbije i. (drugo izdanje). sanu. beograd. 39 – 46. turill, w.b., 1929: the plant life of balkan peninsula. a phytogeographical study. oxford. tutin, t.g., heywood, v.h., burges, n.a., moore, d.m., valentine, d.h., walters, s.m., webb, d.a., eds., 1964-1980: flora europaea, i-v. cambrige university press. london. велчев в., ед., 1982-1989: флора на народна република българия, т. i-x, българската академия на науките, софия. vidanović, g., 1960: vidlič – zabrđe, prilog poznavanju privrednog tipa, ekonomsko – geografska studija, srpska akademija nauka, posebna izdanja geografskog instituta, knjiga 15, beograd. the ecological and floristic characteristics of natural population of micromeria juliana (l.) benth. ex rchb. in bulgaria biologica nyssana 8 (1)  september 2017: 99-103 stojanović, g. et al.  first insight into chemical composition of… 99 original article received: 23 mart 2017 revised: 25 may 2017 accepted: 25 july 2017 first insight into the chemical composition of essential oils and head space volatiles obtained from fresh leaves and flowers of peucedanum longifolium waldst. & kit. gordana stojanović1*, olga jovanović1, bojan zlatković2, snežana jovanović1, ivana zrnzević1, novica ristić3 1university of niš, faculty of science and mathematics, department of chemistry, višegradska 33, niš, serbia 2university of niš, faculty of science and mathematics, department of biology and ecology, višegradska 33, niš, serbia 3university of priština, temporarily settled in kosovska mitrovica, faculty of natural science and mathematics, lole ribara 29, kosovska mitrovica, serbia * e-mail: stgocaus@yahoo.com abstract: stojanović, g., jovanović, o., zlatković, b., jovanović, s., zrnzević, i., ristić, n.: first insight into the chemical composition of essential oils and head space volatiles obtained from fresh leaves and flowers of peucedanum longifolium waldst. & kit.. biologica nyssana, 8 (1), september 2017: 99-103. for the first time, chemical composition of p. longifolium essential oils (eo) and head space (hs) volatiles obtained from the fresh leaves, harvested in different phases of the plant development, and inflorescences was compared. the major contributor of leaves essential oil in vegetative phase (ld) was β-elemene (44.1%). on the contrary, β-elemene (22.5%) was the second most abundant component, while (e)-β-ocimene (26.7%) was the first and cis-lanalool oxide (furanoid, 21.9%) was the third most represented compound in the leaves essential oils collected in blossoming phase (lf). (e)-β-ocimene was the major compound in both hs leaf samples but in very varying amounts (ld 81.8% and lf 27.4%). beside (e)-β-ocimene, p-cymene and limonene were represented more than 10% (13.6% and 15.2% respectively). the essential oil and hs volatiles of the inflorescences was characterized by a high content of β-phellandrene (16.4% and 20.4% respectively), α-phellandrene (22.5% and 13.9% respectively) and myrcene (23.1% and 22.1% respectively). the presented data showed that there was a significant difference in the composition of volatile components from different plant organs and also from the same organ in different stages of development. key words: peucedanum longifolium, essential oil composition, head space volatiles apstrakt: stojanović, g., jovanović, o., zlatković, b., jovanović, s., zrnzević, i., ristić, n.: prvi uvid u hemijski sastav etarskog ulja i "head space" (hs) isparljivih komponenti svežih listova u različitim fazama razvoja biljke i svežih cvetova peucedanum longifolium waldst. & kit.. biologica nyssana, 8 (1), septembar 2017: 99-103. po prvi put je određen i upoređen hemijski sastav etarskog ulja (eo) i "head space" (hs) isparljivih komponenti p. longifolium dobijenih iz svežih listova u različitim fazama razvoja biljke i svežih cvetova. najzastupljenija 8 (1) • september 2017: 99-103 doi: 10.5281/zenodo.964345 biologica nyssana 8 (1)  september 2017: 99-103 stojanović, g. et al.  first insight into chemical composition of… 100 komponenta etarskog ulja listova biljke u vegetativnoh fazi (ld) je bio β-elemen (44.1%). u etarskom ulju listova biljke u fazi cvetanja (lf) redosled zastupljenosti je bio: (e)-β-ocimen (26.7%), β-elemene (22.5%) i cis-lanalol-oksid (furanoid, 21.9%). (e)-β-ocimen je bio najzastupljeniji medju "head space" (hs) isparljivim komponentama oba hs uzorka listova (ld 81.8% i lf 27.4%). pored (e)-β-ocimena, p-cimene i limonene su bili zastupljeni preko 10% (13.6% i 15.2% respektivno). etarsko ulje i hs isparljive komponente cvetova je karakterisao visok sadržaj β-felandrena (16.4% i 20.4%, respektivno), α-felandrena (22.5% i 13.9%, respektivno) i mircena (23.1% and 22.1%, respektivno). prezentovani rezultati pokazuju da postoji razlika u sastavu isparljivih komponenti listova i cvetova p. longifolium kao i listova biljke u fazi razvića i cvetanja. ključne reči: peucedanum longifolium, sastav etarskog ulja, sastav "head space" isparljive komponente introduction about 120 peucedanum l. species are widespread in europe, asia and africa (s p a l i k et al., 2004). of this number, 29 species and subspecies grow in the territory of europe, of which 14 in serbia (n i k o l i ć , 1973). some of the members, e. g. p. longifolium waldst. & kit., p. cervaria (l.) lapeyr., p. officinale l., p. oreoselinum (l.) moench and p. arenarium waldst. & kit. are indicated in cardiovascular, respiratory, genitourinary and nervous system disorders, as used in traditional medicine of some balkan countries (p e t k o v & m a n o l o v , 1978; s a r i ć , 1989; м и т р е в , 1995). peucedanum longifolium (sect. peucedanum) is glabrous perennial plant with solid (60-200 cm high), branched steams and small, yellow flowers arranged in several compound inflorescences. the species inhabits dry grasslands and rocky, usually calcareous slopes of medium altitudes in the c, s & e parts of balkan peninsula, extending to the mountains of c romania (s p a l i k et al., 2004). however, this species is mainly reported for limestone habitats (n i k o l i ć , 1973), while we have surprisingly found it growing over silicate bedrock. previous phytochemical studies found coumarins and flavonoids in the roots, steams, flowers and fruits of p. longifolium (k u z m a n o v et al., 1980). evaluation of antioxidant and antimicrobial activity of essential oils (t e p e et al., 2011; i l i ć et al., 2015) and extracts (m a t e j i ć et al., 2013) was also reported. to the best of the authors’ knowledge, there are only few papers (t e p e et al., 2011; i l i ć et al., 2015; k a p e t a n o s et al., 2008) referring to the chemical composition of essential oil of air-dried aerial parts of p. longifolium in flowering phase and one report related to the volatiles of fresh aerial parts from initial stage of the plant development (j o v a n o v i ć et al., 2015). recently, chemical composition of the essential oil and head space volatiles obtained from the fresh root of p. longifolium was published (s t o j a n o v i ć et al., 2017). the present study reports, for the first time, chemical composition of the essential oil (eo) and head space (hs) volatiles obtained from the fresh leaves and flowers of the p. longifolium growing in unusual natural environments i.e. on silicious substrate. material and methods plant material plant material was collected from thermophilous grasslands at siliceous, rocky grounds of mt. stara planina. all specimens were collected from the same population, habitat and locality (topli do, e serbia, 43°17’59” n and 22°36’42” e). voucher specimens were deposited in the “herbarium moesiacum niš” (hmn), department of biology and ecology, faculty of science and mathematics, university of niš under the acquisition numbers 11780 (vegetative stage; 05.06.2015.) and 11781 (flowering stage; 05.07.2015.). isolation and analysis of volatiles isolation and analysis of volatiles was done as previously described (j o v a n o v i ć et al., 2015). the light yellow oil, which has pleasant odor, was obtained in a yield of 0.09%, 0.07%, and 0.38% from fresh leaves in the developing and flowering phase and from the fresh flowers (respectively). results and discussion oil yield of both leaf samples (0.09% ld and 0.07% lf) was many times lower than the oil yield obtained from the flower (0.38%). a total of 42 compounds, whose representation in at least one sample was greater than or equal 0.1, were identified in six examined samples: essential oils (eo) and head space (hs) volatiles of leaves in vegetative and flowering phase (ld and lf, respectively) as well as inflorescence samples (f) (tab. 1). monoterpenes were predominant in all samples except in sample eo ld which was dominated by sesquiterpene -elemene (44.1%). twenty compounds were identified in the hs ld, biologica nyssana 8 (1)  september 2017: 99-103 stojanović, g. et al.  first insight into chemical composition of… 101 accounting 98.9% of the hs volaties, of which almost four fifth was (e)--ocimene (81.8%). its presence was reduced to 27.4% in the hs lf sample (fig. 1). opposite to that, percentage representation of pinene (1.3% ld, 4.6% lf), sabinene (0.5% ld, 6.0% lf), -elemene (2.2% ld, 3.7% lf), β-pinene (0.6% ld, 8.0% lf), myrcene (1.0% ld, 5.9% l), p-cymene (0.6% ld, 13.6% l) and limonene (0.3% ld, 15.2% l) was increased in lf sample. regarding ld oil, it was characterized by elemene (44.1%) whose participation was approximately twice decreases in the lf sample (22.5%). in contrast to -elemene presence of the following components was greatly increased in the lf sample: (e)--ocimene (8.5% ld, 26.7% lf), cis-linalool oxide (8.2% ld, 21.9% lf), and translinalool oxide (4.5% ld, 11.2% lf). comparing mutual composition of hs and eo, it is evident that the cis-linalool oxide and translinalool oxide were not detected, even as a trace, in samples of hs. further, presence of -elemene was many times more in eo samples than in corresponding hs samples. it can be assumed that the oxides are formed from the acyclic monoterpenes during hydrodistillation (s c h o f i e l d et al., 2002) while lower temperature of preparation of hs samples in relation to the essential oils obtaining (80 °c vs. 100 °c) could be the reason for a lesser presence of -elemene in hs samples. composition of hs f and eo f was different from the corresponding leaves samples, particularly regarding to the -phellandrene. this p-menthane monoterpene was represented with 20.4% and 16.4% in hs f and eo f samples, respectively, while it was not detected in leaf samples at all. -phellandrene was identified in the both flower and leaf samples, but in different quantities (1.2% hs lf, 0.3 % eo lf, 13.9% hs f and 22.5% e of). myrcene was the most abundant compound in the hs f and eo f. comparing mutual composition hs f and eo f it could be noticed a difference in the composition of -pinene (3.8% and 1.8%, respectively) and -pinene (10.9% and 1.4%, respectively) as well as -elemene (0.8% and 5.0%, respectively). previous studies have been done with the entire dry above-ground part on the flowering stage (k a p e t a n o s et al., 2008; t e p e et al., 2011; i l i ć et al., 2015) or fresh above-ground part in the developing stage (j o v a n o v i ć et al., 2015). 8cedren-13-ol (33.7%), myrcene (15.9%), α-pinene (36.3%) and β-elemene (24.9%) (k a p e t a n o s et al., 2008; t e p e et al., 2011; i l i ć et al., 2015; j o v a n o v i ć et al., 2015), respectively, published as the most represented components of essential oils. oil yield of both leaf samples was approximate each other (0.09% ld and 0.07% lf) many times lower than the oil yield obtained from the flower (0.38%). chemical composition of the eo and hs volatiles obtained from the fresh root of p. longifolium growing on the same substrate as here examined sample (s t o j a n o v i ć et al., 2017) was totally different relative to composition of leaves and flowers. namely, α-pinene was the most abundant compound in root eo (60.3%) and root hs volatiles (76.3%) while β-elemene (eo 0.1%; hs bellow fig. 1. variation of the percentage content of the p. longifolium essential oil (eo) and head space volatile (hs) components (ld-leaf, developing phase; lf-leaf, flowering phase; f-flower) biologica nyssana 8 (1)  september 2017: 99-103 stojanović, g. et al.  first insight into chemical composition of… 102 0.1%), (e)-β-ocimene (eo and hs 1.2%) and myrcene (eo 2.3%; hs 2.1%) were manifold less represented comparing to their representation in eo and hs of here examined leaves and flowers. conclusion the results of this and previous studies show that p. longifolium, like most other species, has a different table 1. chemical composition (%) of p. longifolium essential oils (eo) and head space volatile (hs) constituents ri ai compound content (%) ld lf f hs eo hs eo hs eo 1. 802 801 hexanal 0.4 0.5 2. 840 831 (e)-2-hexenal 0.4 3. 854 850 (z)-3-hexenol tr 2.3 6.7 4. 867 863 hexanol 0.9 0.2 tr 5. 929 924 -thujene tr 0.1 3.7 0.1 2.6 1.3 6. 936 932 -pinene 1.3 0.2 4.6 tr 3.8 1.8 7. 951 946 camphene 0.1 0.3 0.9 0.3 8. 975 969 sabinene 0.5 0.3 6.0 0.4 4.5 9. 979 974 -pinene 0.6 0.3 8.0 0.7 10.9 1.4 10. 992 988 myrcene 1.0 0.4 5.9 0.1 22.1 23.1 11. 993 991 trans-dehydroxy-linalool oxide 0.1 12. 1007 1002 -phellandrene 0.2 1.2 0.3 13.9 22.5 13. 1008 1006 cis-dehydroxy-linalool oxide 0.1 14. 1020 1014 -terpinene tr 0.2 tr 15. 1027 1020 p-cymene 0.6 1.8 13.6 0.7 0.5 16. 1031 1024 limonene 0.3 0.9 15.2 2.2 13.2 8.2 17. 1039 1025 -phellandrene 20.4 16.4 18. 1041 1032 (z)-β-ocimene 5.5 0.8 3.2 1.2 1.3 0.8 19. 1050 1044 (e)-β-ocimene 81.8 8.5 27.4 26.7 7.0 7.1 20. 1061 1054 γ-terpinene 0.2 0.5 2.0 2.7 1.4 1.6 21. 1075 1067 cis-linalool oxide (furanoid) 8.2 tr 21.9 tr 1.7 22. 1090 1084 trans-linalool oxide(furanoid) 4.5 11.2 1.1 23. 1091 1086 terpinolene tr 0.4 24. 1101 1095 linalool 0.2 tr 0.1 25. 1180 1174 terpinen-4-ol 0.1 tr 0.1 26. 1389 1389 iso-longifolene 2.4 27. 1397 1389 -elemene 2.2 44.1 3.7 22.5 0.8 5.0 28. 1426 1417 (e)-caryophyllene 0.2 1.5 tr 0.3 0.1 29. 1461 1452 -humulene 0.2 7.9 tr tr 30. 1481 1476 -chamigrene 0.4 31. 1489 1478 γ-muurolene 0.3 32. 1488 1484 germacrene d 3.6 tr 0.4 0.3 0.6 33. 1494 1489 β-selinene 0.3 1.1 tr 0.3 0.7 34. 1502 1498 α-selinene 0.3 0.7 0.3 tr 35. 1503 1500 bicyclogermacrene 0.7 0.1 36. 1514 1508 germacrene a 2.7 5.3 0.6 0.8 0.2 37. 1529 1522 δ-cadinene 0.1 tr 38. 1585 1577 spathulenol 0.3 39. 1591 1582 caryophyllene oxide 0.2 40. 1618 1608 humulene epoxide ii 0.6 41. 1648 1640 epi--murrolol ( τ-muurolol) 0.1 42. 1663 1658 selin-11-en-4-α-ol 1.1 monoterpenoids 91.9 27.2 91.1 68.2 98.1 92.5 sesquiterpenoids 6.2 70.1 4.6 23.7 1.7 6.1 other 0.8 0 3.7 6.9 0 0 total (%) 98.9 97.3 99.4 98.8 99.8 99.2 hs – head space volatiles; eo – essential oil; ld–leaf, vegetative phase; lf – leaf, flowering phase; f – inflorescences; ri – experimental linear retention indices relative to c8-c40 alkanes on the hp-5ms; aiadam’s retention indices; tr– trace (< 0.05%) and not detected (-); the most represented components are in bold. biologica nyssana 8 (1)  september 2017: 99-103 stojanović, g. et al.  first insight into chemical composition of… 103 composition of the volatile components in various organs in the same vegetative phase and in the same organ in different stages of development. in accordance with that, depending on application, should be chosen in which stage of development and which plant organ should be collected. in the case of p. longifolium, if the intention is to use p. longifolium in perfumery, it is best to use its flower because of their pleasing aroma originating from high content of phellandrenes and ocimenes. if there is intention for utilization of p. longifolium in the pharmaceutical industry, it is the best to use leaf, especially in the development phase, because of the high content of βelemene for which benefits for cancer treatment have been previously reported (z h u et al., 2011). acknowledgements. the research was supported by the serbian ministry of education, science and technology development (grant no. 172047). references ilić, b., miladinović, d., kocić , b., miladinović, m., 2015: antibacterial profile of peucedanum longifolium essential oil. acta medica medianae, 54 (1): 20-26. jovanović, o. p., zlatković, b. k., jovanović, s. c., petrović, g., stojanović, g. s., 2015: composition of peucedanum longifolium waldst. & kit. essential oil and volatiles obtained by headspace. journal of essential oil research, 27 (3): 182-185. kapetanos, c., karioti, a., bojović, s., marin, p., veljić, m., skaltsa, h., 2008: chemical and principal-component analyses of the essential oils of apioideae taxa (apiaceae) from central balkan. chemistry and biodiversity, 5 (1): 101119. kuzmanov, b., andreev, n., kozovska, v., 1980: chemotaxonomic study on bulgarian species of peucedanum l. i. anales del jardín botánico de madrid, 37 (2): 779-788. matejić, j. s., džamić, a. m., ćirić, a. d., krivošej, z., ranđelović, v. n., marin, p. d., 2013: antioxidant and antimicrobial activities of extracts of four peucedanum l. species. digest journal of nanomaterials and biostructures, 8(2): 655-665. mitrev, a., 1995. peucedanum arenarium w. et k. in: bondev, i. (ed.), chorological atlas of bulgarian medicinal plants: 141-143. academic publishing house “prof. marin drinov”, sofia. (in bulgarian) nikolić, v., 1973. peucedanum longifolium. in: josifović, m. (eds.), flora of the republic of serbia: 279-294, serbian academy of sciences and arts, belgrade. petkov, v., manolov, p., 1978: pharmacological studies on sub3228 stances of plant origin with coronary dilatating and antiar3229 rhythmic action. comparative medicine east and west, 6: 123-130. sarić, m., 1989: lekovite biljke sr srbije. srpska akademija nauka i umetnosti, posebna izdanja dxcviii, odeljenje prirodno-matematičkih nauka, beograd. schofield, l., kerton, o., mcmorn, p., bethell, d., ellwood, s., hutchings, g., 2002: oxidation of α-hydroxy containing monoterpenes using titanium silicate catalysts: comments on regioselectivity and the role of acidity. journal of the chemical society. perkin transactions, 2, 8: 1475-1481. spalik, k., reduron, j.p., downie, s.r., 2004: the phylogenetic position of peucedanum sensu lato and allied genera and their placement in tribe selineae (apiaceae, subfamily apioideae). plant systematics and evolution, 243 (3): 189–210. stojanović, g., jovanović, o., zlatković, b., jovanović, s., zrnzević, i., ilić, m., 2017: chemical composition of volatiles obtained from fresh root of peucedanum longifolium waldst. & kit.. acta medica medianae, 56 (1):82-85. tepe, b., akpulat, h. a., sokmen, m., 2011: evaluation of the chemical composition and antioxidant activity of the essential oils of peucedanum longifolium (waldst. & kit.) and p. palimbioides (boiss.). records of natural products, 5 (2):108-116. zhu, t., xu, y., dong, b., zhang, j., wei, z., yao, y., 2011: β-elemene inhibits proliferation of human glioblastoma cells through the activation of glia maturation factor β and induces sensitization to cisplatin. oncology reports, 26: 405-413. buenavista, d.p.: contributions to the orchid flora of mindanao long-term ecological research sites, philippines. biologica nyssana, 8 (1), september 2017 biologica nyssana 8 (1)  september 2017: 31-38 buenavista, d.p.  contributions to the orchid flora of mindenao… 31 original article received: 20 july 2017 revised: 12 august 2017 accepted: 15 august 2017 contributions to the orchid flora of mindanao long-term ecological research sites, philippines dave p. buenavista department of biology, central mindanao university, university town, musuan, 8710 bukidnon, philippines (school of environment, natural resources and geography, bangor university, ll57 2ew, wales, united kingdom) * e-mail: davista.cmu@gmail.com abstract: buenavista, d.p.: contributions to the orchid flora of mindanao long-term ecological research sites, philippines. biologica nyssana, 8 (1), september 2017: 31-38. this contribution is based on the field studies on wild orchids conducted from september 2012 to november 2013 in one-hectare plots in five long-term ecological research sites in mindanao island, philippines. the family orchidaceae is the most threatened group of plants in the philippines however, it remains to be poorly known and understudied. this is the first preliminary report on the species richness and distribution of wild orchids in the identified research sites. a list is contains 79 orchid species belonging to 34 genera which represents approximately 7% of the known orchid species in the philippines. a total of 40 endemics and 3 threatened species were recorded in the sites. this list can be used as basis for future biodiversity assessment and conservation initiatives to identify and prioritize areas for immediate conservation of threatened and endemic orchid species as well as the vulnerable mountain ecosystems. key words: inventory, conservation, orchidaceae, mindanao, philippines apstrakt: buenavista, d.p.: prilog flori orhideja mesta dugoročnih ekoloških istraživanja mindanaa, filipini. biologica nyssana, 8 (1), septembar 2017: 31-38. ovaj prilog je zasnovan na terenskim istraživanjima divljih orhideja sprovedenim od septembra 2012 do novembra 2013 na pet površina od po jednog hektara u oblastima obuhvaćenim dugoročnim ekološkim istraživanjima na mindanao ostrvu, filipini. familija orchidaceae je najugroženija grupa biljaka na filipinima ali bez obzira na to, i dalje je slabo poznata i nedovoljno proučavana. ovo je prvi preliminarni izveštaj o bogatstvu vrsta i distribuciji divljih orhideja na identifikovanim lokalitetima istraživanja. lista sadrži 79 vrsta orhideja koje spadaju u 34 roda i predstavljaju približno 7% poznate flore orhideja na filipinima. ukupno 40 endemskih i 3 ugrožene vrste su pronađene na istraživanim lokalitetima. lista može poslužiti kao osnova za dalju procenu biodiverziteta i inicijativu konzervacione inicijative koje bi identifikovale i označile najbitnije lokalitete za hitnu i trenutnu konzervaciju ugroženih i endemičnih vrsta orhideja kao i ranjivih planinskih ekosistema. ključne reči: inventarizacija, konzervacija, orchidaceae, mindanao, filipini 8 (1) • september 2017: 31-38 doi: 10.5281/zenodo.963339 biologica nyssana 8 (1)  september 2017: 31-38 buenavista, d.p.  contributions to the orchid flora of mindenao… 32 introduction the philippines lies in the western pacific ocean and is geographically part of southeast asia, a region that occupies a mere three percent of the earth’s total surface, yet is home to 20 percent of all known species of plants and animals (m i t t e r m e i e r et al., 1999; m y e r s et al., 2000; a m b a l et al., 2012). it has one of the highest orchid floras in the world with approximately 141 genera and 1200 species (c o o t e s , 2001; a g o o et al., 2003; c o o t e s , 2011). about 85-90% of the philippine orchids are found nowhere else in the world (c o o t e s , 2011). by contrast to other countries, canada and the united states, including hawaii, puerto rico, and the virgin islands, have only 325 species among them (h e a n e y & r e g a l a d o , 1998). orchids have also been shown to be excellent indicators of overall biodiversity in an area (n a d k a r n i , 1992; s w a r t s & d i x o n , 2009). orchids are highly evolved with their pollinators and require a specific relationship with mycorrhizal fungi to germinate (l e a k e , 1994; r a s m u s s e n , 2002; o t e r o et al., 2005; m c c o r m i c k et al., 2006; s h e f f e r s o n et al., 2007; r a s m u s s e n & r a s m u s s e n , 2009); therefore they are intimately intertwined with the ecology of their habitat. it is therefore imperative that the conservation of habitat be considered if the orchids had to be conserved. however, habitat loss is one of the major factors that imperiled the philippine biodiversity (p o s a et al., 2008; s o d h i et al., 2010) losing over 80% of its original forest cover since the 16th century (fao, 2005; b a n k o f f , 2007). such threat had direct impact on the orchid flora considering that it has the highest number of threatened species (f e r n a n d o et al., 2008) compared to any plant groups in the philippines. besides the scanty number of orchid studies in the philippines, the assessment of orchid diversity and distributions is further limited by potential problems in nomenclature that are difficult to resolve because of the lack of access to reference specimens, digital imagery, and detailed data collection particularly in mindanao. species richness of endemic species richness is typically used to estimate the biodiversity value of a region (n a g e n d r a , 2002; c l e r g u e et al., 2005; o r m e et al., 2005). one of the most important tasks for conservationists is therefore to find objective methods for assessment of natural areas in terms of their conservation priority (j a n k o w s k i et al., 2009; k i n d l m a n n & v e r g a r a , 2009). defining conservation priorities is essential in minimizing biodiversity loss (b r o o k s et al., 2006; j a c q u e m y n et al., 2007) as it ensures that conservation action focuses on the species at the greatest risk of extinction and on the sites that are most important for their protection. the long-term fig. 1. geographical location of mindanao long-term ecological research sites biologica nyssana 8 (1)  september 2017: 31-38 buenavista, d.p.  contributions to the orchid flora of mindenao… 33 ecological research (lter) sites were established in five mountain regions in mindanao island in order to assess, conserve and protect the increasing number of endangered flora and fauna of the region. to date, knowledge on the orchid flora in the aforementioned lter sites has never been documented; hence, this study was undertaken. material and methods field inventory was conducted from september 2012 to november 2013 in the five long-term ecological research (lter) sites established in the different regions in mindanao island namely (fig. 1), mt. kitanglad, mt. apo, mt. malindang, mt. hamiguitan and mt. musuan. every lter site was delineated into one hectare permanent plot which was surveyed and inventoried. site 1 is mt. apo lter site is located within the vicinity of energy development company (edc), ilomavis, kidapawan city. the site was characterized as a montane forest located at 6°59´47´´n, 125°15´12´´e with an elevation of 19002000 meters above sea level (masl). the site has a close-canopy layer dominated by large trees and shrubs covered by bryophytes. site 2 is mt. hamiguitan lter site in davao oriental. the site is also a closed-canopy montane forest located at 6°43´58´´n, 126°9´58´´e with an elevation of 10001100 masl. site 3 is at the mt. kitanglad lter site situated 8°5´46´´n, 124°55´17´´e in lantapan, bukidnon. the permanent plot is within the montane forest with an elevation of 2100-2200 masl. site 4 is located at the upper montane forest of mt. malindang lter site situated 8°17´45´´n, 123°36´34´´e in misamis occidental at 1600-1700 masl elevation. the slightly open canopy layer of the forest was due to the overlapping crown of various types of trees, palm and shrubs. site 5 is mt. musuan lter site also in bukidnon. the permanent plot is situated at 7°52'36.02”n, 125°04'1.53”e in the lowland secondary, mixed agro-dipterocarp forest which is around 380-480 masl and has slightly to open canopy layer. plant collections were pressed and treated with denatured alcohol after the fieldwork and then further processed in the herbarium. the specimens were allowed to dry up to 75°c using a mechanical drier. since orchid taxonomy relies mainly on the characters of inflorescence, orchids with its flowers were collected whenever available during the field sampling. the inflorescence were dried and kept in separate herbarium pockets as it often wilts and deteriorates easily in the field. the collected plants were classified and identified using taxonomic keys from orchid floras, monographs and published articles of h o l t t u m (1954), d r e s s l e r & d o d s o n (1960); v a l m a y o r (1984); d e v o g e l (1988a,b); p e d e r s e n (1997); f e s s e l & b a l z e r (1999); a g o o et al. (2003); c o o t e s (2001, 2010; 2011) and s u a r e z (2010 & 2011). confirmation of the plant identification was done through direct communication with jim cootes, one of the authorities on philippine orchidaceae. all the collections were then deposited in the university museum of central mindanao university, bukidnon, philippines. results and discussion a total of 79 orchid species belonging to 34 genera was recorded in five lter sites in mindanao (tab. 1). taxonomic identification of the four species were undetermined due to the unavailability of the inflorescence during the survey. in terms of species richness per site, mt. apo had the highest number represented by 31 species belonging to 17 genera. this is followed by mt. kitanglad with 25 species from 16 genera; and mt. hamiguitan with a record of 19 species from 11 genera. the least record in was from mt. malindang with 18 species from 16 genera; and in mt. musuan with only 2 species from 2 genera (fig. 2). fig. 2. bar graph comparing the species richness of orchids in five mindanao lter sites the difference in the species richness between the 5 sites may be explained by the difference in many ecological factors such as elevation, microclimate conditions and presence of pollinators and mycorrhizal associates which have been reported to influence orchid diversity and distribution. j a c q u e m y n et al. (2005) reported that orchid species composition changed continuously with altitude, indicating turnover of species with increasing altitude. analogously, orchid breeding biologica nyssana 8 (1)  september 2017: 31-38 buenavista, d.p.  contributions to the orchid flora of mindenao… 34 table 1. list and distribution of wild orchids in mindanao lter sites species mt. apo mt. hamiguitan mt. kitanglad mt. malindang mt. musuan 1. agrostophyllum longivaginatum ames x x x 2. a. saccatilabium ames & quisumb. x x 3. appendicula laxifolia j.j.sm. x 4. a. malindangensis (ames) schltr. x x x 5. a. tembuyukenensis j.j.wood x 6. arundina graminifolia (d don) hochr. x 7. ascidieria cymbidifolia (ridl.) w.suarez & cootes x 8. bulbophyllum colubrimodum ames x 9. b. escritorii ames x 10. calanthe davaensis ames x 11. calanthe sp. x 12. cephalantheropsis longipes (hook.f.) ormerod x 13. c. obcordata (lindl.) ormerod x 14. ceratostylis latipetala ames x x x x 15. c. wenzelii ames x 16. c. subulata blume x x x 17. cheirostylis octodactyla ames x 18. coelogyne candoonensis ames x 19. c. salvaneraniana w.suarez x 20. crepidium quadridentatum (ames) szlach. x x 21. cryptostylis arachnites (blume) hassk. x x 22. cymbidium pubescens lindl. x 23. dendrobium auriculatum ames & quisumb. x 24. d. crumenatum sw. x 25. d. diffusum lo williams x 26. d. erosum (blume) lindl. x 27. d. phillipsii ames & quisumb. x 28. d. rhombeum lindl. x 29. d. tiongii cootes x 30. d. uniflorum griff. x 31. dendrochilum arachnites rchb.f. x x 32. d. cobbianum rchb.f. x 33. d. coccineum h.a.pedersen & gravend. x 34. d. elmeri ames x 35. d. glumaceum lindl. x x 36. d. kopfii lückel. x 37. d. longifolium rchb.f. x 38. d. macranthum schltr. x 39. d. mearnsii ames x 40. d. tenellum (nees & meyen) ames x 41. d. wenzelii ames 1915 x biologica nyssana 8 (1)  september 2017: 31-38 buenavista, d.p.  contributions to the orchid flora of mindenao… 35 42. epigeneium stella-silvae (loher & kraenzl.) summerh. x 43. e. treacherianum (reichb.f ex hook.f.) summerh. x 44. e. roseum (d.don) lindl. x 45. goodyera clausa (aa eaton ex ames) schltr. x 46. g. viridiflora (blume) blume x 47. habenaria stenopetala lindl. x 48. habenaria sp. x 49. hippeophyllum wenzelii ames x 50. lepidogyne longifolia (blume) blume x 51. liparis amesiana schltr. x 52. l. negrosiana ames x x 53. l. parviflora (blume) lindl. x 54. l. philippinensis (ames) schltr. x 55. mycaranthes candoonensis (ames) cootes & w.suarez x 56. m. gigantea (ames) cootes & w.suarez x x 57. oberonia hispidula ames x 58. octarrhena parvula thwaites x 59. paphiopedilum adductum asher x 60. p. ciliolare (rchb.f) stein x 61. phreatia listrophora ridl. x 62. p. sulcata (blume) j.j.sm. x 63. pinalia bractescens (lindl.) kuntze x 64. p. densa (ridl.) w.suarez & cootes x 65. p. macera (ames) w.suarez & cootes x 66. plocoglottis plicata (roxb.) ormerod 67. rhomboda sp. (lindl.) ormerod x 68. robiquetia cerina (rchb.f) garay x 69. robiquetia sp x 70. spathoglottis kimballiana hook.f. x 71. s. plicata blume x x 72. s. tomentosa lindl. x 73. thelasis pygmaea (griff.) lindl. x 74. trichoglottis latisepala ames x 75. trichotosia ramosii (leavitt) kraenzl x 76. undetermined sp. 1 x 77. undetermined sp. 2 x 78. undetermined sp. 3 x 79. undetermined sp. 4 x total 31 19 25 18 2 systems and floral traits also changed with altitude. relatively more auto-pollinating species were found at high altitudes compared with midand low-altitude sites where animal-pollinated species were most abundant. as such, at very extreme conditions, the absence of specific pollinators may therefore have a possible direct influence that limits their range of dispersal and distribution. furthermore, orchids are highly evolved with their pollinators and require a specific relationship with mycorrhizal fungi to biologica nyssana 8 (1)  september 2017: 00-00 buenavista, d.p.  contributions to the orchid flora of mindenao… 36 germinate (o t e r o et al., 2005, s h e f f e r s o n et al., 2007), therefore they are intimately intertwined with the ecology of their habitat. the findings of v a s q u e z et al. (2003) in bolivia also showed that orchid diversity was mainly concentrated in ecoregions with semi-humid to very humid montane rain forests between 1000 m to 3500 m elevation. the very low species richness observed in mt. musuan (380-480 masl) maybe due to the warm and dry climatic condition which is very unfavorable to some species of epiphytic orchids that heavily relies from air moisture and rainfall (b a r t h l o t t et al., 2001). aside from the fact that mt. musuan is already a secondary forest, the nearby cultivated agricultural areas suggests more anthropogenic disturbance in the area than the other four lter sites. such disturbance was likewise considered to negatively influence the species richness in an area (b a r t h l o t t et al., 2001). a total of 40 philippine endemic species were recorded in all the five lter sites. mt. apo harbors most of the endemics with 18 species, followed by mt. kitanglad with a record of 16 endemic species. the least was in mt. hamiguitan with only 9 endemic species then mt. malindang with 8 endemic species. mt. musuan had no endemic species. highly threatened species was also recorded in the field survey. based on iucn (2017), this includes the critically endangered paphiopedilum adductum asher (fig. 3) and the endangered paphiopedilum ciliolare (rchb.f) stein (fig. 4) from mt. hamiguitan, and the vulnerable epigeneium treacherianum (reichb.f ex hook.f.) summerh (fig. 5) from mt. apo. this reflects that the orchid family remains to be poorly known despite of their dominance in the philippine flora. the national list of threatened plants of f e r n a n d o et al. (2008) assessed only the 5% of philippine orchid flora and the remaining 95% remains to be data deficient or poorly studied. the lack of comprehensive ecological and taxonomic studies in this group of plants. conclusion the lter sites in mindanao harbors a number of endemic and understudied wild fig. 3. critically endangered paphiopedilum adductum asher fig. 4. endangered paphiopedilum ciliolare (rchb.f) stein fig. 5. vulnerable epigeneium treacherianum (reichb.f ex hook.f.) summerh. biologica nyssana 8 (1)  september 2017: 31-38 buenavista, d.p.  contributions to the orchid flora of mindenao… 37 orchids. the species composition in every site maybe influenced by various ecological factors such as elevation, forest types, micro-climate conditions and among others. this benchmark data presented from the lter sites in mindanao island, philippines can be utilized for future monitoring of orchid populations and conservation initiatives of threatened species. acknowledgements. the author would like to express his profound gratitude to jim cootes for validating the identification of specimens, dr. victor amoroso for the help and support, and to jerome ga-as for the company during the field work. references agoo, m.g., schuiteman, a., de vogel, e.f 2003: flora malesiana: orchids of the philippines vol. 1 world biodiversity database cd-rom series. national herbarium of the netherlands. ambal, r.g.r., duya, m.v., cruz, m.a., coroza, o.g., vergara, s.g., de silva, n., molinyawe, n., tabaranza, b. 2012: key biodiversity areas in the philippines: priorities for conservation. journal of threatened taxa, 4(8): 2788–2796. bankoff, g. 2007: one island too many: reappraising the extent of deforestation in the philippines prior to 1946. journal of historical geography, 33: 314-334. barthlott w., schmit-neuerburg, v. , nieder, j., engwald, s. 2001: diversity and abundance of vascular epiphytes: a comparison of secondary vegetation and primary montane rain forest in the venezuelan andes. plant ecology. 152: 145–156. brooks t.m., mittermeier, r.a., fonseca, g.a.b., gerlach, j., hoffmann, m., lamoreux, j.f., mittermeier, c.g., pilgrim, j.d., rodrigues, a.s.l. 2006: global biodiversity conservation priorities. science, 313: 58–61. clergue, b., amiaud, b., pervachon, f., lasserrejoulin, f., plantureux, s. 2005: biodiversity: function and assessment in agricultural areas: a review. agronomy for sustainable development, 25:1–15. cootes, j.e. 2001: the orchids of the philippines. timber press, inc. singapore. 231 p. cootes, j.e. 2011: philippine native orchid species. katha publishing co., inc. quezon city, philippines. 289 p. cootes, j.e. 2010: a new dendrochilum species from the philippines. orchideen journal, 3(3):114-115. de vogel, e. 1988a: orchid monographs vol. 3. revision in coleogyninae (orchidaceae) iii the genus pholidota, leiden, the netherlands. de vogel, e. 1988b: orchid monographs vol. 6. revision in coleogyninae, hortus botanicus, leiden, the netherlands. dressler, r. dodson, c. 1960: classification and phylogeny in orchidaceae. annals of missouri botanical garden, 47: 25-67. [fao] food & agriculture organization of the united nations 2005. global forest resources assessment 2005. rome, forestry department, fao, country report 202: philippines. fernando, e.s., co, l.l., lagunzad, d.a., gruezo, w.s., barcelona, j.f., madulid, d.a., baja-lapis, a., texon, g.i., manila, a.c., zamora, p.m. 2008: threatened plants of the philippines: a preliminary assessment. asia life sciences, supplement, 3: 1–52. fessel, h.h. balzer, p. 1999: a selection of native philippine orchids. times edition, singapore. 192 p. heaney, l.r. regalado, j.c., 1998: vanishing treasure of the philippine rain forest. the field museum. chicago, illinois. u.s.a. 85 p. holttum, r.e. 1954: a revised flora of malaya. vol. i. orchids of malaya, government printing office, singapore. 759 p. iucn 2017: the iucn red list of threatened species. version 2017-1. <www.iucnredlist.org>. downloaded on 20 july 2017. jankowski, j.e., ciecka, a.l., meyer, n.y., rabenold, k.n. 2009: beta diversity along environmental gradients: implications of habitat specialization in tropical montane landscapes. journal of animal ecology, 78: 315–327. jacquemyn, h., micheneau, c., roberts, d.l., pailler, t. 2005: elevational gradients of species diversity, breeding system and floral traits of orchid species on re´union island. journal of biogeography, 32, 1751–1761. jacquemyn h., honnay, o., pailler, t. 2007: range size variation, nestedness and species turnover of orchid species along an altitudinal gradient on re´union island: implications for conservation. biological conservation, 136:388 –397. kindlmann, p. vergara, c. 2009: objective measures of orchid species diversity. in: pridgeon a.m, suarez, j.p. (eds) proceedings of the second scientific conference on andean orchids. universidad técnica particular de loja, loja, ecuador, 83 p. leake, j. 1994: the biology of myco-heterotrophic (saprotrophic) plants. new phytologist, 127:171– 216. mccormick, m.k., whigham, d.f., sloan, d., o’malley, k., hodkinson, b. 2006: orchidhttp://www.iucnredlist.org/ biologica nyssana 8 (1)  september 2017: 00-00 buenavista, d.p.  contributions to the orchid flora of mindenao… 38 fungus fedility: a marriage meant to last. ecology, 87(4): 903–911. myers, n., mittermeier, r.a., mittermeier, c.g., da fonseca, g.a.b., kent, j. 2000: biodiversity hotspots for conservation priorities. nature, 403: 853-858. nadkarni, n.m. 1992: the conservation of epiphytes and their habitats: summary of a discussion at the international symposium on the biology and conservation of epiphytes. selbyana, 12: 140-142. nagendra, h. 2002: opposite trends in response for the shannon and simpson indices of landscape diversity. applied geography, 22: 175–186. orme, c.d.l., davies, r.g., burgess, m., eigenbrod, f., pickup, n., olson, v.a., webster, a.j., ding, t.s., rasmussen, p.c., ridgely, r.s., stattersfield, a.j., bennett, p.m., blackburn, t.m., gaston, k.j., owens, i.p.f. 2005: global hotspots of species richness are not congruent with endemism or threat. nature, 436: 1016-1019. otero, j.t., bayman, p., ackerman, j.d. 2005: variation in mycorrhizal performance in the epiphytic orchid tolumnia variegata in vitro: the potential for natural selection. evolutionary ecology, 19: 29-43. posa, m.r.c., diesmos, a.c., sodhi, n.s., brooks, t.m. 2008: hope for threatened tropical biodiversity: lessons from the philippines. bioscience, 58: 231–240. rasmussen, h.n. 2002: recent developments in the study of orchid mycorrhiza. plant and soil, 244:149–163. rasmussen, h.n., rasmussen, f.n. 2009: orchid mycorrhiza: implications of a mycophagous life style. oikos, 118: 334-345. shefferson, r.p., taylor, d.l., weib, m., garnica, s., mccormick, m.k., adams, s., gray, h.m., mcfarland, j.w., kull, t., tali, k., yukawa, t., kawahara, t., miyoshi, k., lee, y.. 2007: the evolutionary history of mycorrhizal specificity among lady’s slipper orchids. evolution, 61: 1380-1390. sodhi, n.s., posa, m.r.c., lee, t.m., bickford, d., koh, l.p., brook, b.w. 2010: the state and conservation of southeast asian biodiversity. biodiversity conservation, 19: 317–328. suarez, w. 2010: notes on philippine ceraia, cylindrolobus and flickingeria. orchideen journal, 1(1): 10-14. suarez, w. 2011: preliminary studies in the bulbophyllum nymphopolitanum (section lepidorhiza) complex in the philippines. malesian orchid journal, 7: 105-116. swarts, n.d., dixon, k.w. 2009: terrestrial orchids in the age of extinction. annals of botany, 104: 543-556. volatiles of quince fruit and leaf (cydonia oblonga mill.) from serbia biologica nyssana 7 (2)  december 2016: 145-149 veličković, d.t. et al.  volatiles of quince fruit and leaf… 145 original article received: 08 october 2016 revised: 30 october 2016 accepted: 24 november 2016 volatiles of quince fruit and leaf (cydonia oblonga mill.) from serbia dragan t. veličković1*, mihailo s. ristić2, nebojša p. milosavljević1, dejan n. davidović1, dragan m. milenović3, ana s. veličković4 1college of agriculture and food technology, prokuplje, serbia, 2institute for medicinal plant research "dr. josif pančić", belgrade, serebia 3"zdravlje-actavis" company, leskovac, serbia 4out-patient clinic leskovac health center vučje, koste stamenkovića bb, vučje, serbia * e-mail: dvelickovic7@ptt.rs abstract: veličković, d.t., ristić, m.s., milosavljević, n.p., davidović, d.n., milenović, d.m. veličković, a.s.: volatiles of quince fruit and leaf (cydonia oblonga mill.) from serbia. biologica nyssana, 7 (2), december 2016: 145-149. the subject of this study is the chemical composition of volatile fractions from the fruit and leaf of quince (cydonia oblonga mill.). dominant components were ethyl 2-methylbutanoate, (e,e)--farnesene, ethyl(2e,4z)-decadienoate, pentadecanol, -acoradienol, ethyl decanoate, ethyl octanoate, (e)-nerolidol, ethyl dodecanoate, 14-hydroxy-9-epi-(e)-caryophyllene, (2z,6e)-farnesol, -cedrene. volatile fraction of the fruit was also characterized by ethyl-(4z)-decenoate, ethyl 9-dodecenoate, ethyl hexanoate, ethyl nonanoate and cyclocitral, where in the leaf n-octanal, n-hexanol, n-nonanal and benzaldehyde were present. key words: quince fruit, quince leaf, volatiles, 2-methylbutanoate, ethyl-(2e,4z)-decadienoate apstrakt: veličković, d.t., ristić, m.s., milosavljević, n.p., davidović, d.n., milenović, d.m. veličković, a.s.: isparljive supstancije ploda i lista dunje (cydonia oblonga mill.) iz srbije. biologica nyssana, 7 (2), decembar 2016: 145-149. predmet ove studije je hemijski sastav isparljivih frakcija ploda i lista dunje (cydonia oblonga mill.). dominantne komponente su bile etil 2-metilbutanoat, (e,e)--farnezen, etil-(2e,4z)-dekadienoat, pentadekanol, -akoradienol, etil dekanoat, etil oktanoat, (e)-nerolidol, etil dodekanoat, 14-hidroksi-9-epi-(e)kariofilen, (2z,6e)-farnezol, -cedren. isparljivu frakciju ploda karakterišu etil-(4z)-decenoat, etil 9dodecenoat, etil heksanoat, etil nonanoat i -ciklocitral, dok su u listu bili prisutni n-oktanal, n-heksanol, nnonanal i benzaldehid. key words: plod dunje, list dunje, isparljive supstancije, 2-metilbutanoat, etil-(2e,4z)-dekadienoat 7 (2) • december 2016: 145-149 12th sfses • 16-19 june 2016, kopaonik mt doi: 10.5281/zenodo.200413 biologica nyssana 7 (2)  december 2016: 145-149 veličković, d.t. et al.  volatiles of quince fruit and leaf… 146 introduction quince fruit and its products (jam, marmalade, compote, juice, quince-brandy) are highly appreciated in the market. some studies have shown that the quince is an excellent natural source of polyphenols and flavonoids (o l i v e i r a et al., 2007; s i l v a et al., 2008), giving it a very important role and position in traditional medicine. the fruit is used in the treatment of dysentery, the leaf can be used as a sedative and seed as an emulsifying agent in hair-fixing lotions (e l a n d s e n & m a g n e y , 1992; b e r g m a n et al., 1996). in addition to fructose, glucose and sorbitol, malic, tartaric, citric, phytic and quinic acids, and crude fiber (o l i v e i r a et al., 2008; r o d r i g u e z g u i s a d o et al., 2009; s z y c h o w s k i et al., 2014), as main phenolic components in the extract of quince leaf were found 5-o-caffeoylquinic acid and 3-o-caffeoylquinic acid (c o s t a et al., 2009). latest studies show that c. oblonga is a rich source of chlorogenic acids, proanthocyanidins and flavonol c and o-glycosides (k a r a r et al., 2014), as well as the volatile components (s c h m a r r & b e r n h a r d t , 2010). in the seeds of quince linoleic, palmitic, oleic, stearic, eicosanoic, palmitoleic and arachidonic acids were identified (d a n e s h v a n d et al., 2012; s z y c h o w s k i et al., 2014). among a large number of metabolites isolated from the quince fruit skin, a few have been identified and tested on radicalscavenging and antioxidant activities (a l e s i a n i et al., 2010). a l s n a f i (2016) states antioxidant potential, immunological, antiallergic, cardiovascular, reproductive, dermatological, anticancer, antiinflammatory, antidiabetic, protective effects, as well as effect on respiratory smooth muscle contraction. the main goal of this study was to determine quince volatiles of serbian origin, and compare our results with accessible publications. material and methods plant material for this study, it was used the quince cydonia oblonga mill. var. leskovačka, from locality džigolj, municipality prokuplje, central serbia (j o v a n o v i ć , 1972). the authors are very grateful to prof. jugoslav trajković (college of agriculture and food technology) for botanical identification of the plant. ripe fruits were collected in october 2013. isolation of volatiles (vs) volatiles were isolated from small pieces of quince fruit and cut fresh leaves. the materials were put into a round-bottom flask of 2 l, and hydrodistillation process was performed using a clevenger-type apparatus (ph. eur. 4, 2002), with hydromodule 1 : 10. the vs were taken up in cyclohexane (1 ml), which was inserted in a graduated tube of the apparatus. solutions were stored for a few hours at 4 °c in the dark until being analyzed by gc. identification of volatiles (vs) analytical gas chromatography (gc-fid) and combination of gas chromatography and mass spectrometry (gc-ms) were used for the characterization of volatiles trapped in graduated tube of the clevenger type apparatus used. gc-fid analysis was carried out on an agilent technologies, model 7890a gas chromatograph, equipped with split-splitless injector and automatic liquid sampler (als), attached to hp-5ms column (30 m × 320 m, 0.25 m film thickness) and fitted to flame ionization detector (fid). carrier gas flow rate (h2) was 1 ml/min, split ratio 1:30, injector temperature was 250 °c, detector temperature 300 °c, while column temperature was linearly programmed from 40 °c to 260 °c (at a rate of 4 °c/min), and then kept isothermally at 260 °c for 15 min. sample solutions were prepared in cyclohexane (in conc. of approximately 10 g/l), and injected by als (2 l). the same analytical conditions as those mentioned for gc-fid were employed for gc-ms analysis, along with column hp-5ms (30 m × 250 m, 0.25 m film thickness), using hp g 1800c series ii gcd system [hewlett-packard, palo alto, ca (usa)]. helium was used as a carrier gas. transfer line was heated at 260 °c. mass spectra were acquired in ei mode (70 ev) in m/z range 40450. the amounts of 0.2 l of sample solutions were injected. constituents were identified by comparison of their mass spectra with those stored in ms libraries (wiley 275, nist05 and adams 2007), using different search engines (pbm, nist 2.0), as well as using calibrated amdis (ver. 2.64) for determination and comparison of retention indices (a d a m s , 2007). similarly, quantification of present constituents was achieved by normalization method, based upon the area percent report obtained by gc-fid. statistics has been covered by fid specification (results with a range of deviation for the level 1%). biologica nyssana 7 (2)  december 2016: 145-149 veličković, d.t. et al.  volatiles of quince fruit and leaf… 147 table 1. volatiles of different parts of quince (%, w/w) constituents kil fruit leaf ethyl 2-methylbutanoate 864 1.4 23.4 n-hexanol 863 0.3 1.3 2-methylbutyl acetate 875 tr. 2-heptanol 896 0.7 0.7 n.i. 0.4 n.i. 0.3 ethyl pentanoate (ethyl valerate) 901 tr. cumene 924 tr. 0.7 ethyl tiglate 929 0.5 0.2 benzaldehyde 952 1.0 6-methyl-5-hepten-2-one 981 0.2 tr. ethyl hexanoate (ethyl caproate) 997 2.0 tr. n-octanal 998 tr. 2.0 (2e,4e)-heptadienal 1005 tr. hexyl acetate 1007 tr. -phellandrene 1025 tr. 1,8-cineole 1026 tr. ethyl 2-hexenoate 1038 0.1 -terpinene 1054 tr. n-octanol 1063 0.1 cis-linalool oxide 1067 tr. fenchone 1083 tr. linalool 1095 tr. ethyl heptanoate (ethyl enanthate) 1097 0.8 tr. 4-ethyl-2-hexynal n.a. 0.6 n-nonanal 1100 tr. 1.1 (z)-isocitral 1160 0.9 0.6 ethyl 3-(methylthio)-(z)2-propenoate n.a. 0.3 camphor 1141 tr. ethyl-(4e)-octenoate 1184 0.2 -terpineol 1186 tr. ethyl octanoate (ethyl caprilate) 1196 4.3 1.9 n-decanal 1201 0.3 -cyclocitral 1217 1.2 0.6 ethyl-(2e)-octenoate 1245 0.5 ionene 1258 0.6 0.3 vitispirane 1272 1.0 0.6 ethyl nonanoate (ethyl pelargonate) 1294 1.4 0.7 (2e,4e)-decadienal 1315 0.3 n.i. 0.5 n.i. 0.1 ethyl-(4z)-decenoate 1380 2.7 1.9 ethyl-(4e)-decenoate 1388 0.5 ethyl decanoate (ethyl caprinate) 1395 6.3 3.9 (e)--damascone 1413 0.9 0.6 -cedrene 1421 2.5 1.5 constituents kil fruit leaf (e)--farnesene 1454 tr. ethyl-(2e,4z)decadienoate 1467 8.3 4.2 (e)--ionone 1487 tr. tr. ethyl undecanoate 1498 0.7 0.7 (e,e)--farnesene 1505 18.4 16.1 -dehydro-arhimachalene 1516 0.6 megastigmatrienone ** n.a. 0.2 (e)-nerolidol 1561 4.1 2.7 ethyl 9-dodecenoate n.a. 2.2 ethyl dodecanoate (ethyl laurate) 1594 3.2 3.8 -atlantol 1608 tr. n.i. 3.0 1.7 n.i. 0.4 himachalol 1652 0.5 0.3 lyral 1665 tr. 14-hydroxy-9-epi-(e)caryophyllene 1670 3.8 2.3 n.i. 0.4 n.i. 0.2 (2z,6z)-farnesol 1698 0.5 (2e,6z)-farnesal 1713 0.6 0.5 (2z,6e)-farnesol 1722 3.2 1.7 ethyl 3hydroxydodecanoate n.a. 0.2 -acoradienol 1762 7.6 5.1 pentadecanol 1773 7.8 5.2 n.i. 0.1 ethyl tetradecanoate (ethyl myristate) 1795 tr. hexahydrofarnesyl acetone 1838 0.5 n-hexadecanol 1874 0.3 nonadecane 1900 0.3 methyl hexadecanoate (methyl palmitate) 1922 0.5 n.i. 0.2 ambrettolide n.a. 0.2 ethyl hexadecanoate (ethyl palmitate) 1992 tr. eicosane 2000 tr. 0.5 octadecanol 2077 0.1 heneicosane 2100 0.3 1.0 ethyl oleate (ethyl (z)-9octadecenoate) 2179 0.7 0.7 docosane 2200 0.2 n.i. 0.8 tricosane 2300 0.2 0.9 tetracosane 2400 0.2 1.1 n.i. 0.6 pentacosane 2500 0.3 1.4 hexacosane 2600 0.2 1.2 heptacosane 2700 0.3 2.4 % w/w mass percent defined by peak area percent determined by integration (gc-fid) biologica nyssana 7 (2)  december 2016: 145-149 veličković, d.t. et al.  volatiles of quince fruit and leaf… 148 kil kovats (retention) index (adams, 2007) n.a. not available n.i. not identified tr. traces (< 0.1%) ** tentative identification results and discussion table 1. shows a comparative overview of volatiles in the quince fruit and leaf, where 94.8% and 96.2% of the components were identified from the total mass, respectively. components which dominate in the quince fruit and leaf are (e,e)--farnesene (18.4% and 16.1%), ethyl-(2e,4z)-decadienoate (8.3% and 4.2%), pentadecanol (7.8% and 5.2%), -acoradienol (7.6% and 5.1%), ethyl decanoate (6.3% and 3.9%), ethyl octanoate (4.3% and 1.9%), (e)-nerolidol (4.1% and 2.7%) and ethyl dodecanoate (3.2% and 3.8%). the above components are more present in the fruit, with the exception of ethyl dodecanoate. one of the essential components of the leaf is ethyl 2methylbutanoate with 23.4%, which is present in the fruit with only 1.4%. some more components that characterize the volatile fraction of the fruit are 14-hydroxy-9-epi(e)-caryophyllene (3.8%), (2z,6e)-farnesol (3.2%), ethyl-(4z)-decenoate (2.7%), -cedrene (2.5%), ethyl 9-dodecenoate (2.2%), ethyl hexanoate (2.0%), ethyl nonanoate (1.4%) and -cyclocitral (1.2%). the leaf is characterized by 14-hydroxy-9-epi-(e)caryophyllene (2.3%), n-octanal (2.0%), (2z,6e)farnesol (1.7%), -cedrene (1.5%), n-hexanol (1.3%), n-nonanal (1.1%), benzaldehyde (1.0%), as well as the compounds of the cosane group. the presence of fatty acid esters is obvious, especially in the quince fruit: ethyl decanoate (6.3%) > ethyl octanoate (4.3%) > ethyl dodecanoate (3.2%) > ethyl hexanoate (2.0%) > ethyl nonanoate (1.4%) > ethyl heptanoate (0.8%) > ethyl undecanoate (0.7%) > ethyl oleate (0.7%). ethyl pentanoate, ethyl tetradecanoate and ethyl hexadecanoate occur only in traces. s c h m a r r & b e r n h a r d t (2010) identified esters in the quince fruit (ethyl butanoate, ethyl 2methylbutanoate, 2-methylbutyl acetate, butyl acetate, hexyl acetate), alcohols (butanol, 2methylbutanol, hexanol, (e)-2-hexenol), carbonyls (hexanal, (z)-3-hexenal, (e)-2-octenal, (e/z)-2nonenal, 1-octen-3-one), and terpenes ((e)-damascenone, limonene, farnesene, linalool, eugenol). the authors assume that (e,z)and (e,e)ethyl 2,4-decadienoate could be characteristic esters responsible for flavor. s o u s a et al. (2007) identified trans-9-amino-8-hydroxy-2,7-dimethylnona-2,4-dienoic acid glucopyranosyl ester, homomonoterpenic compound which could be chemical marker. also, main volatiles of quince fruit were trans--farnesene, furfural, megastigma-4,6,8-trien3-one, trans-marmelo lactone and cis-marmelo lactone (t s u n e y a et al., 1983). the results of u m a n o et al. (1986) showed that main volatile constituents of quince fruit peel were ethyl propionate, ethyl acetate, ethyl dodecanoate, ethyl octanoate, ethanol, 2-methyl-1-propanol. the newest chemical analysis of c. oblonga showed that it contained phenolics, pectin, essential and volatile oils. the analysis of the leaves essential oils (a l s n a f i , 2016) showed that it contained aromatic aldehyde, fatty acid, oxygenated monoterpene, and sesquiterpene hydrocarbon (benzaldehyde, hexadecanoic acid, linalool, (e)-β-ionone, germacrene d). we have also identified some of those substances, especially regarding the ethyl 2methylbutanoate, ethyl-(2e,4z)-decadienoate, (e,e)-farnesene, ethyl octanoate, and ethyl dodecanoate. conclusion the presented results show that the quince fruit and leaf are an extraordinary source of substances, which from the chemical standpoint, belong to different groups of compounds. quince fruit was rich in fatty acid esters. among the volatiles, ethyl 2methylbutanoate and ethyl-(2e,4z)-decadienoate were the most abundant which could be responsible for flavor. acknowledgements. the ministry of education and science of the republic of serbia supported this study through the projects no. 172047 and 173021. references adams, r.p. 2007: identification of essential oil components by gas chromatography / mass spectrometry. 4th ed. allured publishing corp., carol stream, il 60188 usa. alesiani, d., canini, a., d’abrosca, b., dellagreca, m., fiorentino, a., mastellone, c., monaco, p., pacifico, s. 2010: antioxidant and antiproliferative activities of phytochemicals from quince (cydonia vulgaris) peels. food chemistry, 118: 199-207. al-snafi, a. e. 2016: the medical importance of cydonia oblonga a review. iosr journal of pharmacy, 6 (6): 87-99. bergman, r., afifi, a.k., heidgerip, p.m. 1996: text of histology. 9th ed. saunders wb, montréal, 159 p. biologica nyssana 7 (2)  december 2016: 145-149 veličković, d.t. et al.  volatiles of quince fruit and leaf… 149 costa, r.m., magalhães, a.s., pereira, j.a., andrade, p.b., valentão, p., carvalho, m., silva, b.m. 2009: evaluation of free radical-scavenging and antihemolytic activities of quince (cydonia oblonga) leaf: a comparative study with green tea (camellia sinensis). food and chemical toxicology, 47: 860-865. daneshvand, b., ara, k.m., raofie, f. 2012: comparison of supercritical fluid extraction and ultrasound-assisted extraction of fatty acids from quince (cydonia oblonga miller) seed using response surface methodology and central composite design. journal of chromatography a, 1252: 1-7. elandsen, s., magney, j. 1992: color atlas of histology. 6th ed. mosby, toronto, 104 p. european pharmacopoeia, 2002. 4th edn., strasbourg. jovanović, b. 1972. genus cydonia mill. in: josifović, m. (ed.), flora sr srbije. iv: 130-131, sanu, beograd. karar, e.g.m., pletzer, d., jaiswal, r., weingart, h., kuhnert, n. 2014: identification, characterization, isolation and activity against escherichia coli of quince (cydonia oblonga) fruit polyphenols. food research international, 65: 121-129. oliveira, a.p., pereira, j.a., andrade, p.b., valentão, p., seabra, r.m., silva, b.m. 2007: phenolic profile of cydonia oblonga miller leaf. journal of agricultural and food chemistry, 55: 7926-7930. oliveira, a.p., pereira, j.a., andrade, p.b., valentão, p., seabra, r.m., silva, b.m. 2008: organic acids composition of cydonia oblonga miller leaf. food chemistry, 111: 393-399. rodriguez-guisado, i., hernández, f., melgarejo, p., legua, p., martínez, r., martínez, j.j. 2009: chemical, morphological and organoleptical characterisation of five spanish quince tree clones (cydonia oblonga miller). scientia horticulturae, 122: 491-496. schmarr, h.g., bernhardt, j. 2010: profiling analysis of volatile compounds from fruits using comprehensive two-dimensional gas chromatography and image processing techniques. journal of chromatography a, 1217: 565-574. silva, b.m., valentão, p., seabra, r.m., andrade, p.b. 2008. quince (cydonia oblonga miller): an interesting dietary source of bioactive compounds. in: papadopoulos, k.n. (ed.), food chemistry research developments: 243-266, nova science publishers, inc., new york. sousa, c., silva, m.b., andrade, b.p., valentão, p., silva, a., ferreres, f., seabra, m.r., ferreira, a.m. 2007: homo-monoterpenic compounds as chemical markers for cydonia oblonga miller. food chemistry, 100: 331-338. szychowski, j.p., munera-picazo, s., szumny, a., carbonell-barrachina, a.á., hernández, f. 2014: quality parameters, bio-compounds, antioxidant activity and sensory attributes of spanish quinces (cydonia oblonga miller). scientia horticulturae, 165: 163-170. tsuneya, t., ishihara, m., shiota, h., shiga, m. 1983: volatile components of quince fruit (cydonia oblonga mill.). agricultural and biological chemistry, 47 (11): 2495-2502. umano, k., shoji, a., hagi, y., shibamoto, t. 1986: volatile constituents of peel of quince fruit, cydonia oblonga miller. journal of agricultural and food chemistry, 34 (4): 593-596. terzić, d., đekić, v., milivojević, j., branković, s., perišić, v., perišić, v., đokić, d.: yield components and yield of winter wheat in different years of research. biologica nyssana, 9 (2). december, 2018 biologica nyssana 9 (2) ⚫ december 2018: 119-131 terzić et al. ⚫ yield components and yield of winter wheat in... 119 original article received: 24 august 2018 revised: 23 november 2018 accepted: 10 december 2018 yield components and yield of winter wheat in different years of research dragan terzić1, vera đekić2, jelena milivojević2, snežana branković3, vesna perišić2, vladimir perišić2, dragoslav đokić1 1institute for forage crops, globoder bb, 37000 kruševac, serbia 2small grains research centre, save kovacevica 31, 34000 kragujevac, serbia 3university of kragujevac, faculty of science, institute of biology and ecology, radoje domanović 12, kragujevac, serbia * e-mail: verarajicic@yahoo.com abstract: terzić, d., đekić, v., milivojević, j., branković, s., perišić, v., perišić, v., đokić, d.: yield components and yield of winter wheat in different years of research. biologica nyssana, 9 (2). december, 2018: 119-131. field trial with wheat varieties perfekta, kg 56s, aleksandra and vizija was set on vertisol-type soil during the vegetation season 2010/11 and 2011/12. the aim of the research was to analyse the yield and grain yield components in four varieties of wheat cultivated on acid soil. the highest values of yield components and grain quality were established in the year with moderate temperatures and high precipitation in the vegetation year 2010/11. the kg 56s and vizija varieties had the highest yield of grain, the highest number of plants and spikes per m2. the perfekta variety showed the highest 1000 grain weight and grain weight per spike. the highest number of grains in the spike and the lowest average yield of grain during the research were recorded for the variety aleksandra. a significant positive correlation between grain yield and grain weight per spike, the number of grains in the spike and the number of plants per m2 were established, as well as between the 1000 grain weight and the number of grains in the spike and the grain weight per spike. key words: yield components, grain yield, wheat apstrakt: terzić, d., đekić, v., milivojević, j., branković, s., perišić, v., perišić, v., đokić, d.: komponente prinosa i prinos ozime pšenice u različitim godinama istraživanja. biologica nyssana, 9 (2). decembar, 2018: 119131. poljski ogled sa sortama pšenice perfekta, kg 56s, aleksandra i vizija postavljen je na zemljištu tipa vertisol tokom vegetacionih sezona 2010/11 i 2011/12 godine. cilj istraživanja je bio da se kod četiri sorte pšenice gajene na kiselom zemljištu analizira prinos i komponente prinosa zrna. najveće vrednosti komponenti prinosa i kvaliteta zrna ustanovljene su u godini sa umerenim temperaturama i velikom količinom padavina u vegetacionoj 2010/11 godini. sorte kg 56s i vizija imale su najveći prinos zrna, najveći broj biljaka i klasova po m2. sorta perfekta odlikovala se najvećom masom 1000 zrna i masom zrna po klasu. najveći broj zrna u 9 (2) • december 2018: 119-131 doi: 10.5281/zenodo.2538604 biologica nyssana 9 (2) ⚫ december 2018: 119-131 terzić et al. ⚫ yield components and yield of winter wheat in... 120 klasu i najmanji prosečan prinos zrna tokom istraživanja imala je sorta aleksandra. ustanovljena je značajno pozitivna korelacija između prinosa zrna i mase zrna po klasu, broja zrna po klasu i broja biljaka po m2, kao i između mase 1000 zrna i broja zrna po klasu i mase zrna po klasu. ključne reči: komponente prinosa, prinos zrna, pšenica introduction winter wheat (triticum aestivum l.) is one of the most important field crops in serbia, and it is cultivated on about 530,000 ha per year. average yields of wheat for the last 10 years in the main production areas of serbia have ranged from 4.5-8.0 t/ha. wheat production in serbia depends to a large extent on the environmental factors. the average grain yield of winter wheat and the potential of fertility of cultivated varieties are significantly different, especially in mountainous areas of serbia. hristov et al. (2011) and jocković et al. (2014) state that the yield and grain yield components of winter wheat vary considerably depending on the cultivation system, the applied doses of nitrogen, variety and conditions during the growing year, as well as their complex interactions. in addition to the genotype, the grain yield of winter wheat is largely influenced by the fertilization system, which is one of the key factors affecting the formed yield and its quality, but it must be harmonized with the climatic and soil conditions as well as the requirements of the variety (malešević et al., 2008; jaćimović et al., 2011; popović et al., 2011; đekić et al., 2014; jelic et al., 2015; terzić et al., 2018). research has shown that the yield of wheat depends on a number of components: the number of plants i.e. spikes per unit area, the number of grains in the spike, the weight of the grains in the spike and the absolute grain weight. there are complex interactions between these indicators, because in increasing the value of one parameter, the value of another is often reduced (borojević and williams, 1992; denčić et al., 2000; kandić et al., 2009; dimitrijević et al., 2011; laghari et al., 2011; perišić et al., 2011; đekić et al., 2012; hristov et al., 2012; milovanović et al., 2012; đurić et al., 2013; hagos and abay, 2013; mohamed et al., 2013). the variety as an autonomous genetic, biological and agronomic entity is one of the crucial factors both on quantitative and qualitative production levels (denčić and kobiljski, 2007). the increase in the yield of wheat depends primarily on cultivated variety, climatic conditions and applied cultivation technology (dodig et al., 2008; milovanović et al., 2011; đekić et al., 2012; hristov et al., 2014). the introduction into the production of varieties with increased genetic potential for yield and improved agronomic and technological traits represents the contribution of breeding to achieve higher production per unit area (mladenov et al., 2007; milovanović et al., 2008; luković et al., 2014; przulj et al., 2014; đurić et al., 2018). the genetic potential for yield can be increased in different ways: better use of genetic variability, better utilization of solar energy, increase in number and weight of grain, increase in total biomass of the plant, using heterosis, i.e. wheat hybrid (denčić et al., 2010). choosing the appropriate site/location, i.e. reonization of varieties, will contribute to a less variation in the yields achieved and to achieving better average results (biberdžić et al., 2005; williams et al., 2008; madić et al., 2010; dimitrijević et al., 2011; hristov et al., 2014; perišić, 2016; stevanović et al., 2018; đurić et al., 2018; djuric et al., 2018). bearing all this in mind, it is necessary that climatic conditions are in accordance with the biological requirements of the plants. in the last ten years, extreme temperatures and disturbances in the amount and distribution of precipitation have significantly affected the reduction in overall production of organic matter and yield reduction (hristov et al., 2013; popović et al., 2014). production of winter wheat with high grain yield and appropriate quality is possible only by choosing varieties of good quality with appropriate cultivation conditions and appropriate production technology. during the vegetation years (20102012), in the field trials, four winter wheat varieties were examined in order to determine the selection/breeding of the better varieties for the production conditions of central serbia. material and methods experimental design and soil conditions this study was conducted over a two-year period in the šumadija region, central serbia, on a vertisol soil, at kragujevac location, 173-220 m a. s. l. (44°00′51″ n and 20°54′42″ e), in a temperate continental climate having an average annual temperature of 11.5 °c, typical of šumadija districts in serbia and a rainfall amount of about 550 mm. the basic type of soil on which the trial was carried out in kragujevac was characterized as smonitsa in degradation. the physical properties of this soil are very unfavourable making it the type of heavy clay soil. according to the analysis, this soil was of a medium acid reaction (phkcl = 4.8) and poor in https://tools.wmflabs.org/geohack/geohack.php?pagename=%d0%9a%d1%80%d0%b0%d0%b3%d1%83%d1%98%d0%b5%d0%b2%d0%b0%d1%86&params=44.014167_n_20.911667_e_type:city https://tools.wmflabs.org/geohack/geohack.php?pagename=%d0%9a%d1%80%d0%b0%d0%b3%d1%83%d1%98%d0%b5%d0%b2%d0%b0%d1%86&params=44.014167_n_20.911667_e_type:city biologica nyssana 9 (2) ⚫ december 2018: 119-131 terzić et al. ⚫ yield components and yield of winter wheat in... 121 humus (2.65%). it was very poorly provided with accessible phosphorus (1-2 mg/100 g of soil) and provided with easily accessible potassium (20 mg/100 g of soil). during the 2010/11 and 2011/12 growing seasons, four varieties of winter wheat were investigated (perfekta, kg 56s, aleksandra and vizija) at the small grains research centre in kragujevac (in central serbia). the experiments were conducted in randomized block systems, with a plot size of 50 m2 (5 m x 10 m) in five replications. the sowing was carried out using a machine with row spacing of 12 cm. the soil on which the trial was conducted was uniform and well prepared. the amount of seed per square meter amounted to 400-450 viable seeds, depending on the traits of varieties. it was sown in the third decade of october, with 400 kg/ha of fertilizer npk 15:15:15, which was added in the fall, while during the spring fertilization soil was supplemented with 300 kg/ha (kan 27% n). during the trial, the influence of variety and year on productive yield elements was monitored (number of grains by spike, grain weight per spike, number of plants and spikes per m2 and yield of grain) and grain quality. determination of the number of spikes per m² was done by numbering them in the period of ripening from the surface of 1 m². the analysis of the fertility factors of the spikes (number of grains in the spike and the grain weight per spike) was done by measuring, counting and processing the obtained results. as a material, by random sample, 20 spikes in full maturity were taken from each variant and from all repetitions. after harvest, the yield from each elementary parcel was measured and expressed in kg/ha. the yield was corrected for grain moisture content of 14%. agroecologial conditions data in tab. 1 for the investigated period (20102012) clearly indicate that the years in which the research were conducted differed from the typical multi-annual average for kragujevac region, regarding the meteorological conditions. the average air temperature in 2010/11 was lower by 1.5 c and 2011/12 was lower by 2.1 c as compared to the long-term mean. the sum of rainfall precipitation in 2010/11 was lower by 86.2 mm, where the sum of rainfall in 2011/12 was by 12.5 mm higher than the average of many years and with a very uneven distribution of precipitation per months. compared to the long-term mean, total rainfall values, especially in the first year, second and third year, were considerably higher in february, april and may, whereas total rainfall in april 2010/11 decreased by 31.1 mm. given the high importance of sufficient rainfall amount during the spring months, particularly april and may, for wheat production, the distribution and amount of rainfall over the growing season 2010/11 were considerably more favourable, resulting in increased yields in this year. apart from the rainfall deficiency during the spring months and the non-uniform distribution of rainfall across months, an increase in average air temperatures was also observed. larger precipitation amounts during the spring months (march-may) caused rising groundwater levels and flooding of agricultural land. the excessive amount of moisture influenced the poorer heading and filling of grain, lodging of crops, the abundance of the weeds and the intense occurrence of the disease in the examined wheat and other winter grains. significant deviation of precipitation and temperature from the multi annual average is becoming more pronounced (dodig, 2010; đekić et al., 2015). it was found that newly-produced highyielding wheat varieties are less susceptible to temperature deviation (except for extreme) compared to precipitation (hodson and white, 2009; hristov et al., 2013). namely, the total amount of precipitation is reflected on the multi annual average, but the distribution, especially at critical stages of development, is significantly disturbed. it was found that winter precipitation significantly influences the table 1. mean monthly air temperature and precipitation amount (kragujevac) months interval x xi xii i ii iii iv v vi average mean monthly air temperature (oc) 2010/11 10.2 11.4 2.4 0.9 0.5 7.2 12.0 15.8 20.9 9.0 2011/12 10.4 3.1 4.6 0.7 -3.7 8.1 12.9 16.1 23.0 8.4 average 12.5 6.9 1.9 0.5 2.4 7.1 11.6 16.9 20.0 10.5 the amount of precipitation (mm) 2010/11 86.9 27.9 50.1 29.1 48.5 20.4 20.8 65.8 32.3 381.8 2011/12 33.3 1.3 43.3 117.2 60.1 5.7 74.5 87.3 57.8 480.5 average 45.4 48.9 56.6 58.2 46.6 32.4 51.9 57.6 70.4 468.0 biologica nyssana 9 (2) ⚫ december 2018: 119-131 terzić et al. ⚫ yield components and yield of winter wheat in... 122 realization of the production potential of wheat (malešević et al., 2011). in addition to the necessary reserve for the spring part of the vegetation, winter precipitation greatly influences the distribution of easily accessible nitrogen in the soil (đekić et al., 2014; jelic et al., 2015; terzić et al., 2018). statistical analysis on the basis of achieved research results the usual variation statistical indicators were calculated: average values. experimental data were analysed by descriptive and analytical statistics using the statistics module analyst program sas/stat (sas institute, 2000) for windows. all evaluations of significance were made on the basis of the anova test at 5% and 1% significance levels. relative dependence was defined through correlation analysis (pearson's correlation coefficient), and the coefficients that were obtained were tested at the 5% and 1% levels of significance. results and discussion productive elements of wheat grain yield and quality the average values of productive grain yield and quality traits of the investigated winter wheat varieties grown in the centre for small grains kragujevac are shown in tab. 2. in regard to grain yield, differences were established in the tested varieties. the average yield of wheat in the experiment was 5.091 t/ha, with a variation of 4.630 t/ha in the second year to 5.553 t/ha in the first year of the study. the highest grain yield in the vegetation year 2010/11 was established for the variety vizija (6.127 t/ha) and variety kg 56s (5.863 t/ha). in the second study year, cultivation, the highest average grain yield (5.159 t/ha) was recorded for the variety kg 56s. the highest average grain yield in the two-year period was recorded for kg 56s (5.511 t/ha) and vizija (5.485 t/ha), and the lowest for the variety aleksandra (4.628 t/ha). the weight of 1000 grains in the vegetation year 2010/11 year was significantly higher than in 2011/12 (tab. 2). the average weight of 1000 grains in the study was 43.13 g, with a variation of 42.05 g in the vegetation year 2011/12 to 44.21 g in the vegetation year 2010/11. the highest weight of 1000 grains in the first year was recorded for kg 56s (47.76 g) and perfekta (46.40 g) varieties. in the second year of the study, the highest weight of 1000 grains were achieved by the variety aleksandra (45.80 g). the variety perfekta had the highest average two-year value of the 1000 grain weight (45.00 g) and the lowest the variety vizija (41.98 g). the variety perfekta had the highest number of grains in the spike (45) in the vegetation year 2010/11 year, while in 2011/12 the highest value of the number of grains in the spike was recorded for the variety aleksandra (41). the average two-year value for number of grains in the spike in all wheat varieties examined was 39.65, with a variation of 37.50 in the vegetation year 2011/12 to 41.80 in the vegetation year 2010/11. the data on grain weight per spike, independent of the year, showed that there was a significant difference between the genotypes, with the variety perfekta (1.908 g) showing the highest average weight of grain per spike for the examined years. the average grain weight per spike in the study was 1.725 g, with a variation of 1.423 g to 2.026 g. the highest grain weight per spike in both years was recorded for the varieties perfekta and kg 56s. in regard to the number of plants per m2, significant differences were found in the studied wheat varieties (tab. 2). the average two-year plant count per m2 was 386.9, while the same ranged from 414.6 per m2 in the first year of research to 359.2 per m2 in the second vegetation season. the highest number of plants in vegetation year 2010/11 was established for the varieties vizija (437.2) and kg 56s (426.8). in the second year of testing, the highest number of plants per m2 was found in kg 56s (376.0). the average two-year value of number of spikes per m2 was 518.2, while the same ranged from 564.5 in the first year of research to 472.0 in the second vegetation season. the highest number of spikes in the vegetation year 2010/11 was recorded for the varieties vizija (604.0) and kg 56s (583.6). in the second year of the study, the highest number of spikes was recorded in the variety kg 56s (500.8). analysis of variance between observed traits of wheat the analysis of the variance of productive grain yield and quality traits in the examined kragujevac cultivars of winter wheat, grown in the centre for small grains in kragujevac during the two vegetation seasons, is shown in tab. 3. the estimation of the significance of the obtained results shows that there are statistically highly significant differences between the examined vegetation seasons and grain yield (fexp=14.5689), the number of grains in the spike (fexp=16.0342), the grain weight per spike (fexp=101.1208) and significant for the number of plants per m2 (fexp=8.9838). the statistically significant biologica nyssana 9 (2) ⚫ december 2018: 119-131 terzić et al. ⚫ yield components and yield of winter wheat in... 123 differences were established between the wheat varieties examined and grain yield (fexp=3.3398). the influence of the interaction of agro-climatic conditions and genotypes has had a significant influence on grain yield and grain weight per spike, as well as on the weight of 1000 grains between the tested winter wheat varieties. correlation dependence between tested wheat traits the average values of the pearson’s coefficient of correlation (r) of investigated winter wheat traits are shown in tab. 4. the established correlation coefficients between the yield of grain and the weight of 1000 grains, grain weight per spike and the number of table 2. average values of investigated triticale varieties traits varieties 2010/11 2011/12 average x s sx x s sx x s sx yield, (t/ha) perfekta 5.099 1.001 0.448 4.385 0.678 0.303 4.742 0.890 0.281 kg 56s 5.863 0.537 0.240 5.159 0.956 0.427 5.511 0.820 0.259 aleksandra 5.121 0.421 0.188 4.135 0.256 0.114 4.628 0.615 0.194 vizija 6.127 0.633 0.283 4.843 0.654 0.292 5.485 0.909 0.287 average 5.553 0.780 0.174 4.630 0.747 0.167 5.091 0.887 0.140 1000 grain weight, g perfekta 46.40 2.266 1.013 43.60 1.475 0.660 45.00 2.329 0.737 kg 56s 47.76 0.713 0.319 38.20 1.204 0.538 42.98 5.124 1.620 aleksandra 39.34 3.265 1.460 45.80 2.168 0.969 42.57 4.292 1.36 vizija 43.36 2.611 1.168 40.60 1.673 0.748 41.98 2.528 0.799 average 44.21 3.985 0.891 42.05 3.336 0.746 43.13 3.789 0.599 number of grains per spike perfekta 45.00 0.707 0.316 35.00 4.062 1.817 40.00 5.944 1.880 kg 56s 41.20 1.304 0.583 38.20 2.588 1.158 39.70 2.497 0.789 aleksandra 39.20 2.049 0.916 41.00 3.162 1.414 40.10 2.685 0.849 vizija 41.80 1.923 0.860 35.80 4.147 1.855 38.80 4.392 1.389 average 41.80 2.587 0.579 37.50 4.046 0.905 39.65 3.997 0.632 grain weight per spike, g perfekta 2.186 0.055 0.025 1.630 0.097 0.044 1.908 0.302 0.096 kg 56s 2.138 0.063 0.028 1.500 0.071 0.032 1.819 0.342 0.108 aleksandra 1.810 0.093 0.042 1.287 0.071 0.032 1.548 0.287 0.091 vizija 1.970 0.084 0.037 1.278 0.279 0.125 1.624 0.413 0.131 average 2.026 0.167 0.037 1.423 0.209 0.047 1.725 0.358 0.057 number of plants per m2 perfekta 390.8 44.623 19.956 368.0 88.136 39.416 379.4 66.946 21.170 kg 56s 426.8 65.629 29.350 376.0 49.639 22.199 401.4 61.043 19.303 aleksandra 403.6 81.761 36.564 331.2 39.233 17.545 367.4 71.492 22.608 vizija 437.2 46.981 21.010 361.6 48.711 21.784 399.4 60.193 19.035 average 414.6 59.602 13.327 359.2 57.273 12.807 386.9 64.154 10.144 number of spikes per m2 perfekta 522.0 46.217 20.669 460.8 18.199 8.139 491.4 46.227 14.618 kg 56s 583.6 65.106 29.116 500.8 41.415 18.521 542.2 67.458 21.332 aleksandra 548.4 85.725 38.337 456.0 14.491 6.481 502.2 75.704 23.940 vizija 604.0 22.935 10.257 470.4 32.354 14.469 537.2 75.213 23.785 average 564.5 63.640 14.230 472.0 31.855 7.123 518.2 68.274 10.795 biologica nyssana 9 (2) ⚫ december 2018: 119-131 terzić et al. ⚫ yield components and yield of winter wheat in... 124 plants and spikes per m2 in both vegetation seasons as well as during the two-year research were positive, except between the yield and the weight of 1000 grains in the second year of research in which significantly negative coefficient of correlation (r=0.480*) was established. highly significant and weak correlation was established between grain yield and grain weight per spike (r=0.479**) and medium with number of spikes per m2 (r=0.639*), while significant and weak correlations were established between grain yield and number (r=0.345*) of grains in the spike and number of plants per m2 (r=0.360*). the highest value of the positive and highly significant strong correlation, in the first year of the study, was established between the weight of 1000 grains and the grain weight per spike (r=0.754**), as well as between the number of grains in the spike and grain weight per spike (r=0.770**). the lowest value of the negative correlation in the same year of the study was established between the number of grains per spike and the number of plants per m2 (r=0.190). negative, weak and statistically significant correlation in the second year of the study was determined between grain yield and 1000 grain weight (r=-0.480*). positive and statistically highly significant correlation during the two-year investigation period was established between the grain weight per spike and the yield of grain, the weight of 1000 grains and the number of grains in the spike. positive, weak and significant correlations were found between the number of grains in the spike and the yield (r=0.345*) and the weight of 1000 grains (r=0.372*), as well as between the number of plants per m2 and yield (r=0.360*) and the grain weight per spike (r=0.390*) during the duration of the research. the weight of 1000 grains and the number of plants per m2 in both vegetation seasons, as well as during the duration of the experiment, had a negative coefficient of correlation. correlative dependence between traits showed a large variation depending on the test years which results from the interaction between the properties within each genotype and genotype interactions with environmental factors. this suggests that the selection of genotypes with longer spike (hence higher number of grains per spike) with good resistance to biotic and abiotic stresses contributed to table 3. analysis of variance of the tested parameters (anova) effect of year on the traits analysed traits mean sqr effect mean sqr error f (df 1, 38) p-level grain yield, t/ha 8.50 0.584 14.5689 ** 0.000484 1000 grain weight, g 46.87 13.503 3.4712 0.070187 number of grains per spike 184.90 11.532 16.0342 ** 0.000279 grain weight per spike, g 3.63 .036 101.1208 ** 0.000000 number of plants per m2 30691.60 3416.316 8.9838* 0.004780 number of spikes per m2 85562.50 2532.395 33.7872 ** 0.000001 effect of cultivar on the traits analysed traits mean sqr effect mean sqr error f (df 3, 36) p-level grain yield, t/ha 2.227 0.667 3.3398 * 0.029847 1000 grain weight, g 17.185 14.123 1.2168 0.317626 number of grains per spike 3.500 17.017 0.2057 0.891792 grain weight per spike, g 0.280 0.115 2.4214 0.081864 number of plants per m2 2676.667 4235.600 0.6319 0.599209 number of spikes per m2 6370.767 4518.922 1.4098 0.25577 effect of the year x cultivar interaction traits mean sqr effect mean sqr error f (df 3, 32) p-level grain yield, t/ha 2.227 0.467 4.7713 ** 0.007358 1000 grain weight, g 17.185 4.280 4.0147 * 0.015626 number of grains per spike 3.500 7.563 0.4628 0.710220 grain weight per spike, g 0.280 0.015 18.5725 ** 0.000000 number of plants per m2 2676.667 3666.800 0.7300 0.541718 number of spikes per m2 2302.500 2194.100 1.0494 0.384167 *statistically significant difference (p<0.05); **statistically high significant difference (p<0.01) biologica nyssana 9 (2) ⚫ december 2018: 119-131 terzić et al. ⚫ yield components and yield of winter wheat in... 125 the increase in production and yield stability of wheat in our agroecological conditions. discussion the highest average grain yield of 5.553 t/ha of studied wheat varieties was recorded in vegetation season 2010/11 and it was significantly higher than the yield in 2011/12 (4.630 t/ha), which can mostly be associated with higher precipitation during the first vegetation period. hassan et al. (2013) and perišić et al. (2016) have indicated similar results. after the formation of the number of spikes and the number of grains per spike during the vegetation stage, the yield becomes mainly determined by the weight of the grain (zafaranaderi et al., 2013). the yield of wheat grain, according to many authors, depends on several components: the number of spikes per unit area, the number of grains per spike, the weight of the grains per spike and the plant, as well as the weight of 1000 grains (hristov et al., 2008; petrović et al., 2010; perišić, 2016; djuric et al., 2018). varying of yield below 10% in exceptionally yielding years is not considered a serious problem in intensive wheat production. if these deviations are conditioned solely by the action of climatic factors (without loss during harvest or transport), using varietal agro-technology, it is possible to significantly reduce the disturbance of total grain production in one production area (malešević et al., 2011; jaćimović et al., 2012; đekić et al., 2014; jelić et al., 2015; terzić et al., 2018). in the production of wheat, the correct reonization (regional distribution) of varieties is very important, and it can contribute to a lesser variation in realized yields and achieving better average results (donmez et al., 2001; zečević et al., 2004; dencic and kobiljski, 2007; dodig et al., 2008; madić et al., 2010; dimitrijević et al., 2011; đekić et al., 2012, 2013). bearing all this in mind, it is necessary that climatic conditions are in accordance with the biological requirements of the plants. in the last few years, extreme temperatures and disturbances in the amount and distribution of precipitation have significantly affected the reduction in the total production of organic matter and yield reduction (hodson and white, 2009; đurić et al., 2013; hristov et al., 2013; popović et al., 2013; đekić et al., 2015; jelic et al., 2015). in the continental climate, winter wheat has long been exposed to the influence of weather conditions, and hence the climate extremes. according to the forecasts of the european commission for agriculture, global climate change will cause significant deviations from the average table 4. correlation coefficients by studied environments in winter wheat gy gw ngs gws np ns correlations between the traits analysed in the 2010/11 grain yield, gy 1.00 0.093 0.041 0.137 0.308 0.528* 1000 grain weight, gw 1.00 0.397 0.754** -0.118 -0.094 number of grains per spike, ngs 1.00 0.770** -0.190 -0.190 grain weight per spike, gws 1.00 -0.104 -0.178 number of plants per m2, np 1.00 0.675** number of spikes per m2, ns 1.00 correlations between the traits analysed in the 2011/12 grain yield, gy 1.00 -0.480* 0.113 0.011 0.020 0.350 1000 grain weight, gw 1.00 0.191 0.042 -0.288 -0.316 number of grains per spike, ngs 1.00 -0.356 -0.207 -0.037 grain weight per spike, gws 1.00 0.155 0.276 number of plants per m2, np 1.00 0.348 number of spikes per m2, ns 1.00 correlations between the traits analysed in the 2010-2012 grain yield, gy 1.00 0.022 0.345* 0.479** 0.360* 0.639** 1000 grain weight, gw 1.00 0.372* 0.433** -0.040 0.090 number of grains per spike, ngs 1.00 0.475** 0.091 0.309 grain weight per spike, gws 1.00 0.390* 0.584** number of plants per m2, np 1.00 0.655** number of spikes per m2, ns 1.00 *statistically significant (p<0.05); **statistically high significant (p<0.01) **grain yield, t/ha gy; 1000 grain weight, g gw; number of grains per spike -ngs; grain weight per spike, g gws; number of plants per m2 np; number of spikes per m2 ns. biologica nyssana 9 (2) ⚫ december 2018: 119-131 terzić et al. ⚫ yield components and yield of winter wheat in... 126 values of climatic factors in the field crop areas of serbia (vojvodina, mačva, stig), which is related to the increase in winter and reduction of summer precipitation, increased risk of drought and soil erosion, and to a certain extension of the vegetation period (which would lead to an increase in yield, but with higher variability). temperatures will increase slightly, which will impose changes in the sowing structure and a more careful selection of the variety, i.e. hybrid. according to this scenario, the biggest concern is the increase of winter precipitation (november-march), which will often cause flooding of lower terrains as well as greater possibility of soil erosion. in serbia, there are about 30 percent of the soil with a heavier mechanical composition that is susceptible to compaction and formation of the plow pan. frequent passing of machines and conventional tillage at low depths (due to weaker tractors) leads to the formation of a poorly permeable layer “plow pan” at a small depth and rapid saturation of the surface layer of the soil with water. the soil acidification process has been strengthened in recent times and tackling this problem is an important issue in order to reduce the impact from natural hazards in serbia. decrease of the acidity in acid soils contributes to the improvement of chemical, physical and biological properties of the soil. improvement of these soil properties contributes to better infiltration and water storage and reduction of damage and losses from natural hazards, including floods and droughts. these facts impose the need to change the system of crop fertilization and processing systems as well as land management in general. higher air temperatures in june and july of year 2012, compared to the first year of the study, caused a shortening of the period of filling of the grain, accelerated ripening and reduction of yield. the dependence of the yield on the conditions of the year was also emphasized by other authors (biberdžić et al., 2005; zecevic et al., 2010; perišić et al., 2016). a certain contribution to the continuous increase in grain yield is also interpreted by the increased use of nitrogen fertilizers, pesticide use, improved agro-technology (especially earlier sowing), as well as the positive interaction of these factors (jelić et al., 2012; đekić et al., 2014; terzić et al. 2018). the best genetic predispositions for yield were found in the kg 56s and vizija varieties, while the lowest yield in our study was recorded in the aleksandra variety and it was significantly lower in comparison to the other varieties tested. our results are in agreement with the results of milovanovic et al. (2008), who, based on their research, state the variety vizija as a variety of high genetic potential for yield with the possibility of its manifestation at different sites/locations. the differences in the yields that were observed in the tested varieties in our experiment are the result of varietal specificities, which are mostly genetically conditioned. thus, by analysing the obtained results we can conclude that there is a significant dependence of grain quality components on the genotype, which is in agreement with the results of laghari et al. (2011) and perišić (2016). the weight of 1000 grains in year 2010/11 was significantly higher compared to 2011/12 (44.21 g and 42.05 g). the highest value of the 1000 grain weight was established for the perfekta variety, the variety with the lowest number of grains per spike, and the lowest value for the variety vizija, which had the highest number of grains per spike. vegetation period in year 2011/12, at the time of grain filling, was marked by drought and high temperatures. climatic conditions are especially important during the filling of the grain, since the lack of moisture and high temperature during this period affects the decrease in the weight of 1000 grains (przulj et al., 2014), as confirmed by the results of these studies. perišić (2016) points to significant differences in grain weight between years, irrespective of the genotype. jaćimovic et al. (2012) find that the application of mineral fertilizers has a significant impact on the weight of 1000 grains, i.e. the grain weight is significantly higher in more intensively fertilized variants especially fertilized with nitrogen. the number of grains per spike is a very variable trait of the spike, because it depends on other components of the spike and factors of the external environment. the results of these investigations are in agreement with the previously performed studies by đurić et al. (2018) and established a high variability of the number of grains per spike and grain weight per spike djuric et al., (2018). these spike characteristics have a direct impact on the overall yield of wheat grain. the number of grains per spike is quite variable trait, which largely depends on the agroecological conditions of the year and the applied agro-technology (zecevic et al., 2004). the number of grains per spike is a yield component that is directly dependent on the number of spikelets per spike, the number of flowers per spikelet and the success of pollination and grain formation (borojević and williams, 1992). since these parameters significantly depend on the agroecological conditions of the year and the applied agro-technology, the number of grains per spike is quite variable trait. hristov et al. (2008) point out that the increase in the yield of grain per plant is directly determined by the number of grains per spike and the weight of 1000 grains, which is confirmed by the results of our research. milovanović et al. (2011), based on their research, suggest that better lines of triticale have 7595 grains per spike compared to 40-50 grains in biologica nyssana 9 (2) ⚫ december 2018: 119-131 terzić et al. ⚫ yield components and yield of winter wheat in... 127 wheat. the number of grains per spike for all investigated wheat genotypes ranged from 35 to 45 grains. in the first year of the research, which was more favourable, the number of grains per spike was 42, and in the second year of the research it was 37. the highest number of grains in the spike in the vegetation year 2010/11 was recorded for the perfekta variety (45), while in 2011/12 for the variety aleksandra (41). the average value of the number of grains per spike of 44.80 is established by perišić (2016). the same author states that the number of grains per spike has ranged from 38.61 to 47.76. the number of grains per spike or plant is the result of the number of spikelets and the number of flowers per spikelet, on the one hand, and the success of polination and formating of grains in these flowers, on the other hand, so that the number of grains depends to a large extent on the genetic basis of the variety, i.e. the gene with additive and non-additive effect (perišić et al., 2011; deletić et al., 2012; luković et al., 2014; đurić et al., 2018) and external factors (dodig, 2010; hassan et al., 2013). in the case of wheat genotypes, the increase in the number of grains in the spike was observed during the study in the case of a reduced number of spikes per m2, which is in agreement with the results of perišić (2016). according to garcia del moral et al. (2003), the yield stability under the influence of different environmental conditions is closely related to the number of grains per spike, because a large number of grains per spike allows the achievement of a large number of grains per unit area also in conditions of a smaller number of spikes. donmez et al. (2001) find that significant genetic progress is achieved by improving the yield components formed during the vegetative phase of wheat development (the number of spikes per surface area and the number of grains per spike), compared to the grain weight, a component formed at unfavourable temperature and humidity conditions. the number of spikes per unit area depends on the sowing density, the number of seedlings, the number of plants that managed to survive winter and the number of productive stems. the number of spikes is greatly affected by the conditions of cultivation, which is confirmed by our research. in our study, the highest number of spikes per m2 in the period 2010-2012 was realized by the varieties kg 56s and vizija. the highest number of spikes in the vegetation year 2010/11 was established for the varieties vizija (604.0) and kg 56s (583.6), while in the second year of the study, kg 56s (500.8) had the highest number of spikes. our results are in agreement with perišić (2016), who states that the number of spikes has ranged from 544.4 to 671.1 per m2 and that it was statistically significant among the wheat genotypes examined. biberdžic et al. (2005) pointed out that the number of spikes/m2, the number of grains per spike and the length of spike are important factors that influence grain yield. there are complex interactions between these indicators, as by increasing the value of one parameter, the value of another is often reduced (hristov et al., 2008), which is confirmed by the results of our research. also, it was noticed that in year 2010/11, due to favourable climatic conditions (at the time of emergence and tillering), in almost all genotypes, on average, statistically significantly higher number of spikes per m2 (564.5) was achieved compared to the second year (472.0). obviously, weather conditions (precipitation and temperature) were the least favourable in the second year of testing. it has been confirmed earlier by other authors that meteorological factors, and especially the moisture regime, can influence this property (joshi et al., 2002; janušauskaite and šidlauskas, 2004; erekul and köhn, 2006). in order to be able to perceive which traits have most influenced the formation of the grain yield, as well as the relationship between them, the correlation coefficients between the tested properties have been determined. the correlative dependence of the yield and the weight of the 1000 grains, depending on the investigated vegetation season in wheat, ranged from 0.480* (2011/12) to 0.093 (2010/11). grain yield depends directly on the number of grains per spike and the weight of 1000 grains (hristov, 2008). knowledge of correlation relations between yield components is of practical importance in the breeding process. correlation analysis is the most commonly used method for this purpose (hristov et al., 2011). since yield components directly or indirectly affect the yield, it is necessary to determine their correlation relations. these authors have established a highly significant correlation between the height of the plant and the length of the spike, the number of grains/ spike, the weight of the 1000 grains and the grain weight per plant, the highly significant correlation between the harvest index and plant height and the length of the spike. the weight of the grains per spike was in significant correlation with the yield (r=0.479**), the weight of 1000 grains (r=0.433**) and the number of grains in the spike (r=0.475**). many researchers are of the opinion that an increase in the number of grains per spike leads to an increase in the yield of wheat (zafarnaderi et al., 2013; đurić et al., 2016). many authors consider the number of grains per spike the most important component of the yield. in wheat, the coefficient of correlation showed values from non-significantly positive in 2010/11 (r=0.041) to significantly positive in the two-year investigation period (r=0.345*). many authors cite weak positive (mohammadi et al., 2013) to very positive biologica nyssana 9 (2) ⚫ december 2018: 119-131 terzić et al. ⚫ yield components and yield of winter wheat in... 128 correlations (djuric et al., 2018). according to the majority of researchers, the number of spikes per unit area is in significant positive correlation with the yield of grain (zecevic et al., 2004). iftikhar et al. (2012) find that the number of grains per spike and mass of 1000 grains are in a positive and statistically significant correlation with grain yield and point out that the number of grains per spike and weight of 1000 grains have a direct effect on yield and, therefore, can be used as direct selection criteria. new varieties are characterized by a higher number of spikes per unit area, but their relationship to yield is not always in a positive correlation (deletić et al., 2012). the same authors point out that in the determination of yield, the weight of the grain has the crucial role, and then the number of spikes and the number of grains per spike. perišić (2016) states that a higher number of productive trees cannot guarantee high yields. there are complex interdependent relations between all components of yield. the importance of these components in the formation of grain yield depends on the climatic conditions in critical phases of growth and development, applied agro-technology and various combinations and relationships of npk nutrients (popović et al., 2013; đekić et al., 2014; jelić et al., 2014; terzic et al., 2018). therefore, it is important to know the effect of these properties, i.e. yield components, as well as their interdependence on grain yield. it should be noted that the grain weight per plant or the yield per plant is a direct indicator of total yield, as well as all components of spikes that are in significant positive correlation with this parameter (hristov et al., 2008; đekić et al., 2013). the presence of phenotypic and genotypic correlations between fertility components and fertility itself were studied by kashif and khaliq (2004). they find that at the genotypic level there is a significant positive correlation of the height of the plant, the length of the spike, the number of spikelets per spike, the number of grains per spike, and the weight of 1000 grains with grain yield, while phenotypically there is a very significant association of these properties. due to this association of quantitative properties, it is necessary to select genotypes with a genetic basis in which a balance is achieved between the most important components (number of spikes per unit area, number of grains per spike and grain weight per spike). conclusion in the highest yield of grain, weight of 1000 grains, number of grains per spike, grain weight per spike and number of spikes per m2 in all wheat varieties were in the vegetation period with moderate temperatures at the time of grain filling and large amount of precipitation in the first vegetation period. grain yields in wheat cultivars ranged from 4.628 t/ha (aleksandra) to 5.511 t/ha (kg 56s). the average weight of 1000 grains in the study was 43.13 g, with a variation of 42.05 g in the vegetation year 2011/12 to 44.21 g in the vegetation year 2010/11. the number of grains per spike for all examined wheat genotypes varied from 35 to 45 grains. the coefficient of correlation between grain yield and grain weight per spike, number of grains per spike and number of plants per m2 showed significant positive values over the examined period, while the coefficient between 1000 grain weight and number of grains per spike and grain weight per spike had significant positive values. also, there were significant positive coefficients of correlation between the grain weight per spike with the number of grains per spike and the number of plants per m2. based on the results of the research it can be concluded that a greater number of traits have a decisive role in the formation of grain yield. the contribution of each individual trait may be different in various genotypes and in various environmental conditions so that the correlation between two quantitative traits is not fixed. this suggests that the selection of genotypes with longer spike with good resistance to biotic and abiotic stresses contributed to the increase in production and yield stability of wheat. acknowledgements. investigations necessary for this paper are part of the project tp 31054 and 31057 financed by the ministry of education, science and technology development of republic of serbia. references biberdžić, m., đorđević, m., barać, s., deletić, n., stojković, s. 2005: productivity of some winter wheat genotypes. genetika, 37(2): 131-136. borojević, s., williams, w. 1992: genotype x enviroment interactions for leaf area parametars and yield components and their effects on wheat yield. crop science 22(5): 1020-1025. deletić, n., stojković, s., gudžić, s., đurić, v., aksić, m. 2012: genotypic specificity of some winter wheat traits and their effect on grain yield. genetika, 44(2): 249-258. denčić, s., kastori, r., kobiljski, b., duggan, b. 2000: evaluation of grain yield and its components in wheat cultivars and landraces under near optimal and drought conditions. euphytica, 113: 43-52. dencic, s., kobiljski, b. 2007: organization of wheat genetic resources in collections. uvodno predavanje. 1 st joint psu-uns international biologica nyssana 9 (2) ⚫ december 2018: 119-131 terzić et al. ⚫ yield components and yield of winter wheat in... 129 conference on bioscience: food, agriculture, and the environment. hat yai, thailand. ia 04: 6. denčić, s., kobiljski, b., mladenović, g., jestrović, z., štatkić, s., pavlović, m., orbović, b. 2010: sorta kao faktor proizvodnje pšenice. ratarstvo i povrtarstvo, 47(1): 317-324. dimitrijević, m., knežević, d., petrović, s., zečević, v., bošković, j., belić, m., pejić, b., banjac, b. 2011: stability of yield components in wheat (triticum aestivum l.). genetika, 43(1): 29-39. dodig, d., zorić, m., knežević, d., king, s., šurlanmomirović, g. 2008: genotype x environment interaction for wheat yield in different drought stress conditions and agronomic traits suitable for selection. australian journal of agricultural research, 59: 536-545. dodig, d. (2010): wheat breeding for drought resistance. monografija, društvo genetičara srbije. donmez, e., sears, r., shroyer, j., paulsen, g. 2001: genetic gain in yield attributes of winter wheat in the great plains. crop science, 41: 1412-1419. đekić, v., milovanović, m., staletić, m., stevanović, v., milivojević, j. 2012: influence of growing season on some agronomic characteristics of six winter wheat cultivars grown in acidic soil. proceedings. 47rd croatian and 7rd international symposium on agriculture, 13.-17. februar, opatija, croatia, 478-482. đekić, v., staletić, m., jelić, m., popović, v., branković, s. 2013: the stability properties of wheat production on acid soil. proceedings, 4th international symposium "agrosym 2013", 03-06. oktober, jahorina, 84-89. đekić, v., milovanović, m., popović, v., milivojević, j., staletić, m., jelić, m., perišić, v. 2014: effects of fertilization on yield and grain quality in winter triticale. romanian agricultural research, 31: 175-183. đekić, v., milovanović, m., milivojević, j., staletić, m., popović, v., simić, d., mitrović, m. 2015: impact of year and grain quality of winter wheat. proceedings of research papers pkb agroekonomik, belgarde, 21(1-2): 79-86. đurić, n., trkulja, v., simić, d., prodanović, s., đekić, v., dolijanović, ž. 2013: analiza prinosa zrna i kvaliteta brašna nekih sorata ozime pšenice u proizvodnoj 2011-2012. godini. zbornik naučnih radova instituta pkb agroekonomik, 19(1-2): 15-21. đurić, n., cvijanović, g., dozet, g., matković, m., branković, g., đekić, v. 2016: correlation analysis of more significant production traits of certain winter wheat pkb varieties. agronomy journal, 78(2-3): 85-96. đurić, n., trkulja, v., cvijanović, v., branković, g., đekić, v., spasić, m., ivanović, d. 2018: imperija-nova sorta ozime pšenice stvorena u institutu pkb agroekonomik. zbornik naučnih radova instituta pkb agroekonomik, 24(1-2): 5964. djuric, n., prodanovic, s., brankovic, g., djekic, v., cvijanovic, g., zilic, s., dragicevic, v., zecevic, v., dozet, g., 2018: correlation-regression analysis of morphological-production traits of wheat varieties. romanian biotechnological letters, 23(2): 13457-13465. erekul, o., köhn, w. 2006: effect of weather and soil conditions on yield components and bredmaking quality of winter wheat (triticum aestivum l.) and winter triticale (triticosecale wittm.) varieties in north-east germany. journal of agronomy & crop science, 192(6): 452-464. garcía del moral, l.f., garcía del moral, m.b., molina-cano, j.l., slafer, g.a. 2003: yield stability and development in two-and six-rowed winter barleys under mediterranean conditions. field crops research, 81: 109-119. hagos, h., abay, f. 2013: ammi and gge biplot analysis of bread wheat genotypes in the northern part of ethiopia. journal of plant breeding and genetics, 01: 12-18. hassan, m., mohamed, g., el-said, r. 2013: stability analysis for grain yield and its components of some durum wheat genotypes (triticum durum l.) under different environments. asian journal of crop science, 5(2): 179-189. hodson, d., white, j. 2009: climate change: what future for wheat? in: wheat facts and futures 2009. cimmyt, 52-61. hristov, n., mladenov, n., špika, a.k., štatkić, s., kovačević, n. 2008: direktni i indirektni efekti pojedinih svojstava na prinos zrna pšenice. zbornik radova institut za ratarstvo i povrtarstvo novi sad, 45: 15-20. hristov, n., мladenov, n., кondić-špika, а. 2011: effect of environmental and genetic factors on the correlation and stability of grain yield components in wheat. genetika, 43(1): 141-152. hristov, n., mladenov, n., kondić-špika, a., jocković, b. 2012: novosadske sorte pšenice u agroekološkim uslovima vojvodine. zbornik naučnih radova instituta pkb agroekonomik, 18(1-2): 21-28. hristov, n., mladenov, n., kondić-špika, a., jocković, b. 2013: uticaj padavina i temperature na prinos ozime pšenice pri različitim gustinama setve. zbornik naučnih radova instituta pkb agroekonomik, 19(1-2): 31-38. biologica nyssana 9 (2) ⚫ december 2018: 119-131 terzić et al. ⚫ yield components and yield of winter wheat in... 130 hristov, n., mladenov, n., jocković, b., kondićšpika, a. 2014: uticaj sorte, lokaliteta i godine na prinos ozime pšenice. zbornik naučnih radova instituta pkb agroekonomik, 20(1-4): 33-40. iftikhar, r., khaliq, i., ijaz, m., rashid, rahman, a.m. 2012: association analysis of grain yield and its components in spring wheat (triticum aestivum l.). american-eurasian journal of agricultural and environmental science, 12(3): 289-392. janušauskaite, d., šidlauskas, g. 2004: nitrogen fertilizer efficacy in winter wheat in relation to weather condition in central lithuania. zemdirbyste-agriculture, 88(4): 34-47. jaćimović, g., malešević, m., aćin, v., marinković, b., crnobarac, j., latković, d., bogdanović, d., pejić, b. 2011: efikasnost mineralne ishrane pšenice u zavisnosti od intenziteta đubrenja. letopis naučnih radova, 35(1): 75-86. jaćimović, g., malešević, m., aćin, v., hristov, n., marinković, b., crnobarac, j., latković, d. 2012: komponente prinosa i prinos ozime pšenice u zavisnosti od nivoa đubrenja azotom, fosforom i kalijumom. letopis naučnih radova, 36(1): 72-80. jelić, m., milivojević, j., paunović, a., biberdžić, m., nikolić, o., madić, m., đekić, v. 2012: response of wheat genotypes to liming and fertilization on pseudogley soil. proceedings. 47rd croatian and 7rd international symposium on agriculture, 13.-17. februar, opatija, croatia, 488-491. jelić, m., milivojević, j., đekić, v., paunović, a., tmušić, n. 2014: impact of liming and fertilization on grain yield and utilization of nitrogen and phosphorus in wheat plant grown on soil type pseudogley. proceedings of research papers pkb agroekonomik, belgarde, 20(1-4): 49-56. jelic, m., milivojevic, j., nikolic, o., djekic, v., stamenkovic, s. 2015: effect of long-term fertilization and soil amendments on yield, grain quality and nutrition optimization in winter wheat on an acidic pseudogley. romanian agricultural research, 32: 165-174. jocković, b., mladenov, n., hristov, n., aćin, v., đalović, i. 2014: interrelationship of grain filling rate and other traits that affect the yield of wheat (triticum aestivum l.). romanian agricultural research, 31: 1-7. joshi, s.k., sharma, s.n., singhania, sain, r.s. 2002: genetic analysis of quantitative and quality traits under varying environmental conditions in bread wheat. wheat information service, 95: 510. kandić, v., dodig, d., jović, m., nikolić, b., prodanović, s. 2009: the importance of physiological traits in wheat breeding under irrigation and drought stress. genetika, 41(1): 1120. kasihf, m., khaliq i. 2004: heritability, correlation and path coefficient analysis for some metric traits in wheat. internationl journal agriculture biology, 6(1): 138-142. laghari, g.m., oad, f.c., tunio, s., chachar, q., gandahi, a.w., siddiqui, m.h. 2011: growth and yield attributes of wheat at different seed rates. sarhad journal of agriculture, 27: 177-183. luković, k., milovanović, m., perišić, v., bratković, k., staletić, m. 2014: variety perfekta-another contribution to biodiversity of winter wheat in serbia. proceedings, xviii international ecoconference 2014, 8th eco-conference ® on safe food, 24-27. september 2014, novi sad, 173180. madić, m., paunović, a., đurović, d. 2010: prinos zrna sorti pšenice centra za strna žita kragujevac u različitim agroekološkim uslovima. prvi naučni simpozijum agronoma sa međunarodnim učešćem, agrosym, jahorina, bih, 371-376. malešević, m., starčević, lj., jaćimović, g., đurić, v., šeremešić, s., milošev, d. 2008: prinos ozime pšenice u zavisnosti od uslova godine i nivoa đubrenja azotom. xiii savetovanje o biotehnologiji, čačak, 28-29. mart, 2008, zbornik radova, 13(14): 135-141. malešević, m., jaćimović, g., jevtić, r., aćin, v. 2011: iskorišćavanje genetskog potencijala pšenice u uslovima abiotičkog stresa. zbornik referata sa 45. savetovanja agronoma srbije, 30.01-05.02., zlatibor, 4-14. milovanović, m., perišić, v., bratković, k. 2008: vizija-kg sorta ozime pšenice. zbornik radova više tehničke škole požarevac, 1-2: 66-72. milovanović, m., staletić, m., đekić, v., nikolić, o., luković, k. 2011: seed production and contribution of kg varieties to biodiversity of small grains in the period 2006-2010. 01.-02. decembar 2011, beograd, economics of agriculture, ii(58): 103-111. milovanović, m., staletić, m., rajičić, v., nikolić, o., perišić, v. 2012: actualities of hard winter wheat breeding in center for small grains in kragujevac. xvi international eco-conference, novi sad, proceedings safe food, 115-123. mladenov, n., denčić, s., hristov, n. 2007: oplemenjivanje na prinos i komponente prinosa zrna pšenice. zbornik radova instituta za ratarstvo i povrtarstvo, 43: 21-27. mohamed, n., said, a., amein, k. 2013: additive main effects and multiplicative interaction (ammi) and gge-biplot analysis of genotype × environment interactions for grain yield in bread biologica nyssana 9 (2) ⚫ december 2018: 119-131 terzić et al. ⚫ yield components and yield of winter wheat in... 131 wheat (triticum aestivum l.). african journal of agricultural research, 8(42): 5197-5203. perišić, v., milovanović, m., đekić, v., staletić, m. 2011: nasleđivanje dužine klasa i broja zrna u klasu kod hibrida pšenice. zbornik pkb, 17(1-2): 19-26. perišić, v., perišić, v., živanović, t., milovanović, m., đekić, v., staletić, m., luković, k. 2016: comparative stability analysis of yield components of wheat genotypes. proceedings, xx international eco-conference ® 2016, 9th ecoconference ® on safe food, 28-30. september 2016, novi sad, 63-72. perišić, v. 2016: varijabilnost osobina i stabilnost prinosa i komponenti rodnosti ozime pšenice. doktorska disertacija, poljoprivredni fakultet, beograd.. petrović, s., dimitrijević, m., belić, m., banjac, b., bošković, j., zečević, v., pejić b. 2010: the variation of yield components in wheat (triticum aestivum l.) in response to stressful growing conditions of alkaline soil. genetika, 42(3): 545555. popović, a., babić, v., kravić, n., serčanski, m., prodanović, s. 2014: mogući pravci oplemenjivanja i poljoprivredne mere u cilju prilagođavanja biljaka na klimatske promene u srbiji. selekcija i semenarstvo, 20(2): 59-72. popović, v., glamočlija, đ., malešević, m., ikanović, j., dražić, g., stanković, s. 2011: genotype specificity in nitrogen nutrition of malting barley, genetika, 43(1): 197-204. popović, v., glamočlija, đ., sikora, v., đekić, v., červenski, j., simić, d. 2013: genotypic specificity of soybean [glycine max (l.) merr.] under conditions of foliar fertilization. romanian agricultural research, 30: 259-270. przulj, n., momcilovic, v., simic, j., mirosavljevic, m. 2014: effect of year and variety on barley quality. genetika, 46(1): 59-73. sas/stat 2000: user's guide, version 9.1.3. sas institute inc. stevanović, p., popović, v., jovović, z., ugrenović, v., rajičić, v., popović, s., filipović, v. 2018: kvalitet semena pšenice u zavisnosti od veličine frakcije i lokaliteta gajenja. zbornik naučnih radova instituta pkb agroekonomik, 24(1-2): 6574. terzic, d., đekić, v., jevtic, s., popovic, v., jevtic, a., mijajlovic, j., jevtic, a. 2018: effect of long term fertilization on grain yield and yield components in winter triticale. the journal of animal and plant sciences, 28(3): 830-836. williams, r.m., o’brien, l., eagles, h.a., solah, v.a., jayasenа, v. 2008: the influences of genotype, environment, and genotype х environment interaction on wheat quality. australian journal of agricultural research, 59: 95-111. zafaranaderi, n., aharizad, s., moha-mmadi, s.a. 2013: relationship between grain yield and related agronomic traits in bread wheat recombinant inbred lines under water deficit condition. annals of biological research, 4(4): 711. zecevic, v., knezevic, d., micanovic, d. 2004: genetic correlations and path-coefficient analysis of yield and quality components in wheat (triticum aestivum l.). genetika, 36(1): 13-21. zecevic, v., boskovic, ј., knezevic, d., micanovic, d., madic, м. 2010: ecological and genetic variability of wheat quality components. kragujevac journal of science, 32: 89-94. karalija, e., parić, a.: effects of salicylic acid foliar application on germination, growth and antioxidant potential of basil (ocimum basilicum l.). biologica nyssana, 8 (2) biologica nyssana 8 (2)  december 2017: 145-150 karalija, e., parić, a.  effects of salicylic acid foliar application on… 145 original article received: 24 september 2017 revised: 8 december 2017 accepted: 22 december 2017 effects of salicylic acid foliar application on growth and antioxidant potential of basil (ocimum basilicum l.) erna karalija*, adisa parić biology department, faculty of natural sciences and mathematics, university of sarajevo, zmaja od bosne 33-35, 71000 sarajevo, bosnia and herzegovina * e-mail: erna.k@pmf.unsa.ba abstract: karalija, e., parić, a.: effects of salicylic acid foliar application on growth and antioxidant potential of basil (ocimum basilicum l.). biologica nyssana, 8 (2), december, 2017: 145-150. salicylic acid is one of endogenous plant growth regulators that plays a key role in many physiological processes. the present study analysed the effect of different concentrations (0, 0.01, 0.1, ad 1.0 mm) of salicylic acid on morphological parameters, photosynthetic pigments, protein, proline, total carbohydrates, and secondary metabolites content as well as peroxidase activity. one month after sowing seedlings were replanted in new pots, and salicylic acid was applied in form of a foliar spray. plants were harvested 60 days after salicylic acid application. results showed that foliar application of salicylic acid induces long-term changes in plant growth and metabolism. we recorded increase in leaf area, secondary metabolites and peroxidase activity. reduction in total sugar and proline content is also recorded. decrease in proline content is probably result of degradation of proline in stress induced conditions. key words: chlorophylls; salicylic acid; secondary metabolites; proline; peroxidases; total carbohydrates apstrakt: karalija, e., parić, a.: efekat folijarne primene salicilne kiseline na rast i antioksidativni potencijal bosiljka (ocimum basilicum l.). biologica nyssana, 8 (2), decembar, 2017: 145-150. salicilna kiselina je jedan od endogenih biljnih regulatora rasta sa ključnom ulogom u mnogim fiziološkim procesima. prezentirani rad analizira efekte različitih koncentracija (0.01, 0.1, ad 1.0 mm) salicilne kiseline na morfološke parametre, fotosintetske pigmente, proteine, ukupne ugljikohidrate i sadržaj sekundarnih metabolite kao i peroksidaznu aktivnost. mesec dana nakon setve biljke su presađene u nove posude i salicilna kiselina je aplicirana u formi folijarnog spreja. biljke su prikupljene za analizu 60 dana nakon aplikacije salicilne kiseline. rezultati su pokazali da folijarna aplikacija salicilne kiseline indukuje dugoročne promene u biljnom rastu i metabolizmu. zabeženo je povećanje površine lista, sadržaja sekundarnih metabolita i peroksidazne aktivnosti. također, zabeležena je redukcija u sadržaju ukupnih šećera i sadržaja prolina. redukcija sadržaja prolina verovatno je rezultat degradacije prolina indukovane stresnim uslovima. ključne reči: hlorofili; salicilna kiselina; sekundarni metaboliti; prolin; peroksidaze; ukupni ugljikohidrati 8 (2) • december 2017: 145-150 doi: 10.5281/zenodo.1135966 biologica nyssana 8 (2)  december 2017: 145-150 karalija, e., parić, a.  effects of salicylic acid foliar application on… 146 introduction physiological efficiency of a plant can be improved by plant growth regulators through their effect on photosynthesis, flower and fruit formation and overall productivity of the plant (a s g h a r i & a g h d a m , 2010). foliar application of plant feeding agents usually influences more plant characteristics and yield then those applied in the soil (k a z e m i , 2013). positive effects of salicylic acid (sa) have been recorded in wheat with recorded enhancement of germination rate and seedling growth (s h a k i r o v a , 2007). also activation of antioxidant enzymes such as peroxidases have been reported after plant spraying with salicylic acid, especially in drought stressed plants (h a y a t et al., 2008). although numerous studies have proven positive effects of salicylic acid it is still a subject of interest due to dependence of the salicylic acid effects upon species, developmental stage, application mode as well as concentration of sa used (v a n a c k e r et al., 2001, h o r v á t h et al., 2007). lower concentrations of salicylic acid have been beneficial for plant growth (v i c e n t e & p l a s e n c i a , 2011), while different results for metabolite content have been reported (w a n g et al., 2007, d ’ o n o f r i o et al., 2009). sweet basil (ocimum basilicum l.), member of lamiaceae family, is used as a fresh vegetable for many traditional foods, dressings and salads. its medicinal value and organoleptic quality is a result of basil aroma and essential oil composition. aim of this study was to investigate the long-term effect of short exposure to foliar application of salicylic acid on growth performances and quality of basil plants. material and methods this study was conducted in laboratory for plant physiology at faculty of natural sciences and mathematics, university of sarajevo. plants were grown in controlled growth chambers with artificial light system and main daily temperature of 22 °c and 75% humidity. plant material and treatments the experiment was conducted in a factorial arranged (2 x 2 x 2) randomised block design with five replications with sa (salicylic acid) as main factor in growth chamber of laboratory for plant physiology at department for biology, faculty of natural sciences and mathematics, university of sarajevo during 2014 and 2015. commercial obtained sweet basil (green paradise s.r.l) seeds were planted in trays. one month old seedlings with two developed leaves similar in size were selected and transferred into 10 cm in diameter plastic containers containing about 3 kg of commercial soil. when 4-6 leaves were developed sa was sprayed in ratio 0.01, 0.1 and 1.0 mm until both sides of leaves were wet. two months after salicylic acid application plant material was collected (six plants from each replication) for further analysis. plant mass and leaf area analysis all plants were carefully removed from soil, roots were washed to remove soil residues and fresh and dry weight (dried at 65 °c for 72 hours) was recorded. analysis of water content was calculated according to the formula: %𝑊𝐶 = 𝐴 − 𝐵 𝐴 where: %wc percentage of water content; a fresh mass (g); b dry mass (g) leaves were photographed on millimetre paper and leaf area was determined using imagej software. photosynthetic pigments content photosynthetic pigments were determined from 80% acetone extract by absorbance reading at 663, 646 and 440 nm according to the method of to a r n o n (1949). calculations were made according to the equations proposed by p o r r a et al. (1989) and h o l m (1954): chlorophyll a = 12.25*a663 − 2.55 ∗ a646 (µg/ml) chlorophyll b = 20.31*a646-4.91*a663 (µg/ml) total chlorophylls = 17.76*a646-7.34*a663 (µg/ml) carotenoids = 4.69*a440-0.267×(a663*a640) (µg/ml) proline content proline content was determined according to the method described by c a r i l l o et al. (2008). dried leaf material (50 mg) was homogenized in 5 ml of ethanol : water mixture (60:40) and homogenate was incubated for 24 h at +4 °c, then centrifuged at 10 000 rpm to obtain supernatant. 500 l of supernatant was mixed with 1000 ml of 1% acid ninhydrin. the reaction mixture was placed in water bath for 20 minutes at 95°c, after which was cooled to room temperature and the absorbance was measured at 520 nm. proline standard was used for calibration curve and calculation of proline content in samples and expressed as mg proline per g of dry weight (mg pro/gdw). secondary metabolite analysis phenolics were extracted from aerial parts of plants by maceration in 80% methanol (hplc grade) and biologica nyssana 8 (2)  december 2017: 145-150 karalija, e., parić, a.  effects of salicylic acid foliar application on… 147 incubation for 24h at 4 °c. extracts were centrifuged at 2000 rpm for 15 min, and supernatants were collected for further analysis. total phenolic content was analysed according to w o l f e et al. (2003) using folin-ciocalteu reagent and gallic acid as standard and it was expressed as mg of gallic acid equivalent per g of dry weight (mggae/gdw). total flavonoid content was done using aluminium chloride method (o r d o n e z et al., 2006) with catechin as standard. results were expressed as mg of catechin equivalent per g of dry weight (mgce/gdw). total flavanol content was analysed according to modified method of g a d z o v s k a et al. (2007) using 1% dmaca reagent (p-dimethyl aminocinnamaldehyde in hcl:ch3oh, 8:92) and catechin as standard. results were expressed as mg of catechin equivalent per g of dry weight (mgce/gdw). determination of soluble sugar content dry samples (0.1 g) were grinded and extracted with 2.5 ml of 80% (v/v) ethanol at 90 °c for 60 min, followed by centrifugation at 10 000 g at 4 °c for 10 minutes. the process was repeated for complete extraction. total soluble sugar content was determined using anthrone reagent and glucose as standard (r o e, 1955). results were expressed as mg soluble sugar/g dw. total protein content total protein content was determined according to the method of b r a d f o r d (1976) grinding 50 mg of fresh leaf sample in phosphate buffer (ph 7.0). the extract was centrifuged at 10 000 rpm for 30 min at +4 °c. absorbance of reaction mixture containing supernatant and diluted bradford reagent was done at 765 nm against blank. peroxidase activity extract obtained for protein analysis was used for total peroxidase activity according to g o n z a l e s et al. (1984). the reaction mixture was prepared by mixing 20 l of extract; phosphate buffer (ph 7.0); guaiacol (20 mm) and h2o2 (10 mm). the absorbance of reaction mixture was recorded continuously during 120 s against blank. statistical analysis all obtained data were analysed for significant differences using factorial analysis of variance. statistical analysis was performed using statistica 10 software and the means were compared using newman-keuls test at p<0.05. results ad discussion effect of sa on growth parameters the statistical analysis showed that application of salicylic acid increased leaf area of basil seedlings in concentration dependent manner (tab. 1). dry mass and water content did not differ between different treatments. statistically significant reduction in photosynthetic pigment contents was recorded dependent upon applied concentration indicating reducing effect of salicylic acid on photosynthesis. reduction in photosynthetic pigment content was probably the reason for increase in leaf area as a response to reduced photosynthetic activity. p a n c h e v a et al. (1996) showed that long-term treatment (7 days) of barley seedlings with sa decreased the rate of photosynthesis (chlorophyll content) and our results are in accordance with these findings indicating that this negative effect of salicylic acid on photosynthetic pigments is present also after 2 months. table 1. effect of salicylic acid on growth parameters of basil salicylic acid, mm leaf area, mm2 dry mass, g water content, % chla, mg/gdw chlb, mg/gdw chla+b, mg/gdw car, mg/gdw 0 65.825 ± 4.651d 0.095 ± 0.005a 0.919 ± 0.010a 5.404 ± 0.188a 2.341 ± 0.109a 7.827 ± 0.298a 1.068 ± 0.025a 0.01 83.739 ± 13.972c 0.092 ± 0.005a 0.931 ± 0.004a 5.280 ± 0.033b 2.235 ± 0.022b 7.596 ± 0.055b 1.059 ± 0.002b 0.1 86.749 ± 25.654b 0.107 ± 0.008a 0.925 ± 0.004a 4.650 ± 0.049c 2.026 ± 0.024c 6.746 ± 0.074c 0.935 ± 0.008c 1.0 90.650 ± 15.782a 0.081 ± 0.001a 0.943 ± 0.006a 1.560 ± 0.037d 0.783 ± 0.022d 2.367 ± 0.060d 0.413 ±0.008d values within one parameter not sharing the same letter are significantly different at p<0.05 biologica nyssana 8 (2)  december 2017: 145-150 karalija, e., parić, a.  effects of salicylic acid foliar application on… 148 reduction of chlorophyll content is usual plant response to stress conditions and since sa plays role in stress induced signalling application of sa can result in reduction of chlorophylls (f a r o o q et al., 2009). physiological and biological processes in plants are strongly regulated and sa plays significant role in this regulation. as such sa can be used to improve plant growth under environmental conditions, but the efficiency of exogenous sa can vary dependent upon the species, developmental stage, application method and sa concentration (b o r s a n i et al., 2001). effect of sa on proline content the analysis of variance for proline content showed that lower concentrations of sa induced decrease in proline content while the highest used concentration of sa (1.0 mm) induced statistically significant increase in proline content (tab. 2). in many cases it was recorded that proline content increases under stress conditions (n a y y a r & w a l i a , 2003). also considering that analysis was 2 months after sa application, low proline content in plants sprayed with 0.01 and 0.1 mm sa could be explained by the breakdown of proline upon relief of stress providing sufficient reducing agents for mitochondrial oxidative phosphorylation and atp generation for recovery from stress and alleviating stress-induced damages (h a r e & c r e s s , 1998, h a r e et al., 1998). proline can be considered as one of the protective mechanisms induced by sa application in basil plants, but no long term accumulation is achieved in this experiment. effect of sa on secondary metabolites foliar application of sa induced statistically significant (tab. 2) long-term increase in secondary metabolite content. k a b i r i et al. (2014) recorded no changes in polyphenol content after sa application in control plants presenting only short term effects of sa on polyphenols. in our experiment long-term effects of salicylic acid result in significant increase in polyphenol content dependent upon applied sa concentration similar results of increase in metabolite production was recorded by g h a r i b (2006). increase of secondary metabolite production in basil under stress condition has been previously noted (k a r a l i j a et al., 2016). soluble sugar content the results showed that sugar content decreased dependent upon applied sa concentration (tab. 2). similar sa induced reduction of soluble sugar content in plants that are not under stress conditions was previously recorded in nigella sativa plants (k a b i r i et al., 2014). our results show that this adverse effect of salicylic acid on sugar content is present also after 2 months. protein content and peroxidase activity application of sa induced significant increase in protein content dependent upon applied sa concentration (tab. 2). considering two month period from the sa application, this increase in protein content could be considered as a permanent effect of sa application. considering that protein content is usually decreased when plants are exposed to stress (k a b i r i et al., 2014) plants with higher protein content are primed for upcoming stress conditions. peroxidase activity was significantly higher in plants sprayed with 0.01 and 0.1 mm sa as compared with control plants and plants sprayed with 1.0 mm, even after 2 months (fig. 1). these differences between peroxidase activities are probably result of activation of defence mechanisms. table 2. effect of salicylic acid on total protein, secondary metabolites and total carbohydrates content in basil treatment total proteins, mg/gdw) proline, mg/gdw total flavonoids, mg/gdw total phenols, mg/gdw total flavanols, mg/gdw total carbohydrates, mg/gdw k 0.508 ±0.019a 0.069 ± 0.009b 9.504 ± 0.169c 2.672 ± 0.431cd 3.663 ± 0.146c 41.651 ± 0.774a 001sa 0.351 ± 0.001b 0.028 ± 0.004c 14.909 ± 0.866b 2.981 ± 0.327b 4.383 ± 0.478b 38.776 ± 0.887b 01sa 0.391 ± 0.031b 0.033 ± 0.002c 15.708 ± 0.867b 2.803 ± 0.532cb 4.658 ± 0.014b 38.460 ± 0.742b 1sa 0.551 ± 0.019a 0.107 ± 0.003a 17.609 ± 0.361a 3.528 ± 0.371a 6.249 ± 0.057a 29.370 ± 0.700c values within one parameter not sharing the same letter are significantly different at p<0.05 biologica nyssana 8 (2)  december 2017: 145-150 karalija, e., parić, a.  effects of salicylic acid foliar application on… 149 foliar pre-treatment of plants with abiotic elicitors induce plant resistance for different stressors. one of chemicals responsible for defence induction are phytohormones (v i c e n t e & p l a s e n c i a , 2011; m a f f e i et al., 2007, w a r et al., 2011). induction of plant defence is usually mediated through physiological, biochemical and molecular mechanisms (v i c e n t e & p l a s e n c i a , 2011). salicylic acid is an important endogenous plant growth regulator that induces different metabolic and physiological responses in plants involved in plant defence as well as in plant growth and development (c h e n et al., 2009, h a y a t et al., 2009, z h a o et al., 2009). conclusion foliar application of sa induces growth as well as biochemical changes in basil plants and these changes are evident also after 2 months. having in mind presented results we can conclude that sa induces not only immediate plant defence response but also has long-term effect on plant growth and metabolism. such effect primes the plant also for future upcoming stress conditions. references arnon, d.i. 1949: copper enzymes in isolated chloroplasts. polyphenoloxidase in beta vulgaris. plant physiology, 24 (1): 1. asghari, m., aghdam, m.s. 2010: impact of salicylic acid on post-harvest physiology of horticultural crops. trends in food science & technology, 21: 502-509. borsani, o., valpuesta, v., botella, m.a. 2001: evidence for a role of salicylic acid in the oxidative damage generated by nacl and osmotic stress in arabidopsis seedlings. plant physiology, 126: 1024-1030. bradford, m.m. 1976: a rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. analytical biochemistry, 72 (1-2): 248254. carillo, p., mastrolonardo, g., nacca, f., parisi, d., verlotta, a., fuggi, a. 2008: nitrogen metabolism in durum wheat under salinity: accumulation of proline and glycine betaine. functional plant biology, 35: 412-426. chen, z., zheng, z., haung, j., lai, z., fan, b. 2009: biosynthesis of salicylic acid in plants. plant signaling &behavior, 4: 493-496. d’onofrio, c., cox, a., davies, c., boss, p.k. 2009: induction of secondary metabolism in grape cell cultures by jasmonates. functional plant biology, 36: 323-338. farooq, m., basra, s.m., wahid, a., ahmad, n., saleem, b.a. 2009: improving the drought tolerance in rice (oryza sativa l.) by exogenous application of salicylic acid. journal of agronomy and crop science: 195: 237-246. gadzovska, s., maury, s., delaunay, a., spasenoski, m., joseph, c., hagege, d. 2007: jasmonic acid elicitation of hypericum perforatum l. cell suspensions and effects on the production of phenylpropanoids and naphtodianthrones. plant cell, tissue and organ culture, 89 (1): 1-13. gharib, f.a., 2006: effect of salicylic acid on the growth, metabolic activities and oil content of basil and marjoram. international journal of agriculture & biology, 4: 485-492. gonzales, r., auclair, c., voisin, e., gautero, h., dhermy, d., boivin, p. 1984: superoxide dismutase, catalase, and glutathione peroxidase in red blood cells from patients with malignant diseases. cancer research, 44: 4137-4139. fig. 1. effect of salicylic acid on peroxidase activity in basil two months after treatment 0.307c 0.440a 0.397 b 0.291c 0.165c 0.239a 0.225a 0.190b 0,000 0,100 0,200 0,300 0,400 0,500 0 001sa 01sa 1sa sa60 sa120 0.500 0.400 0.300 0.200 0.100 0.000 biologica nyssana 8 (2)  december 2017: 145-150 karalija, e., parić, a.  effects of salicylic acid foliar application on… 150 hare, p.d., cress, w.a. 1997: metabolic implications of stress-induced proline accumulation in plants. plant growth regulation, 21: 79-102. hare, p.d., cress, w.a., van staden, j. 1998: dissecting the roles of osmolyte accumulation during stress. plant, cell & environment, 21: 535553. hayat, q., hayat, s., irfan, m., ahmad, a. 2009: effect of exogenous salicylic acid under changing environment: a review. environmental and experimental botany, 68: 14-25. hayat, s., hasan, s.a., fariduddin, q., ahmad, a. 2008: growth of tomato (lycopersicon esculentum) in response to salicylic acid under water stress. journal of plant interactions, 3: 297304. holm, g. 1954: chlorophyll mutations in barley. acta agriculturae scandinavica, 4: 457-471. horváth, e., pál, m., szalai, g., páldi, e., janda, t. 2007: exogenous 4-hydroxybenzoic acid and salicylic acid modulate the effect of short-term drought and freezing stress on wheat plants. biologia plantarum: 51: 480-487. kabiri, r., nasibi, f., farahbakhsh, h. 2014: effect of exogenous salicylic acid on some physiological parameters and alleviation of drought stress in nigella sativa plant under hydroponic culture. plant protection science, 50: 43-51. kazemi, m. 2013: foliar application of salicylic acid and calcium on yield, yield component and chemical properties of strawberry. bulletin of environment, pharmacology and life sciences, 2: 19-23. maffei, m.e., mithofer, a., boland, w. 2007: insects feeding on plants: rapid signals and responses preceding the induction of phytochemical release. phytochemistry, 68: 2946-2959. nayyar, h., walia, d.p. 2003: water stress induced proline accumulation in contrasting wheat genotypes as affected by calcium and abscisic acid. biologia plantarum, 46: 275-279. ordonez, a.a.l., gomez, j.d., vattuone, m.a., 2006: antioxidant activities of sechium edule (jacq.) swartz extracts. food chemistry, 97 (3): 452-458. pancheva, t.v., popova, l.p., uzunova, a.n., 1996: effects of salicylic acid on growth and photosynthesis in barley plants. journal of plant physiology, 149 (1-2): 57-63. porra, r.j., thompson, w.a., kriedemann, p.e. 1989: determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectroscopy. biochimica et biophysica acta (bba)-bioenergetics, 975 (3): 384-394. roe, j.h., 1955: the determination of sugar in blood and spinal fluid with anthrone reagent. journal of biological chemistry, 212: 335-343. shakirova, fm. 2007: role of hormonal system in the manifestation of growth promoting and antistress action of salicylic acid. in: hayat, s., ali, b, ahmad a. (eds). salicylic acid: a plant hormone. 69-89, springer, netherlands. vanacker, h., lu, h., rate, d.n., greenberg, j.t. 2001: a role for salicylic acid and npr1 in regulating cell growth in arabidopsis. plant pathology journal, 28: 209-216. vicente, r.s., plasencia, j. 2011: salicylic acid beyond defence: its role in plant growth and development. journal of experimental botany, 62: 3321-3338. wang, y.d., wu, j.c., yuan, y.j. 2007: salicylic acid-induced taxol production and isopentenyl pyrophosphate biosynthesis in suspension cultures of taxus chinensis var. mairei. cell biology international, 31: 1179-1183. war, a.r., paulraj, m.g., war, m.y., ignacimuthu, s. 2011: herbivoreand elicitor-induced resistance in groundnut to asian armyworm, spodoptera litura (fab.) (lepidoptera: noctuidae). plant signaling & behavior, 6: epub. wolfe, k., wu, x., liu, r.h. 2003: antioxidant activity of apple peels. journal of agricultural and food chemistry, 51(3): 609-614. zhao, l.y., chen, j.l., cheng, d.f., sun, j.r., liu, y., tian, z. 2009: biochemical and molecular characterizations of sitobion avenae-induced wheat defense responses. crop protection, 28: 435-442. nikolić, d. et al.  the influence of orographical and bioclimatic factors … biologica nyssana 6 (1)  september 2015: 1-9 nikolić, d. et al.  the influence of orographical and bioclimatic factors … 1 original article received: 15 july 2015 revised: 18 august 2015 accepted: 01 september 2015 the influence of orographical and bioclimatic factors on morphological variability of analyzed characters of jovibarba heuffelii (schott) a. löve & d. löve (crassulaceae) danijela nikolić1*, jasmina šinžar-sekulić2, vladimir ranđelović1, dmitar lakušić2 1university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia 2university of belgrade, faculty of biology, institute of botany and botanical garden „jevremovac”, takovska 43, 11000 belgrade, serbia * e-mail: danid@pmf.ni.ac.rs abstract: nikolić, d., šinžar-sekulić, j., ranđelović, v., lakušić, d.: the influence of orographical and bioclimatic factors on morphological variability of analyzed characters of jovibarba heuffelii (schott) a. löve & d. löve (crassulaceae). biologica nyssana, 6 (1), september 2015: 1-9. the goal of this paper was to determine the extent of morphological variability in species j. heuffelii caused by orographic and bioclimatic factors. samples were collected from 14 populations of species j. heuffelii, from the territories of serbia, macedonia, bulgaria and romania. for this purpose cluster analysis (upgma) on bioclimatic parameters and regression analysis were performed. the cluster analysis of bioclimatic factors has shown that the study area was influenced by semiarid temperate-continental or subcontinental climate, continental mountain climate and humid mountain climate. among the orographic factors, the greatest influence on morphological characters of species j. heuffelii was determined for altitude, exposition and slope of the terrain. the mean temperature of the wettest quartile (bio8) and temperature seasonality (bio4) have shown the greatest influence on the morphological characters of j. heuffelii. key words: jovibarba heuffelii, morphological variability, environmental factors apstrakt: nikolić, d., šinžar-sekulić, j., ranđelović, v., lakušić, d.: uticaj orografskih i bioklimatskih faktora na morfološku varijabilnost analiziranih karaktera jovibarba heuffelii (schott) a. löve & d. löve (crassulaceae). biologica nyssana, 6 (1), septembar 2015: 1-9. cilj ovog rada je bio da se utvrdi u kolikoj meri je velika morfološka varijabilnost kod vrste j. heuffelii uslovljena uticajem orografskih i bioklimatskih faktora. prikupljeni su uzorci 14 populacija vrste j. heuffelii sa područja srbije, makedonije, bugarske i rumunije. urađena je klaster analiza (upgma) sa bioklimatskim parametrima i regresiona analiza. klaster analiza bioklimatskih faktora je pokazala da je istraživano područje pod uticajem tri tipa klime: semiaridne umereno kontinentalne ili subkontinentalne klime, kontinentalne planinske klime i humidne planinske klime. od orografskih faktora pored nadmorske visine, ekspozicija i nagib terena imaju najveći uticaj na morfološke karaktere vrste j. heuffelii. srednja temperatura najvlažnijeg kvartala (bio8) i temperaturna sezonalnost (bio4) najviše utiču na morfološke karaktere j. heuffelii. key words: jovibarba heuffelii, morfološka varijabilnost, ekološki faktori 6 (1) • september 2015: 1-9 biologica nyssana 6 (1)  september 2015: 1-9 nikolić, d. et al.  the influence of orographical and bioclimatic factors … 2 introduction j. heuffelii (crassulaceae) is a succulent xerophyte primarily inhabiting rocky habitats at various substrates (limestone, serpentinite, silicate) in a range of altitudes from the coastline to 2500 m above sea level (b a r c a & n i c u l a e , 2005; l a k u š i ć et al., 2005; d i m i t r i j e v i ć et al., 2011). distribution of this species in balkan peninsula and the southern carpathians indicates that this is a european endemic species (m e u s e l , 1965; j a l a s et al., 1999). according to the climatic division of southeastern europe (h o r v a t et al., 1974) and division into main climate and biome types by w a l t e r and l e i t h (1964), there are two main climate types within the range of j. heuffelii complex: temperate-continental climate (type vi) and mountain climate (type x). the temperatecontinental climate is represented by subtype vi 3 semiarid temperate-continental climate (moesiancarpathian variant). within the mountain climate there are two subtypes: x 2 humid mountain climate of alpine type (illyrian variant) and x 3 – continental mountain climate (moesian-carpathian variant). this species is characterized by a very high morphological variability, as evidenced by recent studies on variability of morphological characteristics of vegetative and reproductive organs (d i m i t r i j e v i ć et al., 2011) and analysis of morphological characteristics of nectaries (n i k o l i ć et al., 2015a). the impact of orographic and bioclimatic habitat factors on morphological variability was analyzed in paper by n i k o l i ć et al. (2015b), while this paper pertains only to populations from identical mountain habitats, while gorge and canyon populations were omitted from the analysis. regression analysis and cluster analysis (upgma) based on matrix of bioclimatic parameters were performed in order to determine the extent of impact of orographic and bioclimatic factors on variability of morphological characteristics and differentiation of all analyzed populations. table 1. the list of population of j. heuffelii used in this study. vouchers are deposited in the herbarium of the institute of botany, faculty of biology, university of belgrade (beou) population coordinate substrate altitude voucher individuals 1. ro-domogled 44°52'41.70"n 22°25'54.11"e limestone 1300 beou16510 20 2. sr-gradac 44°15'17.98"n 19°53'23.00"e limestone 490 beou-16458 21 3. sr-suvaja 44°10'54.98"n 19°52'11.08"e limestone 417 beou-16457 20 4. sr-studenica 43°29'20.56"n 20°32'7.74"e serpentinite 486 beou-16461 20 5. srnebeske stolice 43°15'34.14"n 20°49'33.59"e serpentinite 1907 beou-16468 22 6. sr-treska 43°15'36.31"n 20°47'6.40"e serpentinite 1628 beou-16462 20 7. sr-basarski kamik 43° 9'37.29"n 22°42'9.57"e limestone 1350 beou16460 25 8. sr-radan 42°55'4.99"n 21°33'25.74"e silicate 802 beou-16456 20 9. sr-pljačkovica 42°34'47.20"n, 21°53'31.09"e silicate 674 beou-16465 21 10. srbesna kobila 42°31'45.08"n 22°13'51.10"e silicate 1900 beou16463 30 11. sr-stara planina 43°23'23.41"n 22°38'1.98"e silicate 1840 beou-16459 20 12. bu-trojanski prolaz 42°46'1.62"n 24°37'2.30"e silicate 1400 beou-16509 20 13. ma-treskavec 41°24'14.73"n 21°32'14.44"e silicate 1250 beou16511 20 14. mamavrovo 41°38'14.33"n 20°42'29.90"e limestone 1300 beou16512 20 biologica nyssana 6 (1)  september 2015: 1-9 nikolić, d. et al.  the influence of orographical and bioclimatic factors … 3 table 2. morphological characters investigated using the populations of j. heuffelii described in table 1. morphological characters abbreviations 1 diameter of rosette (mm) ros_d 2 number of leaves in rosette leros_n 3 length of biggest leaf (mm) leros_l max 4 width of biggest leaf (mm) leros_w max 5 distance of widest part of leaf from the top of the leaf (mm) apex_d_ros 6 length of spike (mm) leros_sp_l 7 length of cilia (mm) leros_ci_l 8 width of cartilaginous leaf edge (mm) leros_ed_w 9 height of stem to lowest flower branch (mm) ste_h 10 number of leaves at stem leste_n 11 length of middle leaf on stem (mm) midleste_l 12 width of middle leaf on stem (mm) midleste_w 13 distance of widest part of leaf from the top of the leaf (mm) apex_d_ste 14 number of floral branches flobra_n 15 number of flowers at stage of ripening fruit flo_n 16 length of longest branch in fruit (mm) flobra_l 17 length of sepal (mm) sep_l 18 width of sepal (mm) sep_w 19 length of petal (mm) pet_l 20 width of petal (mm) pet_w 21 length of longest filament (mm) fil_l max 22 height of ovary (mm) ova_h 23 height of stylus (mm) sty_h 24 height of central tooth on petal (mm) centoo_h 25 height of lateral tooth on petal (mm) lattoo_h 26 height of fruit (mm) fru_h 27 width of fruit (mm) fru_w 28 length of rostrum (mm) rost_l 29 total seed length (mm) see_l 30 total seed width (mm) see_w 31 width of central longitudinal fold (costa) (mm) cos_w 32 width of nectary (mm) nect_w 33 height of nectary (mm) nect_h 34 the angle between carpels and nectaries (degree) nect-ang material and methods plant material fourteen populations (299 individuals) of j. heuffelii were collected for morphological analysis. the samples were taken from serbia, macedonia bulgaria and romania (during two growing seasons 2010, 2011). the voucher specimens are deposited in the herbarium of the institute of botany and botanical garden “jevremovac", faculty of biology, university of belgrade beou (tab. 1). morphometric analyses morphometric analyses were performed on dissected plant organs (leaves, stems, flowers, fruits and seeds). leaves and inflorescences were stored in a glycerol: 96% ethanol solution (50:50). for measuring of flowers, the microscope slides were used, where all flower parts (sepals, petals, stamens, carpels) were separated and individually placed on the microscope slide. slides were first scanned (scanexpress a3 usb, mustek) and then measured by using the leicaqwin image analyzing program (leica image software). for purposes of measuring biologica nyssana 6 (1)  september 2015: 1-9 nikolić, d. et al.  the influence of orographical and bioclimatic factors … 4 the smallest details on leaves, flowers and nectaries, these organs were photographed with the leica dm 1000 microscope while seeds were photographed with the leica mz16 a stereomicroscope. thirty four morphological characters were used in these analyses (tab. 2). statistical analysis cluster analysis (upgma) was performed in order to evaluate the bioclimatic differentiation between the habitats of the 14 investigated populations. each location was characterized using 19 bioclimatic parameters, extracted from the worldclim set of global climate layers (h i j m a n s et al., 2005). the extraction of bioclimatic parameters was done with diva-gis 7.5 software (h i j m a n s et al., 2012). regression analysis (linear regression) was performed in order to estimate the correlations between the variation of morphological characters of j. heuffelii and basic orographic, geological, and bioclimatic habitat characteristics, as well as the geographic position of each population. the geographic positions were recorded using a handheld global positioning system (gps garmin etrex vista® c). orographic characteristics including altitude, slope and aspect were calculated from the shuttle radar topography mission (reuter et al., 2007) at an approximate 90 m pixel resolution using arcgis 10 spatial analyst. prior to the regression analysis, habitat characteristics were tested for multicollinearity. bioclimatic predictors that have shown significant correlations with other predictors were not used in regression analysis of morphological characters. all statistical analyses were performed using the package statistica 5.1 (statsoft, 1996). results and discussion cluster analysis of bioclimatic data cluster analysis based on bioclimatic factors showed presence of 3 clusters (fig. 1). the first cluster (c1) included localities with semiarid, temperate-continental climate or subcontinental climates. these habitats appear in gorges of rivers gradac, suvaja and studenica as well as at domogled in romania. this cluster also included populations from bulgaria (bu-trojanski prolaz central part of stara planina) and the population from mt. stara planina in the e part of serbia. the second cluster (c2) contained localities with continental mountain climates (sr-besna kobila, sr-radan, sr-basarski kamik, sr-pljačkovica and ma-treskavec). the third cluster (c3) includes localities with humid mountain climate (sr-treska, sr-nebeske stolice and ma-mavrovo). fig. 1. results of cluster analysis for populations of j. heuffelii based on habitat climatic characteristics (c1, c2, c3, climate type, for details see tab. 2.) biologica nyssana 6 (1)  september 2015: 1-9 nikolić, d. et al.  the influence of orographical and bioclimatic factors … 5 t a b le 3 . m in im a l, a v e ra g e a n d m a x im a l v a lu e o f 1 9 b io c li m a ti c p a ra m e te rs o f lo c a li ti e s st u d ie d . c 1 , c 2 a n d c 3 r e p re se n t c li m a te t y p e s c o rr e sp o n d t o t h e g ro u p in g s o b ta in e d f ro m t h e c lu st e r a n a ly si s o f b io c li m a ti c p a ra m e te rs ( se e f ig . 1 ). t h e b io c li m a ti c v a ri a b le s th a t w e re u se d a s p re d ic to rs i n r e g re ss io n a n a ly si s a re m a rk e d w it h a st e ri sk ( * ). b io c li m a ti c p a r a m e te r s c 1 c 2 c 3 se m i -a ri d m o d e ra te ly c o n ti n e n ta l c li m a te c o n ti n e n ta l m o u n ta in c li m a te h u m id m o u n ta in c li m a te m in a v g m a x m in a v g m a x m in a v g m a x a n n u a l m e a n t e m p e ra tu re ( 1 ) 3 .3 7 7 .7 1 1 1 .3 3 3 .3 0 7 .7 4 9 .7 2 2 .8 7 4 .2 3 4 .9 8 m e a n m o n th ly t e m p e ra tu re r a n g e ( 2 ) * 7 .9 8 9 .1 1 0 .1 8 .7 3 9 .1 1 0 .3 6 7 .4 8 8 .1 6 9 .0 4 is o th e rm a li ty ( 2 /7 ) (* 1 0 0 ) (3 ) * 2 9 .4 9 3 1 .7 4 3 3 .6 7 3 1 .6 4 3 3 .3 0 3 4 .6 4 2 9 .3 4 3 0 .8 8 3 3 .4 9 t e m p e ra tu re s e a so n a li ty ( s t d * 1 0 0 ) (4 ) * 6 7 6 .6 0 7 2 4 .8 4 7 5 5 .9 2 6 6 1 .0 8 7 2 1 .5 2 7 5 0 .3 9 6 4 5 .8 4 6 6 8 .4 9 6 9 1 .6 4 m a x . te m p e ra tu re o f th e w a rm e st m o n th ( 5 ) 1 7 .2 2 2 .6 5 2 6 .8 1 7 .6 2 3 .4 8 2 4 .1 1 6 .4 1 8 .3 1 9 .6 m in . te m p e ra tu re o f th e c o ld e st m o n th ( 6 ) -3 .2 -5 .9 7 -8 .9 -4 .4 -5 .9 3 -9 -7 .4 -8 .1 -9 .1 a n n u a l te m p e ra tu re r a n g e ( 5 – 6 ) (7 ) * 2 6 .1 2 8 .6 2 3 0 .2 2 6 .6 2 9 .4 3 0 .4 2 5 .5 2 6 .4 2 7 m e a n t e m p e ra tu re o f th e w e tt e st q u a rt e r (8 ) 9 .6 1 4 .6 1 1 8 .4 5 3 .9 3 1 0 .2 9 1 3 .5 5 -1 .3 6 .3 6 1 1 .3 2 m e a n t e m p e ra tu re o f th e d ri e st q u a rt e r (9 ) * -0 .9 2 3 .4 5 9 .5 8 1 .8 1 1 .2 7 1 6 .5 2 -2 .7 5 6 .0 6 1 2 .6 3 m e a n t e m p e ra tu re o f th e w a rm e st q u a rt e r (1 0 ) 1 1 .4 8 1 6 .3 6 2 0 .1 7 1 1 .2 5 1 6 .3 5 1 8 .5 8 1 0 .8 5 1 2 .2 6 1 3 .1 2 m e a n t e m p e ra tu re o f th e c o ld e st q u a rt e r (1 1 ) -4 .9 5 -1 .4 2 2 .0 1 -3 .3 -1 .3 4 0 .1 7 -5 .3 8 -4 .0 2 -2 .9 a n n u a l p re c ip it a ti o n ( 1 2 ) * 7 7 8 8 1 5 .1 7 8 4 9 6 0 6 6 6 5 .7 5 7 4 2 9 5 0 1 0 0 1 .6 7 1 0 4 5 p re c ip it a ti o n o f th e w e tt e st m o n th ( 1 3 ) 8 7 1 0 0 .3 3 1 2 1 6 8 7 6 .7 5 8 4 1 0 4 1 1 3 .6 7 1 2 7 p re c ip it a ti o n o f th e d ri e st m o n th ( 1 4 ) * 4 2 5 0 5 5 4 0 4 2 .7 5 4 6 5 6 6 2 .6 7 6 9 p re c ip it a ti o n s e a so n a li ty ( c v ) (1 5 ) * 1 7 .5 7 2 3 .1 3 3 4 .4 0 1 6 .6 7 1 9 .1 0 2 1 .2 9 1 5 .1 3 1 8 .9 2 6 .2 4 p re c ip it a ti o n o f th e w e tt e st q u a rt e r (1 6 ) 2 5 5 2 6 9 .1 3 3 1 0 1 7 2 2 0 0 .7 5 2 3 1 2 8 5 3 1 2 .3 3 3 4 8 p re c ip it a ti o n o f th e d ri e st q u a rt e r (1 7 ) 1 4 7 1 6 2 .1 7 1 7 5 1 2 7 1 3 6 .2 5 1 4 7 1 7 8 2 0 2 2 2 2 p re c ip it a ti o n o f th e w a rm e st q u a rt e r (1 8 ) * 2 2 0 2 4 5 .1 7 2 9 1 1 3 6 1 6 1 .7 5 2 0 4 1 7 9 2 3 5 2 7 2 p re c ip it a ti o n o f th e c o ld e st q u a rt e r (1 9 ) * 1 5 8 1 7 7 .5 1 8 7 1 4 7 1 6 0 1 9 4 2 1 8 2 5 5 3 1 0 biologica nyssana 6 (1)  september 2015: 1-9 nikolić, d. et al.  the influence of orographical and bioclimatic factors … 6 t a b le 4 . s u m a ry s ta ti st ic s o f re g re ss io n ( r 2 ) fo r in d e p e n d e n t b io c li m a ti c , g e o g ra p h ic a l a n d g e o lo g ic a l v a ri a b le s. a c r o n y m a l t a s p s l o b io 2 b io 3 b io 4 b io 7 b io 8 b io 9 b io 1 2 b io 1 3 b io 1 4 b io 1 5 b io 1 8 b io 1 9 r o s_ d 0 .0 7 * * 0 .0 2 0 .0 1 0 .0 2 0 .0 7 * * 0 .0 5 0 .0 0 0 .2 6 0 .1 1 0 .0 1 0 .0 0 0 .0 5 0 .0 1 0 .1 6 0 .0 2 l e r o s_ n 0 .2 6 * 0 .1 3 * 0 .0 2 0 .2 5 * 0 .1 3 * 0 .1 9 * 0 .2 8 * 0 .0 8 * * 0 .0 1 0 .0 2 0 .0 0 0 .0 4 0 .0 2 0 .0 1 0 .0 2 l e r o s_ l m a x 0 .0 9 * 0 .0 2 0 .0 1 0 .0 0 0 .0 5 0 .1 3 * 0 .0 2 0 .2 2 * 0 .0 1 * 0 .0 0 0 .0 3 0 .0 0 0 .0 9 * 0 .1 6 * 0 .0 9 * l e r o s_ w m a x 0 .1 2 * 0 .0 2 0 .0 0 0 .0 0 0 .0 4 0 .1 6 * 0 .0 4 0 .2 0 * 0 .1 9 * 0 .0 1 0 .0 5 0 .0 2 0 .0 3 0 .1 4 * 0 .0 2 a p e x _ d 1 0 .0 6 0 .0 2 0 .0 0 0 .0 0 0 .0 0 0 .0 7 0 .0 2 0 .1 0 * 0 .0 4 0 .0 0 0 .0 0 0 .0 0 0 .0 2 0 .0 4 0 .0 4 l e r o s_ s p _ l 0 .0 0 0 .0 0 0 .0 3 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .0 1 0 .0 2 0 .0 2 0 .0 1 0 .0 0 0 .0 1 0 .0 2 l e r o s_ c i_ l 0 .0 0 0 .0 1 0 .0 7 0 .0 0 0 .0 1 0 .0 4 0 .0 1 0 .0 0 0 .0 1 0 .0 0 0 .0 2 0 .0 2 0 .1 0 * 0 .0 0 0 .0 2 l e r o s_ e d _ w 0 .0 3 0 .0 4 0 .0 4 0 .0 0 0 .0 0 0 .0 1 0 .0 1 0 .0 3 0 .0 1 0 .0 0 0 .0 3 0 .0 7 * * 0 .1 4 * 0 .0 1 0 .0 1 s te _ h 0 .1 7 * 0 .0 4 0 .0 1 0 .0 0 0 .0 4 0 .1 7 * 0 .0 5 0 .3 0 * 0 .1 2 * 0 .0 0 0 .0 0 0 .0 4 0 .0 1 0 .0 8 * * 0 .0 5 l e s te _ n 0 .0 0 0 .0 1 0 .0 0 0 .0 6 0 .1 2 * 0 .0 0 0 .0 1 0 .0 5 0 .0 7 * * 0 .0 3 0 .0 0 0 .1 6 * 0 .1 3 * 0 .0 3 0 .0 0 m id l e s te _ l 0 .0 0 0 .0 1 0 .0 0 0 .0 3 0 .0 5 0 .0 0 0 .0 1 0 .0 4 0 .0 3 0 .0 0 0 .0 2 0 .0 0 0 .0 4 0 .1 0 * 0 .0 2 m id l e s te _ w 0 .0 0 0 .0 0 0 .0 1 0 .0 9 * 0 .1 9 * 0 .0 2 0 .0 2 0 .1 0 * 0 .2 0 * 0 .0 6 0 .0 7 * * 0 .0 9 * 0 .0 0 0 .2 2 * 0 .0 0 a p e x _ d 2 0 .0 5 0 .0 0 0 .0 1 0 .0 0 0 .0 5 0 .0 7 * * 0 .0 1 0 .2 3 * 0 .0 8 * * 0 .0 1 0 .0 2 0 .0 1 0 .0 4 0 .0 7 * * 0 .1 0 * s e p _ l 0 .0 3 0 .0 0 0 .0 9 * 0 .0 0 0 .0 0 0 .0 1 0 .0 0 0 .0 8 * * 0 .0 2 0 .0 6 0 .1 0 * 0 .0 0 0 .0 6 0 .0 0 0 .0 6 s e p _ w 0 .0 2 0 .0 0 0 .0 7 0 .0 0 0 .0 1 0 .0 1 0 .0 0 0 .0 7 * * 0 .0 0 0 .0 1 0 .0 0 0 .0 0 0 .0 0 0 .0 1 0 .0 3 p e t_ l 0 .2 2 * 0 .0 6 0 .0 4 0 .0 2 0 .0 1 0 .1 9 * 0 .0 9 * 0 .3 1 * 0 .0 5 0 .0 1 0 .0 1 0 .0 2 0 .0 3 0 .0 3 0 .0 6 p e t_ w 0 .0 1 0 .0 1 0 .0 4 0 .0 3 0 .0 8 * * 0 .0 1 0 .0 0 0 .0 4 0 .0 4 0 .0 1 0 .0 0 0 .1 1 * 0 .0 9 * 0 .0 1 0 .0 0 f il _ l m a x 0 .2 3 * 0 .1 1 * 0 .0 0 0 .0 2 0 .0 0 0 .1 7 * 0 .0 8 * * 0 .3 4 * 0 .0 8 * * 0 .0 0 0 .0 1 0 .0 5 0 .0 5 0 .0 6 0 .0 3 o v a _ h 0 .2 3 * 0 .1 3 * 0 .0 4 0 .0 1 0 .0 1 0 .1 5 * 0 .0 7 * * 0 .3 1 * 0 .0 4 0 .0 0 0 .0 0 0 .0 3 0 .0 2 0 .0 4 0 .0 3 s ty _ h 0 .2 9 * 0 .1 5 * 0 .0 0 0 .0 4 0 .0 0 0 .2 9 * 0 .1 6 * 0 .3 6 * 0 .1 0 * 0 .0 0 0 .0 0 0 .0 2 0 .0 1 0 .0 9 * 0 .0 7 * * c e n t o o _ h 0 .0 2 0 .0 0 0 .0 5 0 .0 1 0 .0 0 0 .0 0 0 .0 1 0 .0 2 0 .0 1 0 .0 2 0 .0 9 * 0 .0 0 0 .1 2 * 0 .0 3 0 .0 1 l a tt o o _ h 0 .0 1 0 .0 4 0 .0 1 0 .0 0 0 .0 0 0 .0 1 0 .0 0 0 .0 1 0 .0 1 0 .0 1 0 .0 9 * 0 .0 0 0 .1 7 * 0 .0 8 * * 0 .0 0 f ru _ h 0 .3 1 * 0 .1 4 * 0 .0 2 0 .0 3 0 .0 0 0 .2 2 * 0 .1 2 * 0 .4 2 * 0 .0 6 0 .0 0 0 .0 2 0 .0 5 0 .0 7 0 .0 4 0 .0 5 f ru _ w 0 .3 1 * 0 .1 4 * 0 .0 4 0 .0 3 0 .0 0 0 .2 1 * 0 .1 2 * 0 .4 0 * 0 .0 5 0 .0 0 0 .0 2 0 .0 4 0 .0 7 0 .0 3 0 .0 5 r o st _ l 0 .2 5 * 0 .3 7 * 0 .0 8 * * 0 .0 4 0 .0 0 0 .2 6 * 0 .1 3 * 0 .1 9 * 0 .0 0 0 .0 0 0 .0 7 * * 0 .0 0 0 .2 1 * 0 .1 1 * 0 .0 6 f lo b ra _ n 0 .0 3 0 .0 2 0 .0 0 0 .0 1 0 .0 5 0 .0 3 0 .0 0 0 .1 1 * 0 .0 7 * * 0 .0 1 0 .0 1 0 .0 3 0 .0 0 0 .0 9 * 0 .0 0 f lo _ n 0 .0 9 0 .0 5 0 .0 0 0 .0 1 0 .0 0 0 .0 6 0 .0 3 0 .1 0 * 0 .0 4 0 .0 0 0 .0 1 0 .0 0 0 .0 1 0 .0 4 0 .0 1 f il _ l m a x 0 .1 1 0 .0 0 0 .0 0 0 .0 0 0 .0 6 0 .1 3 * 0 .0 2 0 .3 8 * 0 .2 0 * 0 .0 2 0 .0 7 0 .0 2 0 .1 1 * 0 .0 6 0 .1 0 * s e e _ l 0 .0 7 * * 0 .0 8 * * 0 .0 2 0 .0 0 0 .0 0 0 .0 2 0 .0 1 0 .0 7 * * 0 .0 6 0 .0 2 0 .0 6 0 .0 0 0 .0 6 0 .0 9 * 0 .0 0 s e e _ w 0 .1 3 * 0 .0 3 0 .0 0 0 .0 5 0 .0 2 0 .0 5 0 .0 6 0 .0 8 * * 0 .0 4 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 c o s_ w 0 .0 2 0 .0 0 0 .0 2 0 .0 1 0 .0 0 0 .0 1 0 .0 1 0 .0 7 0 .0 1 0 .0 1 0 .1 7 * 0 .0 3 0 .4 1 * 0 .0 1 0 .0 0 n e c t_ w 0 .0 8 * * 0 .1 0 * 0 .0 3 0 .1 7 * 0 .1 3 * 0 .0 5 0 .1 4 * 0 .0 0 0 .0 6 0 .0 6 0 .1 1 * 0 .0 2 0 .0 4 0 .1 2 * 0 .0 2 n e c t_ h 0 .0 4 0 .0 4 0 .0 0 0 .0 0 0 .0 1 0 .0 7 * * 0 .0 3 0 .0 4 0 .0 5 0 .0 3 0 .0 3 0 .0 3 0 .0 0 0 .0 4 0 .0 0 n e c ta n g 0 .0 4 0 .0 1 0 .0 3 0 .0 8 * * 0 .0 8 * * 0 .0 1 0 .0 5 0 .0 0 0 .0 1 0 .0 5 0 .0 4 0 .0 4 0 .0 0 0 .0 5 0 .0 1 a b b re v ia ti o n s: a l t -a lt it u d e , a s p a sp e c t, s l o -s lo p e , b io b io c li m a ti c c h a ra c te ri st ic s (s e e t a b le 3 ) * p < 0 .0 5 , * * p < 0 .0 1 biologica nyssana 6 (1)  september 2015: 1-9 nikolić, d. et al.  the influence of orographical and bioclimatic factors … 7 the analysis of the three clusters regarding the values of bioclimatic parameters (tab. 3) shows that type c1 is characterized by the highest values of bio1 bioclimatic parameter, the mean annual temperature within the range 3.37-11.33 °c. c2 type has shown variation in this character in range of 3.30-9.72 °c, while c3 climate type has shown the lowest values of this parameters, 2.87 4.98 °c (tab. 3). a similar trend was noticed in bioclimatic parameters bio5, bio6, bio8, bio10 and bio11, while in the bioclimatic parameter bio9 (mean temperature of the driest quarter) the highest values were recorded for the second type of climate c2 (1.8-16.52 °c), followed by the third type c3 (–2.75 -12.63 °c) while the lowest values were recorded in the first climate type c1 (–0.92-9.58 °c). the analysis of these three clusters according to bioclimatic factors (tab. 3) indicated that the greatest differences between clusters were present in the amount of precipitation, as measured by the factors annual precipitation (bio12), precipitation of the driest month (bio14) and precipitation of the wettest quarter (bio16). the largest amount of precipitation was present in the third cluster, the second had the lowest amount of precipitation, while the first cluster was intermediate. considering the temperature, the highest values were recorded in canyons and river gorges (c1) and the lowest values in the third cluster (c3). there is an important question: to which extent do bioclimatic factors in analyzed habitats cause the differentiation in populations? the answer to this question may be provided by cluster analysis including both the bioclimatic characters and the morphological characteristics of individuals from the analyzed populations. if the position of populations in the cluster analysis of bioclimatic factors matches the position obtained from morphometric characters, it may be concluded that such grouping is a result of impact by various bioclimatic factors and vice versa (k u z m a n o v i ć et al., 2011). as cluster analysis of bioclimatic factors have shown that the study area is strongly differentiated into three main clusters: one with semiarid temperate continental or subcontinental climate, another with continental mountain climate and the third cluster with humid mountain climate, it might be expected that the cluster analysis of morphological characters would also produce three main clusters. however, the analysis has shown that within the cluster analysis of morphological characters all populations were divided into four clusters (n i k o l i ć et al., 2015b), while the distribution of individual clades (populations) was not matched to the distribution of populations obtained in the cluster analysis of bioclimatic parameters. this type of distribution indicates that bioclimatic factors are not the single reason for such morphological differentiation of populations. results of the cluster analysis performed solely on mountain populations were similar. the effect of microclimatic differences caused by orography on morphological differentiation was avoided in this analysis, as all mountain populations inhabit open grassland habitats from classes festuco-brometea and elyno-seslerietea. although both cluster analyses, of morphological characters and bioclimatic parameters, have shown separation into two groups, the placement of individual populations into these groups is different, indicating that bioclimatic parameters are not crucial in differentiation of populations (n i k o l i ć et al., 2015b). regression analysis (linear regression) regression analysis showed that orographic factors (altitude, aspect and slope) influenced the morphological characters of the species j. heuffelii. the geological substrate was not analyzed, as this factor showed a high level of co-linearity with other factors (tab. 4). altitude had the greatest influence on the following morphological characters: length of rostrum, height of fruit, height of stylus, width of fruit, number of leaves in rosette, length of the longest branch in floral maturation stage, length of the longest filament. exposition aspect is also one of the key abiotic factors, with the greatest impact on morphological characters of reproductive organs (ovary stylus, fruit and nectary). the morphological differentiation of the analyzed populations is also highly influenced by the bioclimatic factors precipitation and temperature. the greatest correlation with the analyzed characters was shown by mean temperature of driest quarter (bio8) and temperature seasonality (bio4). these two bioclimatic factors have the highest influence on the same characters that are highly influenced by altitude. there is a somewhat smaller but still significant impact of precipitation seasonality (bio15), with the greatest impact on following characters: length of petal, height of ovary, height of the stem to the lowest flower branch, width of the nectary, width of the middle leaf on the stem, width of the largest leaf, length of the largest leaf, width of the central longitudinal fold (costa). there is a somewhat smaller but still significant impact of precipitation of warmest quarter (bio18) and mean temperature of driest quarter (bio9). biologica nyssana 6 (1)  september 2015: 1-9 nikolić, d. et al.  the influence of orographical and bioclimatic factors … 8 the geographic variability in plant morphology is the result of phenotypic changes expressed as an answer to local ecological conditions, genetic variability and evolution among the populations, as well as the biogeographic history of species (e l l i s o n et al., 2004). some of the morphological characteristics have genetic origins, for example leaf shape, but they may also be strongly influenced by local conditions in which the plants develop (t h o m p s o n , 1991; s c h l i c h t i n g & p i g l i u c c i , 1998). altitude is a factor with significant impact on variability of morphological characteristics of analyzed populations, as shown by regression analysis. change in altitude causes changes in other factors, including temperature, air humidity, precipitation and partial pressure of gasses in the atmosphere, causing the adaptive response in plants and therefore also changes in morphology and physiology (k ö r n e r , 1999). altitude may have a strong impact on both leaf morphology and physiology in individuals from various populations within the same species (h o v e n d e n & v a n d e r s c h o o r , 2004). with increase of altitude the length and width of leaves generally decrease (k ö r n e r et al., 1986), while thickness of leaves increases (k ö r n e r et al., 1989; r o d e r i c k et al., 2000). regression analysis has supported the hypothesis that altitude has the greatest impact on variability of morphological characters in j. heuffelii populations. conclusion the regression analysis has shown that morphological characteristics are under stronger influence by seasonal dynamics in temperature and precipitation than by the total amount of precipitation or mean annual temperature, which are the most commonly analyzed climatic factors for any geographic area. in addition to altitude, the orographic factors with the strongest impact on morphological characters of species j. heuffelii are exposition and slope of terrain. the bioclimatic parameters may also influence the variability in morphological characters. the greatest impact was recorded in mean temperature of wettest quartile (bio8) and temperature seasonality (bio4). they are mostly influencing the following characters: height and width of fruit, height of stylus, length of rostrum, number of leaves in the rosette, length of the longest flowering branch and length of longest filament of the stamen. the seasonal character of precipitation (bio15) was shown to influence size of leaves, height of stem and reproductive characters: length of petal, height of ovary and width of nectary. these results suggest that temperature conditions and precipitation may contribute to plant phenotype in various habitat types. this trend was also recorded in the populations from canyons and river gorges with c1 type of climate. populations with the highest value of bio15 parameter (seasonality of precipitation) show greater dimensions of leaves than the populations from localities with c2 and c3 types of climate, where values of bio15 parameter are lower. it remains to be determined if the large morphological variability of j. heuffelii populations in the territory of central balkans and southern carpathians was caused by phenotype plasticity or by genetically caused characteristics. the ongoing molecular analyses will help in solving this question. acknowledgements. the ministry of education, science and technological development of the republic of serbia (grant 173030) supported this research. references bârcă, v. & niculae, m. 2005: preliminary data about the chorology of the species jovibarba heuffelii (schott) a. löve & d. löve (crassulaceae) in southern carpatian mountains in romania. contributii botanice, 40: 25-33. dimitrijević, d., šinžar-sekulić, j., ranđelović, v. & lakušić, d. 2011: the nature of the variability of the morphological characteristics of the taxon jovibarba heuffelii (schott) a. löve & d. löve (crassulaceae) in serbia. biologica nyssana, 2: 7-18. ellison, a.m., buckley, l.h., miller, e.t. & gotelli, j.n. 2004: morphological variation in sarracenia purpurea (sarraceniaceae): geographic, environmental and taxonomic correlates. american journal of botany, 91(11): 1930-1935. hijmans, r.j., cameron, s.e., parra, j.l., jones, p.g. & jarvis, a. 2005: very high resolution interpolated climate surfaces for global land areas. international journal of climatology, 25: 1965–1978. hijmans, r.j., guarino, l. & mathur, p. 2012: diva-gis version 7.5. available from: http://www.diva-gis.org/ (accessed 8 january 2013). horvat, i., glavač, v. & ellenberg, h. 1974: vegetation südosteuropas. geobotanica selecta, band iv, gustav fischer verlag, stuttgart. hovenden, m.j. & vander schoor, j.k. 2004: nature vs nurture in the leaf morphology of southern beech, nothofagus cunninghamii biologica nyssana 6 (1)  september 2015: 1-9 nikolić, d. et al.  the influence of orographical and bioclimatic factors … 9 (nothofagaceae). new phytologist, 161: 585594. jalas, j., suominen, j., lampinen, r. & kurtto, a., (eds.), 1999: atlas florae europaeae, distribution of vascular plants in europe, 12. resedeaceae to platanaceae, the committee for mapping the flora of europe & societas biologica fennica vanamo, helsinki, [maps 2928–3270], körner, c. 1999: alpine plant life. germany: springer-verlag, berlin. körner, c., bannister, p. & mark, a.f. 1986: altitudinal variation in stomatal conductance, nitrogen content and leaf anatomy in different plant life forms in new zealand. oecologia, 69: 577–588. körner, c., neumayer, m., menendez-riedl, s. & smeets-scheel, a. 1989: functional morphology of mountain plants. flora, 182: 353-383. kuzmanović, n., šinžar-sekulić, j. & lakušić, d. 2011: ecologically determined variation in leaf anatomical traits of sesleria rigida (poaceae) in serbia –multivariate morphometric evidence. folia geobotanica, 47: 41–57. lakušić, d., blaženčić, j., ranđelović, v., butorac, b., vukojičić, s., zlatković, b. jovanović, s., šinžar-sekulić, j., žukovec, d., ćalić, i. & pavićević, d. 2005: staništa srbije – priručnik sa opisima i opštim podacima. ministartsvo nauke i zaštite životne sredine, uprava za zaštitu životne sredine, institut za botaniku i botanička bašta "jevremovac", biološki fakultet, univerzitet u beogradu, beograd, 684 p. meusel, h., jager, e. & weinert, e. 1965: vergleichende chorologie der zentraleuropäischen flora. jena: gustav fischer verlag. nikolić, d., spasić, m., šinžar-sekulić, j., ranđelović, v. & lakušić, d. 2015a: morphometric analysis of nectaries and their potential use in the taxonomy of the jovibarba heuffelii complex (crassulaceae). archive of biological sciences, 67(2): 511-524. nikolić, d., šinžar-sekulić, j., ranđelović, v. & lakušić, d. 2015b: morphological variation of jovibarba heuffelii (crassulaceae) in the central balkan peninsula the impact of geological, orographical and bioclimatic factors on the differentiation of populations. phytotaxa, 203 (3): 213-230. parkhurst, d.f. & loucks, o.j. 1972: optimal leaf size in relation to the environment. journal of ecology, 60: 505-537. reuter, h.i., nelson, a. & jarvis, a. 2007: an evaluation of void filling interpolation methods for srtm data. international journal of geographical information science, 21: 983– 1008. roderick, m.l., berry s.l. & noble i.r. 2000: a framework for understanding the relationship between environment and vegetation based on the surface area to volume ratio of leaves. functional ecology, 14: 423–437. schlichting, c. & pigliucci, m. 1998: phenotypic evolution: a reaction norm perspective, sinauer associates, inc., sunderland, ma, 387 p. statsoft, 1996: statistica (data analysis software system), version 5.1. statsoft inc., tulsa. available from: www.statsoft.com. (accesed 8 january 2013). thompson, j.d. 1991: phenotypic plasticity as a component of evolutionary change. trends in ecology and evolution, 6: 246–249. thuiller, w., lavorel, s., midgley, g., lavergne, s. & rebelo, t. 2004: relating plant traits and species distributions along bioclimatic gradients for 88 leucadendron taxa. ecology, 85: 16881699. walter, h. & lieth, h. 1967: klimadiagramm weltatlasveb gustav fischer, jena. biologica nyssana 6 (1)  september 2015: 1-9 nikolić, d. et al.  the influence of orographical and bioclimatic factors … 10 spas krumov sotirov (1929-2016) naturalist and biologist biologica nyssana 7 (2)  december 2016: 57-60 ranđelović, n. et al.  in memoriam spas krumov sotirov… 57 in memoriam spas krumov sotirov (1929-2016) naturalist and biologist novica ranđelović1, vlastimir stamenković2, tatjana mihajilov-krstev1 1department of biology and ecology, faculty of sciences and mathematics, university of niš, serbia 2dd “zdravlje” leskovac, serbia * e-mail: tatjanamk@pmf.ni.ac.rs prof. dr spas sotirov was born on 9th january, 1929 in dimitrovgrad, where he had finished both elementary and high school. dr sotirov finished his bachelor biology studies in 1954. at the faculty of biology in belgrade, after which he had finished and defended his master (1962) and phd (1970) theses at the same institution. his professional carrier started in gymnasium in dimitrovgrad where he worked as a professor. during the period from 1958 to 1966, he worked as a teaching assistant at the faculty of veterinary medicine in belgrade and faculty of technology in novi sad. over the following years, during the period 1966-1982, he worked in institute 7 (2) • december 2016: 57-60 12th sfses • 16-19 june 2016, kopaonik mt doi: 10.5281/zenodo.200398 biologica nyssana 7 (2)  december 2016: 57-60 ranđelović, n. et al.  in memoriam spas krumov sotirov… 58 for development of education in niš. his academic carrier started with his promotion into assistant professor in 1982 at the faculty of technology in leskovac, where he was promoted into associate professor (1987) and finally to full professor in 1990. he was involved as an organizer of many symposiums, scientific and specialized workshops. among his organizing activities, one of the most significant place takes symposium entitled one hundred years of “flora of niš surrounding area”, where he was plenary speaker on the subject on dr sava petrović. he gave high contribution to education of the new generations of faculty and other students. as a member of the national education council of the republic of serbia he was working on didactic problems in teaching and as a reviewer on many textbooks written for elementary education. dr sotirov was coauthor of the capital monography “encyclopedia of niš” and worked as a reviewer of special issues of the journals “politika” and “savremena biologija”. also, he was a collaborator and a member of editorial board in the journals “bratstvo”, “most” and “drugarče”. his scientific opus includes working and contribution on many scientific projects on the subject on fundamental and also fieldwork researches on flora and vegetation, as well as resolving of significant problems such as possibilities of using autochthonous plant material in alimentary industry, technology of fruits and vegetables, development of food technology concepts and creation of the modern managing, control and production mechanisms in food industry. he was author of over 50 scientific publications and books. bibliography sotirov, s. 1968: ribe okoline pirota. pirotski zbornik, 1, pirot. sotirov, s. 1973: u dolini jerme susret tradicije prirode sa tradicijom kulture. narodne novine, niš. sotirov, s. 1974: procvat jestvastveništva u starom nišu. gradina, 1-2, niš. sotirov, s. 1977: po dolinata na erma (2. izdanje). bratstvo, niš. ranđelović, n. jovanović, v. sotirov, s. stamenković, v. ružić, m. 1980: campanula versicolor andrews nova vrsta u flori srbije. glasnik prirodnjačkog muzeja u beogradu, 35 (b): 53-56. ranđelović, n., matinović, ž., sotirov, s., jovanović, v., ružić, m., stamenković, v., hill, d.a., krivošej, z., randjelović, v. 1983: prunus laurocerasus l. na ostrozubu cveta. glasnik prirodnjačkog muzeja, b, 38. beograd. fig. 1. the last lecture at the university of niš biologica nyssana 7 (2)  december 2016: 57-60 ranđelović, n. et al.  in memoriam spas krumov sotirov… 59 ranđelović, n., martinović, ž., jovanović, v., ružić, m., stamenković, v., hill, d.a., krivošej, z., ranđelović, v., sotirov, s. 1983: florescencija na ostrozubskoto zeleniče. most, 81: 98-100. sotirov, s., ranđelović, n., stamenković, v. 1984: unapređivanje zakonske regulative u zaštiti ekoloških sistema. iii kongres ekologa jugoslavije, 429-432, sarajevo. sotirov, s., stanković, s., pešić, d. 1985: tehnološke karakteristike ploda džanarike s planinskog područja grdelice. leskovački zbornik, 26, leskovac. sotirov, s., randjelović, n., stamenković, v. 1985: bioresursite na vlasina-kraište. most, 91: 108113, niš. sotirov, s. 1985: istraživanje prirodnih biljnih potencijala na području vlasine. 1. samoniklo jestivo i aromatično bilje. (izveštaj u okviru projekta). leskovac. sotirov, s. 1985: biološko-ekološka edukacija studenata prehrambenih tehnologija. iii kongres ekologa jugoslavije, sarajevo. sotirov, s. 1985: dr sava petrović život i delo, stogodišnjica "flore okoline niša", niš. sotirov, s., ranđelović, n. 1985: prošlost, sadašnjost i budućnost ekosistema na području regiona niš. niški zbornik, 15: 189-197, niš. ranđelović, n., sotirov, s. 1985: rezultati vegetacijskih i florističkih istraživanja jugoistočne srbije i njihov značaj za regionalno planiranje. naučno-istraživački rad i njegova uloga u daljem razvoju regiona niš. niš. ranđelović, n., hill, d.a., sotirov, s. 1985: specifičnosti flore uljanih škriljaca kod aleksinca. stogodišnjica "flore okoline niša", niš. martinović, ž., sotirov, s., randjelović, n. 1985: biljno-geografske karakteristike miljkovačke klisure. zbornik radova stogodišnjica "flore okoline niša”, 89-93, niš. palić, r., anđelković, s., sotirov, s. 1985: prilog taksonomiji satureja montana i satureja kitaibelii w. et k. stogodišnjica "flore okoline niša", niš. sotirov, s., komadinić, v. 1986: gajenje, pomološka i tehnološka svojstva niške "džinke”. (izvod). vii kongres biologa jugoslavije, budva. sotirov, s. 1986: biološko-ekološka zasnovanost industrijske prerade voća i povrća (izvod). vii kongres biologa jugoslavije, budva. sotirov, s. 1986: aktuelni problemi prerade kafe u južnomoravskom regionu. most, 98, niš. sotirov, s. 1986: iskorišćavanje autohtonih biljnih sirovina. most, 99, niš. sotirov, s. 1986: osavremenjivanje upravljačkih mehanizama prehrambene industrije. most, 100, niš. palić, r., sotirov, s., mihajlović, p. 1986: promena količine i sastava etarskog ulja lavandula angustifolia mill. (izvod). glasnik hemičara i tehnologa kosova, 6 (1), priština. sotirov, s., palić, r. 1986: kvalitet lavandinog ulja iz okoline sićeva i mogućnost upotrebe za aromatizaciju prehrambenih proizvoda. (izvod, rukopis). i simpozijum o flori i vegetaciji sr srbije, beograd. ranđelović, n., stamenković, v., sotirov, s. 1986: vegetacija vlasine i krajišta. most, 101-102: 6578, niš. ranđelović, n., hill, d.a., sotirov, s. 1986: specifičnost flore uljnih škriljaca kod aleksinca. i simpozijum o flori i vegetaciji srbije, 27, beograd. ranđelović, n., sotirov, s., jovanović, v., ružić, m., stamenković, v. 1986: rezultati florističkih, vegetacijskih i fitohemijskih istraživanja na području jugoistočne srbije za period 1945-1985 godine. i simpozijum o flori i vegetaciji srbije, 34, beograd. sotirov, s., jakšić, m. 1987: problemi za sotirov, s., jakšić, m. 1987: problemi zaštite i unapređivanja radne i životne sredine u svetlosti sistemskostrukturne koncepcije. zbornik tehnološkog fakulteta, 3-4: 175-180, leskovac. sotirov, s., tasić, s. 1987: korišćenje semena grožđa u proizvodnji kavovina. prethodno saopštenje. zbornik radova tehnološkog fakulteta, 3-4: 201202, leskovac. tasić, s., sotirov, s., ristić, m., palić, r., stanković, m. 1987: ispitivanje sastava semena grožđa. i. sastav frakcije masnih kiselina. xxix savetovanje hemičara sr srbije, beograd. sotirov, s., stojiljković, s., davidović, i. 1988: blanširanje krompira surutkom. prethodno saopštenje. zbornik radova tehnološkog fakulteta, 5-6; 253-256, leskovac. randjelović, n., stamenković, v., sotirov, s. 1988: flora v okolnostitena bosilegrad. most, 112: 4754, niš. stamenković, v., ranđelović, n., sotirov, s. 1989: lekovite biljke užeg područja vlasine. leskovački zbornik, 29: 367-375. ranđelović, n., jovanović, v., ružić, m., sotirov, s., satamenković, v., hill, d.a., ranđelović, v. 1989: lekovite biljke toplice. leskovački zbornik, 29: 375-381. sotirov, s., randjelović, n., stamenković, v. 1990: prilog metodologiji kartiranja i eksploatacije samoniklih resursa lekovitog i začinskog bilja. 2. simpozijum o flori jugoistočne srbije i susednih područja, zbornik radova tehnološkog fakulteta, 7-8: 7-11, leskovac. biologica nyssana 7 (2)  december 2016: 57-60 ranđelović, n. et al.  in memoriam spas krumov sotirov… 60 ranđelović, v., sotirov, s., stamenković, v., redžepi, f., ranđelović, v., zlatković, b. 1990: lekovite biljke subregiona pirot. 2. simpozijum o flori jugoistočne srbije i susednih područja, zbornik radova tehnološkog fakulteta, 7: 29-35, leskovac. ranđelović, n., stamenković, v., sotirov, s. jovanović, v., ranđelović, v. 1990: prirodni resursi divljeg lekovitog bilja niškog subregiona. 2. simpozijum o flori jugoistočne srbije i susednih područja, zbornik radova tehnološkog fakulteta, 7-8: 35-40. leskovac. martinović, ž., ranđelović, n., sotirov, s. 1990: o nalazištu ramondije u dolini toponičke reke. 2. simpozijum o flori jugoistočne srbije i susednih područja, zbornik radova tehnološkog fakulteta, 7: 99-111, leskovac. sotirov, s., popović, r., ranđelović, n., stamenković, v., stojanović, v., anđelković, s., simić, n. 1990: prilog proučavanju ruta graveolens l. iz okoline sićeva. 2. simpozijum o flori jugoistočne srbije i susednih područja, zbornik radova tehnološkog fakulteta, 7-8: 195199, leskovac. đorđević, s., đorđević, i. sotirov, s., gudžić, b. 1990: ispitivanje hemijskog sastava ostrozubske šljive sa posebnim osvrtom na sadržaj minerala. 2. simpozijum o flori jugoistočne srbije i susednih područja, zbornik radova tehnološkog fakulteta, 7-8: 199-204. leskovac. sotirov, s., đorđević, s., tomić, v. zdravković, b. 1990: mineralni sastav droge i infuzuma začinsko-aromatičnih biljaka iz okoline sićeva. 2. simpozijum o flori jugoistočne srbije i susednih područja, zbornik radova tehnološkog fakulteta, 7: 205-209, leskovac. sotirov, s., stamenković, v., ranđelović, n., jančić, n. 1990: dobijanje kavovine od ploda castanea sativa mill. 2. simpozijum o flori jugoistočne srbije i susednih područja, zbornik radova tehnološkog fakulteta, 7-8: 209-213, leskovac. martinović, m.ž., sotirov, s., ranđelović, n., stojanović, d.: uticaj radioaktivnosti na održanje endemoreliktne vrste ramondia serbica. zbornik radova sa 3. simpozijuma o flori jugoistočne srbije i susednih područja, 207-220, pirot. sotirov, s., ranđelović, n., stamenković, v. 1993: prirodnjačke vrednosti pirotskog ponišavlja s posebnim osvrtom na floru i vegetaciju (pregled dosadašnjih istraživanja). plenarni referat. zbornik rezimea, iii simpozijum o flori jugoistočne srbije, 2-10, pirot. ranđelović, n., stamenković, v., sotirov, s, ranđelović, v., zlatković, b. 1993: prilog flori klisure reke jerme. izvodi radova. iii simpozijum o flori jugoistone srbije, 34, pirot. stamenković v., sotirov s., cvetković d., đorđević s., ranđelović n. 1993: dobijanje zelenih začina od svežih plodova paprike i nadzemnih listova crnog luka. izvodi radova. savetovanje o lekovitim i aromatičnim biljkama jugoslavije, 41-42, zlatibor. stamenković v., sotirov s., ranđelović n., cvetanović b., nikolić m. 1993: korišćenje listova vinove loze u prehrambenoj industriji. izvodi radova. savetovanje o lekovitim i aromatičnim biljkama jugoslavije, 39-40, zlatibor. sotirov, s. 1995: poganovskijot manastir i negovotoprirodnoto okruženija. most, niš. sotirov. s. 1995: 600-godišnine na poganovskija manastir (serijal). bratstvo, niš. sotirov, s. 1999: prilog poznavanja života i dela dr save petrovića. acta medica, 3, niš; sotirov, s., zlatković, b., ranđelović, v. 2002: antropogene fitocenoze sa mahonijom (mahonia aquifolium (purch.) nutt.) u okolini dimitrovgrada. vii simpozijum o flori jugoistočne srbije i susednih područja, zbornik rezimea, dimitrovgrad. sotirov, s. 2002: poganovo zveno meždu zemjata i neboto. narodna biblioteka, dimitrovgrad. sotirov, s. 2005: zelene spirale roda ramonda. književna kolonija sićevo, str. 49-77, niš. sotirov, s. 2006: sićevski kolopleti. zograf. sotirov, s. 2010: dr sava petrović život, lik i delo. unus mundus, 35, niš. milosavljević, v., avramović, d., sotirov, s. 2010: treća južnosrpska floristička-fitocenološka škola. 10. simpozijum o flori jugoistočne srbije i susednih regiona, zbornik rezimea, niš. sotirov, s., stanojević, v. 2012: treći alen kamen cerjanska pećina i topilo. academia homo turisticus, niš. sotirov, s. 2012: prodor darvinizma u srbiji. unus mundus, 42, niš. sotirov, s. 2012: u potrazi za panacejom. unus mundus, 42. niš. sotirov, s. 2014: miljenice profesora pančića. univerzitetska biblioteka nikola tesla, niš. madić, v., jovanović, j., stojilković, a., jušković, m., vasiljević, p.: evaluation of cytotoxicity of ‘anti-diabetic’ herbal preparation and five medicinal plants: an allium cepa assay. biologica nyssana, 8 (2) biologica nyssana 8 (2)  december 2017: 151-158 madić, v. et al.  evaluation of cytotoxicity of „anti-diabetic“ herbal… 151 original article received: 14 september 2017 revised: 21 november 2017 accepted: 22 december 2017 evaluation of cytotoxicity of ‘anti-diabetic’ herbal preparation and five medicinal plants: an allium cepa assay višnja madić*, jelena jovanović, aleksandar stojilković, marina jušković, perica vasiljević university of niš, faculty of science and mathematics, department of biology and ecology, višegradska 33, niš, serbia * e-mail: visnja.madic@gmail.com abstract: madić, v., jovanović, j., stojilković, a., jušković, m., vasiljević, p.: evaluation of cytotoxicity of ‘antidiabetic’ herbal preparation and five medicinal plants: an allium cepa assay. biologica nyssana, 8 (2), december, 2017: 151-158. traditional medicine is often used as the treatment for diabetes mellitus, but little is known about potential toxicity of medicinal plants used for this purposes. the cytotoxic effect of aqueous extracts of a traditional ‘anti-diabetic’ herbal preparation, as well as its constituents: rubus fruticosus, vaccinium myrtillus, potentilla erecta, geum urbanum and phaseolus vulgaris was evaluated using the allium cepa assay. onion bulbs were exposed to 400 μg/ml, 800 μg/ml and 1200 μg/ml concentrations of each extract. there was concentration dependent inhibition of root growth by the extracts when compared with the control. all the tested extracts had mitodepressive effect on cell division. ‘anti-diabetic’ herbal mixture (1200 µg/ml) and v. myrtillus (400 µg/ml) caused surprisingly high prophase accumulation. these events manifested the cytotoxic effect of this herbal preparation as well as all the tested medicinal plants at some of the tested concentrations. key words: diabetes, ‘anti-diabetic’ herbal preparation, cytotoxicity apstrakt: madić, v., jovanović, j., stojilković, a., jušković, m., vasiljević, p.: ispitivanje citotoksičnosti “antidijabetičkog biljnog preparata“ i pet medicinskih biljaka: allium cepa test. biologica nyssana, 8 (2), decembar, 2017: 151-158. tradicionalna medicina se često koristi u terapiji dijabetes melitusa, ali malo toga se zna o potencijalnoj toksičnosti medicinskih biljaka korišćenih u ove svrhe. allium cepa testom ispitali smo citotoksični efekat vodenih ekstrakata tradicionalnog “antidijabetičkog” biljnog preparata, kao i njegovih sastojaka: rubus fruticosus, vaccinium myrtillus, potentilla erecta, geum urbanum i phaseolus vulgaris. lukovice su bile izložene trima koncentracijama svakog od ovih ekstrakata, i to: 400 μg/ml, 800 μg/ml and 1200 μg/ml. svi testirani ekstrakti pokazali su mitodepresivni efekat na ćelijsku deobu. “antidijabetički” biljni preparat (1200 µg/ml) i v. myrtillus (400 µg/ml) su, uz to, izazvali iznenađujuće veliku akumulaciju ćelija u profazi. ovi događaji su manifestacija citotoksičnog efekta ovog biljnog preparata i ovih medicinskih biljaka u nekim od ispitivanih koncentracija. ključne reči: dijabetes,”antidijabetički” biljni preparat, citotoksičnost 8 (2) • december 2017: 151-158 doi: 10.5281/zenodo.1135968 biologica nyssana 8 (2)  december 2017: 151-158 madić, v. et al.  evaluation of cytotoxicity of „anti-diabetic“ herbal… 152 introduction the use of medicinal plants has always been part of the human tradition. according to the world health organisation, 80% of the world population relies on the traditional medicine (f a r n s w o r t h et al., 1985). in recent years there has been an increased popularity of traditional medicine globally, mostly due to the high costs of nursing care and international commercial medicines. however, some constituents of medicinal plants have been found to be potentially toxic, mutagenic, carcinogenic and teratogenic (p i n g et al., 2012). also, potentional contraindications between herbal preparations and modern pharmaceutical products are not sufficiently examined, both for short-term and long-term use. for all this, the purpose of this study is to evaluate the toxicological effects of some of the herbal preparations used in traditional medicine. traditional medicine is used as the treatment for a variety of diseases, including diabetes mellitus, commonly referred to as diabetes. in 2015, there were 415 milion adults and half a million children with diabetes globally, most in lowto middle-income countries, predicted to rise to over 642 milion in the next 23 years. every 6 s someone dies from diabetes (international diabetes federation (idf), 2015). uk diabetologist edwin gale’s quoted, ’what is the commonest cause of death in a child with diabetes? the answer – from a global perspective – is a lack of access to insulin’ (g a l e & w a g n e r , 2006). although herbal substituents can not replace insulin, traditional treatments can lead to the development of new hypoglycemic drugs, especially for t2d. there is a variety of herbal mixtures intended for maintaining normal glycemic values, and one of the most commonly used in europe, primarily in the balkan countries, is made of blackberry (rubus fruticosus l., rosaceae) leaves (rfl), blueberry (vaccinium myrtillus l., ericaceae) leaves (vml), tormentil (potentilla erecta uspenski ex ledeb., rosaceae) roots (per), st. benedict's herb (geum urbanum l., rosaceae) aerial parts (gua), and kidney bean (phaseolus vulgaris l., fabaceae) pods (pvp). there is a long history of using these medicinal plants in traditional therapy for diabetes. their hypoglycemic effect was examined by a number of studies (s w a n s t o n -f l a t t et al. 1990; e d d o u k s et al., 2002; t o m c z y k & l a t t é , 2009; k y z n i e t s o v a et al., 2015; k o u p ý et al., 2015; p a u n et al., 2015; j o h n s o n & d e m e j i a , 2016; s i d o r o v a et al., 2017), but a little is known about their potential toxicity. for the most of the substances on this planet there are doses which are toxic and which are nontoxic on the cell level. for this reason, the aim of this study was to investigate cytotoxic effect of aqueous extracts of the entire ‘anti-diabetic’ herbal preparation as well as its individual ingredients and predict their ec50 on the root meristem cells of allium cepa l., fam. amaryllidaceae. allium cepa test is often used in assessing cytological and cellular effects of medicinal plant’s extracts (a k i n b o r o & b a k a r e , 2007; ç e l i k & a s l a n t ü r k , 2010; p i n g et al., 2012). it is a shortterm test with many advantages, such as low cost and good chromosome conditions for the study of disturbance of cell division or chromosome aberrations (f i s k e s j o , 1985). some researchers show certain restriction in regards to using plant test systems for evaluating cytotoxicity of medicinal plants. however, a. cepa test, as a plant test systems in vivo, is validated by several researches, which jointly performed animal testing in vitro and the results obtained were similar (c a m p a r o t o et al., 2002; t e i x e i r a et al., 2003), providing valuable information for human health. moreover, r a n k & n i e l s e n (1994) showed a correlation of 82% between the a. cepa test and the carcinogenicity test in rodents and concluded that the same was even more sensitive than the ames test. material and methods preparation of extracts the medicinal plants utilized in this study, i.e. blackberry (r. fruticosus) leaves (rfl), blueberry (v. myrtillus) leaves (vml), tormentil (p. erecta) roots (per), st. benedict's herb (g. urbanum) aerial parts (gua), and kidney bean (p. vulgaris) pods (pvp), were purchased immediately before evaluation in the certified herbal pharmacy in niš, serbia. originally, these medicinal plants were collected on the mountains of south-eastern serbia. r. fruticosus, v. myrtillus, g. urbanum, p. vulgaris, were collected on the rtanj mt., near soko banja, while p. erecta was collected on the stara planina mt. pirot. rubus fruticosus, v. myrtillus, g. urbanum and p. erecta were found in the wild, while p. vulgaris was organically grown, therefore, the possible impact of pesticide use on results of the a. cepa test was excluded. dried plant material was finely grinded, and 0.48 g of each tested material was separately boiled in 800 ml of distilled water until half of the liquid evaporated. the extract was then filtered and used as a stock solution. stock solutions and dilutions were made immediately before use. three concentrations of each extract, viz: 400 μg/ml, 800 μg/ml and 1200 μg/ml were considered. biologica nyssana 8 (2)  december 2017: 151-158 madić, v. et al.  evaluation of cytotoxicity of „anti-diabetic“ herbal… 153 allium cepa assay small bulbs (1 – 2 cm in diameter) of the common onion, a. cepa (2 n = 16) were purchased at the agricultural pharmacy in niš, serbia. the base of each of the bulbs was suspended on distilled water for 24 h. after that period, the bulbs with satisfactory root lengths were exposed to the tested materials, 5 bulbs per every concentration, for 48 h. test extracts were changed daily. the negative control was 5 bulbs suspended on distilled water for 72 h. the positive control was 5 bulbs suspended 24 h on distilled water, and afterwards, 48 h on 500 µg/ml toluene diluted in 2% dmso. the experiment was performed at 25 ± 1 ºc in the dark. at the end of the exposure period, in order to prevent drying of the material, root length of the whole root bundle was measured outside of the test tube by the ruler, as shown on fig. 1. this method gives one value for each bulb, and permits the experiment to be continued (f i s k e s j o , 1985). fig. 1. measuring the root length of the whole root bundle. root length of the whole root bundle was measured outside of the test tube by the ruler. this method gives one value for each bulb, and permits the experiment to be continued. root tips were cut by scalpel, fixed in methanol / glacial acid (3:1, v/v) for 24 h at 4 ºc. the next day they were placed in 70% ethanol alcohol and refrigerated until used. slides were prepared immediately before microscopic analysis. the root tips were hydrolyzed in 1 n hcl at 60 ºc for 13 minutes and washed three times in distilled water. after that, they were stained with 1% acetocarmine at room temperature for 15 minutes or, alternatively, on a bunsen burner for 1 2 minutes and washed three times in distilled water. the root tips were afterwards transferred onto the cold slides, squashed by cover slips and heat fixed. the following parameters were used for determination of cytotoxicity: treated root growth inhibition (trg), mitotic index (mi) and phase index (pi). for the treated root growth inhibition, from the length of bundle for each concentration (ltr) the percentage of root growth inhibition in relation to the negative control (lnc), i.e. 𝑇𝑅𝐺 = 𝐿𝑇𝑅 (𝑐𝑚) 𝐿𝑁𝐶 (𝑐𝑚) 𝑥100% and the ec50 (the effective concentration where root growth amounts to 50% of the controls) for each extract was determined (f i s k e s j o , 1985). for the mi and pi analysis, for every concentration, as well as for the controls, 5 microscopic slides were made, and 500 cells per slide, i.e. 2500 cells per concentration were observed by leica microscope with 400 x (fig. 2.) and 1000 x magnification (fig. 3.), and analyzed by the same microscope with 400 x magnification. mi was calculated as the total number of cells in prophase (p), metaphase (m), anaphase (a), and telophase (t) in relation to the number of observed cells (t e d e s c o & l a u g h i n g h o u s e iv, 2012). 𝑀𝐼 = 𝑃 + 𝑀 + 𝐴 + 𝑇 500 𝑥100% the proportion of mitotic phases was determined as a percent ratio between cells in specific phase and the dividing cells (f i s k e s j o , 1985). fig. 2. mitosis of a. cepa root meristems. magnification 400 x. leica light microscope. normal stages of mitotic division. (a) interphase (b) prophase (c) metaphase (d) anaphase (e) telophase. statistical analysis statistical analysis was carried out using microsoft excel 2010 and systat 13 with five replicates for each group. data were expressed as mean ± standard deviation (sd) calculated by one way analysis of variance (anova). differences between controls and the individual dosage group of each extract were analyzed by means of the paired t-test, with 95% confidence limits where significance was accepted at p<0.05. biologica nyssana 8 (2)  december 2017: 151-158 madić, v. et al.  evaluation of cytotoxicity of „anti-diabetic“ herbal… 154 results ad discussion the effects of the aqueous extracts of ‘anti-diabetic’ herbal mixture, r. fruticosus, v. myrtillus, p. erecta, g. urbanum and p. vulgaris on root growth of a. cepa are shown on tab. 1. good root growth was achieved in the negative control, while the positive control inhibited root growth. at tested concentrations, root growth was the highest at the 400 µg/ml concentration of all the extracts while it was the lowest at 1200 µg/ml, with the exception of p. vulgaris extract, where the highest root growth was in exposure to 800 µg/ml concentration of extract. inhibition of root growth implying toxicity was concentration dependent and statistically significant (p<0.05) at all tested concentrations of p. erecta and g. urbanum extracts, while extracts of ‘anti-diabetic’ herbal mixture, r. fruticosus and v. myrtillus triggered concentration dependent and statistically significant (p<0.05) inhibition of root growth at higher concentrations (800 µg/ml and 1200 µg/ml). the ec50 for the extracts of ‘anti-diabetic’ herbal mixture, r. fruticosus, v. myrtillus, p. erecta, g. urbanum and p. vulgaris were 1400 µg/ml, 800 µg/ml, 990 µg/ml, 690 µg/ml, 550 µg/ml, and 1590 µg/ml, respectively. the effects of tested extracts on cell division of a. cepa root meristem cells are presented in tab. 2. mitotic index in the negative and the positive control were 20.28 ± 2.17 and 3.88 ± 0.92, respectively. with increasing concentration of the all the tested extracts there was concentration dependent decrease in the mitotic index with the exception of p. erecta extract, where 400 µg/ml concentration did not cause statistically significant decrease of mi compared to the positive control. of all the lowest concentration of tested extracts (400 µg/ml), the fig. 3. stages of mitotic division in cells of a. cepa treated with aqueous extracts of ‘anti-diabetic’ herbal mixture, r. fruticosus, v. myrtillus, p. erecta, g. urbanum and p. vulgaris. magnification 1000 x. leica light microscope. normal stages of mitotic division. (a) interphase (b) prophase (c) metaphase (d) anaphase (e) telophase. table 1. the effects of aqueous extracts of ‘anti-diabetic’ herbal mixture, r. fruticosus, v. myrtillus, p. erecta, g. urbanum and p. vulgaris on root growth of a. cepa. rg (%) of the control, treated root growth expressed as % of the control. mix, ‘anti-diabetic’ herbal mixture. rfl, leaves of r. fruticosus. vml, leaves of v. myrtillus. per, roots of p. erecta. gua, aerial parts of g. urbanum. pvp, pods of p. vulgaris. nc, negative control. pc, positive control. * significant compared to its negative control at 0.05 level. º significant compared to its positive control at 0.05 level. mix rfl vml per gua pvp treatment mean root length (cm) ±sd trg (%) of control mean root length (cm)±sd trg (%) of control mean root length (cm) ±sd trg (%) of control mean root length (cm) ±sd trg (%) of control mean root length (cm) ±sd trg (%) of control mean root length (cm) ±sd trg (%) of control nc 3.04±0.59 100 3.04±0.59 100 3.04±0.59 100 3.04±0.59 100 3.04±0.59 100 3.04±0.59 100 400 µg/ml 2.82±0.46 92.8 º 2.7±0.48 88.8 º 2.44±0.59 80.1 º 2.3±0.21 75.7 * 1.56±0.45 51.3 * 2.9±0.22 95.4 º 800 µg/ml 2.1±0.24 69.1 * 1.52±0.53 50 * 1.7±0.47 55.1 * 1.3±0.41 42.8 * 1.48±0.53 48.7 * 3.12±0.25 102.6 º 1200 µg/ml 1.82±0.49 59.8 * 0.84±0.17 27.6 * 1.5±0.47 49.3 * 1.04±0.11 34.2 * 1±0.14 32.9 * 2.16±0.18 71 * pc 1.38±0.7 45 1.38±0.7 45 1.38±0.7 45 1.38±0.7 45 1.38±0.7 45 1.38±0.7 45 ec50 1400 µg/ml 800 µg/ml 990 µg/ml 690 µg/ml 550 µg/ml 1590 µg/ml biologica nyssana 8 (2)  december 2017: 151-158 madić, v. et al.  evaluation of cytotoxicity of „anti-diabetic“ herbal… 155 highest decrease of mitotic index was influenced by v. myrtillus extract (10.96 %). allium cepa root meristem cells of the control groups displayed the existence of all phases of mitosis. the different concentrations of the tested extracts caused different changes in the proportion of mitotic phase's distribution in comparison to the positive and negative controls, as shown on tab. 3. compared to the positive and negative control, influence of ‘anti-diabetic’ herbal mixture, r. fruticosus, v. myrtillus extracts on phase index was statistically significant concentration depended. prophase index was, as expected, the most frequent one in all of the tested groups. extracts of ‘anti-diabetic’ herbal mixture (400 µg/ml and 800 µg/ml concentrations) and r. fruticosus (800 µg/ml and 1200 µg/ml concentrations) decreased the prophase index of a. cepa root meristem cells, while 400 µg/ml and 800 µg/ml extract of v. myrtillus and 1200 µg/ml of ‘anti-diabetic herbal mixture’ increased the prophase index. the extracts of p. erecta, g. urbanum and p. vulgaris, with the exception of 400 µg/ml concentrated p. erecta which increased prophase index, had no influence on it. extract of ‘anti-diabetic’ herbal mixture did not change metaphase index with statistical significance, while higher concentrations of r. fruticosus (1200 µg/ml) and v. myrtillus (800 µg/ml) increased it. extracts of p. erecta, g. urbanum and p. vulgaris produced no change in metaphase index. none of the extracts had any influence on anaphase index. as expected, telophase index was the least frequent one in all the tested groups. extracts of r. fruticosus (400 µg/ml and 1200 µg/ml), v. myrtillus (all the tested concentrations), g. urbanum (800 µg/ml) and p. vulgaris (1200 µg/ml) decreased this index. higher plants such as a. cepa are accepted as admirable genetic models to evaluate genotoxic effects. results of the current study reflected the utility of root tips of cells of a. cepa for monitoring the cytotoxic effects of medicinal plant extracts. data on the effects of the extracts on root growth of a. cepa showed that there was concentration dependent decrease in root growth. based on the ec50 the order of induction of root growth inhibition was pvp < mix < vml < rfl < per < gua, as shown on tab. 1. as it is already said, little is known about their potential toxicity. our results are consistent with what has been done so far, i.e. a blackberry extract has cytotoxic capabilities as it can induce apoptosis in human leukemia hl-60 cells (s u n et al., 2002); p. erecta rhizome extracts is considered safe with respect to acute toxicity when applied to humans (s h u s h u n o v et al, 2009), and g. urbanum is considered non-toxic as well (p a u n et al., 2015). there is a linear relationship between macroscopic and microscopic parameters for all the extracts. in a. cepa, whenever there is root growth inhibition, there is always reduction in the number of dividing cells (f i s k e s j o , 1985). mitotic index was characterized by the total number of dividing cells in the cell cycle. mitotic index is used as an indicator of cell proliferation biomarkers which measures the proportion of cells in the mitotic phase of the cell cycle. therefore, the decrease in the mitotic index of a. cepa meristem cells could be interpreted as cellular death. low mitotic index may be reflecting a direct cytotoxic effect of the tested extract. (s a s i d h a r a n et al., 2012.) the cells of a. cepa root tips after treatment with extracts of all the tested extracts showed decrease in mitotic index with increasing concentration. there were significant differences (p table 2. cytological effects of aqueous extracts of ‘anti-diabetic’ herbal mixture, r. fruticosus, v. myrtillus, p. erecta, g. urbanum and p. vulgaris on meristematic cells of a.cepa. mi, mitotic index. mix, ‘anti-diabetic’ herbal mixture. rbl, leaves of r. fruticosus. vml, leaves of v. myrtillus. per, roots of p. erecta. gua, aerial parts of g. urbanum. pvp, pods of p. vulgaris. nc, negative control. pc, positive control. * significant compared to its negative control at 0.05 level. º significant compared to its positive control at 0.05 level. mix rfl vml per gua pvp treatment mitotic index (%) ± sd mitotic index (%) ± sd mitotic index (%) ± sd mitotic index (%) ± sd mitotic index (%) ± sd mitotic index (%) ± sd nc 20.28±2.17 20.28±2.17 20.28±2.17 20.28±2.17 20.28±2.17 20.28±2.17 400 µg/ml 13.96±0.64 * º 16.12±1.36 * º 10.96±0.78 * º 16.76±2.67 º 16.92±2.61 * º 16.16±1.38 * º 800 µg/ml 9.8±0.55 * º 12.2±0.6 * º 10.04±1.91 * º 14.68±0.84 * º 12.76±0.48 * º 9.68±0.48 * º 1200 µg/ml 6.4±0.93 * º 9.56±1.08 * º 7.72±1.4 * º 11.84±0.73 * º 9.12±1.47 * º 7.96±0.68 * º pc 3.88±0.92 3.88±0.92 3.88±0.92 3.88±0.92 3.88±0.92 * º 3.88±0.92 * º biologica nyssana 8 (2)  december 2017: 151-158 madić, v. et al.  evaluation of cytotoxicity of „anti-diabetic“ herbal… 156 < 0.05) between treated groups and a control group in mitotic index, as shown on tab. 2. observed mitodepressive effect suggests that tested extracts had some effects on cell division of a. cepa. this may be due to abnormal conditions of the cells induced by the treatments. the reduction of the mitotic index might be explained as being due to the obstruction of the onset of prophase, the arrest of one or more mitotic phases, or the slowing of the rate of cell progression through mitosis (b r i a n d & k a p o o r , 1989). of all the tested aqueous extracts, only ‘antidiabetic’ herbal preparation, r. fruticosus, v. myrtillus showed an influence on the mitotic phases, and ‘anti-diabetic’ herbal preparation (1200 µg/ml) and v. myrtillus (400 µg/ml) caused surprisingly high prophase accumulation. this highly prophase accumulation could be correlated to the blockage of the dividing cells at chfr point which prevents prophase-metaphase transition as proved by s c o l n i c k and h a l a z o n e t i s (2000) who explained that chfr protein delays chromosome condensation and nuclear envelope breakdown in response to drug such as taxol and nocodazole that disrupt microtubule structure. although chromosome abnormalities were not analyzed in this paper, it is observed that ‘anti-diabetic’ herbal mixture, r. fruticosus, v. myrtillus and the positive control caused spindle disturbance, especially c-mitosis, as the most common type of abnormalities, which indicates that these extracts not only caused disruption in the spindle structure, but also caused inhibition of spindle formation and prevented its polymerization (s a l m o n et al., 1984). conclusion the diabetes problem is a global epidemic, mostly in lowto middle income countries. costs of international commercial medicines are high, which caused an increased popularity of traditional medicine. however, some constituents of medicinal plants have been found to be potentially toxic (p i n g et al., 2012). according to our literature review, a little is known about potential toxicity of r. fruticosus, v. myrtillus, p. erecta, g. urbanum and p. vulgaris, medicinal plants used as a traditional remedy for diabetes. table 3. phase index of a. cepa root meristem cells treated with different concentrations of aqueous extracts of ‘anti-diabetic’ herbal mixture, r. fruticosus, v. myrtillus, p. erecta, g. urbanum and p. vulgaris. mix, ‘antidiabetic’ herbal mixture. rfl, leaves of r. fruticosus. vml, leaves of v. myrtillus. per, roots of p. erecta. gua, aerial parts of g. urbanum. pvp, pods of p. vulgaris. nc, negative control. pc, positive control. * significant compared to its negative control at 0.05 level. º significant compared to its positive control at 0.05 level. tested extract phase index (%) ± sd treatment nc 400 µg/ml 800 µg/ml 1200 µg/ml pc mix prophase 52.02±4.17 44.47±5.05 * º 48.91±3.84 º 62.46±6.41 * º 84.0±16.04 metaphase 20.61±4.39 22.06±2 º 22.47±2.3 º 14.93±2.63 º 4.29±4.72 anaphase 14.07±6.41 16.47±5.6 16.76±5.23 13.49±3.8 5.34±5.29 telophase 13.36±2.88 16.99±6.34 11.87±6.48 7.86±4.78 6.29±11.23 rfl prophase 52.02±4.17 55±3.59 * º 48.55±3.59 º 49.36±3.17 * º 84.0±16.04 metaphase 20.61±4.39 24.1±1.56 º 24.85±2.27 º 30.18±2.25 * º 4.29±4.72 anaphase 14.07±6.41 13.41±4.03 22.19±6.42 17.17±3.47 º 5.34±5.29 telophase 13.36±2.88 7.4±3.474 * 7.42±3.61 3.28±1.69 * 6.29±11.23 vml prophase 52.02±4.17 77.18±3.22* º 59.57±1.95 * º 49.66±7.05 º 84.0±16.04 metaphase 20.61±4.39 9.44±1.99 * º 30.84±3.98 * º 17.34±11.53 4.29±4.72 anaphase 14.07±6.41 7.15±2.8 8.44±2.27 23.8±10.91 º 5.34±5.29 telophase 13.36±2.88 6.23±1.75 * 1.14±1.07 * 9.19±2.9 * 6.29±11.23 per prophase 52.02±4.17 58.25±2.55* º 51.42±7.98 º 48.62±10.12 º 84.0±16.04 metaphase 20.61±4.39 18.26±3.12 º 19.56±3.47 º 26.11±14.82 º 4.29±4.72 anaphase 14.07±6.41 13.14±2.21 º 15.13±3.04 º 12.15±4.89 º 5.34±5.29 telophase 13.36±2.88 10.34±3.49 13.9±5.18 13.13±5.88 6.29±11.23 gua prophase 52.02±4.17 53.28±5.81 º 56.17±6.35 º 50.24±4.65 º 84.0±16.04 metaphase 20.61±4.39 19.31±3.19 º 18.14±5.1 º 22.49±4.1 º 4.29±4.72 anaphase 14.07±6.41 15.07±5.89 º 17.88±3.13 º 17.45±3.31 º 5.34±5.29 telophase 13.36±2.88 12.23±4.58 8.78±2.97 * 9.82±4.35 6.29±11.23 pvp prophase 52.02±4.17 58.12±4.96 º 55.46±3.92 º 65.94±10.26 84.0±16.04 metaphase 20.61±4.39 23.23±1.69 º 21.09±2.61 º 16.29±5.38 º 4.29±4.72 anaphase 14.07±6.41 9.95±2.87 13.62±2.16 º 10.79±4.29 5.34±5.29 telophase 13.36±2.88 8.7±3.78 9.82±4.88 6.97±1.67 * 6.29±11.23 biologica nyssana 8 (2)  december 2017: 151-158 madić, v. et al.  evaluation of cytotoxicity of „anti-diabetic“ herbal… 157 the results presented in this paper are therefore important since they suggest the cytotoxic effect of these medicinal plants in some concentrations. exposure to the high concentrations of all the tested extracts, especially to ‘anti-diabetic’ herbal mixture, r. fruticosus and v. myrtillus, initiates a cascade of deleterious changes within a. cepa root cells, visible as the inhibition of root growth and the reduction of the mitotic index. moreover, ‘anti-diabetic’ herbal preparation (1200 µg/ml) and v. myrtillus (400 µg/ml) caused surprisingly high prophase accumulation as well as spindle disturbance, especially c-mitosis, indicating that these extracts at these concentrations have a toxic effect. further cytogenetic studies dealing with clastogenicity and genotoxicity of these extracts with more comprehensive genotoxicity assessment in animal model may reveal further interesting results for their usage as a traditional medicine. references akinboro, a., bakare, a.a. 2007: cytotoxic and genotoxic effects of aqueous extracts of five medicinal plants on allium cepa linn. journal of ethnopharmacology, 112 (3): 470-475. briand, c.h., kapoor, b.m. 1989: the cytogenetic effects of sodium salicylate on the root meristem cells of allium sativum l. cytologia, 54: 203–209. camparoto, m.l., teixeira, r.o., mantovani, m.s., vicentini, v.e.p. 2002: effects of maytenus ilicifolia mart. and bauhinia candicans benth infusions on onion root-tip and rat bone-marrow cells. genetics and molecular biology, 25: 85-89. çelik, t.a., aslantürk, ö.s. 2010: evaluation of cytotoxicity and genotoxicity of inula viscosa leaf extracts with allium test. journal of biomedicine and biotechnology: 1-8. eddouks, m., maghrani, m., lemhadri, a., ouahidi, m.l. 2002: ethnopharmacological survey of medicinal plants used for the treatment of diabetes mellitus, hypertension and cardiac diseases in the south-east region of morocco (tafilalet). journal of ethnopharmacology, 82 (2-3): 97-103. farnsworth, n.r., akerele, o., bingel, a.s., soejarto, d.d., guo, z. 1985: medicinal plants in therapy. bulletin of the world health organization, 63 (6): 965–981. fiskesjo, g. 1985: the allium test as a standard in environmental monitoring. hereditas, 102: 99112. gabriela, p., neagu, e., albu, c., radu, g.r. 2015: inhibitory potential of some romanian medicinal plants against enzymes linked to neurodegenerative diseases and their antioxidant activity. pharmacognosy magazine, 11 (1): s110116 gale, e.a.m., wagner, t.h. 2006: dying of diabetes. bmj publishing group limited and rps publishing, lancet, london. 368 (9548): 1626-1628. international diabetes federation (idf). 2015: idf diabetes atlas 7th edition. international diabetes federation. 47-65 johnson, m.h., de mejia, e.g. 2016: phenolic compounds from fermented berry beverages modulated gene and protein expression to increase insulin secretion from pancreatic cells in vitro. journal of agricultural and food chemistry, 65 (30): 6211-6221. koupý, d., kotolová, h., kučerová, j. 2015: effectiveness of phytotherapy in supportive treatment of type 2 diabetes mellitus billberry (vaccinium myrtillus). ceska a slovenska farmacie, 64 (1-2): 3-6. kyznietsova, m. y., halenova, t. i., savchuk, o. m., vereschaka, v. v., ostapchenko, l. i . 2015: carbohydrate metabolism in type 1 diabetic rats under the conditions of the kidney bean extract application. fiziolohichnyi zhurnal, 61 (6): 96– 103. paun, g., neagu, e., albu, c., radu, g. l., 2015: inhibitory potential of some romanian medicinal plants against enzymes linked to neurodegenerative diseases and their antioxidant activity. pharmacognosy magazine, 11 (1): 110– 116. ping, k.y., darah, i., yusuf, u.k., yeng, c., sasidharan, s. 2012: genotoxicity of euphorbia hirta: an allium cepa assay. molecules, 17 (7): 7782-7791. rank, j., nielsen, m.h. 1994: evaluation of the allium anaphase-telophase test in relation to genotoxicity screening of industrial wastewater. mutation research, 312 (1): 1724. salmon, e.d., mckeel, m., hays, t. 1984: rapid rate of tubulin dissociation from microtubules in the mitotic spindle in vivo measured by blocking polymerization with colchicine. the journal of cell biology, 99 (3): 1066–1075. sasidharan, s., ping, darah, i., yusuf, u.k., yeng, y., 2012: genotoxicity of euphorbia hirta: an allium cepa assay. molecules, 17: 7782-7791. scolnick, d.m., halazonetis, t.d. 2000: chfr defines a mitotic stress checkpoint that delays entry into metaphase. nature, 406 (6794): 430–435. sidorova, y., shipelin, v., mazo, v., zorin, s., petrov, n., kochetkova, a. 2017: hypoglycemic and hypolipidemic effect of vaccinium myrtillus l. leaf and phaseolus vulgaris l. seed coat extracts in diabetic rats. nutrition, 41: 107–112. biologica nyssana 8 (2)  december 2017: 151-158 madić, v. et al.  evaluation of cytotoxicity of „anti-diabetic“ herbal… 158 shushunov, s., balashov, l., kravtsova, a., krasnogorsky, i., latté, k.p., vasiliev, a. 2009: determination of acute toxicity of the aqueous extract of potentilla erecta (tormentil) rhizomes in rats and mice. journal of medicinal food, 12 (5): 1173-1176 sun, j., chu, y.f., wu, x., liu, r.h. 2002: antioxidant and antiproliferation activities of fruits. journal of agricultural and food chemistry, 50: 7449–7454. swanston-flatt, s.k., day, c., bailey, c.j., flatt, p. r. 1990: traditional plant treatments for diabetes. studies in normal and streptozotocin diabetic mice. diabetologia, 33 (8): 462–464. tedesco, s.b., laughinghouse iv, h. d. 2012: bioindicator of genotoxicity: the allium cepa test. in: strivastava, j.k.: environmental contamination, 137-156. intech. teixeira, r.o., camparoto, m.l., mantovani, m.s., vicentini, v.e.p. 2003: assesment of two medicinal plants psidium guajava l. and achillea millefolium l., in in vitro and in vivo assays. genetics and molecular biology, 26 (4): 551-555. tomczyk, m., latté, k. p. 2009: potentilla a review of its phytochemical and pharmacological profile. journal of ethnopharmacology, 122 (2): 184– 204. anticancer compounds from medicinal plants biologica nyssana 4 (1-2)  december 2013: 41-48 atanacković v. et al.  morphological and anatomical observatoris on … 41 original article morphological and anatomical observations on ranunculus auricomus l. var. biformis l. in vlasina lake, serbia valentina atanacković 1 , vladimir ranđelović 2 , rashid ismael hag ibrahim 3 1 university of lleida, etsea, department hbj, av. alcalde rovira roure 191, 25198 lleida, spain; 2 university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia 3 department of horticulture, gyeongnam national university of science and technology, jinju 660-758, republic of korea * e-mail: valentina.atanackovic@gmail.com abstract: atanacković, v., ranđelović, v., ibrahim, r.i.h.: morphological and anatomical observatoris on raunculus auricomus l. var. biformis l. in vlasina lake, serbia . biologica nyssana, 4 (1-2), december 2013: 41-48. r. auricomus var. biformis l. (r. binatus kit.) grows mainly in the suburbs of vlasina plateau beside a variety of other species and forms one part of the peat-land association. specimens were collected from the northeast coastal flooded area of vlasina lake (latitude: 42° 41' 46 n, longitude: 22° 22' 14 e). in this study, some morphological and anatomical characteristics were investigated. samples for anatomical analysis were prepared according to the standard methods described by ruzin (1999) though, slightly modified (atanackovic et al., 2012). morphological features such as leaf, petiole, stem, and root have been described. the anatomy presented in this work form the first of its kind available in the literature about this variety found in the vlasina lake. transverse sections of ground-leaf, upper-leaf, petiole, stem and root have been examined and photographed. the observed morphological and anatomical feauters showed that the species grows in these flooding conditions. key words: anatomy, leaf, morphology, petiole, ranunculus, root, stem introduction ranunculus l. or buttercup is the largest genus of ranunculaceae that contains around 600 species and described as cosmopolitan (t a m u r a , 1993) that mostly distributed in all continents throughout temperate, arctic, sub-antarctic and rarely in high elevated areas of the tropic zones excluding the antarctic (e r s t a n d s u k h o r u k o v , 2011). ranunculus grows in a wide range of habitats such as rivers, lakes, wet soils, damp/dry meadows and forests, and shows several morphological adaptations to different habitats (p a u n et al., 2006). ranunculus is well established in central europe where the yellow buttercups are found in meadows, pastures, or ruderal places during spring and summer (s t e i n b a c h a n d g o t t s b e r g e r , 1994). r. auricomus complex (aggregate) represents one of the larger clades within ranunculus (h ö r a n d l et al., 2005) that is distributed throughout europe to western siberia, from the arctic zone to the mediterranean region, greenland and alaska (j a l a s a n d s u o m i n e n , 1989). r. auricomus complex is highly diverse in morphotypes, cytotypes and ecotypes, which were the reasons to be considered as a separate section (l o o s , 1997) and often subdivided into four morphological groups; auricomus, cassubicus, fallax and monophyllus sensu (e r i c s s o n , 1992; 2001). 11 th sfses • 13-16 june 2013, vlasina lake 4 (1-2) • december 2013: 41-48 biologica nyssana 4 (1-2)  december 2013: 41-48 atanacković v. et al.  morphological and anatomical observatoris on … 42 h ö r a n d l et al. (2005) reported that various morphological adaptations and reproductive strategies such as vegetative reproduction (stolons), self compatibility (in water-buttercups), and likely agamospermy (r. auricomus complex) could play important role in the ranunculus ability to colonize different habitats, altitudes, and latitudes. despite the morphological adaptations, r. auricomus var. biformis is in an extremely high risk of extinction at the regional scale and it is recognized as a critically endangered variety (r a n đ e l o v i ć et al., 2010). temporary or continuous flooding of soil with fresh water occurs as a result of overflowing of the lake (r a n đ e l o v i ć , z l a t k o v i ć , 2010). the mechanisms by which flood-tolerant plants survive water-logging are complex and involve interactions of morphological, anatomical, and physiological adaptations (k o z l o w s k i , 1997; s t e v e n s et al., 2002). generally, adverse effects of flooding include changes in plant distribution and composition as well as changes in shoot growth of many plants due to suppressed leaf formation and expansion of leaves and internodes. soil inundation leads to increased length of the first internode and furthermore, flooding may also increases intercellular spaces in the tissues of flood-tolerant plants (a t a n a c k o v i c et al., 2012). certain terrestrial plant species respond to poor aeration in flooded areas and produce aerenchyma mainly in the cortical tissues of the stem and root (j a c k s o n a n d c o l m e r , 2005; s t e v e n s et al., 2002; jung et al., 2008). thus, flooded habitats may change the anatomical structure such as the cuticle layer and phloem in leaves, pith cavity area and vessel lumen in stem (t a o et al., 2009). different plants express different morphological or anatomical adjustments in response to environmental changes, which act as mechanisms of survival and reproduction under a wide range of conditions (b e n z et al., 2007). we assumed that r. auricomus var. biformis adapted and survived in temporal flooded peat bog habitat due to morphological and/or anatomical adjustments. hence, its morphological and anatomical features of leaf, petiole and stem and anatomical adaptations of root were investigated. material and methods sampling of plant material plant material were collected from the northeast coastal flooded area of vlasina lake at latitude 42° 41' 46 n and longitude 22° 22' 14 e (fig. 1). the variety was identified by professor vladimir stevanovic. plant samples were fixed in 50% ethanol and samples were also deposited and recorded at the herbarium and wet collection of the department of ecology and geography of plants, institute of botany and botanical garden “jevremovac”, faculty of biology, university of belgrade, serbia. fig. 2. r. auricomus var. biformis plant growing in a peat bog habitat fig. 1. geographical position of investigated area biologica nyssana 4 (1-2)  december 2013: 41-48 atanacković v. et al.  morphological and anatomical observatoris on … 43 preparation of plant material for anatomical analysis anatomical analysis of leaf, petiole, stem and root were performed. samples were prepared according to the standard methods described by r u z i n (1999), though slightly modified (a t a n a c k o v i ć et al., 2012). morphological observations and anatomical analysis under light microscope certain quantitative measures were carried out for leaf, stem and root of r. auricomus var. biformis as well as description of petioles. morphometric measures were taken for leaves that included; leaf length, leaf length from base to the widest part, leaf width, leaf perimeter, and leaf area, while those for the plant included; length of each of the first three internodes, stem length and total number of internodes. measurements performed on the cross section of leaf included; leaf thickness, width and thickness of epidermal cells and surface, total thickness of mesophyll including thickness of spongy and palisade tissues and number of layers for each tissue. the diameter of primary cortex and central cylinder at the radius of root cross section were also measured. statistical analysis to increase the accuracy of data and statistical analysis, each character was measured on 25 samples of leaf, stem and root of individuals from the plant population. all measurements of quantitative characters were performed on an “image analyzer” software package (qwin, leica microsystems imaging solutions ltd, cambridge, uk), and then the measurement results were processed by the software spss 15 for windows (spss 15.0. 2006). for each character, basic parameters were statistically analyzed such as mean, minimum value, maximum value and standard errors. results and discussion in serbia, r. auricomus var. biformis grows in light and moist deciduous forests, bushes, thickets, damp meadows, fields from lowland regions to alps belt, but grows mainly in the suburbs of vlasina plateau beside a variety of other species. r. auricomus var. biformis became part of the peatland association that includes caricetum goodenowii and caricion cannescentis-nigrae, which belong to the class caricetalia fusca and scheuchzerio-caricetea fusca (r a n đ e l o v i ć et al., 2010). r. auricomus var. biformis is a perennial plant that grows during spring-summer in a halfrosette and hemicryptophyte (fig. 2). ground leaves are rounded, kidney-shaped and partially divided into lobes. the stem is erect, in the range of 9.5 cm high and rarely covered with trichomes. the root is short and vertical with numerous hair roots. morphological characteristics leaf r. auricomus var. biformis has two heterophyllous leaves here named as upper and ground leaves, which differ in shape and size. upper leaves are sessile and cut into 4-8 linear-lanceolate lobes. these leaves are fan-like-shaped, soft, shiny, smooth and dark green, while the reverse side is light green and the length of leaf is nearly six times its width (fig. 3a). there are two types of ground leaves namely ground leaves that are not lobed (fig. 3b) and ground leaves that are divided into lobes (fig. 3c). deep ground leaves have larger lobes with rounded tips, sharply-toothed secondary parts and sometimes appear jagged with lateral lobes. ground leaves are kidney-shaped, shallow, soft, smooth, and glossy green with long petioles. the length of leaf is slightly less than or equal to its width (table1). fig. 3. morphological features of upper (a) and ground leaves; not lobed (b) and lobed (c) from r. auricomus var. biformis. a b c biologica nyssana 4 (1-2)  december 2013: 41-48 atanacković v. et al.  morphological and anatomical observatoris on … 44 stem r. auricomus var. biformis is an erect plant, branched and can reach 9.5 cm tall (fig. 2). two or three internodes of various lengths were observed and while the first internode is the longest, the second and third were slightly shorter (table 2). anatomical characteristics upper leaf the upper leaf of r. auricomus var. biformis is thinner compared with the ground leaf (table 1). the adaxial layer of the epidermis consists of larger cells and a few stomata were observed (fig. 4a). the mesophyll composed of the palisade tissue of 2-3 layers and the spongy tissue of 4 layers of cells and large intercellular spaces, but cells are smaller than in the ground leaves. the palisade tissue is slightly thicker than the spongy tissue (fig. 4b). the vascular bundle is collateral and surrounded by sclerenchyma tissue (fig. 4c). ground leaf the two types of ground leaves are not different in structure as seen from the cross sections. the epidermis was observed as one layer of large cells on the adaxial side of leaf and the exterior walls are thicker and slightly prominent. stomata were seen on both adaxial and abaxial surfaces or leaves are amfistomatic. similar to the upper leaves, the mesophyll is divided into the palisade tissue that consists of 2-3 layers of extremely large cells, but the first layer of cells is slightly longer than other layers and smaller intercellular spaces were observed. the spongy tissue of 4-6 layers of cells that are irregular in shape, thick-walled and separated by large intercellular spaces (fig. 5a). the vascular bundles are collateral, enclosed in sclerenchyma tissue (fig. 5b) and the number of vascular bundles is between 28 and 30 (table 1). petiole the cross section of the petiole of ground leaf; lobed or not, is kidney-shaped and showed clearly distinct epidermis, primary cortex, central cylinder and pith cavity on one side towards the centre. the epidermis of the petiole consisted mostly of one layer, but two layers in some parts, covered with a well-developed cuticle and stomata were observed. the primary cortex consists of small parenchyma cells that are densely packed in groups of 6-7 layers separated by large intercellular spaces and sometimes there might be one or two large voids. five vascular bundles were seen and the central bundle is the largest, while the marginal were the least developed. all bundles are partially enveloped in sclerenchyma that was observed on both sides of the phloem and xylem and the cambium was visible in the central bundle. the pith cavity is irregular in shape (saddle-shaped) and broken in some areas and remains of the pith could be seen (fig. 6). stem the cross section of stem clearly showed distinct epidermis, primary cortex, vascular tissue and pith cavity located at the centre. the epidermis of stem consists of mostly two layers covered with a well-developed cuticle and stomata were also observable in some areas (fig. 7a). the primary cortex consists of slightly thicker-walled small parenchyma cells and densely packed but in some areas large intercellular spaces were visible. the chloroplasts and starch reserves were observable in parenchyma cells (fig. 7b). the vascular tissue consists of 9-10 vascular bundles partially enveloped in sclerenchyma tissue, which is found only on the phloem side of the vascular bundle. in addition to the basic parenchyma tissue, the vascular tissue contains xylem vessels and phloem in a collateral arrangement and remains of the cambium could be seen (fig. 7c). the central cavity forms the pith cavity and remains of the pith were observed in some areas (fig. 7a). the thickness of stem cortex is ¾ of the radius of pith cavity (table 2). root the cross section of root showed the primary cortex and central cylinder (fig. 8a). the parenchyma of primary cortex consists of thinwalled, densely packed cells filled with reserve materials and intercellular spaces between cells scarcely observable. the endodermis forms the last layer of the primary cortex. the vascular tissue consists of the pericycle of 1 or 2 layers of cells, the xylem and phloem that were arranged in radials (fig. 8b). the xylem consists of 3-4 bundles each contains 4-10 vessels and typical to the normal root with a central stele. the plates of phloem are radial in the vascular bundles. the protophloem mesh tubes were seen larger and located on the periphery, while the metaphloem is near the centre (fig. 8c). the primary cortex is five times larger than the central cylinder (table 2). biologica nyssana 4 (1-2)  december 2013: 41-48 atanacković v. et al.  morphological and anatomical observatoris on … 45 fig. 4. a cross section through the upper leaf of r. auricomus var. biformis (a) shows the upper and lower epidermis, mesophyll tissue; palisade and spongy tissue, vascular bundle of mid-rip, and stomata (b) and close up of the vascular bundle of mid-rip (c). ep; epidermis, p; palisade tissue, s; spongy tissue, vb; vascular bundle of mid-rip, st; stoma, x; xylem vessel. table 1. morphological and anatomical features of the upper and ground leaf from r. auricomus var. biformis 1 mean ± standard error fig. 5. a cross section through a ground leaf of r. auricomus var. biformis (a) shows a vascular bundle (b). ep; epidermis, st; stoma, p; palisade tissue, s; spongy tissue, x; xylem vessel, ph; phloem, sc; sclerenchyma. upper leaf ground leaf (not lobed) 1 ground leaf (lobed) morphology leaf length (mm) 06.20 ± 0.20 / / length from the base to widest part (mm) 03.07 ± 0.10 05.8 ± 0.30 05.5 ± 0.2 leaf width (mm) 01.40 ± 0.40 15.6 ± 0.60 18.2 ± 0.3 leaf perimeter (mm) 71.20 ± 0.20 51.7 ± 0.30 31.4 ± 0.1 leaf blade area (cm²) 09.80 ± 0.01 14.3 ± 0.01 03.1 ± 0.1 anatomy leaf thickness (µm) 106.813 ± 0.4 120.80 ± 0.2 114.0 ± 0.2 width of upper epidermis (µm) 014.300 ± 0.6 012.40 ± 0.4 012.4 ± 0.3 thickness of upper epidermis (µm) 010.900 ± 0.5 010.40 ± 0.3 010.6 ± 0.4 mesophyll tissue thickness (µm) 086.700 ± 0.4 099.90 ± 0.2 098.9 ± 0.3 palisade tissue thickness (µm) 056.800 ± 0.2 056.50 ± 0.2 045.7 ± 0.2 spongy tissue thickness (µm) 046.900 ± 0.3 055.00 ± 0.1 055.1 ± 0.1 the number of layers of palisade tissue 002.000 ± 0.0 003.00 ± 0.0 003.0 ± 0.0 the number of layers of spongy tissue 004.000 ± 0.0 006.00 ± 0.2 005.0 ± 0.2 width of lower epidermis (µm) 010.900 ± 0.5 009.20 ± 0.3 008.8 ± 0.3 thickness of lower epidermis (µm) 009.200 ± 0.4 008.92 ± 0.3 008.5 ± 0.2 ep p s vb a b c st x st ep sc x ph p s a b biologica nyssana 4 (1-2)  december 2013: 41-48 atanacković v. et al.  morphological and anatomical observatoris on … 46 fig. 6. a cross section of the leaf petiole of r. auricomus var. biformis. ep; epidermis, vb; central vascular bundle, pc; pith cavity. fig. 7. a cross section through the stem of r. auricomus var. biformis shows a distinct epidermis, primary cortex, vascular tissue and pith cavity located at the centre (a) a vascular bundle in a parenchyma tissue (b) and details of a vascular bundle (c). ep; epidermis, co; cortex, vp; vascular bundle, pc; pith cavity, sc; sclerenchyma, pa; parenchyma, x; xylem vessel, ca; cambium residues, ph; phloem. fig. 8. a cross section through the root of r. auricomus var. biformis (a) shows the central cylinder and cortex (b) and details of the central cylinder (c). co; cortex, cc; central cylinder, en; endodermis, x; xylem vessel, ph; phloem. vb pc ep a b a b c sc pa ca ph x pc ep co vb a b c cc co x en ph biologica nyssana 4 (1-2)  december 2013: 41-48 atanacković v. et al.  morphological and anatomical observatoris on … 47 table 2. morphological and anatomical features of the stem and root of r. auricomus var. biformis feature mean ± se 1 stem morphology stem length (cm) 9.40 ± 0.005 length of first internode 1(cm) 5.72 ± 0.400 length of second internode (cm) 1.96 ± 0.400 length of third internode 3(cm) 3.07 ± 0.400 total number of internodes 2.30 ± 0.900 stem anatomy stem cortex thickness (µm) 303.1 ± 0.1 radius of pith cavity of stem (µm) 399.9 ± 0.9 root anatomy cortex diameter of root (µm) 527.9 ± 0.1 radius of central cylinder (µm) 126.5 ± 0.5 1 mean ± standard error discussion r. auricomus var. biformis is characterized by a terrestrial life form. despite this terrestrial mode of life, r. auricomus var. biformis managed to adapt and survive during periods of drought in mostly wet meadows. the durability of this modified form of life varies and depends on microclimatic changes in a given habitat (personal observation during this study). morpho-anatomical analysis indicated that r. auricomus var. biformis considers specific structure and typical terrestrial/aquatic forms. r. auricomus var. biformis belongs to the ecological group of hygromesophytes and grows as a terrestrial plant in temperate and moist areas on the slopes around vlasina lake that is characterized by slight changes of humidity. on this terrestrial type and according to the position on plant, two types of ground kidney-shaped leaves are formed. these leaves are characterized by one layer of epidermis on the adaxial surface, large cells, elongated palisade tissue cells and large intercellular spaces, which can be correlated with significant surface moisture due the wet habitat. the plant is characterized by a small diameter of the primary cortex of stem. morphological characteristics of leaves and stem two kinds of heterophyllous leaves were observed, namely; upper linear-lanceolated-lobed and sessile leaves and ground leaves that were lobed or not. the length of the internodes varied and the first internode was the longest that could be a consequence of the wet habitat (table 2), which is in contrary to the shortest first internode of r. lingua from the same area (atanackovic et al., 2012). anatomical characteristics of leaf, petiole, stem and root the upper leaf of r. auricomus var. biformis was thinner compared with the ground leaf (table 1). the three types of leaves contained one layer of epidermis and the stomata were rarely observed, which could be an adaptation to the habitat. smaller intercellular spaces were seen in the mesophyll of the upper leaves compared with the ground leaves. large intercellular spaces between cells in the spongy tissue and long palisade cells are characteristics influenced by water availability on leaves. the plant stem showed structural features of the genus ranunculus that consists of primary cortex, vascular tissue and pith cavity located at the centre (fig. 7). one layer of epidermal cells contains only few stomata, primary cortex composed of small cells that are separated by large intercellular spaces, which could be a characteristic anatomical feature of a plant living in a wet habitat. the central cylinder consists of 9-10 vascular bundles. the petiole of ground leaves showed similar structure as the stem, but less number of vascular bundles, i.e. around 5. the root consisted of primary cortex and central cylinder, though the structure was not different from other ranunculus species. conclusion the goal of this study was to observe the morphological characteristics of leaves and stem and anatomical characteristics of leaves, petiole, stem and root of r. auricomus var. biformis in peat bog habitat, in se serbia. the wet habitat partially influenced the anatomical structure of leaf, stem and root, while the morphological characteristics did not show any influence by the habitat except for the length of first internode of the stem. nevertheless, the constraints in a term of temporal flooding could not affect the species due the stable structure of plant organs. acknowledgements. this study was supported by the project of ministry of education, science and technological development of republic of serbia (project no. 173030). we wish to thank vladimir stevanovic for the identification of the species and variety in the laboratory at the department of ecology and geography of plants, university of belgrade. we are also thankful to branka stevanovic for her useful comments on the anatomy part of this study. biologica nyssana 4 (1-2)  december 2013: 41-48 atanacković v. et al.  morphological and anatomical observatoris on … 48 references atanacković, v., ranđelović, v., ibrahim, r.i.h. 2012: morphological and anatomical observations on ranunculus lingua l. under flooding conditions in vlasina lake (se serbia), biologica nyssana, 3 (2): 97-103. benz b.r., rhode j.m., cruzan m.b. 2007: aerenchyma development and elevated alcohol dehydrogenase activity as alternative responses to hypoxic soils in the piriqueta caroliniana complex. amer j of bot 94(4): 542-550. debell d.s., hook d.d., mckee jr w.h. 1984: growth and physiology of loblolly pine roots under various water table level and phosphorous treatments. for sci 30:705-714. ericsson s. 1992: the microspecies of the ranunculus auricomus complex treated at the species level. ann bot fenn 29: 123-158. ericsson s. 2001: ranunculus auricomus complex. in: jonsell b. and karlsson t. (eds.), flora nordica, chenopodiaceae to fumariaceae. vol. 2, pp. 237-255. the bergius foundation and the swedish royal academy of sciences, stockholm. erst a.s. and sukhorukov a.p. 2011: records of ranunculus (ranunculaceae) from nepal. ecology 56(3): 218-222. hörandl e., paun o., johansson j.t., lehnebach c., armstrong t., chen l., lockhart p. 2005: phylogenetic relationships and evolutionary traits in ranunculus s.l. (ranunculaceae) inferred from its sequence analysis. mol phyl and evol 36(2): 305-327. jackson m.b. and colmer t.d. 2005: response and adaptation by plants to flooding stress. annals of bot 96: 501-505. jalas j., and suominen j. 1989: atlas florae europaeae, vol. 8, nymphaeaceae to ranunculaceae. helsinki: the committee for mapping the flora of europe and societas biologica fennica vanamo. jung f., böhning-gaese k., prinzing a. 2008: life history variation across a riverine landscape: intermediate levels of disturbance favor sexual reproduction in the ant-dispersed herb ranunculus ficaria. ecography 31: 776-786. kozlowski t.t. 1997: responses of woody plants to flooding and salinity. tree physiology monograph (1): 1-29. lieffers v.j. and rothwell r.l. 1986a: effects of water table and substrate temperature on root and top growth of picea mariana and larix laricina seedlings. can j for res 16:1201-1206. lieffers v.j. and rothwell r.l. 1986b: rooting of peatland black spruce and tamarack in relation to depth of water table. can j bot 65:817-821. loos g.h. 1997: contribution to a supraspecific structure of ranunculus sect. ranunculus. thaiszia j bot 7: 1-7. mckevlin m.r., hook d.d., mckee jr w.h. 1995: growth and nutrient use efficiency of water tupelo seedlings in flooded and well-drained soils. tree physiol 15:753-758. paun o., greilhuber j., temsch e.m., hörandl e. 2006: patterns, sources and ecological implications of clonal diversity in apomictic ranunculus carpaticola (ranunculus auricomus complex, ranunculaceae). mol ecol 15(4): 897910. ranđelović v., zlatković b., dimitrijević d., vlahović t. 2010: phytogeographical and phytocoenological analysis of the threatened plant taxa in the flora of the vlasina plateau (se serbia ). biologica nyssana 1: 1-7. ruzin s.e. 1999: plant microtechnique and microscopy. oxford university press, new york, oxford. spss 15.0. (2006) command syntax reference, spss inc., chicago ill. steinbach k. and gottsberger g. 1994: phenology and pollination biology of five ranunculus species in giessen, central germany. phyton 34: 203-218. stevens k.j., peterson r.l., reader r.j. 2002: the aerenchymatous phellem of lythrum salicaria (l.): a pathway for gas transport and its role in flood tolerance. annals of bot 89(5): 621-625 tamura m. 1993: ranunculaceae. in: kubitzki k, rohwer jg, bittrich v (eds.). the families and genera of vascular plants vol. 2, pp. 563-583. springer-verlag, berlin. tao y., chen f., wan k., li x., li j. 2009: the structural adaptationof aerial parts of invasive alternanthera philoxeroides to water rgime. j plant biol 53: 403-410. trends in biological activity research of wild-growing aromatic plants from central balkans biologica nyssana 7 (2)  december 2016: 61-73 džamić, a. et al.  trends in biological activity research of wild… 61 review article received: 10 october 2016 revised: 02 november 2016 accepted: 07 november 2016 trends in biological activity research of wild-growing aromatic plants from central balkans ana m. džamić1*, jelena s. matejić2, petar d. marin1 1university of belgrade, faculty of biology, institute of botany and botanical garden “jevremovac”, 11.000 belgrade, serbia 2university of niš, faculty of medicine, 18.000 niš, serbia * e-mail: simicana@bio.bg.ac.rs abstract: džamić, a.m., matejić, j.s., marin, p.d.: trends in biological activity research of wild-growing aromatic plants from central balkans. biologica nyssana, 7 (2), december 2016: 61-73. flowering plants consists of more than 300.000 species around the world, out of which a small percentage has been sufficiently investigated from phytochemical and biological activity aspects. plant diversity of the balkans is very rich, but still poorly investigated. the aim of this paper is survey of current status and trends in research of wild-growing aromatic plants from central balkans. many aromatic plants are investigated from morphological, physiological, ecological, systematic and phytochemical aspects. however, traditionally used medicinal and aromatic plants can also be considered from applicative aspects, concerning their health effects, and from wide range of usage in cosmetics, and as food, agrochemical and pharmaceutical products. in order to achieve all planned objectives, following methodology has been applied: field research, taxonomic authentication and, comparative biologically assayed phytochemical investigations. the total herbal extracts, postdistillation waste (deodorized) extracts, essential oils and individual compounds of some autochthonous plants have been considered as potential source of antibacterial, antifungal, anti-biofilm, antioxidant and cytotoxic agents. in this manuscript, composition of essential oils and extracts were evaluated in a number of species, from the apiaceae, lamiaceae, rosaceae and asteraceae families. extracts which were rich in phenols mostly of flavonoids, often showed high antioxidant potential. also, phenolic compounds identified in essential oils and extracts were mostly responsible for expected antimicrobial activity. current worldwide demand is to reduce or, if possible, eliminate chemically synthesized food additives. plant-produced compounds are becoming of interest as a source of more effective and safe substances than synthetically produced antimicrobial agents (as inhibitors, growth reducers or even inactivators) that control growth of microorganisms. many different pathogens have developed resistance toward synthetic antibiotics and mycotics, so, there is a need for discovering a new antimicrobials. it is worth noting that synergistic effect of components found in essential oils or in various extracts may pay key role in its biological activities. key words: antibacterial, antifungal, antioxidant, cytotoxic activity, plant extracts, essential oil 7 (2) • december 2016: 61-73 12th sfses • 16-19 june 2016, kopaonik mt doi: 10.5281/zenodo.200401 biologica nyssana 7 (2)  december 2016: 61-73 džamić, a. et al.  trends in biological activity research of wild… 62 apstrakt: džamić, a.m., matejić, j.s., marin, p.d.: trendovi u istraživanjima biološke aktivnosti divljerastućih aromatičnih biljaka centralnog balkana. biologica nyssana, 7 (2), decembar 2016: 61-73. broj cvetnica u svetu se procenjuje na više od 300.000 vrsta, od kojih je mali procenat u potpunosti proučen sa fitohemijskog i sa aspekta biološke aktivnosti. biljni diverzitet balkana je bogat, ali je i dalje slabo proučen. cilj ovog rada je pregled trenutnog stanja i trendova u proučavanje samoniklih aromatičnih biljaka sa područja centralnog balkana. mnoge aromatične biljke su proučavane sa morfološkog, fiziološkog, ekološkog, sistematskog i fitohemijskog aspekta. međutim, lekovite i aromatične biljke koje se koriste u narodnoj medicini mogu se razmatrati i sa stanovišta njihove primenljivosti, imajući u vidu njihov efekat na zdravlje i široku upotrebu u kozmetici, kao hrane, i u agrohemijskim i farmaceutskim proizvodima. u cilju postizanja planiranih zadataka, primenjena je sledeća metodologija istraživanja: terenski rad, taksonomska autentifikacija i komparativna biološka i fitohemijska istraživanja. ukupni biljni ekstrakti, ekstrakti postdestilacionih ostataka (deodorisani), etarska ulja i pojedinačne komponente autohtonih biljka su razmatrani kao potencijalni izvori antibakterijskih, antifungalnih, anti-biofilm, antioksidativnih i citotoksičnih agensa. u ovom radu prikazan je hemijski sastav etraskih ulja i ekstrakata brojnih vrsta iz familija apiaceae, lamiaceae, rosaceae and asteraceae. ekstrakti bogati fenolima, naročito flavonoidima često poseduju visok antioksidativni potencijal. takođe, fenolne komponente idetifikovane u etarskim uljima i ekstraktima se smatraju odgovornim za antimikrobnu aktivnost. širom sveta se trenutno radi na smanjenju ili potpunom eliminisanju hemijski sintetisanih aditiva u hrani. biljni proizvodi su postali značajni kao izvor efikasnijih i bezbednih supstanci nego što su sintetički antimikrobni agensi koji kontrolišu rast mikroorganizama (inhibitori, regulatori rastenja ili inaktivatori rasta). mnogi različiti patogeni su postali rezistentni na sintetičke antibiotike i mikotike, pa je potrebno pronaći nove efikasne antimikrobne supstance. potrebno je istaći da sinergistički efekat između komponenti u etarskom ulji ili različitim ekstraktima može imati ključni efekat za njegovu biološku aktivnost. ključne reči: antibakterijska, antifungalna, antioksidativna, citotoksična aktivnost, biljni ekstrakti, etarska ulja introduction plant kingdom, with over 300.000 higher species, represents a source of new chemical agents for active pharmaceutical ingredients and lead compounds. only a small percent 5% to15% of these plants have been chemically and pharmacologically investigated. approximately 10.000 to 15.000 of the world’s plants have documented medicinal uses and roughly 150-200 have been incorporated in western medicine (k r a u s e & t o b i n , 2013). the balkan flora is not only the richest in europe, but comprises also many endemics. according to contemporary assessments, vascular flora of the balkans comprises almost 8.000 taxa (s t e v a n o v i ć et al., 2007). in flora of sebia 3.562 species and subspecies are recorded and among them 32.12% are protected (i g i ć et al., 2010). plants which contain bioactive compounds are often categorised as medicinal or poison plants, and their consumation should have adverse or beneficial result which often depends on the dose of taking. secondary metabolites are small organic molecules and specific biologically active compouds that are produced by plants. they pay important role in the function of plant organisms and are important for long-term survival and defence. nowadays, it is known that production of secondary metabolites are the rule rather than exception. secondary metabolites represent essential source for discovery of a novel compounds because of great diversity of their structures (b e r n h o f t , 2010). modern approach adopted to explore plant and its bioactive constituents involve interdisciplinary work in botany, pharmacognosy, chemistry and toxicology (h o s t e t t m a n n et al., 1995) and should take following steps: 1. selection, collection, botanical identification, preparation of plant material 2. extraction with suitable solvents and preliminary analysis 3. biological and pharmacological screening of crude extracts 4. chromatographic separation of pure bioactive constituents guided by bioassay 5. structure determination 6. analyses and pharmacological profile of pure compounds 7. toxicological testing if plants show no signs of being attacked by pests and neither has pieces eaten out of the leaves, there is a good chance that some metabolites are present which act as insecticides or antimicrobial agents. the evaluation of plant extracts to ensure efficacy and safety is followed by identification of active principles, dosage formulations, efficacy and pharmaco-kinetic profile of the new drug. many plants have been used because of their antibacterial, antifungal, anti-biofilm, antioxidant, cytotoxic, antiinflammatory, antidiabetic, anti-cholinesterase, antinociceptive, anti-proliferative, genotoxic, antigenotoxic, neuroprotective, herbicidal, enzyme biologica nyssana 7 (2)  december 2016: 61-73 džamić, a. et al.  trends in biological activity research of wild… 63 inhibition, anti-alzheimer properties and have been investigated by a number of researchers worldwide. ethnopharmacologists, botanists, microbiologists, natural product chemists, and many others are searching for phytochemicals which could be developed for some applications (d a s et al., 2010). it has to be pointed out that biological activity data for majority of chemically investigated wild growing plants are still missing (č a v a r z e l j k o v i ć & m a k s i m o v i ć , 2014). this study gives an overview of current status and trends in research of potential of wild-growing aromatic plants of different biological aspects and methods in vitro. extracts and essential oils plants are complex matrices, which produce a wide range of secondary metabolites with different functional groups and polarities. categories of natural products commonly encountered include waxes and fatty acids, polyacetylenes, terpenoids (e.g. monoterpenoids, iridoids, sesquiterpenoids, diterpenoids, triterpenoids), steroids, essential oils (lower terpenoids and phenylpropanoids), phenolics (simple phenolics, phenylpropanoids, flavonoids, tannins, anthocyanins, quinones, coumarins, lignans), alkaloids and glycosidic derivatives (e.g. saponins, cardiac glycosides, flavonoid glycosides). for preliminary investigation of biological properties the most important is to test complex mixture as total plant extracts. several approaches can be employed to extract the plant material. although water is used as an extractant in many traditional protocols, organic solvents of varying polarities are generally selected in modern methods of extraction to exploit a solubility of plant constituents. solvent extraction procedures applied for the initial extraction plant natural products include maceration, percolation, soxhlet extraction, pressurized solvent extraction, ultrasound assisted solvent extraction, and steam distillation. terpenoids and essential oils are secondary metabolites that are highly enriched in compounds based on an isoprene structure. the most abundant terpenes in essential oils are monoterpenes (c10) and sesquiterpenes (c15) while diterpenes (c20), as well as aliphatic hydrocarbons, acids, alcohols, aldehydes, acyclic esters or lactones (s e i d e l , 2006; b o h l i n et al., 2012) are often present. antimicrobial activity current standard antimicrobial methods approved by various organizations such as clinical and laboratory standards institute (clsi), and the european committee for antimicrobial susceptibility testing (eucast) exist, for guidelines of antimicrobial susceptibility testing of convenient drugs, these might not be exactly applicable to plant extracts and modifications have to be made. in general, antimicrobial methods are broadly classified into diffusion and dilution methods. diffusion tests include agar well diffusion, agar disk diffusion, poison food technique, and bioautography, while dilution methods include agar dilution, broth microdilution and broth macrodilution technique (d a s et al., 2010). agar well diffusion and disk diffusion have been applied in the first screening, where extract or test substance are placed into agar or applied on the filter disc. zone of inhibition have been measured. bioautography is also employed as a preliminary phytochemical screening technique, by bioassay guided fractionation, to detect active components. bioautography is a very convenient way of testing plant extracts and pure phytochemical compounds for their effect on both human pathogenic and plant pathogenic microorganisms. it can be employed in the target directed isolation of active constituents (c h o m a & g r z e l a k , 2011). however, the most preferred method is broth microdilution assay (or micro-well dilution assay), using microtitration plates, by making the serial twofold dilutions of test substances in corresponding medium. using this method, minimum inhibitory concentrations (mic) and minimum bactericidal/fungicidal concentrations (mbc/mfc) are recorded. minimal inhibitory concentration (mic) is defined as the lowest concentration of the samples inhibiting visible growth. minimal bactericidal/fungicidal concentration (mbc/mfc) is defined as the lowest sample’s concentration that kills 99.9% of bacterial/fungal cells. the main advantages of broth methods, especially microdilution which is carried out in microtiter trays are lower workloads for a larger number of replicates and the use of small volumes of the test substance and growth medium. in this method dilution of the oil is better and there is no agar in the medium, which both enable better diffusion through the liquid medium (s o k o v i ć et al., 2011). antimicrobial activity of wild growing apiaceae species the in vitro antimicrobial activity of essential oils and various extracts of wild growing apiaceae has been investigated by the microdilution method, where a panel of pathogenic bacteria and microfungi was tested (m i l o s a v l j a v i ć et al., 2007; s o k o v i ć et al., 2009; s t o j k o v i ć et al., 2009; biologica nyssana 7 (2)  december 2016: 61-73 džamić, a. et al.  trends in biological activity research of wild… 64 g l a m o č l i j a et al., 2011; m a t e j i ć et al., 2012, 2013, 2014, 2015; m a r č e t i ć et al., 2014a; 2014b; m i l e s k i et al., 2014, 2015; p o p o v i ć et al., 2015a, 2015b) (tab. 1). essential oil analyses of inflorescence and aerial parts of endemic species, ferulago macedonica showed α-pinene as the main compound (43% and 23%, respectively). both oils demonstrated strong antibacterial activity. the essential oil of echinophora spinosa, containing δ-3carene (60.86%) as the dominant component, was the most prominent against escherichia coli and pseudomonas aeruginosa while the most resistant bacterial species against the oil was the staphylococcus aureus (g l a m o č l i j a et al., 2011). essential oil of echinophora sibtorpiana (characterized by presence of methyl eugenol (60.40%), followed by p-cymene (11.18%) and αphellandrene (10.23%) demonstrated considerable antimicrobial activity. the lowest antimicrobial activity was detected for aqueous extracts. the most resistant were fungus aspergillus niger and bacteria micrococcus flavus and escherichia coli (m i l e s k i et al., 2014). essential oil of seseli rigidum, with dominance of α-pinene (48.5%), showed antimicrobial activity. the most resistant bacterial strains were micrococcus flavus and staphylococcus epidermidis, and the most resistant micromycete was aspergillus niger (s t o j k o v i ć et al., 2009). methanol extracts of various seseli taxa revealed activity against different bacteria and yeast candida albicans (with mics of 0.78-12.5 mg/ml) (matejić et al., 2012). methanol and ethyl acetate extracts of cacrys cristata rare and critically endangered species in the flora of serbia, exhibited similar antibacterial activity. methanol extract from aerial parts, inflorescence and fruits of opopanax hispidus showed significant antimicrobial effect (m a t e j i ć et al., 2014, 2015). antimicrobial activity of wild growing lamiaceae species the in vitro preliminary antimicrobial tests were carried out by disc diffusion method. the results of the antimicrobial activity obtained by disc-diffusion assay showed that the diethyl ether extracts of stachys inhibited the growth of all the tested bacteria, and s. plumosa exhibited significant antimicrobial activity against pathogens e. coli, p. aeruginosa and s. aureus (comparable to antibiotics used as the positive controls (l a z a r e v i ć et al., 2010). in last decade majority of antimicrobial experiments used microdilution method. it is well known, that essential oils from lamiacae family showed high antimicrobial potential. antibacterial and antifungal activity of essential oils of wild growing thymus, mentha, mellisa, hyssopus, salvia, satureja, sideritis and stachys species were analyzed (tab. 1). essential oils of salvia sclarea (linalyl acetate and linalool) and mentha longifolia with dominance of cisand trans-dihydrocarveol and piperitone, were analyzed for their antifungal potential. the most susceptible was phytopathogen phomopsis helianthi, while the most resistant was trichoderma viride (d ž a m i ć et al., 2008, 2010). essential oil of satureja kitaibelii characterized by dominance of pcymene showed strong antimicrobial activity (mic values of 0.10-25 mg/ml) (k u n d a k o v i ć et al., 2011; m i h a j i l o v -k r s t e v et al., 2011), while essential oil of hyssopus officinalis subsp. pilifer (1,8-cineole, β-pinene and isopinocamphone) showed lower antimicrobial potential (d ž a m i ć et al., 2013). methanolic extract of satureja kitaibelii and its major compound rosmarinic acid possessed strong antimicrobial activity against tested bacteria and microfungi (s t a n o j k o v i ć et al., 2013). the components with phenolic structure are known to be highly active antimicrobial agents. thymol, a main constituent of various thymus essential oils, was responsible for wide spectrum of antibacterial and antifungal activity (s o k o v i ć et al., 2009; v l a d i m i r -k n e ž e v i ć et al., 2012; p e t r o v i ć et al., 2016). carvacrol and thymol are recognized as strong antimicrobial agents able to disintegrate the outer membrane of gram-negative bacteria, releasing lipopolysaccharides and increasing the permeability of the cytoplasmic membrane. antimicrobial activity is associated with chemical structure of compounds. the most important is lipophilic character of hydrocarbon skeleton and hydrophilic character of functional group. antimicrobial activity should be arranged in this order: phenols > ketones > alcohols > ethers > aldehydes > hydrocarbons (dorman & deans, 2000). antimicrobial activity of wild growing rosaceae species antibacterial activity of three wild fruits, red wild berry fruit (cornus mas), blackthorn (prunus spinosa) and wild blackberry (rubus fruticosus) extracts was analyzed using disc-diffusion and microdilution methods against 12 bacterial strains (tab. 1). the antimicrobial activity of wild berry fruits extracts was high against almost all the tested bacterial strains (r a d o v a n o v i ć et al., 2013). antimicrobial activity of wild growing asteraceae species essential oil of helichrysum arenarium in bioautography assay showed clear inhibition zones which were indication of strong antimicrobial activity gram (+) (micrococcus luteus, biologica nyssana 7 (2)  december 2016: 61-73 džamić, a. et al.  trends in biological activity research of wild… 65 staphylococcus aureus, staphylococcus epidermidis) and gram (-) bacteria (escherichia coli, pseudomonas tolaasii, salmonella enteritidis, salmonella typhimurium) and yeast candida albicans (r a n č i ć et al., 2005). the most rarely used was micro-atmosphere assay. the essential oil evaporated and the activity by vapor contact has been measured. due to the high lipophilic nature of mycelia coupled with a large surface area relative to the volume of a fungus, vapours of essential oils may act mainly by accumulation on mycelia than in the agar (i n o u y e et al., 2000). s t u p a r et al. (2014) investigated antifungal activity of the essential oil of helichrysum italicum using micro-atmosphere method. the main components of the oil were γcurcumene, α-pinene, and neryl acetate. the essential oil of h. italicum showed moderate antifungal activity against fungi isolated from cultural heritage objects. the most susceptible fungi to oil treatment were epicoccum nigrum and penicillium sp., while the most resistant was trichoderma viride. pathogenic strain pseudomonas aeruginosa causes pathological changes on vegetables, and it is considered as conditional parasite of plants, but also as a human parasite because it causes infections of urinary and respiratory tract. the effect of the water and ethanol plant extract was tested against pathogenic strains pseudomonas aeruginosa and p. fluorescens using disc diffusion method. water and ethanol extract of achillea millefolium and salvia officinalis showed the greatest inhibitory effect on pseudomonas aeruginosa, (the water extract of achillea). great inhibitory effect of the ethanol extract had helichrysum arenarium, rosmarinus officinalis, satureja montana (p e t r o v i ć et al., 2003). in general, microdilution assay is the most rewarding method for different analyses. the several strains of microorganisms should be tested on one plate, and small amount of tested sample could be used. nowadays this technique is the most frequently used. the chemical composition and the potential to have antimicrobial activity depend highly on the plant species, habitat and ontogeny phase as well as of methods of processing, extraction solvent and extract dose. many times the values of the inhibition zones were proportional to the concentration of the extracts and essential oils. sometimes dominant compounds are crucial for the biological activity. syinergistic effect of the compounds are also important. antioxidant activity compounds with antioxidant activity are mainly phenolic acids, flavonoids and polyphenols (d i l l a r d & g e r m a n , 2000). phenols provide the plants with defense mechanisms to neutralize reactive oxygen species (ros) in order to survive and prevent molecular damage and damage by microorganisms, insects, and herbivores. flavonoids show a wide range of biological activities such as inhibition of cell-proliferation, induction of apoptosis, inhibition of enzymes and other antibacterial and antioxidant effects (p a n d e y & g u p t a , 2014). antioxidants may directly react with the reactive radicals to destroy them by accepting or donating electron(s) or they may indirectly decrease the formation of free radicals by inhibiting the activities or expressions of free radical generating enzymes or by enhancing the activities and expressions of other antioxidant enzymes. many research models have been established in chemical and/or biological systems for studying the mechanisms of action of antioxidants and for identifying new antioxidants (lu et al., 2010) and it is suggested to test potential antioxidants by different methods. important step for determination of the antioxidant capacity is quantification of phenolic compounds in samples by using total phenol content (tpc) assay with folin–ciocalteu reagent, as well as quantification of total flavonoids (tfc). the most frequently used in vitro antioxidant methods are: 1,1-diphenyl-2-picrylhydrazyl (dpph) radical scavenging assay, 2,2-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid (abts) radical scavenging activity, ferry reducing power (frap) assay, total reducing power (trp) assay, β-carotenelinoleic acid assay etc. available literature data of in vitro antioxidant activity of wild growing species from central balkans are given in tab. 1. antioxidant activity of wild growing apiaceae species methanolic extract of seseli taxa tested for its antioxidant potential (total phenols, total flavonoids dpph and abts assays) revealed seseli libanotis ssp. libanotis for its highest reducing power (m a t e j i ć et al., 2012). essential oil of echinophora sibthorpiana possessed high radical scavenging capacity in dpph and abts assays. aqueous extract of aerial parts was the strongest tested extract. extracts of aerial parts expressed higher radical scavenging activity in comparison to the root extracts. all extracts had lower antiradical activity compared to bha and vitamin c (m i l e s k i et al., 2014). in the case of various extracts of ferulago macedonica, it was found that there is no strong relationship between total phenolic and flavonoid contents and radical scavenging activities biologica nyssana 7 (2)  december 2016: 61-73 džamić, a. et al.  trends in biological activity research of wild… 66 (dpph, abts). these results can be explained since the antioxidant property of a plant extract is generally considered as the result of the combined activity of a wide range of compounds including, not only phenols, but also peptides, organic acids and other components. both, phenols and flavonoids were present in higher amounts in f. macedonica extracts of inflorescence in comparison to the aerial parts (m i l e s k i et al, 2015). antioxidant activity of wild growing lamiaceae species extracts of waste water after hydrodistillation of satureja montana and s. cuneifolia, showed better ability to reduce stable dpph radical than the standards essential oils or thymol, carvacrol, and thymoquinone. this is probably due to the high concentration of other active compounds, such as (e)-coniferyl alcohol. to compare these results, total antioxidant activity of these two satureja species was also tested using the phosphomolybdenum method. this test is based on the reduction of mo(vi) to mo(v) by the extract and subsequent formation of a green phosphate/mo(v) complex at acid ph. total antioxidant activity of the phosphomolybdenum model evaluates both water-soluble and fat-soluble antioxidants, i. e. total antioxidant capacity. the highest activity in this method was found for essential oil of s. cuneifolia (č a v a r et al., 2013). for scavenging ability on dpph radicals, various extracts of hissopus officinalis subsp. pilifer were effective in the order of: deodorized aqueous extract > deodorised metanol extract > deodorised ethyl acetate extract. the total phenol content, determined by folin–ciocalteau reagent was in the same order. essential oil of h. officinalis subsp. pilifer possessed the lowest activities compared to other extracts and control substances (d ž a m i ć et al., 2013). antioxidant potential of melittis melissophyllum various hydro alcoholic, ethanol and methanol extracts revealed a high scavenging activity by dpph and abts methods. reducing power of extracts were evaluated through the frap assay. the highest phenol and flavonoid content were identified in the 96% ethanol extract and the lowest were in the ethanol 10% solution. also, the highest correlation was observed between total phenol and flavonoid contents. statistically significant correlations were registered for dpph and abts assays which is logical due to their similar mechanism of action as radical scavengers. a hierarchical cluster analysis (hca) was performed on the total phenols and total flavonoids content dpph, abts, and frap assay. the dendrogram (fig. 1) shows that total phenols and total flavonoids content are quite homogeneous, while dpph was placed in separate cluster (g r u j i ć et al., 2014). tree diagram for 5 variables ward`s method euclidean distances 0 10 20 30 40 50 60 70 80 90 100 110 (dlink/dmax)*100 ic50 dpph, (mg/ml) frap, (µmolfe/mg) abts, mg vit c/g total flavonoids (que/g) total phenols (gae/g) fig. 1. dendrogram of applied assays for antioxidant characteristics evaluation for melittis melissophyllum extracts (g r u j i ć et al., 2014). biologica nyssana 7 (2)  december 2016: 61-73 džamić, a. et al.  trends in biological activity research of wild… 67 table 1. antimicrobial and in vitro antioxidant activity of extracts and essential oils of wild-growing species from central balkans fam species extracts eo composition antimicrobial methods antioxidant methods references a p ia c e a e cachrys cristata dc. methanol, ethyl acetate, acetone, aqueous aerial parts, fruits antibacterial antifungal microdilution total phenols total flavonoids dpph abts matejić et al.,2012, 2014 daucus carota l. fruits, flowers, root, leaves and stem antibacterial antifungal microdilution soković et al., 2009 echinophora sibthorpiana guss. echinophora spinosa l. methanol, ethanol, aqueous inflorescence aerial parts antibacterial antifungal microdilution total phenols total flavonoids dpph abts mileski et al., 2014 glamočlija et al., 2011 eryngium palmatum pančić and vis. methanol chloroform root microdilution antibacterial anticandidal total phenols dpph frap marčetić et al., 2014a; 2014b ferulago macedonica micevski and e. mayer methanol, ethanol, aqueous antibacterial antifungal microdilution total phenols total flavonoids dpph abts mileski et al., 2015 laserpitium latifolium l. l. zernyi hayek l. ochridanum micevski chlorophorm underground parts aerial parts fruits antibacterial antifungal microdilution antibiofilm popović et al, 2015 a,b opopanax hispidus (friv.) griseb. methanol, ethyl acetate antibacterial anticandidal microdilution total phenols total flavonoids dpph abts matejić et al., 2015 seseli pallasii besser, seseli libanotis (l.) koch ssp. libanotis seseli libanotis ssp. intermedium (rupr.) p. w. ball seseli rigidum waldst. & kit. seseli globiferum vis. seseli annuum l. methanol aerial parts fruits antibacterial antifungal microdilution total phenols total flavonoids dpph abts frap matejić et al., 2012 ilić et al., 2014; 2015 janacković et al, 2011 stojković et al, 2009 milosavljević et al., 2007 tordylium maximum l. methanol, aqueous antibacterial anticandidal microdilution total phenols total flavonoids dpph abts matejić et al., 2013 l a m ia c e a e hyssopus officinalis ssp. pilifer deodorized aqueous, methanol and ethyl acetate aerial parts antifungal microdilution total phenols dpph džamić et al., 2013 mentha longifolia l. aerial parts antifungal microdilution dpph džamić et al., 2010 mellisa officinalis l. aerial parts antibacterial antifungal microdilution dpph oh radical mimica-dukić et al., 2004 melittis melissophyllum l. methanol, ethanol (hydroalcoholic) total phenols total flavonoids dpph abts frap grujić et al., 2014 salvia nemorosa l. ssp. nemorosa salvia sclarea l. aerial parts antibacterial antifungal microdilution dpph phosphomolybd enum božin et al., 2011 džamić et al., 2013 satureja montana l. satureja cuneifolia ten satureja montana ssp. pisidica (wettst.) šilić satureja montana l. ssp. montana aerial parts antibacterial antifungal microdilution dpph orac trp apf čavar et al., 2013 kundaković et al., 2014 mihajilov-krstev et al., 2014; 2011 biologica nyssana 7 (2)  december 2016: 61-73 džamić, a. et al.  trends in biological activity research of wild… 68 table 1. continuation of the table fam species extracts eo composition antimicrobial methods antioxidant methods references l a m ia c e a e satureja kitaibelii wierzb. ex heuff. satureja horvatii šilić bukvički et al., 2014 lakušić et al., 2008 sideritis montana l. aerial parts antibacterial microdilution miladinović et al., 2012 stachys germanica ssp. heldreichii (boiss) hayek stachys iva griseb. stachys plumosa griseb. stachys scardica griseb. diethyl ether, ethyl acetate antibacterial antifungal disk-diffusion phosphomolybd enum lazarević et al., 2010 thymus praecox opiz ssp. polytrichus thymus longicaulis c. presl thymus serpillum l. thymus tosevii velen supercritical extracts methanol, ethanol, aqueous aerial parts antibacterial antifungal microdilution macrodilution disk-diffusion dpph petrović et al., 2016 a, b vladimir-knežević et al., 2012 soković et al., 2009 r o sa c e a e crataegus oxyacantha l. antibacterial disk-diffusion total phenols anthocyan content flavonoid content dpph kostić et al., 2012 cornus mas l. prunus spinosa l. acidified methanol solution antibacterial disk-diffusion microdilution dpph radovanović et al., 2013 rosa canina l. methanol antibacterial antibiofilm microdilution živković et al., 2015 rubus caesius l. var. aquaticus weihe and nees rubus fruticosus l. methanol, ethanol, acetone, aqueous acidified methanol solution antibacterial disk-diffusion microdilution total phenols total flavonoids dpph abts frap trc veličković, et al., 2015; radovanović et al., 2013 a st e r a c e a e achillea collina becker ex heimerl s.l. achillea pannonica scheele aerial parts disk-diffusion dpph božin et al., 2008 ambrosia artemisiifolia l. 70% aqueous acetone extracts total phenols total flavonoids dpph frap maksimović, 2008 artemisia alba turra artemisia absinthium l. ethanol aerial parts antibacterial anticandidal microdilution total phenols total flavonoids dpph frap abts đorđević et al., 2013 mihajilov-krstev et al., 2014 helichrysum arenarium (l.) moench helichrysum italicum (roth) g. don aerial parts antibacterial antifungal microdilution bioautography rančić et al., 2005 stupar et al., 2014 hieracium pillosela l. methanol, dychlorometane, ethyl acetate, dychlorometane:me thanol antibacterial disk-diffusion stanojević et al., 2008 xeranthemum annuum l methanolic acetone ethyl acetate antibacterial antifungal microdilution total phenols total flavonoids dpph stanković et al., 2011 biologica nyssana 7 (2)  december 2016: 61-73 džamić, a. et al.  trends in biological activity research of wild… 69 antioxidant activity of wild growing rosaceae species r a d o v a n o v i ć et al. (2013) found that in polyphenolic extracts of fruits from tree rosaceae species main compounds were gallic acid, caffeic acid, p-coumaric acid, ferulic acid, (+)-catechin, procyanidin b2, (-)-epicatechin, quercetin, rutin and quercetin-3-glucoside. all extracts showed high scavenging effect on dpph radical. extracts obtained from hieracium pilosella have significant free radical scavenging activity on stable dpph• and high reactive hydroxyl radical. the data suggest that aqueous, ethanolic and methanolic extracts of h. pilosella from southeastern serbia are a potential source of natural antioxidants. chlorogenic acid was detected in the highest quantities in all investigated extracts (s t a n k o v i ć et al., 2009). antioxidant activity of wild growing asteraceae species according to b o ž i n et al. (2008) the main detected compounds in essential oil of achillea callina were β-pinene, chamazulene and e-caryophyllene, while in essential oil of a. pannonica 1,8-cineole and camphor were the dominant components. authors represented that both oils possess strong antioxidant effects in vitro and in vivo assays. antioxidant capacities are influenced by many factors, which cannot be fully described with a single method. the most commonly used method for assessment of antioxidant properties of natural products is dpph radical assay. the dpph radical assay overcomes the limitations of monitoring the activity of the numerous samples over a specified period of time. it is reproducible and strongly correlated with phenolic compounds (m i l i a u s k a s , 2004). cytotoxic activity brine shrimp, artemia salina, is the test organism that is used for evaluation of possible cytotoxic activity of plant extracts or essential oils. brine shrimp assay is simple, inexpensive method and results are obtained quickly. in the study represented by j a n a ć k o v i ć et al. (2008) a brine shrimp assay was used to examine potential cytotoxic activity of different centaurea species, measuring lethal concentration (lc). cytotoxic activity of methanol and ether extracts was tested. the methanol extract of c. arenaria showed very significant activity while the lowest activity was found with c. chrysolepis methanol extract. among ether extracts, the most active was c. splendens (and the lowest activity was recorded for the ether extract of c. scabiosa. toxicity test on drosophila melanogaster showed that the essential oil of a. absinthium is toxic for developing insect larvae. starting with the concentration of 0.38 % of essential oil in medium, significant mortality of larvae exposed to the oil was noted when compared to the control. the essential oil also affected the development of d. melanogaster larvae and significantly delayed achievement of the pupa stadium (m i h a j i l o v -k r s t e v et al., 2014). many plant extracts and natural products, especially phenols, with high antioxidant activity have shown cytotoxic effects in different cell lines. flavonoid anticancer activities include inhibition of cell growth, inhibition of protein kinase activities, and induction of apoptosis. popular and broadly used for cytotoxity examination is the mtt (3-[4,5dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay which is based on the conversion of mtt into formazan crystals by living cells, which determines mitochondrial activity. methanolic extract of origanum vulgare showed significant antiproliferative activity in both colon cancer (hct116) and breast cancer (mda-mb-231) cell lines. inhibitory activity appeared to be particularly conspicuous in treated hct-116 cell lines, whereas at the highest concentration (500 µg/ml), only 10% of the cells remained viable. the results indicate that o. vulgare is considered to be a particularly valuable source of effective anti-proliferative and cytotoxic substances (g r b o v i ć et al., 2013). the methanol extract of satureja kitaibelii exhibited strong activity against fem-x human malignant melanoma cells, and moderate activity against breast cancer cell lines (s t a n o j k o v i ć et al., 2013). the cytotoxic effect of the essential oils from satureja montana ssp. pisidica from two localities (mountains korab and galičica) was tested against cancer cell lines mdamb-361, mda-mb-453, hela, ls174 and mrc5 cells. the essential oil from korab demonstrated significantly better results than the oil from galičica, particularly against hela and mda-mb-453 cell lines, while the oil from galičica was the most active on the human epithelial cervical cancer hela cells (k u n d a k o v i ć et al., 2014). other activities most studies describe the action of essential oil and extracts for other biological activities. strong inhibition of human serum cholinesterase by essential oil of satureja montana, suggests this as a natural source of compounds that can be used in the treatment of foodborne and neurological diseases, wound and other infections, as well as for general health improvement (m i h a j i l o v -k r s t e v et al., 2014). considering human and horse serum biologica nyssana 7 (2)  december 2016: 61-73 džamić, a. et al.  trends in biological activity research of wild… 70 cholinesterase inhibition, both essential oils acted as strong inhibitors, even higher than effect of their dominant constituent α-pinene, leading to the conclusion that the other constituents of the essential oil manifested their action toward cholinesterase (ilić et al., 2015). thyme essential oil, as well as its major phenolic constituent’s thymol and carvacrol were researched for application in alzheimer’s disease treatment or as remedies for cognitive disorders. it was noted that phenolic chemotypes possess higher antioxidant activity than non-phenolic ones, as well as acetylholinesterase inhibitory activity (č a v a r z e l j k o v i ć & m a k s i m o v i ć , 2014). conclusion the reported results from in vitro studies hint the potential antimicrobial and antioxidant value of the wild growing plants from central balkan. it was prepared based on plenty literature search of biological activity of extracts and essential oils. the microdilution method was found as the mostly used in antimicrobial assay. dpph method is the most frequently used in antioxidant assay. the traditional use of many aromatic species leads to discovering the unknown biological potential of related species. the most important approach is combination of phytochemical investigation and bioassay. essential oil and extracts without identifying of compounds responsible for this activity are also insufficient for preclinical trials. in spite the fact that a lot of studies have been reported so far, many of wild-growing plants are still waiting to be explored. acknowledgements. this work was founded by the ministry of education, science and technological development of the republic of serbia for financial support (grant no. 173029). references bernhoft, a. 2010. a brief review on bioactive compounds in plants. in: bernhoft, a. (ed.), bioactive compounds in plants – benefits and risks for man and animals 11-17, the norwegian academy of science and letters, oslo. bohlin, l., alsmark, c., göransson, u., klum, m., wedén, c., backlund, a. 2012: strategies and methods for a sustainable search for bioactive compounds. planta medica, 78-op7. božin, b., lakić, n., srđenović čonić, b., kladar, n., orčić, d., mimica-dukić, n. 2012: antioxidant and antimicrobial properties of a new chemotype of woodland sage (salvia nemorosa l. subsp. nemorosa, lamiaceae) essential oil. biologica serbica, 34: 51-60. božin, b., mimica-dukić, n., bogavac, m., suvajdžić, lj., simin, n., samojlik, i., couladis, m. 2008: chemical composition, antioxidant and antibacterial properties of achillea collina becker ex heimerl s.l. and a. pannonica scheele essential oils. molecules, 13: 2058-2068. bukvički, d.r., stojković, d.s., soković, m.d., vannini, l., montanari, c., pejin, b, savić, a., veljić, m. m, grujić, s.m., marin p.d 2014: satureja horvatii essential oil: in vitro antimicrobial and antiradical properties and in situ control of listeria monocytogenes in pork meat. meat science, 96(3): 1355-1360. čavar, s., edita šolić, m., maksimović, m. 2013: chemical composition and antioxidant activity of two satureja species from mt. biokovo. botanica serbica, 37(2): 159-165. čavar zeljković, s., maksimović, m. 2015: chemical composition and bioactivity of essential oil from thymus species in balkan peninsula. phytochem review, 14: 335–352. choma, i.m., grzelak, e.m. 2011: bioautography detection in thin-layer chromatography. journal of chromatogrphy a, 1218(19): 2684-91. das, k., tiwari, r.k.s., shrivastava, d.k. 2010: techniques for evaluation of medicinal plant products as antimicrobial agent: current methods and future trends. journal of medicinal plants research, 4(2): 104-111. dillard, c.j., german, j.b. 2000: review phytochemicals: nutraceuticals and human helth. journal of the science of food and agriculture, 80: 1744–1756. đorđević, s., stanisavljević, d., ristića, m., milenković, m., veličković, d., stojičević, s., zlatković, b. 2013: chemical, antioxidant and antimicrobial analysis of the essential oil and extract of artemisia alba tura. digest journal of nanomaterials and biostructures, 8(4): 1377– 1388. doroman, h.j.d., deans s.g. 2000: antimicrobial agents from plants: antibacterial activity of plant volatile oils. journal of applied microbiology, 88: 308-316. džamić, a., soković, m.d., ristić, m.s., novaković, m., grujić-jovanović, s., tešević, v., marin p.d. 2010: antifungal and antioxidant activity of mentha longifolia (l.) hudson (lamiaceae) essential oil. botanica serbica, 34(1): 57-61. džamić, a., soković, m., ristić, m., grujićjovanović, s., vukojević, j., marin, p.d. 2008: chemical composition and antifungal activity of salvia sclarea (lamiaceae) essential oil. archives of biological sciences, 60: 233-237. džamić, a.m., soković, m.d., novaković, m., jadranin, m., ristić, m.s., tešević v., marin, biologica nyssana 7 (2)  december 2016: 61-73 džamić, a. et al.  trends in biological activity research of wild… 71 p.d. 2013: composition, antifungal and antioxidant properties of hyssopus officinalis l. subsp. pilifer (pant.) murb.essential oil and deodorized extracts. industrial crops and products, 51: 401–407. glamočlija, j.m., soković, m.d., šiljegovic, j.d., ristić, m.s., ćiric, a.d., grubišić, d.v. 2011: chemical composition and antimicrobial activity of echinophora spinosa l. (apiaceae) essential oil. records of natural products, 5(4): 319-323. grbović, f., stanković, m. s., ćurčić, m., đorđević, n., šeklić, d., topuzović, m., marković, s. 2013: in vitro cytotoxic activity of origanum vulgare l. on hct-116 and mda-mb-231 cell lines. plants, 2(3): 371-378. grujić, s., stojanović, g., mitić, v., stankovjovanović, v., džamić, a., alimpić, a., marin p. 2014: evaluation of antioxidant activity of melittis melissophyllum l. extracts. archives of biological sciences, 66(4): 1401-1410. hostettmann, k., marston, a., wolfender, j.-l. 1995. strategy in the search for new biologically active plant constituents. in: hostettmann, k., marston a., millard m. and hamburger m. (eds.), phytochemistry of plants used in traditional medicine 17-45, oxford science publications. oxford university press inc., new york. igić, r., vukov, d., božin, b., orlović, s. 2010: lekovite biljke. prirodni resursi vojvodine. društvo za zaštitu životne sredine “vrelo”, novi sad, srbija. ilić, m.d., stankov jovanović, v.p., mitić, v.d., jovanović, o.p., mihajilov-krstev, t.m., marković, m.s., stojanović, g.s. 2015: comparison of chemical composition and biological activities of seseli rigidum fruit essential oils from serbia. open chemistry, 13: 42–51. inouye, s., tsuruoka, m., watanabe, m., takeo, k., akao, m., nishiyama, y., yamaguchi h. 2000: inhibitory effect of essential oils on apical growth of aspergillus fumigatus by vapour contact. mycoses, 43: 17–23. janaćković, p., tešević, v., marin, p., milosavljević, s., duletić-laušević, s., janaćković, s., veljić, m. 2008: brine shrimp lethality bioassay of selected centaurea l. species (asteraceae). archives of biological sciences, 60(4): 681-685. janaćković, p.t., soković, m.d., vujisić, lj.v., vajs, v.e., vućkovic, i.m., krivošej, z.dj., marin, p.d. (2011): composition and antimicrobial activity of seseli globiferum essential oil. natural product communications, 6(8): 1163-1166. kostić, d.a., velicković, j.m., mitić, ss., mitić, m.n., randelović, s.s 2012: phenolic content, and antioxidant and antimicrobial activities of crataegus oxyacantha l. (rosaceae) fruit extract from southeast serbia. tropical journal of pharmaceutical research, 11(1): 117-124. krause, j., tobin g. 2013: discovery, development, and regulation of natural products. in: using old solutions to new problems natural drug discovery in the 21st century. 3-35. kundaković, t., milenković, m., zlatković, s., kovacević, n., goran, n. 2011: composition of satureja kitaibelii essential oil and its antimicrobial activity. natural product communications, 6(9): 1353-1356. kundaković, t., stanojković, t., kolundzija, b., marković, s., sukilović, b., milenković, m., lakušić, b. 2014: cytotoxicity and antimicrobial activity of the essential oil from satureja montana subsp. pisidica (lamiceae). natural product communications, 9(4): 569-572. lakušić, b., ristić, m., slavkovska, v., antić stanković, j., milenković, m. 2008: chemical composition and antimicrobial activity of the essential oil from satureja horvatii šilić (lamiaceae). journal of serbian chemical society, 73(7) 703–711. lazarević, j.s., palić, r.m., radulović, n.s., ristić, n.r., stojanović, g.s. 2010: chemical composition and screening of the antimicrobial and antioxidative activity of extracts of stachys species. journal of serbian chemical society, 75(10): 1347–1359. lu, j.-m., lin, p.h., yao, q., chen, c. 2010: chemical and molecular mechanisms of antioxidants: experimental approaches and model systems. journal of cellular and molecular medicine, 14(4): 840-860. maksimović, z. 2008: in vitro antioxidant activity of ragweed (ambrosia artemisiifolia l., asteraceae) herb. industrial crops and products, 28(3): 356– 360. marčetić, m., petrović, s., milenković, m., niketić, m.s. 2014a: composition, antimicrobial and antioxidant activity of the extracts of eryngium palmatum pančić and vis. (apiaceae). central european journal of biology, 9(2): 149-155. marčetić, m., petrović, s., milenković, m., vujisić, lj. v., tešević, v.v., niketić, m.s 2014b: composition and antimicrobial activity of root essential oil of balkan endemic species eryngium palmatum. chemistry of natural compounds, 49(6): 1140-1142. matejić, j., džamić, a., mihajilov-krstev, t., ranđelović, v., krivošej, z., marin, p. 2012: total phenolic content, flavonoid concentration, antioxidant and antimicrobial activity of methanol biologica nyssana 7 (2)  december 2016: 61-73 džamić, a. et al.  trends in biological activity research of wild… 72 extracts from three seseli l. taxa. central european journal of biology, 7(6): 1116-1122. matejić, j.s., džamić, a.m., mihajilov-krstev, t.m., ranđelović, v.n., mileski, k.s., marin, p.d. 2014: total phenolic and flavonoid contents and biological activities of cachrys cristata dc. extracts. archives of biological science, 66(3): 1117-1123. matejić, j., džamić, a., mihajilov-krstev, t., ranđelović, v., krivošej, z., marin, p. 2013: total phenolic and flavonoid content, antioxidant and antimicrobial activity of extracts from tordylium maximum. journal of applied pharmaceutical science, 3(1): 055-059. matejić, j.s., džamić, a.m., mihajilov-krstev, t.m., ranđelović, v.n., marin, p.d. 2015: antioxidant and antimicrobial potential of opopanax hispidus (apiaceae) extracts. lekovite sirovine, 35: 141150. mihajilov-krstev, t.m., kitić, d.v., radnović, d.v., ristić, m.s., mihajlović-ukropina, m., zlatković, b.k. 2011: chemical composition and antimicrobial activity of satureja kitaibelii essential oil against pathogenic microbial strains. natural product communications, 6(8): 11671172. mihajilov-krstev t.m., jovanović b., jović j. l., ilić b.s., miladinović d.l., matejić j.s., rajković j.s., djordjević lj.b., cvetković v.j., zlatković b.k. 2014: antimicrobial, antioxidative, and insect repellent effects of artemisia absinthium essential oil. planta medica, 80(18): 1698-1705. mihajilov-krstev, t., radnović, d., kitić, d., stankov-jovanović, v., mitić, v., stojanovićradić, z., zlatković, b. 2014: chemical composition, antimicrobial, antioxidative and anticholinesterase activity of satureja montana l. ssp montana essential oil. central european journal of biology, 9(7): 668-677. miladinović, d.l., ilić, b.s., mihajilov-krstev, t.m., jović, j.l., marković, m.s. 2014: in vitro antibacterial activity of libanotis montana essential oil in combination with conventional antibiotics. natural product communications, 9(2): 281-286. miladinović, d.l., ilić, b.s., mihajilov-krstev, t.m., nikolić, n.d., milosavljević, v.n., nikolić, d.m. 2012: antibacterial potential of the essential oil from sideritis montana l. (lamiaceae). hemijska industrija, 66(4): 541–545. mileski, k., džamić, a., ćirić, a., grujić, s., ristić, м., matevski, v., marin, p.d. 2014: radical scavenging and antimicrobial activity of essential oil and extracts of echinophora sibthorpiana guss. from macedonia. archives of biological sciences, 66(1): 401-413. mileski, к., džamić, а., ćirić, а., ristić, м., grujić, s., matevski, v., marin, p.d. 2015: composition, antimicrobial and antioxidant properties of endemic species ferulago macedonica micevski and e. mayer. records of natural products, 9(2): 208-223. miliauskas, g., venskutonis, p.r., van beek, t.a. 2004: screening of radical scavenging activity of some medicinal and aromatic plant extracts. food chemistry, 85: 231-237. milosavljević, s., tešević, v., vučković, i., jadranin, m., vajs, v., soković, m., janaćković, p., jovanović, a. 2007: composition and antifungal activity of the essential oil of seseli annuum wildgrowing in serbia. fitoterapia, 78(4): 319–322. mimica-dukić, n.m., božin, b.n., soković, m.d., simin, n.d. 2004: antimicrobial and antioxidant activities of melissa officinalis l. (lamiaceae) essential oil. journal of agricultural and food chemistry, 52(9): 2485-2489. pandey s., gupta r.k. 2014: screening of nutritional, phytochemical, antioxidant and antibacterial activity of chenopodium album (bathua). journal of pharmacognosy and phytochemistry, 3(3): 1-9. petrović, j., stanojković, a., čomic, lj., ćurčić, s. 2003: biotic activity of some aromatic plants on pseudomonas aeruginosa and pseudomonas fluorescens. lekovite sirovine, 23: 31-36. petrović, n.v., petrović, s.s., džamić, а.m., ćirić, а.d., ristić, м.s., milovanović, s.l., petrović, s.d. 2016a: chemical composition, antioxidant and antimicrobial activity of thymus praecox supercritical extracts. journal of supercritical fluids, 110: 117–125. petrović, n.v., petrović, s.s., ristić, м.s., džamić, а.m., ćirić, а.d. 2016b: chemical composition and antimicrobial activity of thymus praecox opiz ssp. plytrychus essential oil from serbia. records of natural products, 10(5): 633-638. radovanović, b., milenković-anđelković, a.s., radovanović, a.b., anđelković, m.z. 2013: antioxidant and antimicrobial activity of polyphenol extracts from wild berry fruits grown in southeast serbia. tropical journal of pharmaceutical research, 12(5): 813-819. seidel, v. 2006. initial and bulk extraction. in: sarker, s.d., latif, z., gray, a.i. (ed.), methods in biotechnology vol. 20, natural products isolation, 2nd ed. humana press inc., totowa, nj. soković, m.d., stojković, d.s., glamočlija, j.m., ćirić, a.d., ristić, m.s., grubišić, d.v. 2009: susceptibility of pathogenic bacteria and fungi to essential oils of wild daucus carota. pharmaceutical biology, 47(1): 38-43. biologica nyssana 7 (2)  december 2016: 61-73 džamić, a. et al.  trends in biological activity research of wild… 73 soković, m.d., vukojević, j., marin, p.d., brkić, d.d., vajs, v., van griensven, l.j. 2009: chemical composition of essential oils of thymus and mentha species and their antifungal activities. molecules, 14: 238-249. stanković, m.s., radojević, i.d., stefanović, o.d., topuzović, m.d., čomić, lj.r., branković, s.r. 2011: immortelle (xeranthemum annuum l.) as a natural source of biologically active substances. excli journal, 10: 230-239. stanojević, lj.p., stanković, m.z., nikolić, v.d., nikolić, lj.b. 2008: anti-oxidative and antimicrobial activites of hieracium pillosela l. extracts. journal of serbian chemical society, 73(5): 531-540. stanojević, lj., stanković, m., nikolić, v., nikolić, lj., ristić, d., čanadanović-brunet, j., tumbas v. 2009: antioxidant activity and total phenolic and flavonoid contents of hieracium pilosella l. extracts. sensors, 9(7): 5702–5714. stanojković, a., ceković, j., pivić, r., stanojković, a. 2008: in vitro antibacterial activity of thymus serpyllum extracts. proceedings 43rd croatian and 3rd international symposium on agriculture, 516-519. stanojković, t., kolundžija, b., ćirić, a., soković, m., nikolić, m., kundakovića, t. 2013: cytotoxicity and antimicrobial activity of satureja kitaibelii wierzb. ex heuff (lamiaceae). digest journal of nanomaterials and biostructures, 8(2): 845 – 854. stevanović, v, tan, k, petrova, a. 2007: mapping the endemic flora of the balkans—a progress report. bocconea, 21: 131–137. stojković, s., petrović, s., kukić, j., džamić, a., ristić, m., milenković, m., glamočlija, j., soković, m., stojković, d. 2009: chemical composition and antimicrobial and antioxidant actyvity of essential oil from the flowers of sesili rigidum. chemistry of natural compounds, 4: 253-256. stupar, m., ljaljević grbić, m., džamić, a., unković, n., ristić, m., vukojević, j. 2014: antifungal activity of helichrysum italicum (roth) g. don (asteraceae) essential oil against fungi isolated from cultural heritage objects. archives of biological science, 66(4): 1539-1545. vladimir-knežević, s., kosalec, i., babac, m., petrović, m., ralić, j., matica, b., blažeković b. 2012: antimicrobial activity of thymus longicaulis c. presl essential oil against respiratory pathogens. central european journal of biology, 7(6): 1109-1115. živković, j., stojković, d., petrović, j., zdunić, g., glamočlija, j., soković, m. 2015: rosa canina l. – new possibilities for an old medicinal herb. food and function, 6(12): 3687-3692. orchid flora of the muntele mic (caraş – severin country, romania) biologica nyssana 7 (2) ⚫ december 2016: 107-112 milanovici, s. ⚫ the orchid flora of the muntele mic… 107 original article received: 25 june 2018 revised: 28 sptember 2018 accepted: 18 november 2018* the orchid flora of the muntele mic (caraş – severin county, romania) sretco milanovici natural science section, banat national museum, huniade square no. 1, timișoara city, timiș county, romania * e-mail: milanovici.sretco@wildcarpathiangarden.com abstract: milanovici, s.: the orchid flora of the muntele mic (caraş – severin county, romania). biologica nyssana, 7 (2), december 2016: 107-112. muntele mic mountain is located in the southwestern part of romania and belongs to the southern carpathians. although relatively small, muntele mic contains most of typical mountain and high-mountain habitats. the field research regarding the orchid’s family in the muntele mic area, have started in the summer of 2009. owing to easy access (asphalt road that goes to the tourist center of muntele mic), although it is classified as part of the european natura 2000 network (rosci0126 munţii ţarcu), the area is influenced by negative anthropogenic factors. although considered to be a very anthropized area, the field research concluded that there are 10 species of orchids growing in this location, of which three: gymnadenia frivaldii hampe ex griseb., dactylorhiza fuchsii (druce) soó and dactylorhiza saccifera (brongn.) soó), were not mentioned in the literature data. key words: orchids, conservation, threats, muntele mic, romania apstrakt: milanovici, s.: flora orhideja planine muntele mic (caraş – severin county, romania). biologica nyssana, 7 (2), decembar 2016: 107-112. planina muntele mic nalazi se u jugozapadnom delu rumunije i pripada južnim karpatima. iako relativno male površine, muntele mic sadrži većinu karakterističnih planinskih i visokoplaninskih staništa. sistematska terenska istraživanja porodice orhideja planine muntele mic, započeta su u proleće 2009 godine. zahvaljujući lakom pristupu (asfaltni put koji ide do turističkog centra muntele mic), iako svrstan kao deo mreže natura 2000 rosci0126 munţii ţarcu, pod snažnim je udarom negativnih faktora antropogenog porekla. iako se smatra veoma antropizovanim, terenskim istraživanjem konstatovano je da ovde raste 10 vrsta orhideja, od čega se tri: gymnadenia frivaldii hampe ex griseb., dactylorhiza fuchsii (druce) soó i dactylorhiza saccifera (brongn.) soó), ne pominju u stručnoj literaturi. ključne reči: orhideje, problemi zaštite, muntele mic, rumunija introduction according to “flora româniei” (vol. vii, p a u c a et al., in s a v u l e s c u , 1972) there are 56 orchid species growing in romania. in the last decades, some taxa, formerly classified as subspecies, became species per se (a typical example is several species of the epipactis genus). in 2009 ciocârlan mentioned 7 (2) • december 2016: 107-112 12th sfses • 16-19 june 2016, kopaonik mt doi: 10.5281/zenodo.2528264 biologica nyssana 7 (2) ⚫ december 2016: 107-112 milanovici, s. ⚫ the orchid flora of the muntele mic… 108 58 orchid species recorded for the romanian flora (c i o c â r l a n , 2009), while according to sârbu this number reaches 60 (s â r b u et al., 2013). three more species are to be added to the last figure mentioned: epipactis guegelii robatsch (r o b a t s c h , 1996), epipactis persica (soó) hausskn. ex nannf. (w u c h e r p f e n n i g , 2008) and epipactis greuteri h. baumann et künkele (a r d e l e a n , 2011). so far, the total amount of orchid species recorded in romania is 62. we do not regard this figure as final. in his reference work b o ş c a i u (1971) mentions 16 orchid species for the muntele mic area. the geographical unity of ţarcu mountains, whose part is the muntele mic, cover the northwestern region of meridional carpathian mountains. material and methods the field studies on the orchidaceae l. family in the muntele mic mountain area, regarding the species richness, distribution, size and dynamics of populations as well as the acknowledgment of the threats with (direct and indirect) impact upon the orchid species and populations have started in 2009 and ended in 2015 (fig. 1, 2b). the main bibliography related to the muntele mic mountain, started with the basic studies: b o ș c a i u (1971) and p a u c a et al., in s a v u l e s c u – “flora r. s. românia”, vol. xii, (1972). to understand the biology and ecology of terrestrial orchid species, literature data of d r e s s l e r (1981, 1983) and r a s m u s s e n (1995) were used. the determination of taxa up to the species level has been done based on the data provided by “flora of the s. r. romania” vol. xii (p a u c a et al., in s a v u l e s c u , 1972) and the nomenclature of used taxa has been harmonized to world checklist of monocotyledons database (2003). also, other relevant literary sources used (s o ó , 1973; m o o r e , 1980; d e l f o r g e , 2006; c i o c â r l a n , 2009) and related websites have been used. the area where the field studies were conducted covers only the zone of the alpine meadows, spruce and beech forests, along the mixed ones, mesophilic mountain meadows (especially those located in the upper part of the craiu valley) to the lowest altitude of 600 m. results and discussion the herein presented studies on the orchid species comprised the area of muntele mic, from an altitude of 600 m to the top of the mountain. although in his reference work b o ş c a i u (1971) mentioned 16 orchid species for the muntele mic zone, we have no fig. 1. geographical position of the muntele mic investigated area. biologica nyssana 7 (2) ⚫ december 2016: 107-112 milanovici, s. ⚫ the orchid flora of the muntele mic… 109 field confirmation (in the researched zone above 600 m) for seven species, namely: listera ovata (l.) r.br., platanthera chlorantha (custer) rchb., anacamptis palustris subsp. elegans (heuff.) r.m.bateman, pridgeon & m.w.chase, anacamptis coriophora (l.) r.m.bateman, pridgeon & m.w.chase, orchis mascula subsp. signifera (vest) soó and anacamptis morio (l.) r.m.bateman, pridgeon & m.w.chase, neotinea ustulata (l.) r.m.bateman, pridgeon & m.w.chase. instead, in the lower meadows area (borlova zone) and beech forests, below 600 m, one can notice the presence of most of the species mentioned above. the fieldwork recorded the presence of three orchid species, not mentioned in the relevant literature (esp boşcaiu, 1971) for the muntele mic zone, specifically leucorchis friwaldskiana (hampe) fuss, dactylorhiza fuchsii (druce) soó and dactylorhiza saccifera (brongn.) soó. in the following text, a description is given for each species confirmed in the fieldwork of the present study (muntele mic zone, above 600 m elevation): the genus dactyloriza neck. ex nevski. dactylorhiza cordigera (fr.) soó habitat: it sprouts individually or in groups of up to dozens of specimen on the eastern slope of the mountain, on wet meadows, on the banks of the torrents coming down from the mountain or in bogs, at an altitude between 1450 and 1600 m (fig. 2a); status of population: population is relatively large and stabile. following the multiannual countering, one can find at least 500 blooming specimens (mostly on the eastern slope of muntele mic); threats: over pasturing, leveling of the meadows for skiing tracks, off road motorcycling. dactylorhiza fuchsii (druce) soó habitat: sporadic and very rare; it had been found in a small number of specimens on the upper part of valea craiu, at the confluence of craiu and cuntu streams, at the edge of the forest, 610 m in altitude; status of population: population comprises around 20 specimens; threats: deforestation along removal of timber logs and their storage along the road; observation: newly found species for the muntele mic zone. dactylorhiza maculata (l.) soó habitat: sporadic and very rare; it had been found in a small number of specimens in the mesophilic meadow at the confluence of craiu and cuntu streams, 610 m in altitude (fig. 2c); status of population: population comprises around 20 specimens; threats: deforestation along removal of timber logs and their storage along the road. dactylorhiza viridis (l.) r.m.bateman, pridgeon & m.w.chase (syn. coeloglossum viride (l.) hartm.) habitat: very rare; a number of 5 specimens had been found, on shadowy rocks (northern exposure), on the eastern slope of the mountain; altitude: 1600 m; status of population: population comprises maximum 10 specimens; threats: intensive pasture. dactylorhiza saccifera (brongn.) soó habitat: found individually or in groups of up to ten specimens at the forest edge (near the paved road that climbs toward muntele mic resort), from valea sebeşului (600 m) along wet meadows from climbing road and the bottom of meadows on the eastern slope of the mountain, on an altitude between 600 and 1.400 m (fig. 2g); status of population: a fairly numerous population, comprising around 200 specimens; threats: intensive pasture, deforestation along removal of timber logs and their storage along the road, off road motorcycling; observation: newly found species for the muntele mic zone. the genus neottia guett. neottia nidus-avis (l.) rich. habitat: found individually or in groups of up to ten specimens in beech or mixed beech – spruce forests, preponderantly below 600 m altitude. as the altitude grows, the specimen became increasingly rare. some rare specimen have been detected on the eastern slope of muntele mic, about 800 m altitude , in the mixed beech – spruce forest; status of population: a very numerous population, comprising around 300 specimens (above 600 m); threats: deforestation along removal of timber logs, pasture on forest ground. the genus gymnadenia rich. gymnadenia conopsea (l.) r.br. habitat: very rare; a few specimens were found on the alpine meadows on the eastern slope, at an altitude between 1.300 and 1.500 m; status of population: given the reduced number of found specimen, one cannot correctly assess the size biologica nyssana 7 (2) ⚫ december 2016: 107-112 milanovici, s. ⚫ the orchid flora of the muntele mic… 110 fig. 2: a) dactylorhiza cordigera (fr.) soó (photo: 18.06.2009); b) muntele mic – general view (photo: 05.06.2015); c) dactylorhiza maculata (l.) soó (photo: 18.06.2009); d) gymnadenia frivaldii hampe ex griseb. (photo: 18.06.2009); e) pseudorchis albida (l.) á. löve & d. löve (photo: 18.06.2009); f) epipactis helleborine (l.) crantz (photo: 20.08.2015); g) dactylorhiza saccifera (brongn.) soó (photo: 18.06.2009). biologica nyssana 7 (2) ⚫ december 2016: 107-112 milanovici, s. ⚫ the orchid flora of the muntele mic… 111 of the population (taking also into account the dormancy, frequently met with the terrestrial orchid species); threats: over pasturing, leveling of meadows for skiing tracks, off road motorcycling. gymnadenia frivaldii hampe ex griseb. habitat: a relatively large population was identified in a single area on muntele mic (on a surface of about 50 sq m), on the eastern slope, in a mesophyllic alpine meadow, 1.460 m altitude (fig. 2d); status of population: the population is relatively numerous, concentrated on a very small surface and comprises some 200 bloomy specimens; threats: extremely threatened by over pasture; not far from the habitat of this species, a farm was built two years ago; the species is also threatened by off road motorcycling. observation: newly found species for the muntele mic zone. the genus pseudorchis ség. pseudorchis albida (l.) á. löve & d. löve habitat: a very small number of specimens were found on the eastern slope, 1400 m altitude (fig. 2e); status of population: very rare; a total of six bloomy specimens, in different locations and different years; threats: extremely threatened by over pasture, the more so as closely to its habitat a sheepfold was installed 2 years ago; the species is also threatened by off road motorcycling. the genus epipactis zinn epipactis helleborine (l.) crantz habitat: a relatively large population of some 200 specimens was found in a small spruce forest, near a torrent, on the border of the alpine meadows. it is interesting that in the lower areas, although the habitat is friendlier to this species, not a single specimen had been identified (fig. 2f); status of population: relative large population, comprising some 200 specimens (around 1200 m altitude); the entire population is concentrated on some 100 sq m; threats: pasturing on forest ground (closely to its habitat a sheepfold was installed 2 years ago) and technical improvements for a skiing track. conclusion although relatively isolated from tarcu mt range (to which it belongs to), muntele mic mt contains most of the typical high-mountain habitats. field studies on the orchid species and their populations started in 2009 and ended in 2015. since the investigated area comprised area above 600 m a.s.l. up to the highest peak of the muntele mic mt (1802 m a.s.l.), the number of recorded species was 10, which was lower than number recorded by boșcaiu (1971). however, the herein presented results revealed the existance of three new species for muntele mic mt, namely gymnadenia frivaldii, dactylorhiza fuchsii and dactylorhiza saccifera. the dactylorhiza cordigera species, according to multiannual monitoring, presents the largest population (at least 500 bloomy specimens, most of them on the east slope of muntele mic). the rarest species are: pseudorchis albida, gymnadenia conopsea (!?), dactylorhiza viridis, dactylorhiza fuchsii and dactylorhiza maculata, as the number of specimens for each of these species is up to 20. considering the easy access up the mountain (asphalted road to muntele mic resort), the existing habitats are subject to an extremely severe negative anthropogenic impact. the greatest threats to the species and their habitats come from leveling of the meadows for skiing tracks and adjacent infrastructure, unfortunately highly aggressive and irreversible (including the south western part). it is followed by the over pasturing, due to existence of at least three sheepfolds on muntele mic. another threat comes from the illegal off-road motorcycling races. references ardelean, corina, 2011: epipactis greuteri (orchidaceae) a new species for romanian flora. journal europäischer orchideen, 43 (3): 527534. boşcaiu, n. 1971: flora şi vegetaţia munţilor ţarcu, godeanu şi cernei. edit. academiei republicii socialiste românia, bucureşti. 494 p.. chase, m. w., cameron, k. m., barrett, r. l., freudenstein, j. v. 2003: dna data and orchidaceae systematics: a new phylogenic classification. in: dixon k.w., kell, s.p., barrett, r.l., cribb, p.j., eds. orchid conservation. kota kinabalu, sabah: natural history publications, 69–90. ciocârlan, v. 2009: flora ilustrată a româniei. pteridophyta et spermatophyte, ed. ceres, bucureşti. 1141 p.. cribb, p.j., kell, s.p., dixon, k.w., barrett, r.l. 2003: orchid conservation: a global perspective. in: dixon k. w., kell, s. p., barrett, r. l., cribb, p.j., eds. orchid conservation. kota kinabalu, sabah, natural history publications, 1–24. biologica nyssana 7 (2) ⚫ december 2016: 107-112 milanovici, s. ⚫ the orchid flora of the muntele mic… 112 delforge, p. 2006: orchids of europe, north africa and the middle east. a&c black, london. 640 p.. dressler, r. l. 1981: the orchids, natural history and classification. cambridge, ma: harvard university press. 332 p.. dressler, r. l. 1993: phylogeny and classification of the orchid family, dioscorides press. 314 p.. govaerts, r. 2003: world checklist of monocotyledons database in access: 1-71827. the board of trustees of the royal botanic gardens, kew. nyárády, e. i. 1958: flora şi vegetaţia munţilor retezat. edit. acad. r.p.r. bucurești. 195 p.. oltean, m., negrean, g., popescu, a., roman, n., dihoru, g., sanda, v., mihăilescu, s. 1994: lista roşie a plantelor superioare din românia, st. sin. doc. ec., 1/1994, bucureşti. pócs, t. 1957: contributions á la flore des carpathes orientaux et meridionaux. annales historiconaturales musei nationalis hungarici, 8: 205-217. robatsch, k. 1996: epipactis guegelii k. robatsch spec. nov., eine neue epipactis-art aus rumänien. journal europäischer orchideen, 28 (4):765-772. rogobete, gh., grozav, adia, beutură, d., nemeş i. 2007: soil acidification by ferrolysis in a pedological sequence of muntele mic, caraşseverin county. factori şi procese pedogenetice din zona temperată 6 s. nouă, 67-74. sanda, v., öllerer, kinga, burescu, p. 2008: fitocenozele din românia. sintaxonomie, structură, dinamică şi evoluţie. ed. ars docendi, universitatea din bucureşti. 570 p.. sârbu, i., ştefan, n., oprea, a. 2013: plante vasculare din românia. determinator ilustrat de teren. ed. victor b victor, bucureşti. 1320 p.. savulescu, t., (ed) 1972: flora republicii socialiste românia, vol. xii. editura academiei republicii socialiste românia, bucureşti. soó, r. 1973: a magyar flóra és vegetáció rendszertani – növényföldrajzi kézikonyve v. akadémiai kiadó, budapest. 723 p.. tutin, t.g., heywood, v.h., burges, n.a., valentine, d.h., eds. 1980: flora europaea, vol. v. cambridge university press, cambridge. urdea, p., törok-oance, m., ardelean, m., vuia, f., voiculescu, m. 2009: geomorphological aspects of the human impact in the alpine area of southern carpathians (romania). hrvatski geografski glasnik, 71 (1): 19-32. wucherpfennig, w. 2008: epipactis persica and andere orchideen des donaudeltas (rumänien). – ber. arbeitskrs. heimische orchideen, 25 (1):85110. http://www.theplantlist.org http://apps.kew.org/wcsp/home.do http://rbg-web2.rbge.org.uk/fe/fe.html  note: this article has been retracted due to technical and grammar errors and plagiarisms in introduction, which were not influenced by the originality and importance of research results in 23 aprile 2018. in agreement with the reviewers, the editor decided to approve resubmission of the manuscript and publication in the same issue of the journal. oxidative stress in bromus (bromus mollis l.) seedlings treated with clary sage (salvia sclarea l.) aqueous extract biologica nyssana 7 (2)  december 2016: 141-144 šućur, j. et al.  the effect of satureja montana l. aqueous… 141 original article received: 01 july 2016 revised: 11 november 2016 accepted: 24 november 2016 oxidative stress in bromus (bromus mollis l.) seedlings treated with clary sage (salvia sclarea l.) aqueous extract jovana šućur1, dejan prvulović1, goran anačkov2, đorđe malenčić1 1 department of field and vegetable crops, faculty of agriculture, university of novi sad, trg dositeja obradovića 8, novi sad, serbia 2 department of biology and ecology, faculty of science, university of novi sad, trg dositeja obradovića 3, novi sad, serbia * e-mail: jovana.sucur@polj.edu.rs abstract: šućur, j., prvulović, d., anačkov, g., malenčić, đ.: oxidative stress in bromus (bromus mollis l.) seedlings treated with clary sage (salvia sclarea l.) aqueous extract. biologica nyssana, 7 (2), december 2016: 141-144. extensive use of synthetic pesticides has negative effects on the environment and on human and animal health. knowledge on allelopathic interactions could provide effective tools for a better exploitation of natural resources in the management of weeds without using herbicides. one of highly resistant weed species is bromus. the effects of two concentrations (0.1% and 0.2%) of salvia sclarea l. aqueous extract on the activity of the antioxidant enzymes, superoxide dismutase (sod) and catalase (cat) in leaves and roots of bromus (bromus mollis l.) seedlings, were examined. our results showed that both concentrations of the extract used (0.1% and 0.2%) stimulated the significant increase of the superoxide dismutase activity in leaves and roots of bromus 72 hours and 120 hours after the treatment. the significant increase of the catalase activity was recorded in roots of bromus 72 h after the treatment. two tested extract concentrations affected activity of the antioxidant enzymes in the same way, but the higher activity was observed in the roots treated with higher concentration (0.2%). the increase of the activities of antioxidant enzymes, in response to stress induced by s. sclarea aqueous extract, indicate that the plant extract possesses allelopathic activity on treated plant. key words: allelopathy, biopesticides, bromus mollis l., salvia sclarea l. apstrakt: šućur, j., prvulović, d., anačkov, g., malenčić, đ.: oksidativni stres u sadnicama klasače (bromus mollis l.) tretiranim vodenim ekstraktom muskatne žalfije (salvia sclarea l.). biologica nyssana, 7 (2), decembar 2016: 141-144. upotreba sintetičkih pesticida ima negativan efekat na životnu sredinu, kao i zdravlje ljudi i životinja. poznavanje alelopatskih interakcija može da obezbedi efikasniji način eksploatacije prirodnih resursa u suzbijanju korova bez upotrebe sintetičkih herbicida. jedna od otpornijih vrsta korova je korov klasača. u ovom radu je ispitan uticaj dve koncentracije (0,1% i 0,2%) vodenog ekstrakta s. sclarea na aktivnost antioksidantnih enzima superoksid-dismutaze (sod) i katalaze (cat) u listu i korenu klasače (bromus mollis l.). dobijeni rezultati su pokazali statistički značajno povećanje aktivnosti enzima superoksid-dismutaze u 7 (2) • december 2016: 141-144 12th sfses • 16-19 june 2016, kopaonik mt doi: 10.5281/zenodo.200411 biologica nyssana 7 (2)  december 2016: 141-144 šućur, j. et al.  the effect of satureja montana l. aqueous… 142 listu i korenu klasače, 72 i 120 časova nakon tretmana za obe primenjene koncentracije ekstrakata (0,1% i 0,2%). statistički značajno povećanje aktivnosti katalaze uočeno je u korenu klasače 72 časa nakon tretmana. obe ispitane koncentracije vodenih ekstrakata uticale su na aktivnost antioksidantnih enzima, pri čemu je veća aktivnost uočena u korenu klasače tretirane sa višom koncentracijom (0,2%). povećanje aktivnosti antioksidantnih enzima, kao odgovor na stres prouzrokovan vodenim ekstraktom s. sclarea, pokazuje da ekstrakt poseduje alelopatski uticaj na tretiranu biljku. ključne reči: alelopatija, glycine max (l.) merr., satureja montana l. introduction the flowering plant family of lamiaceae is very important since its highly diverse species are potent source of secondary metabolites (f a k o o r z i b a et al., 2014). numerous species of the genus salvia, belonging to lamiaceae, have been used in traditional medicine (n a s e r m o a d e l i & r o w s h a n , 2013). salvia sclarea l., known as clary sage, a plant native to southern europe, is one of the most important aromatic plants cultivated world-wide as a source of essential oils and many other compounds derived from different parts of the plant (h u d a i b et al., 2001, n a s e r m o a d e l i & r o w s h a n , 2013). plant-derived chemical compounds are also involved in communication between plants (w e i r et al., 2004). allelopathy is an interference mechanism in which plants release chemical substances, called allelochemicals, from leaves, stems, roots, flowers and seeds which inhibit or stimulate plant growth (g e l l a et al., 2013). accordingly, allelopathy is a natural and an environment-friendly technique which may prove to be also a unique tool for weed control (s h a r m a & s a t s a n g i , 2013). allelochemicals are highly attractive as new classes of herbicides due to a variety of advantages. most of them are water-soluble which makes them easier to apply and their chemical structures are more environmentally friendly than synthetic ones (s o l t y s et al., 2013). the aim of this study was to examine the effect of two concentrations (0.1% and 0.2%) of salvia sclarea l. aqueous extract on the activity of the antioxidant enzymes superoxide dismutase (sod) and catalase (cat) in leaves and roots of bromus (bromus mollis l.) seedlings and to explore the biopesticidal potential of this species in weed control. material and methods the wild, aromatic plant, s. sclarea l., was collected on mt rujan, in the south of serbia (n 42º22ʹ40.44ʹʹ, e 21º53ʹ09.23ʹʹ, 494 m.a.s.l.) in july of 2012. voucher specimens of collected plant was confirmed and deposited at the herbarium of the department of biology, faculty of natural sciences, university of novi sad (buns 2-1545). the aqueous extract of s. sclarea was prepared with air-dried plant material (10 g) in boiling distilled water (100 ml). after 24 h, the extract was filtered through whatman no.4 filter paper and kept at 4 °c in the fridge until application. the experiment was performed at the laboratory of biochemistry, faculty of agriculture, novi sad and conducted under controlled conditions (28 oc, 60% relative humidity, a photoperiod of 18 h, and a light intensity of 10.000 lx). the bromus (bromus mollis l.) seeds were grown in plastic pots containing sterile sand. after 30 days, the plants were transplanted in plastic pots containing 700 ml of hoagland’s solution and 7 and 14 ml of s. sclarea aqueous extract, while pots of control contained just the same volume of hoagland’s solution (10% mgso4 x 7h2o, 10% ca(no3)2 x 4 h2o, 10% kh2po4, 10% kno3, microelements, 7.5% feedta). plants were harvested for further biochemical analyzes 24, 72 and 120 h after the treatments. fresh leaves and roots of bromus plants (2 g each) were homogenized in 10 ml of phosphate buffer (0.1 m, ph 7.0). homogenates were centrifuged for 20 min at 10.000 x g (boeco, germany) and filtered. the supernatants were used for determining the investigated biochemical parameters (activity of the antioxidant enzymes superoxide dismutase (sod) and catalase (cat)). biochemical analyses were carried out spectrophotometrically using an uv/vis spectrophotometer model thermo scientific evolution 220 (usa). the catalase (cat) (ec 1.11.1.6) activity was determined according to s a t h y a & b j o r n , (2010). the decomposition of h2o2 was followed as a decrease in absorbance at 240 nm. the enzyme extract was added to the assay mixture containing 1 ml of 50 mm potassium phosphate buffer (ph 7.0) and 10 mm h2o2 (centrohem, serbia). the activity of the enzyme is expressed as u per 1 g of protein (u mg–1 protein). superoxide dismutase (sod) (ec 1.15.1.1) activity was assayed according to a slightly modified method of mandal et al. (2008) by measuring its ability to inhibit photochemical reduction of nitro blue tetrazolium (nbt) chloride. the reaction mixture contained 50 mm phosphate buffer (ph 7.8), 13 mm l-methionine (sigma (st. biologica nyssana 7 (2)  december 2016: 141-144 šućur, j. et al.  the effect of satureja montana l. aqueous… 143 louis, mo.)), 75 μm nbt (sigma (st. louis, mo.)), 0.1 mm edta, 2 μm riboflavin (sigma (st. louis, mo.)) and 0.02 ml of the enzyme extract. it was kept under a fluorescent lamp for 30 min, and then the absorbance was read at 560 nm. one unit of the sod activity is defined as the amount of enzyme required to inhibit the reduction of nbt by 50%. the activity of the enzyme is expressed as u per 1 mg of protein (u mg–1 protein). values of the biochemical parameters were expressed as means ± standard error (se) of determinations made in triplicates and tested by anova followed by comparison of the means by the duncan multiple range test (p < 0.05). data were analyzed using statistica for windows, version 11.0. the results are presented as histograms (values marked with different letter differ significantly at p < 0.05). results and discussion in the leaves of bromus seedlings, the significant increase in activity of cat was detected in the treatment with lower concentrations of the s. sclarea aqueous extract 120 h after the treatment (fig. 1). the highest activity of the sod was observed in plants 120 h after the treatment with 0.2% s. sclarea aqueous extract (fig. 2). in the roots of bromus, the significant increase in cat activity was recorded 72 and 120 h after the treatment (fig. 1). the activity of sod was significantly increased 72 and 120 h after the treatments with s. sclarea aqueous extract (fig. 2). numerous growth inhibitors identified in some plants are responsible for their allelopathic properties and may be a useful source for the future development of bioherbicides (x u a n et al., 2005). in the research of s h a r m a & s a t s a n g i (2013) fig. 1. activity of cat in leaves and roots of bromus seedlings 24, 72 and 120 h after the treatment with different concentrations (%) of s. sclarea aqueous extracts (v/v) and in control (c). fig. 2. activity of sod in leaves and roots of bromus seedlings 24, 72 and 120 h after the treatment with different concentrations (%) of s. sclarea aqueous extracts (v/v) and in control (c) 0 5 10 15 20 25 30 c 0.1 0.2 c 0.1 0.2 c 0.1 0.2 24 h 72 h 120 h u /m g p ro te in s extract concentration (%) leaves roots a a a,b a,b a a c b c b c a a b c a a,b c 0 10 20 30 40 50 60 70 80 c 0.1 0.2 c 0.1 0.2 c 0.1 0.2 24 h 72 h 120 h u /m g p ro te in s extract concentration (%) leaves roots a b b c b b c b a a a,b c b c c b c c biologica nyssana 7 (2)  december 2016: 141-144 šućur, j. et al.  the effect of satureja montana l. aqueous… 144 the aqueous leaf extract of the sunflower (helianthus annuus l.) showed an inhibitory effect on germination and growth of parthenium hysterophorus. this negative effect came from allelochemicals present in the aqueous extract of the sunflower. h u s s a i n & r e i g o s a (2011) reported that allelochemical cinnamic acid, widespread phenolic acid, was phytotoxic to c3 perennial plant species (dactylis glomerata, lolium perenne and rumex acetosa). the toxicity of the extracts varied with the concentration and soaking periods. the extract with higher concentration and longer soaking periods were more inhibitory then lower (h u s n a et al., 2016). further, lower concentrations of plant’s aqueous extracts could be more inhibitory in sterile soil than in nonsterile soil (b a r n e s & p u t n a m , 1986). our results showed that both concentrations of s. sclarea extract used (0.1% and 0.2%), stimulated the significant increase of the superoxide dismutase activity in leaves and roots of bromus 72 and 120 h after the treatment. the two tested extract concentrations affected the activity of the catalase in the bromus roots in the same way, but a higher activity was observed in the treatment with the higher concentration of the s. sclarea aqueous extracts (0.2%). the increases in the activities of antioxidant enzymes probably occur in response to stress induced by s. sclarea aqueous extract. conclusion in conclusion, our results showed that s. sclarea aqueous extract stimulates an increase of the catalase activity in the roots of bromus seedlings while both tested concentrations caused a significant increase of the superoxide dismutase activity in bromus leaves and roots. the results indicate that the plant extract possesses allelopathic activity and increases in activity of antioxidant enzymes probably occur in response to stress. acknowledgements. this study was carried out within a project of the ministry of education, science and technological development, republic of serbia, grant no tr-31022. references barnes, j.p., putnam, a.r. 1986: evidence for allelopathy by residues and aqueous extracts of rye (secale cereale). weed science, 34(3): 384– 390. fakoorziba, m.r., moemenbellah-fard, m.d., azizi, k., shekarpoor, h., alipoor, h. 2014: excitorepellency effects of salvia sclarea l. (lamiaceae) extracts on adult house flies, musca domestica l. (diptera: muscidae). journal of health sciences and surveillance system, 2 (1): 1–7. gella, d., ashagre, h., negewo, t. 2013: allelopathic effect of aqueous extracts of major weed species plant parts on germination and growth of wheat. journal of agricultural and crop research, 1(3): 30–35. hudaib, m., grazia bellardi, m., rubies-autonell,c., fiori, j., cavrini, v. 2001: chromatographic (gcms, hplc) and virological evaluations of salvia sclarea infected by bbwv-i. il farmaco, 56: 219–227. hisna, shah, m., sayyed, a., shabeena, aziz, l., ismail, gul., h. 2016: allelopathic effect of salvia plebia r. brown on germination and growth of zea mays var. 30-25 hybrid, triticum astivum var. pirsabak-04 and sorghum bicolor l. journal of applied environmental and biological sciences, 6(4): 93–104. hussain, m.i., reigosa, m.j. 2011: allelochemical stress inhibits growth, leaf water relations, psii photochemistry, non-photochemical fluorescence quenching, and heat energy dissipation in three c3 perennial species. journal of experimental botany, doi:10.1093/jxb/err161. nasermoadeli, s., rowshan, v. 2013: comparison of salvia sclarea l. essential oil components in wild and field population. international journal of agriculture and crop sciences, 5(8): 828–831. sharma, m., satsangi, g.p. 2013: potential allelopathic influence of sunflower (helianthus annuus l.) on germination and growth behavior of two weeds in-vitro condition. international journal of biotechnology and bioengineering research, 4(5):421–426. soltys, d., krasuska, u., bogatek, r., gniazdowska, a. 2013: allelochemicals as bioherbicidespresent and perspectives, herbicides-current research and case studies in use, doi: 10.5772/56185. weir, t.l., park, s., vivanco, j.m. 2004: biochemical and physiological mechanisms mediated by allelochemicals. current opinion in plant biology, 7: 472–479. xuan, t.d., shinkichi, t., khanh, d.t., min, c.i. 2005: biological control of weeds and plant pathogens in paddy rice by exploiting plant allelopathy: an overview. crop protection, 24: 197–206. genetic diversity of the critically endangered verbascum davidoffii murb. (scrophulariaceae) and implications for conservation biologica nyssana 7 (2)  december 2016: 101-106 petrova, g. et al.  genetic diversity of the critically endangered verbascum… 101 original article received: 08 october 2016 revised: 30 october 2016 accepted: 05 november 2016 genetic diversity of the critically endangered verbascum davidoffii murb. (scrophulariaceae) and implications for conservation galya petrova1*, stefan petrov2, svetlana bancheva3 1laboratory “photosynthesis activity and regulation”, institute of plant physiology and genetics, bulgarian academy of sciences, “acad. g. bonchev” str. 21, 1113 sofia, bulgaria 2gene regulation department, institute of molecular biology “roumen tsanev”, bulgarian academy of sciences, “acad. g. bonchev” str. 21, 1113 sofia, bulgaria 3department of plant and fungal diversity and resources, institute of biodiversity and ecosystem research, bulgarian academy of sciences, “acad. g. bonchev” str. 21, 1113 sofia, bulgaria * e-mail: galiaty@abv.bg abstract: petrova, g., petrov, s., bancheva, s.: genetic diversity of the critically endangered verbascum davidoffii murb. (scrophulariaceae) and implications for conservation. biologica nyssana, 7 (2), december 2016: 101-106. verbascum davidoffii murb. (scrophulariaceae), one of the rarest plant species in bulgarian flora, is a local endemic, protected by the national biodiversity act, included in the red list of vascular plants, as well as in the red data book of bulgaria with conservation status “critically endangered”. its distribution is limited due to anthropogenic pressure, specific ecological requirements and low reproductive capability. in this study, we aimed to measure the genetic diversity level in the unique single world population of verbascum davidoffii located in pirin national park, bulgaria. we found high genetic diversity in the excitant population of the species. the present study indicates that the primary objective in conservation of verbascum davidoffii is to preserve as much as possible of its evolutionary potential. key words: verbascum davidoffii, endemic species, genetic diversity, conservation apstrakt: petrova, g., petrov, s., bancheva, s.: genetički diverzitet kritično ugrožene vrste verbascum davidoffii murb. (scrophulariaceae i implikacije za konzervaciju. biologica nyssana, 7 (2), decembar 2016: 101-106. verbascum davidoffii murb. (scrophulariaceae), jedna je od najređih biljaka flore bugarske i lokalni endemit koji je zaštićen nacionalnim zakonom o biodiverzitetu, uključen u crvenu listu vaskularnih biljaka, kao i u crvenu knjigu bugarske sa konzervacionim statusom “kritično ugrožena”. njeno rasprostranjenje je ograničeno zbog antropogenog pritiska, specifičnih ekoloških prohteva i niske reproduktivne sposobnosti. u ovom istraživanju, naš cilj bio je da merimo nivo genetičkog diverziteta u jedinstvenoj populaciji verbascum davidoffii na svetu, lociranoj u nacionalnom parku pirin, bugarska, gde je utvrđen visok genetički diverzitet. 7 (2) • december 2016: 101-106 12th sfses • 16-19 june 2016, kopaonik mt doi: 10.5281/zenodo.200406 biologica nyssana 7 (2)  december 2016: 101-106 petrova, g. et al.  genetic diversity of the critically endangered verbascum… 102 ovo istraživanje je ukazalo na to da je primarni cilj u konzervaciji vrste verbascum davidoffii da se njen evolucioni potencijal sačuva u što većoj meri. ključne reči: verbascum davidoffii, endemična vrsta, genetički diverzitet, konzervacija introduction bulgarian vascular flora consists of more than 4000 species. this high level of plant diversity is determined by various factors and the geographical position of the country is a main factor, allowing the distribution of diverse floristic elements on a relatively restricted territory. the richest in endemic species are the genera centaurea (cornflower), verbascum (mullein), anthemis (dog fennel), silene (catchfly), etc. (b i s e r k o v et al., 2015). the genus verbascum comprises of about 360 species of flowering plants in the scrophulariaceae family (f i r a t , 2015). verbascum davidoffii murb. (scrophulariaceae), one of the rarest plant species in bulgarian flora, is a local endemic, protected by the national biodiversity act, included in the red list of vascular plants and in the red data book of bulgaria with conservation status “critically endangered”. the unique single world population of the species is located between the valleys of river bunderitsa and razlozhki suhodol, pirin national park, bulgaria (fig. 1). its distribution is limited due to anthropogenic pressure, specific ecological requirements and low reproductive capability (a s s y o v & d e n c h e v , 2015). genetic diversity is of fundamental importance in the continuity of a species as it provides the necessary adaptation to the prevailing biotic and abiotic factors, and enables changes in the genetic composition to cope with environmental changes (r a o & h o d g k i n , 2002). understanding of the genetic variation within and between populations is essential for the establishment of efficient conservation practices for rare plants. small populations are expected to demonstrate lower genetic diversities than larger populations and vice versa (w i l l i et al., 2006; l i et al., 2012). because of the small size of v. davidoffi population, we hypothesized that the genetic diversity of the species will be low. the loss of genetic diversity is one of the key aspects in many conservation programmes of threatened plant species (h a m r i c k & g o d t , 1996). thus, the present study was designed as a first attempt towards investigating the genetic diversity of v. davidoffii. the increasing availability of pcrbased molecular markers allows the detailed analyses and evaluation of genetic diversity in plants (i d r e e s & i r s h a d , 2014). currently, there is no an universal method for all applications and each technique has its own advantages and limitations. among the various types of available molecular fig. 1. geographic location of verbascum davidoffii population in bulgaria. biologica nyssana 7 (2)  december 2016: 101-106 petrova, g. et al.  genetic diversity of the critically endangered verbascum… 103 markers, inter-simple sequence repeats (issrs) do not need prior knowledge of the genome to be analyzed (z i e t k i e w i c z et al., 1994). the issr analysis requires comparatively low amount of dna, the utilization of long primers allows more stringent annealing temperatures and reveals more polymorphic fragments (f a n g & r o o s e , 1997; w o l f e & l i s t o n , 1998; c a m a c h o & l i s t o n , 2001). considering these advantages of issr primers, we attempted to use this marker system in order to analyze the pattern of genetic diversity of v. davidoffii, given that such knowledge will be important for more efficient planning of the strategies for conservation of this endangered plant species. material and methods species analyzed morphology and biology. biennial herbaceous plant. stem up to 80 cm high, usually unbranched. basal leaves obovate, densely hairy. flowers in groups of 2–4 in the upper part of the stem. calyx 5–merous, with black glandular hairs. corolla 5-merous, golden yellow, up to 3.5 cm in diameter. fruit spherical to ovate capsule. fl. vi–vii, fr. vii–ix. reproduction by seeds (a s s y o v & d e n c h e v , 2015). distribution in bulgaria and description of species’ habitat. the population of v. davidoffii consists of very low number of individuals distributed between the valleys of river bunderitsa and razlozhki suhodol, pirin national park. the park encompasses over 40 000 ha of unique nature, at an altitude between 1008 and 2914 m in the pirin mountains, southwest bulgaria. pirin national park comprises diverse limestone mountain landscapes with glacial lakes, waterfalls, caves and predominantly coniferous forests. v. davidoffii inhabits grassy openings and light, rocky places in forests of pinus heldreichii on calcareous soils (a s s y o v & d e n c h e v , 2015). plant material fourteen individuals were sampled during the flowering stage from the v. davidoffii population in pirin national park (latitude (n): 41°76'82''; longitude (e): 23°42'63''). dna extraction genomic dna was extracted from young leaves following the modified ctab procedure of d o y l e & d o y l e (1987). issr analysis issr primers (microsynth, balgach, switzerland) in the initial screening that had a high level of polymorphism, repeatability, and the best scorability were selected, after the screening of 35 primers on the subset of collected samples (tab. 1). polymerase chain reactions were performed in a volume of 25 µl, containing a final concentration of 1 × pcr buffer (fermentas, vilnius, lithuania), 1 u taq dna polymerase (fermentas, vilnius, lithuania), 100 µm of each dntp, 1 µm of each primer and 50 ng of extracted dna. pcr cycling conditions were as follows: 5 min. initial denaturation at 95 °c, 35 cycles of amplification [45 s at 94 °c, 1 min. at the annealing temperature (ta), 2 min. elongation at 72 °c] and a final elongation step of 5 min. at 72 °c. pcr experiments were performed with a tc-5000 gradient thermal cycler (techne, staffordshire, uk). to determine the optimal annealing temperature for each primer, an interval of 10 °c around the melting temperature (tm) was tested. the temperatures leading to clear patterns were then repeated until the optimal ta was selected for each primer for routine issr fingerprinting. the reproducibility of the table 1. sequences, annealing temperature, total number and number of polymorphic bands for selected issr primers in the present study of verbascum davidoffii. primer sequence (5'→3') total number of bands number of polymorphic bands percent of polymorphic loci (%) annealing temperature (ºc) (ga)8t 12 12 100 60 (ga)8a 13 13 100 60 (ca)8g 11 11 100 60 (ac)8t 17 17 100 60 (ac)8c 12 12 100 60 (ac)8g 9 9 100 60 (ag)8yt 9 9 100 55 (ag)8yc 5 5 100 55 (ga)8yg 5 5 100 55 (ac)8yt 10 10 100 55 103 103 100 biologica nyssana 7 (2)  december 2016: 101-106 petrova, g. et al.  genetic diversity of the critically endangered verbascum… 104 technique was tested by replicating each amplification reaction twice. to further ensure the quality, pcr reactions were performed with one positive and one negative control. the pcr products were analyzed on 2% agarose gels (fermentas, vilnius, lithuania) in 0.5 × tbe buffer. a 100-bp plus dna ladder size standard (fermentas, vilnius, lithuania) was used to estimate the length of pcr products. the gels were stained by incorporating 1.5 µl of ethidium bromide (0.5 mg/ml) in 100 ml agarose. electrophoresis was run for 1.5 h at 150 v, the issr profiles were visualized with a uv transilluminator (tfp-m/wl, vilber lourmat, eberhardzell, germany) and further analyzed with a video image analyzer. software data analysis the issr band profiles were treated as dominant markers and each locus was considered as a bi-allelic locus with one amplifiable and one null allele. the well resolved and consistently reproducible amplified dna fragments as bands were scored with regards to their presence (1) or absence (0). the assignment of issr bands to genetic loci was performed semi-automatically using the gelanalyzer 2010a image analysis software (http://www.gelanalyzer.com). the genetic diversity was measured using genalex v.6.5 (p e a k a l l & s m o u s e , 2012). results and discussion genetic diversity the low level of genetic diversity is considered to be a common feature of endemic plant species and it is generally attributable to the small size of their populations (h u a n g et al., 2009; z h u et al., 2009; p e t r o v a et al., 2014). however, in contrast to our expectations, the genetic pattern of v. davidoffii revealed in this study was characterized with a high level of genetic diversity. ten of 35 tested issr primers resulted in polymorphic issr profiles including 4 poly (ac), 3 poly (ga), 2 poly (ag) and 1 poly (ca) dinucleotide primers (tab. 1). the number of bands produced by the used primers ranged between 5 (primers (ag)8 yc and (ga)8yg) and 17 for the primer (ac)8t. the total number of alleles produced by the ten primers is 103. a 100% polymorphism was scored for all primers. examples of photographs illustrating the issr fingerprinting by selected primers are shown in fig. 2. these include issr fingerprinting revealed by primers (ca)8g (fig. 2a) and (ga)8yg (fig. 2b). the data from the issr-analysis were combined in a single matrix and the genetic coefficients (shannon’s information index, si; nei’s genetic diversity, h and unbiased diversity, hu) were calculated to describe the genetic pattern of the excitant population of v. davidoffii. the shannon’s information index (si) was estimated to be 0.406 and the issr derived unbiased diversity (hu = 0.266) from the present study was close to the average value of narrow and outcrossing plant species according to the nybom statistic, where estimates derived by the dominantly inherited markers are very similar and may be directly comparable (n y b o m , 2004). in summary, our study indicated that the critically endangered bulgarian endemic v. davidoffii is able to maintain high level of genetic diversity even its small population size. the observed maintenance of genetic diversity in v. davidoffii is surprising, considering the limited area of distribution of the species, as well as its "critically endangered" status. in recent years, a lot of studies revealed high levels of genetic variability in rare or narrow endemic plants (h e l e n u r m , 2001; z a w k o et al., 2001; x u e et al., 2004; j i a et al., 2016; t u r c h e t t o et al., 2016). among the various factors, the breeding system is an important factor that may be invoked to explain high levels of genetic diversity in rare plants (h a m r i c k & g o d t , 1996). previous studies on the mating system of v. davidoffii suggested that the species is predominantly outcrossing (s t e f a n o v a g a t e v a , 1995). the outcrossing and long-lived figure 2. examples of photographs illustrating issr fingerprints of verbascum davidoffii by issr primers (ca)8g (a) and (ga)8yg (b). lanes 1 ÷ 14 stand for individual samples from the population of the species. m: 100 bp plus dna ladder. biologica nyssana 7 (2)  december 2016: 101-106 petrova, g. et al.  genetic diversity of the critically endangered verbascum… 105 plants commonly have higher levels of genetic diversity than selfing plants (n y b o m , 2004)). therefore, we assume that the high level of genetic load maintained in v. davidoffii is probably due to its reproductive system and may be contributed to the outcrossing behavior of the species. generally, mountain plants are among the species most vulnerable to global warming, because of their isolation and narrow geographic distribution. altitudinal gradients in mountains modify environmental conditions and put populations under different selective pressures that can easily be linked to ongoing climate changes (w a l t h e r et al., 2005; g o n z a l o t u r p i n & h a z a r d , 2009). verbascum davidoffii is distributed in a single high altitude population (a s s y o v & d e n c h e v , 2015). in the present study, we did not observe a correlation between the issr derived genetic diversity of the species and the size of its population despite the theoretical prediction that small populations might lose genetic variation. thus, we hypothesize that the genetic diversity pattern of v. davidoffii may be a result from recently dispersed individuals from different population sources, and it may not respond immediately to the reduction in population size. conservation implications the high genetic diversity of v. davidoffii indicates that the major factors that threaten the persistence of its population are ecological factors rather than genetic. the main risk is that this species is found in only one population and no other natural populations were found. in order to protect it against extinction and loss of its available genetic resources, we recommend collection of seeds from as many individuals as possible and storage in seed banks and living collections. we suggest that conservation programs should be improved to protect its natural habitat. pirin national park was established in 1962 and it was added to the world heritage list in 1983. although, it has long been a subject to tourism pressure, largely caused by the development of ski facilities. such activities may affect the values and integrity of the property and therefore require rigorous control. the development of sustainable forms of tourism would guarantee preservation of ecosystems in the park. we recommend the establishment of variety of educational programs that directly present conservation and biodiversity issues. the key outcomes include enhanced visitor experience, administrative support, volunteer activities and ecological restoration. conclusion the high level of genetic diversity enables v. davidoffi to adapt to changing environments. the maintenance of genetic diversity in the excitant population of the species is an indicator that its current endangered status is not caused by genetic factors. the results reported herein suggest that the high genetic diversity of the species may derive from an ancestral population. however, the ecological requirements of the species, as well as its origin and relationship with other related species should be further elucidated. acknowledgements. the authors wish to thank chief assistant stoyan stoyanov (iber-bas) and dr. boryana sidjimova (iber-bas) for the samples of v. davidoffii used in this work. this work was supported by the bulgarian nsf under grant dfni-bo2/18. references assyov, b., denchev, c. 2015: verbascum davidoffii murb. in: biserkov, v. et al. (eds.), digital edition of red data book of republic of bulgaria. bas & moew, sofia. http://e-ecodb.bas.bg/rdb/en/vol1/verdavid.html. biserkov, v. et al. (eds.) 2015: digital edition of red data book of republic of bulgaria. bas & moew, sofia. http://e-ecodb.bas.bg/rdb/en/. camacho, f.j., liston, a. 2001: population structure and genetic diversity of botrychium pumicola (ophioglossaceae) based on inter-simple sequence repeats (issr). american journal of botany, 88 (6): 1065-1070. doyle, j.j., doyle, j.l. 1987: a rapid dna isolation procedure for small quantities of fresh leaf tissue. phytochemical bulletin, 19: 11-15. fang, d.q., roose, m.l. 1997: identification of closely related citrus cultivars with inter-simple sequence repeat genetics. theoretical and applied genetics, 95 (3): 408-417. firat, m. 2015: verbascum kurdistanicum (scrophulariaceae), a new species from hakkari, turkey. phytokeys, 52: 89-94. gonzalo-turpin h., hazard, l. 2009: local adaptation occurs along altitudinal gradient despite the existence of gene flow in the alpine plant species festuca eskia. journal of ecology, 97 (4): 742-751. hamrick, j.l., godt, m.j.w. 1996: conservation genetics of endemic plant species. in: avise, j.c., hamrick, j.l. (eds.), conservation genetics: case histories from nature. chapman and hall, new york, pp. 281-304. helenurm, k. 2001: high levels of genetic polymorphism in the insular endemic herb http://e-ecodb.bas.bg/rdb/en/ biologica nyssana 7 (2)  december 2016: 101-106 petrova, g. et al.  genetic diversity of the critically endangered verbascum… 106 jepsonia malvifolia. journal of heredity, 92 (5): 427-432. huang, y., zhang, c.q., li, d.z. 2009: low genetic diversity and high genetic differentiation in the critically endangered omphalogramma souliei (primulaceae): implications for its conservation. journal of systematics and evolution, 47 (2): 103–109. idrees, m., irshad, m. 2014: molecular markers in plants for analysis of genetic diversity: a review. european academic research, 2 (1): 1513-1540. jia, j., zeng, l., gong, x. 2016: high genetic diversity and population differentiation in the critically endangered plant species trailliaedoxa gracilis (rubiaceae). plant molecular biology reporter, 34 (1): 327-338. li, y.y., guan, s.m., yang s.z., luo, y., chen, x.y. 2012: genetic decline and inbreeding depression in an extremely rare tree. conservation genetics, 13 (2): 343-347. nybom, h. 2004: comparison of different nuclear dna markers for estimating intraspecific genetic diversity in plants. molecular ecology, 13 (5): 1143-1155. peakall, r., smouse, p.e. 2012: genalex 6.5: genetic analysis in excel. population genetic software for teaching and research an update. bioinformatics, 28 (19): 2537-2539. petrova, g., dzhambazova, t., moyankova, d., georgieva, d., michova, a., djilianov, d., moeller, m. 2014: morphological variation, genetic diversity and genome size of critically endangered haberlea (gesneriaceae) populations in bulgaria do not support the recognition of two different species. plant systematics and evolution, 300 (1): 29-41. rao, v.r., hodgkin, t. 2002: genetic diversity and conservation and utilization of plant genetic resources. plant cell, tissue organ culture, 68 (1): 1-19. stefanova-gateva, b. 1995: verbascum l. in: kožuharov, s. (ed.), flora republicae popularis bulgaricae 10: 26-100, acad. "prof. m. drinov", serdicae, sofia, bulgaria (in bulgarian). turchetto, c., segatto, a.l.a., mäder, g., rodrigues, d.m., bonatto, s.l., freitas, l.b. 2016: high levels of genetic diversity and population structure in an endemic and rare species: implications for conservation. aob plants 8: plw002; doi:10.1093/aobpla/plw002. walther, g.-r., beißner, s., burga, c.a. 2005: trends in the upward shift of alpine plants. journal of vegetation science, 16 (5): 541-548. willi, y., van buskirk, j., hoffmann, a.a. 2006: limits to the adaptive potential of small populations. annual review of ecology, evolution and systematics, 37: 433-458. wolfe, a.d., liston, a. 1998: contributions of pcrbased methods to plant systematics and evolutionary biology. in: soltis, p.s., soltis, d.e., doyle, j.j. (eds.), molecular systematics of plants: dna sequencing. kluwer, new york, pp. 43-86 xue, d.-w., ge, x.-j., hao, g., zhang, c.-q. 2004: high genetic diversity in a rare, narrowlu endemic primrose species: primula interjacens by issr analysis. acta botanica sinica, 46 (10): 11631169. zawko, g., krauss, s.l., dixon, k.w., sivasithamparam, k. 2001: conservation genetics of the rare and endangered leucopogon obtectus (ericaceae). molecular ecology, 10 (10): 23892396. zhu, y., geng, y., tersing, t., liu, n., wang, q., zhong, y. 2009: high genetic differentiation and low genetic diversity in incarvillea younghusbandii, an endemic plant of qinghaitibetan plateau, revealed by aflp markers. biochemical systematics and ecology, 37 (5): 589-596. zietkiewicz, e., rafalski, a., labuda, d. 1994: genome fingerprinting by simple sequence repeat (ssr)-anchored polymerase chain reaction amplification. genomics, 20 (2): 176-183. anticancer compounds from medicinal plants biologica nyssana 7 (1)  september 2016: 41-46 čubrić, t.  species distribution modeling techniques as a tool in… 41 original article received: 30 mart 2016 revised: 27 april 2016 accepted: 01 june 2016 species distribution modeling techniques as a tool in preliminary assessment of special nature reserve “goč-gvozdac” tijana čubrić* university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia * e-mail: tijanacubric@hotmail.com abstract: čubrić, t.: species distribution modeling techniques as a tool in preliminary assessment of special nature reserve “goč-gvozdac”. biologica nyssana, 7 (1), september 2016: 41-46. effective conservation actions such as defining new nature reserve require accurate estimates of the spatial distributions of the target species. species distribution models provide habitat suitability maps for studied species. in this paper we used maxent software to estimate the distribution and extent of potential suitable habitat of five amphibian and reptilian species (mesotriton alpestris, bombina variegata, testudo hermanni, lacerta viridis and vipera ammodytes) in the special nature reserve “goč-gvozdac” (central serbia) in order to assess how much of the potential habitats is included in this reserve. comparing produced suitable habitat maps of the species with a map of the special nature reserve “goč – gvozdac” we concluded that the reserve boundaries do not coincide with the proposed distribution of suitable habitats for m. alpestris, t. hermanni, l. viridis and v. ammodytes, and therefore this reserve does not contribute much to the protection of local populations of these species. key words: species distribution modeling, maxent, nature reserve, reptiles, amphibians apstrakt: čubrić, t.: modelovanje prostornog rasprostranjenja vrsta kao tehnika u preliminarnom ocenjivanju specijalnog rezervata prirode „goč-gvozdac“. biologica nyssana, 7 (1), septembar 2016: 41-46. efektivne konzervacione mere kao što je definisanje novog zaštićenog područja zahtevaju precizne procene prostornog rasprostranjenja vrsta koja se štite. uz pomoć modelovanja prostornog rasprostranjenja vrsta, dobijaju se mape pogodnog staništa za istraživane vrste. u ovom radu korišćen je programski paket maxent sa ciljem procene rasprostranjenja pogodnih staništa za pet vrsta vodozemaca i gmizavaca (mesotriton alpestris, bombina variegata, testudo hermanni, lacerta viridis i vipera ammodytes) u specijalnom rezervatu prirode „goč-gvozdac“ (centralna srbija), kako bi se procenilo koliko se potencijalnih pogodnih staništa za ove vrste nalazi unutar teritorije ovog rezervata. poređenjem dobijenih mapa pogodnih staništa sa mapom specijalnog rezervata prirode „goč-gvozdac“ zaključili smo da se granice ovog rezervata ne poklapaju sa predloženim rasprostranjenjem pogodnih staništa za vrste m. alpestris, t. hermanni, l. viridis i v. ammodytes, te stoga ovaj rezervat ne doprinosi zaštiti lokalnih populacija ovih vrsta. key words: modelovanje prostornog rasprostranjenja vrsta, maxent, rezervati prirode, gmizavci, vodozemci 7 (1) • september 2016: 41-46 doi: 10.5281/zenodo.159102 biologica nyssana 7 (1)  september 2016: 41-46 čubrić, t.  species distribution modeling techniques as a tool in… 42 introduction the most effective way to preserve species diversity is conservation of viable populations in their natural habitats through establishment of network of protected areas (r o d r i g u e z -r e y et al., 2013). knowing where species are likely to occur is a basic part of natural resource management (r u s t o n et al., 2004). effective conservation actions such as defining new nature reserve require accurate estimates of the spatial distribution of the target species (h e r n a n d e z et al., 2006). with this information in hand conservationists can also predict species’ response in form of change in it’s local distribution due to landscape alteration and environmental change (h e r n a n d e z et al., 2006). species distribution models (sdms) are empirical models that relate field observations to environmental predictor variables based on statistically or theoretically derived response surfaces (g u i s a n & z i m m e r m a n n , 2000). species distribution modeling can provide a measure of a species occupancy potential in areas which are not covered by biological surveys and therefore these techniques are becoming an indispensable tool in conservation planning (g u i s a n & z i m m e r m a n n , 2000; c o r s i , 2000; l o i s e l l e et al., 2003), especially when funding and time are limited. also, predicted species distribution data from sdms are commonly used for conservation planning because the alternatives (e.g. survey data) are often incomplete or spatially biased (a n d e l m a n & w i l l i n g , 2002). one of the sdm software is maxent (p h i l l i p s et al., 2006). maxent is a generalpurpose method for characterizing probability distributions from incomplete information (p h i l l i p s et al., 2006). maxent combines distribution data with environmental factors and assesses the probability of presence of one species in a given cell on the basis of environmental features in that cell. maxent calculates the range of species distribution in order to find the species distribution of maximum entropy – closest to the uniform (p h i l l i p s et al., 2006). distribution based on maximum entropy means that the species are evenly distributed through environmental variables. mean task in this modeling program is to predict suitable habitat in relation to environmental variables. it requires only presence data. in this paper maxent was used in predicting suitable habitats for two amphibian and three reptilian species: mesotriton alpestris (amphibia: urodela: salamandridae), bombina variegata (amphibia: anura: bombinatoridae), testudo hermanni (reptilia: chelonia: testudines: testudinidae), lacerta viridis (reptilia: squamata: sauria: lacertidae) and vipera ammodytes (reptilia: squamata: serpentes: viperidae). nine amphibian and twelve reptilian species inhabits special nature reserve "goč gvozdac". the five chosen species are the most easy to spot while performing transects. in addition, all five species are protected under national law. also, they are listed under the iucn red list (www.iucnredlist.org) and under the annexes of bern convention (www.coe.int). m. alpestris, b. variegata, l. viridis and v. ammodytes are listed in the last concern iucn red list category and t. hermanni is listed in the near threatened category (www.iucnredlist.org). also, except m. alpestris, all the other selected species are considered of community interest in need of strict protection (annex iv) in habitat directive (a n o n y m o u s , 1992); additionally, b. variegata and t. hermanni are considered also a species which conservation requires the designation of special areas of conservation (annex ii in habitat directive). the objectives of this study are: 1) to use habitat suitability models to determine potential current distribution of chosen species throughout the study area 2) to estimate the distribution and extent of species potential habitat in the special nature reserve “goč-gvozdac” and 3) to assess how much of the potential habitats is included in this reserve. the basic question was whether the border of the reserve is set up in a way to contribute to the protection of the most important local habitats of these species. material and methods study area as the aim of this study was to evaluate special nature reserve “goč-gvozdac”, study area was wider than the reserve itself (fig. 1) and included the nature reserve (positioned in the middle of study area), the valleys of the ibar and west morava rivers and slopes of kopaonik and golija mountains. special nature reserve “goč – gvozdac” is located in central serbia, between 43° 31' and 43° 34' north latitude and 20° 37' and 20° 47' east longitude. it represents a forested mountainous area with its highest peak of 1484 m. and covers 3957 ha in total. the field research lasted from april to september 2014. the survey was carried out through 49 transects of the average length of 5 km set in randomly selected locations. the geographic coordinates of the localities where the species were found were collected and they included 10 records of m. alpestris, 22 records of b. variegata, 11 records of t. hermanni, 25 records of l. viridis and 10 records of v. ammodytes. biologica nyssana 7 (1)  september 2016: 41-46 čubrić, t.  species distribution modeling techniques as a tool in… 43 fig. 1. study area (red polygon with dimensions 50x50km). in the middle of the study area is located special nature reserve “goč-gvozdac” table 1. climatic variables used in models bio1 annual mean temperature bio2 mean diurnal range (mean of monthly (max temp min temp)) bio3 isothermality (bio2/bio7) (* 100) bio4 temperature seasonality (standard deviation *100) bio5 max temperature of warmest month bio6 min temperature of coldest month bio7 temperature annual range (bio5bio6) bio8 mean temperature of wettest quarter bio9 mean temperature of driest quarter bio10 mean temperature of warmest quarter bio11 mean temperature of coldest quarter bio12 annual precipitation bio13 precipitation of wettest month bio14 precipitation of driest month bio15 precipitation seasonality (coefficient of variation) bio16 precipitation of wettest quarter bio17 precipitation of driest quarter bio18 precipitation of warmest quarter bio19 precipitation of coldest quarter environmental variables climate variables were collected from worldclim databank while corine map was used for land cover data. worldclim base is a collection of maps having medium, minimum and maximum monthly temperature and precipitation for the entire land area of the planet. the maps are of high resolution (30 arc sec) which corresponds to a resolution of approximately 1 km2. the worldclim databank consists of 19 climatic variables (tab. 1). corine digital map is created for 29 european countries with a scale 1: 100 000 or 25 ha minimum size of the unit. resolution of all variables was set to a resolution of corine land cover maps (about 100x100 m). the variables were prepared using esri arcmap 9.3. species distribution modeling following basic parameters of the model maxent were used: calibration (training) data were generated by random selection of 75% occurrence records and 25% for testing. the chosen maxent default settings were: maximum number of iterations = 500, convergence threshold = 10-5, maximum number of background points = 10000. we used a regularization multiplier = 2. in order to validate the obtained models, the predicted values of habitat suitability assigned to presence and pseudo-absence of the data in the test subset were compared by producing the ‘receiver operating characteristic’ (roc) plots (fielding and bell, 1997) and deriving the relative ‘area under curve’ (auc) value (faraggi and reiser, 2002) through a jack-knife procedure. the main positive feature of this method consists of being a single threshold-independent measure for model performance. maps of suitable habitat (raster maps) were made in maxent. the maps of suitable habitats were then overlapped with the map of the special nature reserve using diva-gis 7.5. maps were compared in order to determine which parts of the reserve are suitable. we analyzed the binary maps with suitable and unsuitable habitats for the species. this allowed the calculation of the suitable habitat area. then, we analyzed the size of suitable habitat area of each species in the reserve and the relation between the area of suitable habitat in the reserve and the total area of suitable habitat in the study area. results model performance the models got relatively good performance. training auc was 0.924 for m. alpestris, 0.887 for b. variegata, 0.870 for t. hermanni, 0.863 for l. viridis and 0.754 for v. ammodytes, comparing binary maps of suitable habitats with the map of special nature reserve “goč-gvozdac” comparing the distribution of suitable habitats with a map of the reserve revealed that the suitable habitats for m. alpestris (fig. 2), t. hermanni (fig. 4), l. viridis (fig. 5) and v. ammodytes (fig. 6) are mostly located outside the borders of the reserve and that biologica nyssana 7 (1)  september 2016: 41-46 čubrić, t.  species distribution modeling techniques as a tool in… 44 very small part of the suitable habitat occurs on the northern border of the reserve. the large part of the reserve (75%) has suitable habitats for b. variegata (fig. 3), and the surface of suitable habitat in the study area is greater than the surface of the reserve. consequently, the largest portion of the surface of a suitable habitat in the reserve in relation to the total area of suitable habitat had b. variegata (tab. 2), and the lowest m. alpestris. discussion one of the most important aspects of amphibian and reptile biology that should be clarified for the efficient planning of conservation measures is represented by the patterns of species distribution, as well as by the environmental factors that influence such patterns (b o m b i , 2010). comparing the distribution of suitable habitat maps of the studied species with a map of the special nature reserve "goč gvozdac" we can conclude that the reserve boundaries do not coincide with the distribution of suitable habitats for m. alpestris, t. hermanni, l. viridis and v. ammodytes. therefore we can conclude that this reserve does not contribute much to the protection of local populations of these species. on the contrary, the area of suitable habitat for b. variegata occupies 75% of the reserve and the proposed reserve boundaries contribute to its protection. for m. alpestris it would be good to expand the northern boundaries of the reserve in a way to protect also potentially important habitats north of the reserve. given the fact that the area of suitable habitat for other analyzed species exceeds the area of the reserve, one of the potential measures to protect their local populations would be the establishment of additional new protected areas. areas for protection are often selected to cover the maximum number of species (c h u r c h et al. 1996; k i e s t e r et al., 1996). various authors (p r e s s e y & n i c h o l l s , 1989) recommend maximizing biodiversity in a protected area in order to be able to protect them with limited financial resources. therefore, it would be more economical to establish one large protected area by expanding the boundaries of the existing one, or to design a connected network of small protected areas. fig. 2. comparison of binary map of suitable habitat with a map of the reserve. the dark polygon represents the territory of the special nature reserve “goč-gvozdac” and hatched part represents the suitable habitat for species mesotriton alpestris. fig. 3. comparison of binary map of suitable habitat with a map of the reserve. the green polygon represents the territory of the special nature reserve “goč-gvozdac” and hatched part represents the suitable habitat for species bombina variegata. fig. 4. comparison of binary map of suitable habitat with a map of the reserve. the green polygon represents the territory of the special nature reserve “goč-gvozdac” and hatched part represents the suitable habitat for species testudo hermanni. biologica nyssana 7 (1)  september 2016: 41-46 čubrić, t.  species distribution modeling techniques as a tool in… 45 protected parts of nature that have been identified on the basis of their range and predictions of species distribution are likely to be more comprehensive and more representative than those that are identified solely on the basis of available presence data of the species. this is because the species presence is limited to the area where field research was conducted while species distribution models predict species distribution in and out of the researched area (r o n d i n i n i at al., 2006). also, simple probability-based approaches may be considered an alternative to more complex population viability analysis when conservation decisions involve large number of species and there is little time and few resources available (a r a ú j o et al. 2002). in this study it is achieved to evaluate the suitability of the total special reserve for selected species in regard to the wider area, although field work has not been conducted in all spatial segments. obviously, this study could be improved. first, vegetation map with better resolution would be more appropriate because finer resolution usually provides better predictions for fixed or very locally mobile organisms (g u i s a n & t h u i l l e r , 2005) such as species in this study. also, researched area was relatively small for resolution of variables. further, identifying small habitat features (such as cave openings, vegetation patches) is also important as v. ammodytes and l. viridis use their environments at a relatively fine spatial scale. new types of remotely sensed data such as laser altimetry or light detection and ranging (lidar) (l e f s k y et al., 2002) should prove useful for generating detailed habitat maps. various authors (r o n d i n i n i at al., 2005) concluded that there are no available maps showing presence of small, temporary water bodies that are important for certain species (e.g. m. alpestris and b. variegata) and that represents an obstacle in sdm. it may also happen that the resolution of variables does not match the scale at which species use environment. also, the relatively small number of samples should be taken into account in interpreting the results of this study, although maxent represents the best of the available programs for modeling with quantity of samples up to 5,10, 15 and 20 (h e r n a n d e z et al., 2006). also, fig. 5. comparison of binary map of suitable habitat with a map of the reserve. the green polygon represents the territory of the special nature reserve “goč-gvozdac” and hatched part represents the suitable habitat for species lacerta viridis. fig. 6. comparison of binary map of suitable habitat with a map of the reserve. the green polygon represents the territory of the special nature reserve “goč-gvozdac” and hatched part represents the suitable habitat for species vipera ammodytes. table 2. area of suitable habitats for analyzed species (snr-special nature reserve) species size of suitable habitat within the study area size of suitable habitat within the snr % of the size of suitable habitat within the snr in relation to the size of suitable habitat within the study area % of the size of suitable habitat within the snr in relation to the size of the reserve (3957 ha) mesotriton alpestris 2916,8171 ha 1,8224 ha 0,062 0,046 bombina variegata 70980,2766 ha 2998,6221 ha 4,22 75,78 testudo hermanni 57018,9638 ha 130,9016 ha 0,23 3,30 lacerta viridis 57559,7044 ha 224,1334 ha 0,39 5,66 vipera ammodytes 120566,0616 ha 1086,9871 ha 0,9 27,46 biologica nyssana 7 (1)  september 2016: 41-46 čubrić, t.  species distribution modeling techniques as a tool in… 46 the model is more accurate for species with a small distribution range and narrow ecological valence (h e r n a n d e z et al., 2006). conclusion because contemporary research in the field of conservation planning has focused on the development of theories and tools to design reserve networks that protect biodiversity in an efficient and representative manner (a r a u j o & w i l l i a m s , 2000; a r a u j o et al., 2002; c a b e z a et al., 2004), sdms (like models in this study) can be used as a foundation for the development of finer scale models, usable in a) definition of suitable habitat, b) identification of boundaries for new conservation areas and c) assessment of existing area to secure the protection of amphibian and reptilian species. acknowledgements. i am grateful to dimitrije radišić for support and help for realizing my ideas and to nikola vuletić for a company in the field. references andelman, s.j., willing, m.r. 2002: alternative configurations of conservation reserves for paraguayan bats: considerations of spatial scale. conservation biology, 16: 1352-1363. anonymous, 1992: council directive on the conservation of natural habitats and of wild fauna and flora 92/43/eec. araujo, m.b., williams, p.h., fuller, r.j. 2002: dynamics of extinction and the selection of nature reserves. proceedings of the royal society of london series b-biological sciences 269: 1971– 1980. araújo, m. b., williams, p. h. 2000: selecting areas for species persistence using occurrence data. biological conservation, 96 (3): 331-345. bombi p. 2010: toward a new instrument for identifying the italian hotspots of biodiversity: a case study of the amphibians and reptiles of sicily. italian journal of zoology, 77: 453-459. church, r. l., stoms, d. m., davis, f. w. 1996: reserve selection as a maximal covering location problem. biological conservation, 76 (2): 105-112. cabeza, m., araújo, m. b., wilson, r. j., thomas, c. d., cowley, m. j., moilanen, a. 2004: combining probabilities of occurrence with spatial reserve design. journal of applied ecology, 41 (2): 252-262. corsi, f. 2000: modelling species distribution with gis. in: boitani, l. and fuller, t. k. (eds), research techniques in animal ecology; controversies and consequences. columbia univ. press, pp. 389-434. faraggi d, reiser b. 2002: estimation of the area under the roc curve. statistics in medicine, 21: 3093–3106. fielding ah, bell jf. 1997: a review of methods for the assessment of prediction errors in conservation presence/absence models. environmental conservation, 24: 38–49. guisan, a., thuiller, w. 2005: predicting species distribution: offering more than simple habitat models. ecology letters, 8 (9): 993-1009. guisan, a., zimmermann, n. e. 2000: predictive habitat distribution models in ecology. ecological modelling, 135: 147-186. hernandez, p. a., graham, c. h., master, l. l., albert, d. l. 2006: the effect of sample size and species characteristics on performance of different species distribution modeling methods. ecography, 29 (5): 773-785. kiester, a. r., scott, j. m., csuti, b., noss, r. f., butterfield, b., sahr, k.,white, d. 1996: conservation prioritization using gap data. conservation biology, 10 (5): 1332-1342. lefsky, m.a., cohen w.b., parker g.g., harding d.j. 2002: lidar remote sensing for ecosystem studies. bioscience, 52: 19-30. loiselle, b. a., howell, c. a., graham, c. h., goerck, j. m., brooks, t., smith, k. g., williams, p. h. 2003: avoiding pitfalls of using species distribution models in conservation planning. conservation biology, 17 (6): 1591-1600. phillips, s.j., anderson, r.p., schapire, r.e. 2006: maximum entropy modelling of species geographic distributions. ecological modelling, 190: 231–259. pressey, r. l., nicholls, a. o. 1989: efficiency in conservation evaluation: scoring versus iterative approaches. biological conservation, 50 (1): 199-218. rodríguez-rey, m., jiménez-valverde, a., acevedo, p. 2013: species distribution models predict range expansion better than chance but not better than a simple dispersal model. ecological modelling, 256: 1-5. ruston, s.p., ormerod s.j., kerby g. 2004: new paradigms for modeling species distributions? journal of applied ecology, 41: 193 –200. rondinini, c., stuart, s., boitani, l. 2005: habitat suitability models and the shortfall in conservation planning for african vertebrates. conservation biology, 19 (5): 1488-1497. rondinini, c., wilson, k. a., boitani, l., grantham, h., possingham, h. p. 2006: tradeoffs of different types of species occurrence data for use in systematic conservation planning. ecology letters, 9 (10): 1136-1145 jakšić et al., 2019, biologica nyssana 10(1) 10 (1) september 2019: 43-48 doi: 10.5281/zenodo.3464006 further notes on zygaenidae (lepidoptera) from montenegro original article predrag jakšić čingrijina 14/25, zvezdara, 11000 beograd, serbia jaksicpredrag@gmail.com gerhard m. tarmann tiroler landesmuseen, ferdinandeum, naturwissenschaftliche abteilung, sammlungsund forschungszentrum, krajnc-straße 1, 6060 hall, austria g.tarmann@tiroler-landesmuseen.at ana nahirnić national museum of natural history, tsar osvoboditel blvd. 1, 1000 sofia, bulgaria ananahirnic@nmnhs.com (corresponding author) received: april 23, 2019 revised: july 17, 2019 accepted: august 13, 2019 abstract: faunistic data on 11 species of zygaenidae from seven pre-selected research areas in montenegro obtained during several trips in the year 2017 are presented. photographs of adults of jordanita notata, zygaena punctum, z. viciae, z. ephialtes and z. filipendulae are shown. interspecific mating attempts by males of z. filipendulae with females of z. ephialtes and z. viciae are discussed. key words: zygaenidae, interspecific mating, montenegro apstract: novi podaci o ziganidama (insecta, zygaenidae) crne gore predstavljeni su faunistički podaci o 11 vrsta zygaenidae iz sedam prethodno odabranih i istraženih područja u crnoj gori, dobijeni tokom više obilazaka terena 2017. godine. prikazane su fotografije adulta vrsta jordanita notate, zygaena punctum, z. viciae, z. ephialtes i z. filipendulae. diskutovani su pokušaji interspecijskog parenja mužjaka z. filipendulae sa ženkama z. ephialtes i z. viciae. ključne reči: zygaenidae, interspecijsko parenje, crna gora introduction as montenegro intends to become a member of the eu, data about regional species distributions are of great importance, specifically as regards annex ii species of the habitats directive. on the other hand, species of the lepidopterous family zygaenidae are excellent indicators of environmental conditions. during the last decades more than 300 papers were published dealing with the moths and butterflies of montenegro (jakšić & nahirnić, 2017). according to nahirnić & tarmann (2014) there are about 30 publications known which contain data about zygaenidae for montenegro. in 2017 & 2018 the first author had the opportunity to participate in the ipa project ‘establishment of natura 2000 network, montenegro’. some of the results are presented in this paper. materials and methods specimens were collected with a butterfly net. research was done in preselected areas (key biodiversity areas kbas) some of which should be proposed as potential natura 2000 sites. the coordinates of the localities where zygaenidae were collected were determined by using garmin e-trex vista gps device (tab. 1). the photos in situ of specimens were taken using nikon camera with af-s micro nikkor lens. after preparation, we determined the specimens on the base of habitus and male genitalia. the preparations were carried out following the well-known standard procedure: maceration by boiling in potash, dissecting and cleaning, clearing in xylolum and mounting in canada balsam. abdomen and genitalia of zygaena purpuralis were mounted in euparal on glass slide or were after cleaning placed in glycerol © 2019 jakšić et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 43 filled microvials. all the material is deposited in the collection of p. jakšić. the taxonomic order follows nieukerken et al. (2011). results zygaenoidea latreille, 1809 zygaenidae latreille, 1809 subfam. procridinae boisduval, 1828 jordanita notata (zeller, 1847) podgorica, cijevna (restaurant “niagara”), 38 m, 9. may 2017, 1♂, jakšić p. leg. et coll. genitalia checked, slide: cg-2871. (fig. 1); ćemovsko polje, 40 m, 2. june, 2017, 2♀, jakšić p. leg et coll. third record for montengro! so far, this species was only mentioned once by jakšić (1990) on durmitor (crno jezero, čeline). it has been recollected on durmitor in 2015 (tepca, tara river, 6. june 2015; leg. p. jakšić, 1♂, genitalia checked, slide: cg 2684). an early flying green “forester moth” (= procridinae). the preferred habitats are semidry and dry grassy places with centaurea-species, the larval host-plants. this species is not common but widespread throughout southern europe and can be found from the sea shore up to lower mountain meadows. adscita geryon (hübner, 1813) durmitor mt., žabljak, meždo, 1376 m, 21 july, 2017, 1♂, jakšić p. leg et col., genitalia checked: slide cg-2912. a widespread species that is most common in higher elevations. the preferred habitats are open mountain grassland, grassy ravines, often on steep, rocky ground and semidry and dry meadows. the larvae feed on helianthemum spp. (cistaceae). adscita mannii (lederer, 1853) bukovica, in the vicinity of tušina, 999 m, 21 july 2017, 1♂, jakšić p. leg. et coll. genitalia checked, slide: cg-2906; bistrica, đalovića klisura gorge, 780 m, 20 july, 2017, 1♀, jakšić p. leg et col. gen44 habitats and localities elevation (m) coordinate latitudeφ (n) longitude λ (e) 4070 bushes with pinus mugo and rhododendron hirsutum (mugo-rhododendretum hirsuti) prutaš, durmitor mt. 2262 43° 07' 47,3'' 19° 00' 04,9'' veliki štuoc, durmitor mt. 1880 43° 11' 28'' 19° 03' 25'' 6210 semi-natural dry grasslands and scrubland facies on calcareous substrates (festuco-brometalia) meždo, žabljak, durmitor mt. 1376 43° 09' 54,8'' 19° 09' 02,0'' 6520 mountain hay meadows tepca village, tara river canyon 880-920 43° 12' 19'' 19° 04' 37'' 8210 calcareous rocky slopes with chasmophytic vegetation todorov do, durmitor mt. 1820 – 1900 43° 07' 40'' 18° 59' 15'' 9110 luzulo-fagetum beech forests (meadow on forest edge) monastery st. nikola, bistrica, đalovića klisura 780 43° 04' 14'' 19° 54' 12'' vrelo bukovice, durmitor mt. 1350 43° 03' 28,8'' 19° 06' 37,4'' bukovica, in the vicinity of tušina village 999 42° 57' 07'' 19° 10' 19,7'' sušičko jezero lake sušica river canyon 1180 43° 11' 46'' 19° 00' 18'' komarnica river, before nevidio canyon 981 42° 59' 17'' 19° 04' 02'' 9280 quercus frainetto woods cijevna river, podgorica (restaurant “niagara”) 38 42° 22' 50'' 19° 16' 28'' 9530 (sub-) mediterranean pine forests with endemic black pines sušičko jezero, sušica river canyon, mala crna gora 1554 43° 11' 45,8'' 19° 00' 18,5'' 6410 molinia meadows on calcareous, peaty or clayeysilt-laden soils (molinion caeruleae) veliko pošćensko jezero, pošćenska jezera lakes 1010 42° 59' 03'' 19° 04' 04'' table 1. list of annex i habitat types according to the “catalogue of habitat types of eu importance of montenegro” (petrović et al., 2012) and collecting localities in montenegro 2017 biologica nyssana ● 10 (1) september 2019: 43-48 jakšić et al. ● further notes on zygaenidae (lepidoptera) from montenegro 45 fig. 1. jordanita notata (zeller, 1847), male, podgorica, cijevna (restaurant “niagara”), 38 m, 9. may 2017, photo p. jakšić biologica nyssana ● 10 (1) september 2019: 43-48 jakšić et al. ● further notes on zygaenidae (lepidoptera) from montenegro italia checked, slide: cg-2913. this adriato-mediterranean species is widespread along the adriatic coast inhabiting various types of biotopes like mediterranean bushland near the sea, semidry and dry meadows, forest clearings and rocky ground with grassy spots between the rocks. in montenegro this species occurs at the coast and in the mountains. the larvae feed on helianthemumand cistus-species (cistaceae). only a few records are known for this species in montenegro but it is expected to be more widespread. subfam. zygaeninae latreille, 1809 zygaena punctum ochsenheimer, 1808 podgorica, cijevna (restaurant “niagara”), 38 m, 9. may 2017, 4♂, 2♀, jakšić p. leg. et coll. (fig. 2). all populations of zygaena punctum in montenegro belong to ssp. dalmatina boisduval, 1834. this population group is distributed from southern croatia and western bosnia and herzegovina southwards as far as greece. one of the main characters of this subspecies is its small size (compared with the nominotypical z. punctum punctum from eastern central europe) and the tendency to confluence of the red pattern on the forewing upperside. the larva of z. punctum feeds on eryngium species, mainly on e. campestre (apiaceae). the preferred habitats are dry, undisturbed meadows, rocky slopes with grassy spots and abandoned cultures with low or medium high vegetation. zygaena purpuralis (brünnich, 1763) bukovica near tušina village, 900 m, 6. june, 2017, 2♀, jakšić p. leg et col.; meždo, žabljak, 1376 m, 21-24. july 2017, 2♂, 1♀, jakšić p. leg. et coll.; veliki štuoc, 1880 m, 22. july 2017, 1♂, 1♀, jakšić p. leg et col.; mala crna gora, right bank of sušica river canyon, 1554 m, 22. july, 2017, 2♂, 5♀, jakšić p. leg et coll., genitalia checked: glycerin and slide anz573♀; sušičko jezero (sušica lake), 1180 m, 22. july 2017, 3♂, 2♀, jakšić p. leg. et coll.; todorov do, 1900 m, 23. july, 2017, 2♂, 2♀, jakšić p. leg et coll., genitalia checked: glycerin and slide anz574♂; prutaš, 2262 m, 22. july 2017, 1♂, 1♀, jakšić p. leg et coll. in montenegro only z. purpuralis lathyri boisduval, 1828 can be found. it inhabits various biotopes in hilly and mountainous areas. zygaena minos ([denis & schiffermüller], 1775), a species which is indistinguishable from z. purpuralis by habitus of imago, occurs in montenegro as well (nahirnić et al., 2013). correct determination of these species must be based on genitalia or larvae (nahirnić & tarmann, 2016). fig. 2. zygaena punctum ochsenheimer, 1808, female, podgorica, cijevna, 38 m, 9. may 2017, photo p. jakšić 46 zygaena carniolica (scopoli, 1763) meždo, žabljak, 1376 m, 21-24. july 2017, 3♂, 2♀, jakšić p. leg. et coll.; tepca village, 1260 m, mt., 22. july 2017, 6♂, 2♀, jakšić p. leg. et coll.; komarnica: nevidio and pošćenska jezera, 1010 m, 24. july 2017, 3♂, 2♀, jakšić p. leg. et coll.; vrelo bukovice, 1350 m, 24. july 2017, 3♂, 1♀, jakšić p. leg. et coll. the populations of z. carniolica of montenegro belong to the nominotypical subspecies z. carniolica carniolica. this population group is distributed in the balkans from the south-eastern alps and the karst areas in slovenia throughout the dinaric arc to northern albania. the larva feeds mainly on onobrychis spp. (fabaceae). the preferred habitats are undisturbed and unfertilized semidry and dry meadows. this species is known as a perfect indicator for air pollution by pesticides. it has disappeared in many agricultural and industrial areas in europe where it was still common half a century ago (tarmann, 2009). zygaena viciae ([denis & schiffermüller], 1775) bukovica, in the vicinity of tušina, 999 m, 21. july 2017, 1♀ in copula with ♂ of z. filipendulae, jakšić p. leg. et coll. (fig. 5); veliki štuoc mt., 1880 m, 22. july 2017, 3♂, jakšić p. leg. et coll. all populations of z. viciae in montenegro belong to ssp. bosniensis reiss, 1922. this subspecies differs from the nominotypical z. viciae viciae from central europe by its much darker appearance and the broad dark margin on the hindwing. zygaena viciae has so far only been found in the mountains of montenegro around durmitor. its preferred habitats are humid meadows. the larval host-plants in europe are fabaceae (preferably vicia and lathyrus species) but nothing is known on the life history of the populations of montenegro up to this date. zygaena ephialtes (linnaeus, 1767) monastery st. nikola, bistrica, đalovića klisura, 780 m, 20. july 2017, 12. ♂, 7♀, jakšić p. leg. et coll.; bukovica, in the vicinity of tušina, 999 m, 21. july 2017, 12♂, 4♀, jakšić p. leg. et coll.; 1554 m, 22. july 2017, 9♂, 2♀, jakšić p. leg. et coll.; komarnica: nevidio, pošćenska jezera, 1010 m, 24. july 2017, 4♂, 3♀, jakšić p. leg. the specimens of zygaena ephialtes collected during this project belong to ssp. istoki silbernagel, 1944. this subspecies, consisting of black ephialtoid morphs only, with a mixture of red and yellow individuals that can be 5or 6-spotted, is distributed throughout large parts of the southern balkans from bosnia and herzegovina and southern serbia to greece and eastwards to the black sea. there is another subspecies of z. ephialtes known from montenegro, viz. z. ephialtes rauchi hofmann, 2003. this population group is restricted to the surroundings of the bay of kotor and the coastal areas of montenegro. it is significantly different from ssp. istoki (see hofmann, 2003). zygaena ephialtes prefers bushy habitats with presence of the larval hostplant securigera varia l. (lassen) (fabaceae) and good nectar resources for the adults. it is often found on clearings, near forest roads and in river valleys with flowering rubus bushes where the adults obtain nectar. zygaena angelicae ochsenheimer, 1808 bistrica, đalovića klisura gorge, 780 m, 20. july, 2017, 1♀, jakšić p. leg et coll., durmitor mt., todorov do, 1900 m, 23. july 2017, 1♀, jakšić p. leg et col. a widespread species on the whole balkans from lowland to elevations above 2000 meters. larvae on fabaceae, mainly on hippocrepis comosa l. this species is represented in montenegro by its subspecies z. angelicae herzegowinensis reiss, 1922 that inhabits the whole mountainous part of the balkans from slovenia to greece and the black sea. zygaena filipendulae (linnaeus, 1758) bukovica, in the vicinity of tušina, 999 m, 21. july 2017, 4♂, 3♀ , jakšić p. leg.; meždo, žabljak, 1376 m, 21-24. july 2017, 6♂, 3♀, jakšić p. leg. et coll.; 1554 m, 22. july 2017, 8♂, 4♀., jakšić p. leg. et coll.; vrelo bukovice, 1350 m, 24. july 2017, 1♂, 1♀, jakšić p. leg. et coll. zygaena filipendulae is common on the whole balkans and occurs here in a number of different subspecies. montenegro is inhabited by z. filipendulae illyrica holik, 1943. this species has a wider ecological tolerance than most other zygaena species and can inhabit various biotopes from wetlands to dry environment, open meadows to forest clearings and roadside habitats. it can be observed in the lowlands but also in the mountains up to more than 2000 meters. the larvae live on various fabaceae, mainly on lotus species. zygaena lonicerae (scheven, 1777) monastery st. nikola, bistrica, đalovića klisura, 780 m, 20. july 2017, 4♂, 1♀, jakšić p. leg. et coll.; mala crna gora, right edge of sušica river canyon, 1554 m, 21. july, 2017, 1♂, jakšić p. leg et coll. zygaena lonicerae is widespread and common in the balkans and in montenegro. its preferred habitats are meadows near forest or forest clearings. the larvae feed on lotus and trifolium (fabaceae). biologica nyssana ● 10 (1) september 2019: 43-48 jakšić et al. ● further notes on zygaenidae (lepidoptera) from montenegro 47 fig. 4. interspecific mating attempt by male of zygaena filipendulae and female of z. ephialtes in the wild. bukovica river, near tušina village, 1006 m, 21. july 2017. photo p. jakšić fig. 3. zygaena filipendulae male x z. viciae female, found in copula on durmitor mt., veliki štuoc, 1930 m, 22. july, 2017. jakšić p. leg. fig. 5. interspecific mating attempt by male of zygaena filipendulae and female of z. ephialtes in the wild. mala crna gora village, right bank of sušica river canyon, 1554 m, 22. july 2017. photo p. jakšić. discussion and conclusion in all seven pre-selected research areas zygaenidae species could be found. this is an important proof that these biotopes are in a good environmental condition. as stated by šašić, nahirnić & tarmann (2016), zygaenidae are extremely stenoecious insects and are therefore good indicator species for environmental changes. they can show us perfectly whether a biotope is in a good‚ natural condition or somehow disturbed or even contaminated. special studies on this topic have been undertaken in northern italy in val venosta (vinschgau) (huemer & tarmann, 2001; tarmann, 2000, 2009, 2016). it has been shown that destructive poisons from apple plantations (some of them commonly used plant protection substances) were transported by wind into the environment over kilometres into earlier habitats and that zygaenidae were amongst the first insects that disappeared from seemingly completely intact habitats. several of such habitats are annex i habitat types according to the habitats directive. more intensive studies on montenegrin zygaebiologica nyssana ● 10 (1) september 2019: 43-48 jakšić et al. ● further notes on zygaenidae (lepidoptera) from montenegro 48 nahirnić, a., tarmann, g.m. 2016: on the early stages of species of the zygaena purpuralis – complex on the balkan peninsula and adjacent regions (zygaenidae, zygaeninae). xv international symposium on zygaenidae, mals/malles, südtirol/alto adige, italy: 30. nahirnić, a., tarmann, g.m., jakšić, p. 2011: a review of faunistical data on zygaenidae (lepidoptera) in the central-western balkan peninsula. in: bulić z. (ed.), international conference, nature protection in xxi century, proceedings of the conference, thematic sessions, abstracts, poster presentations. book no 2: 451–455. nahirnić, a., tarmann, g.m., jakšić, p. 2013: new data on rare zygaenidae from the balkan peninsula. xviii european congress of lepidopterology, blagoevgrad, bulgaria: 58. petrović, d., hadžiablahović, s., vuksanović, s., mačić, v., lakušić, d. 2012: katalog tipova staništa crne gore značajnih za evropsku uniju. podgorica – beograd zagreb. 116 p. šašić, m., nahirnić, a., tarmann, m.g, 2016: zygaenidae (lepidoptera) in the lepidoptera collections of the croatian natural history museum. natura croatica, 25(2): 233-248. tarmann, g. 1999: tagfalter und widderchen in südtiroler wiesen. ein fallbeispiel einer schleichenden umweltkatastrophe. in gepp, j., entomologische forschung in den alpen, öeg-fachgespräch 16. oktober 1999, dornbirn. kurzfassung der vorträge: 15–16. tarmann, g.m. 2009: die vinschger trockenrasen – ein zustandsbericht auf basis der bioindikatoren tagfalter und widderchen (lepidoptera: rhopalocera, zygaenidae). wissenschaftliches jahrbuch der tiroler landesmuseen, 2: 306–350. tarmann, g.m. 2016: the decline of zygaenidae in the valleys of the alps during the last 100 years, p. 39. in: tarmann, g. m., tremewan, w. g. & spalding, a. (eds), abstracts of the xv international symposium on zygaenidae, mals, 11–18 september 2016: i–xx, 1–47. tremewan, w.g. 2006: ecology, phenotypes and the mendelian genetics of burnet moths (zygaena fabricius, 1775). gem publishing company, wallingford. 190 p. nidae and especially their ecology and biology are needed as there is little known about their exact distribution in the country, local larval food plants, their nectar resources, preferred habitat requirements, parasites and predators. during this study three interspecific copulae could be observed. this is a well-known phenomenon in zygaenidae and has been studied by various authors. the reason is a somehow unspecific pheromone system that leads to the attraction of males of various species by females. however, hybrids can only be produced between extremely closely related species. such hybrids are known only for a few species. they can not only be produced by artificial rearing experiments but are also found in nature. the species combinations found in copula in montenegro in 2017 (figs. 3-5) cannot produce fertilized eggs as they are not very closely related species. in his monographic study, tremewan (2006) provided data for 200 literature sources that provide examples of this phenomenon. in these data 73 different combinations of species are listed. references hofmann, a. 2003: zygaena (zygaena) ephialtes (linnaeus, 1767) in südlichen balkan peninsula nebst anmerkungen zur entstehung von polymorphismus sowie melanistischer zygaena-formen im mittelmeerraum (lepidoptera: zygaenidae). entomologische zeitschrift mit insekten-börse, 2: 50-54; 3: 75-86; 4: 108-120. huemer, p., tarmann, g. 2001: artenvielfalt und bewirtschaftungsintensität: problemanalyse am beispiel der schmetterlinge auf wiesen und weiden südtirols. gredleriana, 1: 331–418. jakšić, p. 1990: zygaenidae (insecta, lepidoptera). in: nonveiller, g. (ed.), fauna durmitora. crnogorska akademija nauka i umjetnosti, titograd 3: 203– 232. jakšić, p., nahirnić, a. 2017: istorijat lepidopteroloških (insecta, lepidoptera) istraživanja u crnoj gori sa bibliografijom. tokovi, 37–81. nahirnić, a., tarmann, g.m. 2014: zygaena brizae (esper, 1800), a new species for montenegro (lepidoptera: zygaenidae). acta entomologica serbica, 19(1/2), 79-82. biologica nyssana ● 10 (1) september 2019: 43-48 jakšić et al. ● further notes on zygaenidae (lepidoptera) from montenegro matijević, m., popović, i., stepić, m., nešić, m., radoičić, m., stanković, m., šaponjić, z., petković, m.: inorganic nanoparticles in biology: drug carriers and auxiliary tools in bioimaging and bioanalytics. biologica nyssana, 9 (1). september, 2018: 1-19. biologica nyssana 9 (1) ⚫ september 2018: 1-19 matijević et al. ⚫ inorganic nanoparticles in biology: drug carriers… 1 review article received: 13 april 2018 revised: 27 june 2018 accepted: 22 august 2018 inorganic nanoparticles in biology: drug carriers and auxiliary tools in bioimaging and bioanalytics milica matijević1, iva popović1, milutin stepić1, maja nešić1, marija radoičić1, maja stanković2, zoran šaponjić1, marijana petković1 1“vinča” institute of nuclear sciences, university of belgrade, belgrade, serbia; 2faculty of natural sciences, university of niš, niš, serbia * e-mail: milica.m@vin.bg.ac.rs abstract: matijević, m., popović, i., stepić, m., nešić, m., radoičić, m., stanković, m., šaponjić, z., petković, m.: inorganic nanoparticles in biology: drug carriers and auxiliary tools in bioimaging and bioanalytics. biologica nyssana, 9 (1). september, 2018: 1-19. among various nano-scaled materials composed from a spectrum of chemical compounds, inorganic nanoparticles are very attractive due to their physico-chemical properties, as well as their availability, simplicity, possibility of modifications, stability and biocompatibility. they are, on the one hand, an useful tool in advanced analytical chemistry, in particular for studying of biologically-relevant processes, but also important as functional parts of the systems designed for controlled and targeted delivery of medicaments for treatment of a variety of diseases and for imaging. so far, thousands of compounds and systems have been developed for the above-mentioned purposes, but there are only a few reviews dealing with these topics. the aim of this review is, thus, to summarize recent applications of nano-structured inorganic materials in the field of drug delivery, bioimaging and bioanalytics, and to give a prospective from the standpoint of biology-related applications. key words: inorganic nanoparticles, photodynamic therapy, imaging apstrakt: matijević, m., popović, i., stepić, m., nešić, m., radoičić, m., stanković, m., šaponjić, z., petković, m.:: neorganske nanočestice u biologiji: nosači lekova i pomoćni alat u bioimidžingu i bioanalitici. biologica nyssana, 9 (1). septembar, 2018: 1-19. među različitim nanomaterijalima, neorganske nanočestice privlače posebnu pažnju zbog svojih fizičkohemijskih osobina, kao i dostupnosti, jednostavnosti, mogućnosti modifikacije, stabilnosti i biokompatibilnosti. neorganske nanočestice su korisna pomoćna sredstva u analitičkoj hemiji, posebno za ispitivanje biološki-relevantnih procesa, ali su i značajan funkcionalni deo sistema za kontrolisanu i ciljanu dostavu medikamenata za terapiju raznih oboljenja, kao i za medicinsku imidžing dijagnostiku. do sada je razvijen veliki broj jedinjenja i sistema za gore-navedenu svrhu, ali se može naći samo nekoliko preglednih članaka na ovu temu. cilj ovog revijalnog rada je da dâ zbirni pregled primene nano-strukturnih neorganskih 9 (1) • september 2018: 1-19 doi: 10.5281/zenodo.1470841 2 materijala u oblasti dostave medikamenata, bioimidžinga i bioanalitike, kao i da pruži buduće smernice sa stanovišta primene u oblasti biologije. ključne reči: neorganske nanočestice, fotodinamička terapija, imidžing introduction nanomaterials are attracting more and more attention for use in biological systems. the number of their potential applications in biology causes an expansion of new scientific discipline, so called nanobiology. this term refers not only to the application of nanomaterials for investigations of important biological processes in health and in pathological states but also to the utilization of various nanomaterials as auxiliary tools in different analytical methods for investigations of biological systems and their components. in this work, we are focusing on the application of inorganic nanoparticles for drug delivery, imaging and analytical chemistry, to cover the units which are interconnected and can be merged in practical work. apart from those, there are also other numerous applications of nanoparticles, which are important from the standpoint of advances in technology, medicine and science in general. for instance, nanoparticles are widely used in diagnostics (imaging diagnostics, laboratory diagnostics, genetic diseases` and tumor diagnostics), radiosensitization, tissue engineering, drug delivery, therapeutic drugs, to the specific medical and other instrumentations such are nanoprobes, nanosensors and nanorobots (su h. et all., 2017). other application areas that are not covered in this review, but should be mentioned are the areas of the molecular biology as a bio-labels (kumar & sophia, 2018), surface chemistry, protein corona and intracellular nanoparticles pathways. the literature data suggest that term “nano” in biology mostly refers to the particles smaller than 5 fig. 1. trajectory of peptide-conjugated gold nps (gnps) through the cell. a, schematic of a functionalized gnp used in the study b. trajectory of peptide-conjugated gnps through the cell. c-f, path of the nps was captured using tem images and is as follows: c, gnp-peptide complex bound to the plasma membrane for entry into the cell via the endocytosis process, d, internalized nps were localized in vesicles, such as, endosomes and lysosomes, e, escaping of nps from vesicles into the cytoplasm, f, entering the nucleus through nuclear pore complex (npc) (scale bars = 100nm). reproduced with permission from yang et al. (2014). biologica nyssana 9 (1) ⚫ september 2018: 1-19 matijević et al. ⚫ inorganic nanoparticles in biology: drug carriers… 3 µm (singh & lillard, 2009), which can be, with minimum risk, administered into the blood circulation. comparing with the smallest human cells, i.e. erythrocytes, the dimension of a typical nanoparticle is about 5 times smaller, whereas in comparison to oocytes, the largest nanoparticle could be more than 120 times smaller. that indicates that nanoparticles (nps), speaking in physical terms, can be ingested by a cell, reach cellular interior and become available for the interaction with intracellular organelles or to target specific biomolecules. nps can be ingested by a cell through utilizing the existing cellular pathways for ingestion, and/or diffusion through the membrane in the case of smaller nps. another way, i.e. endocytosis, or a receptor-mediated pathway is illustrated in fig. 1, on the example of a peptide-bound to the np (yang et al., 2014). based on that, nps can be designed for intracellular imaging, to target particular biomolecule or an organelle and to interfere with the intracellular processes/signaling, either for the diagnostic or the therapeutic processes. in terms of a therapy, nps as carriers for targeted and controlled drug delivery have attracted much attention. they have functions to preserve a drug until it reaches target tissue/organ and to secure targeted delivery and control a drug concentration (pandey, 2017). after activation by various mechanisms, the drug is released and can act on desired site, thus minimizing side effects on healthy tissues. although numerous medicaments have been designed to use intrinsic properties of diseased tissues, such as low intrinsic ph, due to a low oxygen concentration in tumor cells (casciari et al., 1992), nanoparticle-based carriers are still attractive and promising approach for targeted therapy. in analytical (bio)chemistry, nps are used as auxiliary tools in various techniques. thanks to their properties, they enable better precision of the method (frigerio et al., 2012; will et al., 2006; lucena et al., 2011), and its increased selectivity and sensitivity (ling et al., 1991; arakawa & kawasaki, 2010; thompson et al., 2008). a waste amount of biologically important molecules are qualitatively and quantitatively analyzed with the assistance of nps (kawasaki et al., 2007; chiang et al., 2010a; popović et al., 2016; popović et al., 2016a; popović et al., 2016b). some examples of methods which exploit the advantageous properties of nps are mass spectrometry, chromatography, but also mass spectrometry imaging, and their application will be discussed in more details in this review. physical and chemical properties of nanoparticles nps can be fabricated from various materials, and coarse classification involves organic and inorganic, and commonly used types are shown at the fig. 2. based on their size and composition, there are several groups of nano-structured materials: nps (1-100 nm), nanocapsules (nanoscale shells made of a polymer), fig. 2. types of nps commonly used for biomedical applications: (a) polymer, (b) liposomes, (c) amphiphilic cyclodextrines, (d) dendrimers, (e) gold nps, (f) micelles, (g) carbon nanotubes and (h) quantum dots (qds), modified form (mccarthy et al., 2014). 4 fullerenes (made of a carbon and with various shapes), dendrimers (repetitively branched molecule), quantum dots (qds), (a semiconductor nanostructures), nanostructures (size between intermediate and microscopic particles) and nanopores (with tiny hole in a thin membrane) (akeson et al., 1999). in line with the topic of this review, the following part will summarize some biologically-relevant properties of organic vs. inorganic nps. each of them, organic and inorganic, has its own advantages and disadvantages. organic materials are made mostly from biodegradable materials, and they can be metabolized in the organism, finally resulting with co2 and water. as disadvantage, there is always a risk of premature degradation, which leaves the medicament exposed to enzymes and immunological system, causing too early activation of a medicament, which leads to the expressed side effects. this disadvantage could be avoided, or minimized, through the encapsulation of a medicament in the nps (dobrovolskaia & mcneil, 2007; singh & lillard, 2009). monitoring of the destiny/biodistribution of organic nps in organism is sometimes difficult, due to limited possibilities to distinguish them in the surrounding organic milieu. the labeling techniques, for instance by some voluminous fluorescence dyes, might prevent the interaction with target molecules/tissue leading to the misinterpretation of the data. the application of inorganic materials for fabrication of nps gives much more possibilities for surface functionalization, and much broader spectrum of methods which can be used for the monitoring of nps` biodistribution. modification of nps allows controlled changes of the interface properties, which affects the dissolution and degradation rates, catalytic activity and possibility of stabilization through the adsorption of macromolecules (stark, 2011). on the other hand, precaution is needed because inorganic material must be selected by making a compromise between biological inertness, availability and potential of surface modification. for biological applications, selection of inorganic materials for fabrication of nps should be based on the information of their potential biological role. for instance, selected material should not contain a metal which might be a co-factor for any enzyme in the cells. in other case, the final concentration of metal should be under the enzyme-activating level (chen et al., 2012; lee & yeo, 2015). the presence of higher concentrations of the enzyme cofactors, might lead to the enzyme activation and the interference with the intracellular signaling processes. inorganic materials made of non-biological metals enable also easier detection by instrumental methods, which utilize either properties of their atoms (magnetic or paramagnetic properties, for instance) or intrinsic fluorescence, i.e. optical properties. for example, titanium is not biologically abundant, and it can be detected with methods which differentiate inorganic from organic materials, i.e. nmr (padro et al., 2000), or other approaches. some of most frequently used nps in medicine are those made of gold, which could be visualized by microscopy, or fluorescence labeling, or other method (gonciar, 2014). having in mind that the inorganic nps are the focus of this review, the summarized list of the different types of inorganic nps that are relevant for this review, is shown in the tab. 1. metal oxide semiconductor-based nanoparticles a number of metals can form oxides that can conduct an electrical current, and nps made of them are used as drug carriers, for imaging purposes and in analytical chemistry. the most commonly used are zno, cuo, tio2, zro2 or mgo. besides electroconductivity, properties which are advantageous for the variety of applications are photo-responsivity, magnetism, possibility of surface modification, etc. metal nps have large surface-to-volume ratio and due to their high density and limited size of corner or edge on the surface site, metal oxide nps exhibit unique chemical and physical properties. for instance, for the application in anticancer drug delivery systems, the size of metal nps should be larger than 10 nm in order not to be cleared by the kidney excretion and captured by macrophages (lee & yeo, 2008), and on the other hand, to be smaller than 100 nm to reach the tumor tissue and enter the cell (cho et al., 2008). even within the range of 10100 nm, small alteration in size of nps makes a big difference regarding their properties, i.e., as the size decreases, the number of surface and interface atoms generates strain or stress and concomitant structural perturbations (gleiter, 1995; trudeau & ying, 1996; valden et al., 1998; rodriguez et al., 2002; song et al., 2003; schoiswohl et al., 2004). in terms of the nps uptake by mammalian cells, oxides of zinc, iron, manganese, and cobalt exhibit nonclassical interaction – the trojan-horse-type, which enables diffusion of the toxins by packing it in the small particles (stark, 2011). surface of inorganic nps can be easily modified, and thanks to that property, they are often considered for drug discovery and therapy development. they can be modified for targeted drug biologica nyssana 9 (1) ⚫ september 2018: 1-19 matijević et al. ⚫ inorganic nanoparticles in biology: drug carriers… 5 delivery for various significant biomolecules (nucleic acids, proteins or peptides) or for specific tissues. such targeted nanocarriers decrease overal drug toxicity and offer more effective biodistribution, as discussed above. the conjugate of a drug and nanocarrier can pass some natural body barriers, such as blood or brain barriers (rawat et al., 2006) and deliver drug to the target tissue. there are several external activators, which employ inner properties of inorganic nanoparticle as a carrier, eventually leading to cytotoxic effects at the target tissue, which will be discussed more in the next chapter. stimulus or activators can be high magnetic field or ultrasound applied locally, which increase the temperature of diseased tissue, due to the accumulated nps in the tissue (arruebo et al., 2007; singh & lillard, 2009; lee and yeo, 2015). light as activator attracted much attention in later decades, because it can be easily table 1. overview of different types of inorganic nps from cited literature in this review system structure characteristics references aunps semiconductor nps: cdse or cdte doped oxide materials, y2o3 fe2o3, conps colloidal effective phototermal destruction of cancer cells and tissues photoluminiscence in the form of fluorescence phosphorescence magnetic moment sperling & parak, 2010 feridex colloid with low molecular weight dextran coating, with a particle size of 120180 nm magnetic moment diagnostic agent in use cherukuri et al., 2010 cobalt ferrite, cofe2o4 magnetic moment binding to serum albumin proteins amiri et al., 2017 fe3o4 nps fe3o4-peg nps naked, 80±5 nm functionalization with polyethylene glycol 600 diacid, 46±0,6 nm magnetic moment higher accumulation in the lungs due to their in vivo agglomeration magnetic moment potential in hyperthermia based cancer treatmens radović et al., 2015 platinum-tethered gold nps-peg au nps functionalized with thiolated poly(ethylene glycol) (peg monolayer capped with a carboxylate group of [pt(r,r-dach)] nps functionalized with peg do not make aggregate higher uptake than pt(r,r-dach) nps are capable of delivering [pt(r,r-dach)] to the cell and then releasing it brown et al., 2010 scg8-auag nanoroads gold-silver-(auag-) nanoroads labeled with molecular aptamers higher hyperthermia efficiency and selectivity to cem cells than aptamer alone conde et al., 2012 carbon nanotubes carbon cylinders composed of benzene ring linked to the methotrexate attached to medicament-methotrexate water-soluble and biocompatible through chemical modification pastorin et al., 2006 s1mps, s2mps mesoporous sio2 nps, s1mps which large pores are loaded with s2mps high biocompatibility high surface areas and pore volumes controlled release dynamic of medicament tasciotti et al., 2008 tio2 nps colloidal tio2 nps attached to potentional medicament – ru bis-bipyrydil complex minimal dark cytotoxicity high surface to volumes relation the surface of tio2 can be modified controlled release dynamic of medicament nešić et al., 2016, 2017 6 controlled and focused to minimize influence on the healthy tissue. light can be used in a combination of photo-responsive drugs or nanoparticle/drug system, which is then called a photosensitizer. among inorganic nps, we have selected for our investigations, those made of tio2, due to their availability, stability, non-toxicity, potential to modify particles surface, but also photo-responsivity, and its properties will be discussed in the next paragraph, with the focus on the photodynamic therapy. the inorganic nps, which are photoresponsive, can serve as a source of photogenerated charges that interact with the electronic properties of the biomolecules. the linking of inorganic nps with (bio)molecules facilitate hole transfer across the interface, establishing efficient crosstalk between the (bio)molecule and metal oxide nps. these photoactive bioinorganic conjugates have properties that make them good for light induced manipulation of biomolecules and their switching functions (rajh et al., 2004). tio2, which will be presented also later in this review, is typical n-type semiconductor material, with many good properties, such as costeffectiveness, chemical and photo-stability, and excellent biocompatibility in the dark. besides photo catalysis, energy storage system development and energy conversion, sun screening and sensor research, tio2 has a wide application in medical fields, too. it can be applied as anti-cancer agent, implant and a substrate for stem cell expansion (hamidi et al., 2017; oliveira et al., 2017; sims et al., 2017). quantum dots qds are semiconductors nanocrystals with size between 1-10 nm, which have unique and tunable absorption and emission properties, depending on their size and shape. for instance, they have large transition dipole moment, so can be precisely tuned from the uv to the infrared region (lucky et al., 2015). some of them exhibit photoluminescence or fluorescence (semiconductor quantum dots, e.g., cdse, cdte, cdtese/zns) (gozuacik et al., 2014). structurally, qds consist of a metalloid crystalline core and a “cap” or “shell” that shields the core, and further assignation of coatings or functional groups to the qd core–shell can give qds a desired bioactivity. qds` core consists of the metal complex which defines the structure group that particular qd presents. for example, the group iii–v series qds are composed of indium phosphate (inp), indium arsenate (inas), gallium arsenate (gaas) and gallium nitride (gan) metalloid cores, and group ii–iv series qds, of zinc sulfide (zns), zinc–selenium (znse), cadmium–selenium (cdse), cadmium–tellurium (cdte) cores and heavier structures such are cdte/cdse, cdse/znte) and hybrids composed of lead–selenium (pbse). regarding qd toxicity, it depends on multiple factors: qd size, charge, concentration, outer coating bioactivity (capping material, functional groups), and oxidative, photolytic, and mechanical stability have each been shown to be determining factors in qd toxicity (hardman, 2006). in biological systems, qds have been investigated for the application mostly in cancer treatment, theranostics, cancer drug delivery, photothermal and photodynamic therapy. the ability of fluorescence emission of qds, make some of them good imaging agents. furthermore, photoluminescent properties of qds have been exploited in the other fields of application, such as in analytical chemistry in environmental monitoring, pharmaceutical and clinical analysis and food quality control (frigerio et al., 2012). magnetic nanoparticles the advances in the synthesis of biocompatible magnetic nps in a reproducible way, allowed extensive research and further development of drug delivery systems based on magnetic field as an external driving and control force. applications of magnetic nps in biomedical areas, on the other hand, require the use of magnetic colloids, which consist of a suspension of magnetic particles of nano sizes in a carrier liquid like water, with usual particle concentrations in the range of 1021-1023 particles/m3 (goya et al., 2008). magnetic iron oxide nps are the most widely used nps with magnetic properties, and there are various trade names for super oxide and ultra-small super oxide variations in structure (shadab et al., 2015). usually, inorganic nanoparticle core is coated by a suitable coating material, which increases the stability and solubility of the nano-drug conjugate, leading to higher rates of biocompatibility and aqueous stability in the saline environment of biological tissues. feridex is an example of this commercially available iron nps, which are coated with sugars (e.g. dextran) (cherukuri et al., 2010). the enrichment of magnetic np surface, for instance with sio2, can allow direct functionalization making it more suitable for biomedical application (radović et al., 2015). the other group of compounds with magnetic properties, facile synthesis and chemical stability, are ferrites of the general formula mfe2o4 where m = fe, mn and co. some ferrites exhibit additional biologica nyssana 9 (1) ⚫ september 2018: 1-19 matijević et al. ⚫ inorganic nanoparticles in biology: drug carriers… 7 properties making them even more suitable for application in the biological systems, for instance, cobalt ferrite, cofe2o4 binds strongly to serum albumin proteins (amiri et al., 2017). inorganic nanoparticles controlled drug delivery systems: applications for anticancer therapy in recent years, a combined nanotechnology – drug delivery systems have been developed by two components within the general formulation, nanocarrier/drug complex. the purpose of the nanodrug conjugation is to allow modification of the pharmacokinetics and metabolism of the drugs, particularly regarding reducing the side effects, such as non-selective toxicity to tumor cells and inactivity against drug-resistant cell lines (llevot & astruc, 2012). the drug delivery system with nanocarrier has even more advantageous properties, such as better encapsulation, bioavailability, lower toxic effects to the healthy tissues and possibility for controlled release (pandey, 2017). in such manner, some of these features are related to each other. as instance, the encapsulation of the drug in the nanocarrier prevents quick elimination by the reticuloendothelial system, which consequently leads to higher levels of blood circulation and to increased rates of the transport through biological barriers. the final outcomes of these processes are better availability of drug at the targeted tissue and lower toxic effects to the healthy tissues. moreover, in the focus of the researches is to develop the drug delivery system which is capable of maintaining constant concentration gradients by continuous and controlled release of therapeutic drugs. these unique chemical and biological properties of the nanocarriers are linked to the nps large surface area-to-volume ratio, as discussed above. this property allows them to combine, absorb and hold other compounds like drugs molecules, dna, rna, proteins (shadab et al., 2015). additionally, surface modification can enable the increase of the circulation time in the body, cell specific targeting and membrane permeability. by interacting with biomolecules placed on the cells surface and inside the cells, penetration into the tissues is promoted with the higher levels of specificity. furthermore, it is possible to develop the advanced nps drug delivery systems for the spatially and temporally controlled release of drugs in response to specific stimuli within the tumor microenvironment (yin et al., 2013). a stimuli-responsive delivery system improves the efficacy of the drug, and can be designed to react to the disease specific property (ph level, redox property or enzyme levels). drug accumulation and release at the tumor site can be additionally intensified by external forces like magnetic field (arruebo et al., 2007; shadab et al., 2015), light (mari et al., 2014; el-hussein et al., 2015; nešić et al., 2017) and/or heat (cherukuri et al., 2010). to date, there are only a few clinically approved nanocarriers that incorporate molecules to selectively bind and target cancer cells, and these drug delivery systems are mostly based on organic nanocarriers, such as liposomes and polymers, free or attached to proteins (lópez-dávila et al., 2012). inorganic nanocarriers, on the other hand, are much smaller, and the active form is usually simple metal based structure with gold (brown et al., 2010), silver (conde et al., 2012), carbon (pastorin et al., 2006; cho et al., 2008), oxides (mesoporous sio2, tio2, fe2o3, graphene oxide) (goya et al., 2008; tasciotti fig. 3. schematic representation of various stages involved in pdt. reproduced with permission from the authors avirah et al. (2012) 8 et al., 2008; nešić et al., 2016; nešić et al., 2017), or in the form of qds (selenides, sulfides or tellurides of metals like cadmium, lead or zinc) (sperling & parak, 2010; chen et al., 2014). different designs varying in size, shape, and porosity allow easy conjugation with drugs for cancer therapy (bhattacharyya et al., 2011) and they are more stable over large ranges of ph and temperature (shadab et al., 2015). photo-sensitive systems for drug delivery optical energy can trigger a variety of photochemical processes useful for therapies. it is the raising field in the development of new therapeutic approaches in the last decades. photodynamic therapy (pdt) involves the administration of a tumor-localizing photosensitizer (ps), usually np, which undergoes reversible changes upon light exposure of a specific wavelength. the induced changes in such manner, can lead to the release of therapeutic drugs, thereby providing spatio-temporal control of drug release (shadab et al., 2015), or the excited ps can transfer its energy to molecular oxygen, thus generating cytotoxic reactive oxygen species (ros) and singlet oxygen, which can modify cellular macromolecules leading to tumor cell ablation (lucky et al., 2015). simplified schematic representation of the stages involved in pdt, is shown in fig. 3. inorganic, metallic, or composite nps have been investigated as multifunctional carriers for pdt due to their unique characteristics such as optical properties and tunability in its shape, size, porosity, and that they may not degrade readily in the biological systems. it is also reported that the degree of drug resistance is far less than those encountered in chemotherapy, and that there is no cross-resistance between pdt and chemotherapy (kim et al., 2015) the most promising pss are nanosized oxides, which exhibit distinctive optical absorption properties, transparency of the matrix to light absorption and chemical inertness. they can be easily attached to therapeutic medicament in the form of nanocomposite system, which can exhibit greater cytotoxicity (nešić et al., 2017) and higher therapeutic effectiveness for cancer than the corresponding free drugs (chen et al., 2011; wang, y. et al., 2015). the most investigated nps/metal oxides are mesoporous silica (teng et al., 2013), titanium dioxide (nešić et al., 2016) and zinc oxide (zhang et al., 2014). tio2 nps are specially interesting as, upon ultraviolet (uv) light excitation, they absorb the energy higher than its own band gap energy (3.2 ev), which leads to excitation of the electrons from the valence to the conduction band, creating the electron/hole pairs and further generating active free radicals, thus allowing oxidation/reduction of species in surrounded medium. tio2 itself was reported to have a good anticancer effect in response to the light, owing to the production of active free radicals under uv irradiation (wang, t. et al., 2015). in addition, the incomplete coordination sphere of the surface metal atoms exhibits the high affinity of tio2 nps for linking with biomolecules and oxygen-containing ligands (rajh et al., 2004). while tio2 nps with the anatase crystalline phase absorb uv light and consequently generate ros, great chemical stability and minimal cytotoxicity of tio2 nps are detected in the dark. in comparison with traditional organic photosensitizers, tio2 nps can be maintained for a longer time in the body. even tio2 nps possess high photosensitization activity toward various cancer cell lines, for the suppression of in vivo tumors there are characteristics that need to be improved, as low penetration into tissue and the damage to normal tissue (hou et al., 2015). in the study of nanocomposite system based on tio2 nps and daunorubicin, mtt assay have confirmed significant cytotoxicity when leukemia cell line, k562, was treated with the nanocomposite system, while low cytotoxicity was shown when k562 cells were treated solely with tio2 nps, and thus tio2 nps demonstrated important properties of a drug carrier and showed good potential for the applications in cancer therapy (zhang et al., 2012). in a similar study, it was demonstrated that the nanocomposite system made of pt(nh3)4cl2 transitional metal complex and tio2 nps, as well as individual components, lead to a significant decrease in c6 glioma cell proliferation; the reduction in the tumor volume was also confirmed (lópez et al., 2008). in our previous work we studied in vitro light stimuli-responsive properties of nanocomposite system based on the colloidal tio2 nps and transitional metal complex, cis-dichlorobis(2,2′bipyridyl-4,4′-dicarboxylic acid)ruthenium(ii). the system had the light controlled release properties, as the release of the complex was facilitated upon illumination with uv light, and sustained upon illumination with visible light. results obtained by the investigation of the nanocomposite system cellular cytotoxicity towards melanoma cancer cells line a375 were in agreement with in vitro drug release test: while the nanocomposite system and its components exhibited the significant decrease of the cell viability upon uv irradiation, red light irradiation showed an extremely low anti-cancer effect (nešić et al., 2017). biologica nyssana 9 (1) ⚫ september 2018: 1-19 matijević et al. ⚫ inorganic nanoparticles in biology: drug carriers… 9 inorganic nanoparticles and tissue imaging inorganic nps including semiconductor qds, iron oxide nps, and gold nps have been developed as contrast agents for diagnostics by molecular imaging (huang et al., 2011; yu & zheng, 2015). compared to traditional contrast agents, nps offer several advantages: their optical and magnetic properties can be tailored by engineering the composition, structure, size, and shape; their surfaces can be modified with ligands to target specific biomarkers of disease, as mentioned in the introduction section. the contrast enhancement provided can be equivalent to millions of molecular counterparts; and they can be integrated with a combination of different functions for multimodal imaging (cho et al., 2010). broadly, the field of biomedical imaging can be divided into categories based upon the electromagnetic spectrum as shown in fig. 4. molecular imaging is a new frontier of biomedical research for visualizing, characterizing, and monitoring biological processes in cells, tissues, and organisms using sensitive instrumentation and contrast mechanisms. molecular imaging differs from traditional imaging in that way in which the contrast agents are typically utilized to help identify particular biomarkers or pathways with high sensitivity and selectivity (cho et al., 2010), and for that purposes, inorganic nps is an actively explored technology for the development of contrast agents. this imaging function based on interrogation of biological processes to report on and reveal the molecular abnormalities that might point to a disease, being thus a powerful tool for the diagnosis of cancer, cardiovascular syndrome, or neurological disorders. imaging based on ionizing radiation generally refers to the detection of high frequency emissions from radioactive elements such as the gamma ray emitters 111in or 99mtc (metastable nuclear isomer of technetium-99) or the passage of x-rays through the body. the main technologies involved are positron emission tomography (pet), single-photon emission computed tomography (spect), and x-ray computed tomography (ct). magnetic resonance imaging (mri) tends to operate on the other end of the spectrum in the mhz frequency range, relying upon contrast agents such as gadolinium or superparamagnetic iron oxide to modify the relaxivity of water molecules to provide soft tissue contrast (pansare et al., 2012). compared with other imaging approaches, optical imaging is a versatile, economic and efficient modality for bio-imaging with high sensitivity, great choice for probe selection and it does not require precaution, like in the case of application of x-rays or other ionizing radiation. various fluorescent molecules or nanomaterials, such as conventional organic fluorescein, semiconductor qds, rare-earth co-doped up-conversion nanophosphors, etc., have been explored to label and image cancer cells both in vitro and in vivo. integrating mesoporous silica with fluorescent materials towards multifunctionalization could endow them with simultaneous optical bioimaging and drug transport capabilities (chen et al., 2013). there are numerous efforts to combine the unique advantages which nanoprobes have in cancer imaging, and to create a “universal” nanoprobe. such nanoprobe should be multimodal, have a strong epr (electron paramagnetic resonance) effect, able to circulate in a body for a long time (bouccara et al., 2015), be stable in physiological conditions, specific for target tissue, and not to interfere with physiological processes. most inorganic nps can meet some criteria, but one of the most challenging is the avoidance of renal clearance, which enables the prolonged blood circulation, and afterwards, accumulation in target, or tumor tissues. some examples of inorganic nps for various molecular imaging purposes are given below in the text, classified according to their purpose, i.e. imaging technique (yu & zheng, 2015). fig. 4. types of medical imaging: ionizing radiation (x-ray and ct), magnetic resonance imaging (mri), ultrasound and optical imaging 10 major inorganic nanoparticles for molecular imaging contrast agents for various imaging purposes should be easy to design but it is required for them to have particular electrical, magnetic and optical properties. their properties can be fine-tuned by tailoring nps` composition, geometry and structure. therefore, particles can be composed of metals, metal oxides, semiconductors, they can vary in size and shape, and be solid, or hollow. some of nps used in imaging techniques are iron oxide nps, qds, gold nps, and others, which can be used in different imaging approaches (cho et al., 2010; huang et al., 2011; chen et al., 2013; bouccara et al., 2015; yoon et al., 2017), and most inorganic nps possess great potential for noninvasive and real-time in vivo diagnosis of disease (chen et al., 2013). in terms of magnetic resonance imaging (mri), both, magnetic or superparamagnetic nps can be used as contrast agents. among them, iron/based nps are routinely applied because they were approved by fda for human use (yoon et al., 2017). however, a caution is needed regarding the size of nps for mri. in general, reducing a size of ferroor ferrimagnetic particles below critical, could lead to randomization of the magnetic dipoles in a short period of time, as a thermal energy becomes comparable to what is needed for spins to flip. those nps are superparamagnetic, because they do not have permanent magnetic moments in the absence of an external field, but they can quickly respond to it when applied (cho et al., 2010). qds are currently replacing fluorescence dyes, because they have high absorption coefficients and broad emission range in visible and near infrared region (cho et al., 2010). gold nps are also utilized in optical imaging, thanks to their optical properties, i.e. presence of strong extinction peaks in visible and near ir region (pansare et al., 2012). those extinction peaks are caused by the collective oscillations of conduction electrons in the presence of an incident light. this phenomenon is known as localized surface plasmon resonance. in addition, gold nps can be used as contrast agents for ct imaging, due to their absorption coefficient for the xrays, but also, they are capable of photoluminescence. finally, gold nps can enhance the intensity of raman-active molecules and be used for the surface-enhanced raman spectroscopy (cho et al., 2010; huang et al., 2011). a particular class of nps are rare-earth doped nps, which exhibit sharp emission peaks, long fluorescence lifetimes, high quantum yields, and excellent photostability (cho et al., 2010; bouccara et al., 2015). applications of inorganic nanomaterials in analytical (bio)chemistry nps have been used as an auxiliary tool to increase sensitivity and specificity of standard analytical methods. one of earlier applications of inorganic particles, was the use of magnetic particles (beads) in the affinity chromatography, in which the antigen was bound to the surface of a particle, and after specific and selective interaction with a component from a complex solution magnetic nps could be easily collected and separated from the mixture (chen & chen, 2005). also, nps are used as the stationary phase in several chromatographic techniques, where they increase the active surface available for the interaction with analytes. in this chapter, the overview of inorganic nps and their application in chromatography and mass spectrometry will be given and discussed. chromatography the application of nps and nanomaterials in chromatography has been of great interest in recent years due to the unique physical properties of these substances, their large surface area-to-volume ratios, and their ability to be employed with a variety of surface chemistries (guihen, 2013; tang et al., 2014; zhang & qiu, 2015; castillo-garcía et al., 2016). a number of carbon-based nanomaterials and inorganic nanomaterials have been considered for use in lc, some examples are: carbon nanotubes, fullerenes and nanodiamonds (valcárcel et al., 2007; scida et al., 2011; guihen, 2013; pyrzynska, 2013; speltini et al., 2013; zhang et al., 2013), metal nps and nanostructures based on silica, because of their relatively high surface area, availability in various particle sizes and pore diameters, and the ease with which surface can be modified to contain a wide range of functional groups to increase specificity and sensitivity of detection and separation (zhang et al., 2006). these properties have led to the use of silica nps and related materials (e.g., silica nanofibers) in making stationary phases or supports for such methods as reversed-phase chromatography, hilic, and utlc (ge et al., 2006; jim et al., 2011; newsome & olesik, 2014; aydoğan & el rassi, 2016), alumina, zirconia and titanium dioxide (zhang et al., 2006; nesterenko et al., 2013). application of these nanomaterials have included the reversed-phase, normal-phase, ionexchange, and affinity modes of lc, as well as related methods such as chiral separations, ion-pair chromatography and hydrophilic interaction liquid chromatography. biologica nyssana 9 (1) ⚫ september 2018: 1-19 matijević et al. ⚫ inorganic nanoparticles in biology: drug carriers… 11 mass spectrometry small molecules – metabolites (molecular mass less than 1000 da) are significant for understanding of cellular processes and metabolism, and their importance is reflected in the development of relatively new discipline, called metabolomics. the metabolome represents the large scale qualitative and quantitative changes of all metabolites in a biological cell, tissue, organ or organism, which are the end products of cellular processes. this is of high importance for the understanding of biological processes. metabolomics combine strategies for identification and quantification of metabolites with different analytical approaches and statistical methods, and the great efforts are made for their development. metabolites are involved in different cell processes e.g. cell signaling which controls cell division, growth and differentiation. another very important and diverse group of small molecules are drugs. qualitative and quantitative analysis of drugs (especially their biodistribution) is essential for the drug discovery, monitoring and understanding of their mechanism of action and pharmacological and toxicological effects (monro, 1994; mizojiri et al., 1996). method that is routinely applied for detection and analysis of drugs is autoradiography, which for qualitatively and quantitatively investigation of the drugs in the tissues requires radioactive materials, and the procedure is rather time consuming. other methods that are in use for the detection of small molecules are: high-performance liquid chromatography, hplc (araki & sako, 1987; rafii et al., 2007), capillary electrophoresis, се (caussé et al., 2000; bayle et al., 2002), gas chromatography/mass spectrometry, gc/ms (larsson & lindgren, 2005), sers (pietzsch et al., 1997) and fluorescence spectroscopy (ling et al., 1991). these techniques are time consuming, and additional methods for the sample preparation and additional derivatization are needed. some of these methods have additional disadvantages such as low selectivity and low sensitivity for the detection of small molecules in biological samples. matrix assisted laser desorption and ionization time of flight mass spectrometry (maldi tof ms) is powerful technique that is traditionally used for the analysis of large, non-volatile, and termolabile biomolecules and biopolymers (proteins, dna, saccharides). in this method, organic matrices are traditionally in use, like 2,5-dihydroxybenzoic acid (dhb), 9-aminoacridine (9-aa), 2′,4′,6′trihydroxyacetophenone (thap), α-cyano-4hydroxycinnamic acid (chca), sinapic acid (sa). in theory, maldi tof can be used for the semiquantitative mass analysis of all kind of molecules, but in practice that is not a case because of numerous disadvantages which are consequence of the use of organic matrices. firstly, inhomogeneous distribution of samples molecules between matrix molecules (hot spots, cold spots) (tholey et al., 2006; jaskolla et al., 2009; kuzema, 2011) lead to the mass spectra with poor quality, and eventually the reproducibility and the repeatability of a method is low. second drawback refers to the analysis of small molecules; organic matrices have low molecular masses and the high number of signals in the low mass range (usually less than 500 da) which interfere with sample signals. matrices are usually organic acids and they have intensive signals in negative ion mode, so it is very difficult to detect the negative ions of samples (petković et al., 2001a; petković et al., 2001b). another disadvantage is that the organic matrices do not tolerate high concentrations of salts. biological samples have high concentrations of inorganic salts, and because of that the great number of matrix signals are present in spectra and the signals arising from the analytes are with underestimated intensity if not completely suppressed. to overcome listed disadvantages of organic matrices, the inorganic matrices/substrates are much more in use. wei et al. (1999), were synthesized porous nanostructures based on silicon (desorption and ionization on silicon, dios). the term saldi the first was used by sunner et al. (1995), when replaced organic matrices with graphite, and refers to the techniques that use nanostructured substrates instead of organic matrices. different inorganic materials which absorb ultraviolet light were used as substrates for saldi: carbon (chen & chen, 2006; kawasaki et al., 2009; tang et al., 2009; dong et al., 2010), silicon (hu et al., 2007; northen et al., 2007; teruyuki et al., 2007; guénin et al., 2009; walker et al., 2010; cheng et al., 2012), nanomaterials based on metals and metal oxide (chen & chen, 2004; chen & chen, 2005; mclean et al., 2005; okuno et al., 2005; chen & chen, 2006; huang & chang, 2006; wu et al., 2007; kailasa et al., 2008; kawasaki et al., 2008; sherrod et al., 2008; chen et al., 2009; chiang et al., 2010b). substrates based on metals and metal oxides have additional advantages compared to substrates based on silicon and carbon. these substrates are more chemically stable in the air and have high conductivity (chen & chen, 2004; arakawa & kawasaki, 2010), the samples are nps based on au, pt, ag, zno, fe and mno2/mno3. different inorganic nps are in use in analytics of different biological molecules and samples: sherrod et al. (2008) describe selective ionization of olefin compounds (cholesterol and carotenoids) direct from 12 the mixture with silver nps. wu et al. (2007) were analyzed and quantified small, neutral carbohydrates from urine with gold nps. lee at al. (2007) found that тio2 nps with diameter less than 20 nm interact with enediol compounds, which have an impact on increase of absorptivity in uv-vis spectra of light. chiu et al. (2008) analyzed urine samples for the detection of estrogen hormones – estrone, estradiol and estriol with silver nps of a diameter (34 ± 3) nm. watanabe et al. (2008) were used zno nps that have anisotropic shapes, such as cubic or rectangular parallelepiped, for the detection of verapamil hydrochloride, testosterone, phospholipids, oligosaccharides and synthetic polymers. semiconductor zns nps with surface modification with different functional groups were used for the analysis of cyclodextrin and small proteins (kailasa et al., 2008). arakawa & kawasaki, (2010) used oxidized graphitized carbon black (gcb) particles for the analysis of pharmaceutically active compounds. a common pharmaceutical compound, propranolol, was successfully extracted from baltic sea blue mussels and quantified using oxidized gcb. tio2 is a good candidate for saldi mass spectrometry, because it is readily available, nontoxic and efficiently absorb uv light of n2 laser in maldi instrument. it is first shown applicability of sol-gel tio2 films (chen & chen, 2004), and then applicability of tio2 nps (radisavljević et al., 2012), nanotubes (lo et al., 2008) and nanowires (kim et al., 2014) in saldi analysis of biomolecules. the example of positive ion spectra obtained by saldi method in which tio2 served as a substrate is shown in fig. 5. scarce data were found in literature about tolerance of substrate on high concentration of salts in samples solutions. popović et al. (2015) have shown that the quality of mass spectra of transition metal complexes obtained with tio2 nps was idependent of the high concentration of sodium and potassium ions. size and shape of nanocrystals have impact on analytical performances and ionization efficiency in saldi mass spectrometry (popović et al., 2016; popović et al., 2016a; popović et al., 2016b). tio2 nanocrystals with various shapes and sizes, such colloidal tio2 nps (nps, average diameter ~ 5 nm), prolate nanospheroids (pnss, length: 40–50 nm, the lateral dimension: 14–16 nm) and nanotubes (nts, length: 100-150 nm, average diameter 11 nm), were investigated substrates for saldi-tof ms for quantitative analysis of small biomolecules such as various sex hormones (testosterone and progesterone), vitamins (vitamin a and e), carbohydrates and others (popović et al., 2016; popović et al., 2016a). in general, the best reproducibility was obtained with the larger nanocrystals, pnss and nts, making them good candidates for the quantitative determination of small molecules. however, for analysis of citric acid, dexasone, vitamins e and a, pnss provided the highest sensitivity and reproducibility (popović et al., 2016b). future use of nanomaterials in analytics of (bio)molecules – a summary inorganic particles often exhibit improved and some novel properties as their size approaches nanometer scale dimensions. the unique electronic and optical properties of nanocrystals may lead to future advances of analytical (bio)chemistry, biology, for investigation of biological processes and in medicine. their additional and important application is the development of novel therapeutical approaches. in terms of therapy, novel nps and activators are expected to be exploited. the challenge is to further minimize the side effects and involve fig. 5. positive ion saldi tof mass spectra of inorganic complexes: the upper spectrum represents the spectrum of rucomplex, whereas the bottom is the spectrum of pt-complex. the spectra are acquired with the assistance of tio2 nps with diameter of 5 nm. transmission electron micrograph of tio2 nps is given in the upper left corner of the figure. reproduced with permission (popović et al., 2016). biologica nyssana 9 (1) ⚫ september 2018: 1-19 matijević et al. ⚫ inorganic nanoparticles in biology: drug carriers… 13 light (and potentially other stimuli) in therapy of deep tumors. in terms of imaging, the future challenge is to target specifically a single molecule by a np. one of advanced imaging approaches is mass spectrometry imagining which is a rapidly growing field, which uses nanotechnology (either for production of targets or for the interaction with molecules within the cell/tissue) for the visualization of molecules in tissues, based on the identification of molecules on ions generated. one of the problems with which is this approach facing is lateral resolution in cells and still, despite of the creativity of scientists, is not easy to visualize the intracellular content by this method. however, the potential of new materials and nanocomposites in those fields is yet expected to be exploited in the future. acknowledgements. this work was funded by the serbian ministry of education, science and technological development, grants oi 172011, oi 172056 and iii 45010. references akeson, m., branton, d., kasianowicz, j.j., brandin, e., deamer, d.w. 1999: microsecond time-scale discrimination among polycytidylic acid, polyadenylic acid, and polyuridylic acid as homopolymers or as segments within single rna molecules. biophysical journal, 77: 3227–3233. amiri, m., akbari, a., ahmadi, m., pardakhti, a., salavati-niasari, m. 2017: synthesis and in vitro evaluation of a novel magnetic drug delivery system; proecological method for the preparation of cofe2o4 nanostructures. journal of molecular liquids, 249: 1151-1160. arakawa, r., kawasaki, h. 2010: functionalized nanoparticles and nanostructured surfaces for surface-assisted laser desorption/ionization mass spectrometry. analytical sciences, 26: 1229– 1240. araki, a., sako, y. 1987: determination of free and total homocysteine in human plasma by highperformance liquid chromatography with fluorescence detection. journal of chromatography b: biomedical sciences and applications, 422: 43–52. arruebo, m., fernández-pacheco, r., ibarra, m.r., santamaría, j. 2007: magnetic nanoparticles for drug delivery. nano today, 2: 22–32. avirah, r.r., jayaram, d.t., adarsh, n., ramaiah d. 2012: squaraine dyes in pdt: from basic design to in vivo demonstration. organic & biomolecular chemisrty. 10 (5): 911-920. aydoğan, c., el rassi, z. 2016: monolithic stationary phases with incorporated fumed silica nanoparticles. part ii. polymethacrylate-based monolithic column with “covalently” incorporated modified octadecyl fumed silica nanoparticles for reversed-phase chromatography. journal of chromatography a, 1445: 62–67. bayle, c., issac, c., salvayre, r., couderc, f., caussé, e. 2002: assay of total homocysteine and other thiols by capillary electrophoresis and laserinduced fluorescence detection: ii. pre-analytical and analytical conditions. journal of chromatography a, 979: 255–260. bhattacharyya, s., kudgus, r.a., bhattacharya, r., mukherjee, p. 2011: inorganic nanoparticles in cancer therapy. pharmaceutical research, 28: 237–259. bouccara, s., sitbon, g., fragola, a., loriette, v., lequeux, n., pons, t. 2015: enhancing fluorescence in vivo imaging using inorganic nanoprobes. current opinion in biotechnology, 34: 65–72. brown, s.d., nativo, p., smith, j.-a., stirling, d., edwards, p.r., venugopal, b., flint, d.j., plumb, j.a., graham, d., wheate, n.j. 2010: gold nanoparticles for the improved anticancer drug delivery of the active component of oxaliplatin. journal of the american chemical society, 132: 4678–4684. casciari, j.j., sotirchos, s. v., sutherland, r.m. 1992: variations in tumor cell growth rates and metabolism with oxygen concentration, glucose concentration, and extracellular ph. journal of cellular physiology, 151: 386–394. castillo-garcía, m.l., aguilar-caballos, m.p., gómez-hens, a. 2016: nanomaterials as tools in chromatographic methods. trac trends in analytical chemistry, 82: 385–393. caussé, e., issac, c., malatray, p., bayle, c., valdiguié, p., salvayre, r., couderc, f. 2000: assays for total homocysteine and other thiols by capillary electrophoresis–laser-induced fluorescence detection: i. preanalytical condition studies. journal of chromatography a, 895: 173– 178. chen, c.t., chen, y.c. 2004: desorption/ionization mass spectrometry on nanocrystalline titania sol– gel-deposited films. rapid communications in mass spectrometry, 18: 1956–1964. chen, c.-t., chen, y.-c. 2005: fe3o4/tio2 core/shell nanoparticles as affinity probes for the analysis of phosphopeptides using tio2 surface-assisted laser desorption/ionization mass spectrometry. analytical chemistry, 77: 5912–5919. 14 chen, w.-y., chen, y.-c. 2006: affinity-based mass spectrometry using magnetic iron oxide particles as the matrix and concentrating probes for saldi ms analysis of peptides and proteins. analytical and bioanalytical chemistry, 386: 699–704. chen, y., chen, h., shi, j. 2013: in vivo bio-safety evaluations and diagnostic/therapeutic applications of chemically designed mesoporous silica nanoparticles. advanced materials, 25: 3144–3176. chen, y., chen, h., shi, j. 2014: inorganic nanoparticle-based drug codelivery nanosystems to overcome the multidrug resistance of cancer cells. molecular pharmaceutics, 11: 2495–2510. chen, y., wan, y., wang, y., zhang, h., jiao, z. 2011: anticancer efficacy enhancement and attenuation of side effects of doxorubicin with titanium dioxide nanoparticles. international journal of nanomedicine, 6: 2321–2326. chen, z., geng, z., shao, d., mei, y., wang, z. 2009: single-crystalline euf 3 hollow hexagonal microdisks: synthesis and application as a background-free matrix for maldi-tof-ms analysis of small molecules and polyethylene glycols. analytical chemistry, 81: 7625–7631. chen, z., penet, m.-f., nimmagadda, s., li, c., banerjee, s.r., winnard, p.t., artemov, d., glunde, k., pomper, m.g., bhujwalla, z.m. 2012: psma-targeted theranostic nanoplex for prostate cancer therapy. acs nano, 6: 7752– 7762. cheng, y.-c., chen, k.-h., wang, j.-s., hsu, w.-l., chien, c.-c., chen, w.-y., tsao, c.-w. 2012: rapid analysis of abused drugs using nanostructured silicon surface assisted laser desorption/ionization mass spectrometry. analyst, 137: 654–661. cherukuri, p., glazer, e.s., curley, s.a. 2010: targeted hyperthermia using metal nanoparticles. advanced drug delivery reviews, 62, 339–345. chiang, c.k., chiang, n.c., lin, z.h., lan, g.y., lin, y.w., chang, h.-t. 2010a: nanomaterialbased surface-assisted laser desorption/ionization mass spectrometry of peptides and proteins. journal of the american society for mass spectrometry, 21: 1204–1207. chiang, c.k., yang, z., lin, y.w., chen, w.t., lin, h.j., chang, h.-t. 2010b: detection of proteins and protein−ligand complexes using hgte nanostructure matrixes in surface-assisted laser desorption/ionization mass spectrometry. analytical chemistry, 82: 4543–4550. chiu, t.-c., chang, l.-c., chiang, c.-k., chang, h.t. 2008: determining estrogens using surfaceassisted laser desorption/ionization mass spectrometry with silver nanoparticles as the matrix. journal of the american society for mass spectrometry, 19: 1343–1346. cho, e.c., glaus, c., chen, j., welch, m.j., xia, y., 2010. inorganic nanoparticle-based contrast agents for molecular imaging. trends in molecular medicine. 16, 561–573. cho, k., wang, x., nie, s., chen, z.g., shin, d.m. 2008: therapeutic nanoparticles for drug delivery in cancer. clinical cancer research, 14: 1310– 1316. conde, j., doria, g., baptista, p. 2012: noble metal nanoparticles applications in cancer. journal of drug delivery, 2012: 1–12. dobrovolskaia, m.a., mcneil, s.e. 2007: immunological properties of engineered nanomaterials. nature nanotechnology, 2: 469– 478. dong, x., cheng, j., li, j., wang, y. 2010: graphene as a novel matrix for the analysis of small molecules by maldi-tof ms. analytical chemistry, 82: 6208–6214. el-hussein, a., mfouo-tynga, i., abdel-harith, m., abrahamse, h. 2015: comparative study between the photodynamic ability of gold and silver nanoparticles in mediating cell death in breast and lung cancer cell lines. journal of photochemistry and photobiology b: biology, 153: 67–75. frigerio, c., ribeiro, d.s.m., rodrigues, s.s.m., abreu, v.l.r.g., barbosa, j.a.c., prior, j.a.v., marques, k.l., santos, j.l.m. 2012: application of quantum dots as analytical tools in automated chemical analysis: a review. analytica chimica acta, 735: 9–22. ge, j., li, y., chen, l. 2006: characterization of tio2 /sio2 based stationary phase for rp‐hplc. journal of liquid chromatography & related technologies, 29: 2329–2339. gleiter, h. 1995: nanostructured materials: state of the art and perspectives. nanostructured materials, 6: 3–14. gonciar, a. 2014: detection of intracellular gold nanoparticles. biotechnology, molecular biology and nanomedicine, 2: 21–25. gozuacik, d., yagci-acar, h. f., akkoc, y., kosar, a., isin dogan-ekici a., ekici s. 2014: anticancer use of nanoparticles as nucleic acids carriers. journal of biomedical nanotechnology, 10: 1751–1783 goya, g., grazu, v., ibarra, m. 2008: magnetic nanoparticles for cancer therapy. current nanoscience, 4: 1–16. guénin, e., lecouvey, m., hardouin, j. 2009: could a nano-assisted laser desorption/ionization target improve the study of small organic molecules by laser desorption/ionization time-of-flight mass biologica nyssana 9 (1) ⚫ september 2018: 1-19 matijević et al. ⚫ inorganic nanoparticles in biology: drug carriers… 15 spectrometry. rapid communications in mass spectrometry, 23: 1395–1400. guihen, e. 2013: nanoparticles in modern separation science. trac trends in analytical chemistry, 46: 1–14. hamidi, m.f.f.a., harun, w.s.w., samykano, m., ghani, s.a.c., ghazalli, z., ahmad, f., sulong, a.b. 2017: a review of biocompatible metal injection moulding process parameters for biomedical applications. materials science and engineering: c, 78: 1263-1276. hardman, r. 2006: a toxicologic review of quantum dots: toxicity depends on physicochemical and environmental factors. environmetal health perspective, 114: 165-172. hou, z., zhang, y., deng, k., chen, y., li, x., deng, x., cheng, z., lian, h., li, c., lin, j. 2015: uvemitting upconversion-based tio2 photosensitizing nanoplatform: near-infrared light mediated in vivo photodynamic therapy via mitochondria-involved apoptosis pathway. acs nano, 9: 2584–2599. hu, l., xu, s., pan, c., zou, h., jiang, g. 2007: preparation of a biochip on porous silicon and application for label-free detection of small molecule-protein interactions. rapid communications in mass spectrometry, 21: 1277–1281. huang, h.c., barua, s., sharma, g., dey, s.k., rege, k. 2011: inorganic nanoparticles for cancer imaging and therapy. journal of controlled release, 155: 344-357. huang, y.-f., chang, h.-t. 2006: nile red-adsorbed gold nanoparticle matrixes for determining aminothiols through surface-assisted laser desorption/ionization mass spectrometry. analytical chemistry, 78: 1485–1493. jaskolla, t.w., papasotiriou, d.g., karas, m. 2009: comparison between the matrices α-cyano-4hydroxycinnamic acid and 4-chloro-αcyanocinnamic acid for trypsin, chymotrypsin, and pepsin digestions by maldi-tof mass spectrometry. journal of proteome research, 8: 3588–3597. jim, s.r., oko, a.j., taschuk, m.t., brett, m.j. 2011: morphological modification of nanostructured ultrathin-layer chromatography stationary phases. journal of chromatography a, 1218: 7203–7210. kailasa, s.k., kiran, k., wu, h.-f. 2008: comparison of zns semiconductor nanoparticles capped with various functional groups as the matrix and affinity probes for rapid analysis of cyclodextrins and proteins in surface-assisted laser desorption/ionization time-of-flight mass spectrometry. analytical chemistry, 80: 9681– 9688. kawasaki, h., sugitani, t., watanabe, t., yonezawa, t., moriwaki, h., arakawa, r. 2008: layer-bylayer self-assembled mutilayer films of gold nanoparticles for surface-assisted laser desorption/ionization mass spectrometry. analytical chemistry, 80: 7524–7533. kawasaki, h., takahashi, n., fujimori, h., okumura, k., watanabe, t., matsumura, c., takemine, s., nakano, t., arakawa, r. 2009: functionalized pyrolytic highly oriented graphite polymer film for surface-assisted laser desorption/ionization mass spectrometry in environmental analysis. rapid communications in mass spectrometry, 23: 3323–3332. kawasaki, h., yonezawa, t., watanabe, t., arakawa, r. 2007: platinum nanoflowers for surface-assisted laser desorption/ionization mass spectrometry of biomolecules. the journal of physical chemistry c, 111: 16278–16283. kim, j.i., park, j.m., hwang, s.j., kang, m.j., pyun, j.c. 2014: top-down synthesized tio2 nanowires as a solid matrix for surface-assisted laser desorption/ionization time-of-flight (salditof) mass spectrometry. analytica chimica acta, 836: 53–60. kim, y.r., kim, s., choi, j.w., choi, s.y., lee, s.h., kim, h., hahn, s.k., koh, g.y., yun, s.h. 2015: bioluminescence-activated deep-tissue photodynamic therapy of cancer. theranostics, 5: 805–817. kumar, m.r. , sophia, p.j. 2018: nanoparticles as precious stones in the crown of modern molecular biology. trends in insect molecular biology and biotechnology, 331-351. kuzema, p.a. 2011: small-molecule analysis by surface-assisted laser desorption/ionization mass spectrometry. journal of analytical chemistry, 66: 1227–1242. larsson, m., lindgren, j. 2005: analysis of glutathione and immunoglobulin g inside chromatographic beads using surface-enhanced raman scattering spectroscopy. journal of raman spectroscopy, 36: 394–399. lee, j.h., yeo, y. 2015: controlled drug release from pharmaceutical nanocarriers. chemical engineering science, 125: 75–84. lee, k.h., chiang, c.k., lin, z.h., chang, h.t. 2007: determining enediol compounds in tea using surface-assisted laser desorption/ionization mass spectrometry with titanium dioxide nanoparticle matrices. rapid communications in mass spectrometry, 21: 2023–2030. ling, l. b., baeyens, w.r.g., dewaele, c. 1991: capillary zone electrophoresis with ultraviolet 16 and flourescence detection for the analysis of thiols. application to mixtures and blood. analytica chimica acta, 255: 283–288. llevot, a., astruc, d. 2012: applications of vectorized gold nanoparticles to the diagnosis and therapy of cancer. chemical society reviews, 41: 242–257. lo, c.y., lin, j.y., chen, w.y., chen, c.t., chen, y.c. 2008: surface-assisted laser desorption/ionization mass spectrometry on titania nanotube arrays. journal of the american society for mass spectrometry, 19: 1014–1020. lópez-dávila, v., seifalian, a.m., loizidou, m. 2012: organic nanocarriers for cancer drug delivery. current opinion in pharmacology, 12: 414–419. lópez, t., recillas, s., guevara, p., sotelo, j., alvarez, m., odriozola, j.a. 2008: pt/tio2 brain biocompatible nanoparticles: gbm treatment using the c6 model in wistar rats. acta biomaterialia, 4: 2037–2044. lucena, r., simonet, b.m., cárdenas, s., valcárcel, m. 2011: potential of nanoparticles in sample preparation. journal of chromatogaphy a, 1218: 620–637. lucky, s.s., soo, k.c., zhang, y. 2015: nanoparticles in photodynamic therapy. chemical reviews, 115: 1990–2042. mari, c., pierroz, v., rubbiani, r., patra, m., hess, j., spingler, b., oehninger, l., schur, j., ott, i., salassa, l., ferrari, s., gasser, g. 2014: dna intercalating ruii polypyridyl complexes as effective photosensitizers in photodynamic therapy. chemistry a european journal, 20: 14421–14436. mc carthy, d.j., malhotra, m., o'mahony, a.m., cryan, j.f., o'driscoll, c.m. 2014: nanoparticles and the blood-brain barrier: advancing from invitro models towards therapeutic significance. pharmaceutical research, 32: 1161-1185 mclean, j.a., stumpo, k.a., russell, d.h. 2005: size-selected (2−10 nm) gold nanoparticles for matrix assisted laser desorption ionization of peptides. journal of the american chemical society, 127: 5304–5305. mizojiri, k., shindo, h., ohno, y. 1996: the possibility of predicting tissue accumulation after repeated dosing using a single-dose tissue distribution study. the journal of toxicological sciences, 21: 523–527. monro, a.m. 1994: are routine tissue distribution studies justifiable for approval of human drugs? drug metabolism and disposition, 22: 341-342. nešić, m., popović, i., leskovac, a., šaponjić, z., radoičić, m., stepić, m., petković, m. 2016: testing the photo-sensitive nanocomposite system for potential controlled metallo-drug delivery. optical and quantum electronics, 48: 119–126. nešić, m., žakula, j., korićanac, l., stepić, m., radoičić, m., popović, i., šaponjić, z., petković, m. 2017b: light controlled metallo-drug delivery system based on the tio2nanoparticles and rucomplex. journal of photochemistry and photobiology a: chemistry, 347: 55–66. nesterenko, e.p., nesterenko, p.n., connolly, d., he, x., floris, p., duffy, e., paull, b. 2013: nanoparticle modified stationary phases for highperformance liquid chromatography. analyst, 138: 4229. newsome, t.e., olesik, s. v. 2014: silica-based nanofibers for electrospun ultra-thin layer chromatography. journal of chromatography a, 1364: 261–270. northen, t.r., yanes, o., northen, m.t., marrinucci, d., uritboonthai, w., apon, j., golledge, s.l., nordström, a., siuzdak, g. 2007: clathrate nanostructures for mass spectrometry. nature, 449: 1033–1036. okuno, s., arakawa, r., okamoto, k., matsui, y., seki, s., kozawa, t., tagawa, s., wada, y. 2005: requirements for laser-induced desorption/ionization on submicrometer structures. analytical chemistry, 77: 5364–5369. oliveira, w.f., arruda, i.r.s., silva, g.m.m., machado, g., coelho, l.c.b.b., correia, m.t.s. 2017: functionalization of titanium dioxide nanotubes with biomolecules for biomedical applications. materials science and engineering: c, 81: 597-606. padro, d., howes, a.p., smith, m.e., dupree, r. 2000: determination of titanium nmr parameters of atio3 compounds: correlations with structural distortion. solid state nuclear magnetic resonance, 15: 231–236. pandey, a. 2017: an overview on advances in the nanocarriers drug delivery systems. emr/esr/epr spectroscopy for characterization of nanomaterials, 62: 65–76. pansare, v., hejazi, s., faenza, w., prud ’homme, r.k. 2012: review of long-wavelength optical and nir imaging materials: contrast agents, fluorophores and multifunctional nano carriers. chemistry of materials, 24: 812-827. pastorin, g., wu, w., wieckowski, s., briand, j.-p., kostarelos, k., prato, m., bianco, a. 2006: double functionalisation of carbon nanotubes for multimodal drug delivery. chemical communications, 1182-1184. petković, m., schiller, j., müller, j., müller, m., arnold, k., arnhold, j. 2001a: the signal-tonoise ratio as the measure for the quantification of biologica nyssana 9 (1) ⚫ september 2018: 1-19 matijević et al. ⚫ inorganic nanoparticles in biology: drug carriers… 17 lysophospholipids by matrix-assisted laser desorption/ ionisation time-of-flight mass spectrometry. analyst, 126: 1042–1050. petković, m., schiller, j., müller, m., benard, s., reichl, s., arnold, k., arnhold, j. 2001b: detection of individual phospholipids in lipid mixtures by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry: phosphatidylcholine prevents the detection of further species. analytical biochemistry, 289: 202–216. pietzsch, j., julius, u., hanefeld, m. 1997: rapid determination of total homocysteine in human plasma by using n(o,s)-ethoxycarbonyl ethyl ester derivatives and gas chromatography-mass spectrometry. clinical chemistry, 43: 2001–2004. popović, i., milovanović, d., miletić, j., nešić, m., vranješ, m., šaponjić, z., petković, m. 2016a: dependence of the quality of saldi tof ms analysis on the tio2 nanocrystals’ size and shape. optical and quantum electronics, 48: 113. popović, i., nešić, m., nišavić, m., vranješ, m., radetić, t., šaponjić, z., masnikosa, r., petković, m. 2015: suitability of tio2 nanoparticles and prolate nanospheroids for laser desorption/ionization mass spectrometric characterization of bipyridine-containing complexes. material letters, 150: 84–88. popović, i., nešić, m., vranješ, m., šaponjić, z., petković, m. 2016b: saldi-tof-ms analyses of small molecules (citric acid, dexasone, vitamins e and a) using tio2 nanocrystals as substrates. analytical and bioanalytical chemistry, 408: 7481–7490. popović, i., nešić, m., vranješ, m., šaponjić, z., petković, m. 2016: tio2 nanocrystals – assisted laser desorption and ionization time-of-flight mass spectrometric analysis of steroid hormones, amino acids and saccharides. validation and comparison of methods. rsc advances, 6: 1027– 1036. pyrzynska, k. 2013: use of nanomaterials in sample preparation. trac trends in analytical chemistry, 43: 100–108. radisavljević, m., kamčeva, t., vukićević, i., radoičić, m., šaponjić, z., petković, m. 2012: colloidal tio2 nanoparticles as substrates for m(s)aldi mass spectrometry of transition metal complexes. rapid communications in mass spectrometry, 26: 2041–2050. radović, m., calatayud, m.p., goya, g.f., ibarra, m.r., antić, b., spasojević, v., nikolić, n., janković, d., mirković, m., vranješ-đurić, s. 2015: preparation and in vivo evaluation of multifunctional 90 y-labeled magnetic nanoparticles designed for cancer therapy. journal of biomedical material research part a, 103: 126–134. rafii, m., elango, r., courtney-martin, g., house, j.d., fisher, l., pencharz, p.b. 2007: highthroughput and simultaneous measurement of homocysteine and cysteine in human plasma and urine by liquid chromatography–electrospray tandem mass spectrometry. analytical biochemistry, 371: 71–81. rajh, t., šaponjić, z., liu, j., dimitrijević, n.m., scherer, n.f., arroyo, v.m., zapol, p., curtiss, l.a., thurnauer, m.c. 2004: charge transfer across the nanocrystalline-dna interface: probing dna recognition. nano letters, 4: 1017– 1023. rawat, m., singh, d., saraf, s. 2006: nanocarriers: promising vehicle for bioactive drugs. biological and pharmaceutical bulletin, 29: 1790–1798. rodriguez, j.a., liu, g., jirsak, t., hrbek, j., chang, z., dvorak, j., maiti, a. 2002: activation of gold on titania: adsorption and reaction of so2 on au/tio2 (110). journal of the american chemical society, 124: 5242–5250. schoiswohl, j., kresse, g., surnev, s., sock, m., ramsey, m.g., netzer, f.p. 2004: planar vanadium oxide clusters: two-dimensional evaporation and diffusion on rh(111). physical review letters, 92: 206103. scida, k., stege, p.w., haby, g., messina, g.a., garcía, c.d. 2011: recent applications of carbonbased nanomaterials in analytical chemistry: critical review. analytica chimica acta, 691: 6– 17. shadab, m., shalini, g., sana, f., saurabh, s. 2015: nanotechnology as carriers for chemotherapeutics: future of drug delivery. world journal of pharmaceutical research, 4: 923–946. sherrod, s.d., diaz, a.j., russell, w.k., cremer, p.s., russell, d.h. 2008: silver nanoparticles as selective ionization probes for analysis of olefins by mass spectrometry. analytical chemistry, 80: 6796–6799. sims, c.m., hanna, s.k., heller, d.a., horoszko, c.p., johnson, m.e., montoro bustos, a.r., reipa, v., riley, k.r., nelson, b.c. 2017: redoxactive nanomaterials for nanomedicine applications. nanoscale, 9: 15226–15251. singh, r., lillard, j.w. 2009: nanoparticle-based targeted drug delivery. experimental and molecular pathology, 86: 215–223. song, z., cai, t., chang, z., liu, g., rodriguez, j.a., hrbek, j. 2003: molecular level study of the formation and the spread of moo3 on au (111) by scanning tunneling microscopy and x-ray photoelectron spectroscopy. journal of the american chemical society, 125: 8059–8066. 18 speltini, a., merli, d., profumo, a. 2013: analytical application of carbon nanotubes, fullerenes and nanodiamonds in nanomaterials-based chromatographic stationary phases: a review. analytica chimica acta, 783: 1–16. sperling, r.a., parak, w.j. 2010: surface modification, functionalization and bioconjugation of colloidal inorganic nanoparticles. philosophical transactions of the royal society a mathematical, physical and. engineering sciences, 368: 1333–1383. stark, w. 2011: nanoparticles in biological systems. angewandte chemie international edition, 50 (6): 1242-1258. su, h., wang, y., gu, y., bowman, l., zhao, j., ding, m., 2018: potential applications and human biosafety of nanomaterials used in nanomedicine. journal of applied toxicology, in press sunner, j., dratz, e., chen, y. c. 1995: graphite surface-assisted laser desorption/ionization timeof-flight mass spectrometry of peptides and proteins from liquid solutions. analytical chemistry, 67: 4335–4342. tang, h.w., ng, k.m., lu, w., che, c.m. 2009: ion desorption efficiency and internal energy transfer in carbon-based surface-assisted laser desorption/ionization mass spectrometry: desorption mechanism(s) and the design of saldi substrates. analytical chemistry, 81: 4720–4729. tang, s., guo, y., xiong, c., liu, s., liu, x., jiang, s. 2014: nanoparticle-based monoliths for chromatographic separations. analyst, 139: 4103. tasciotti, e., liu, x., bhavane, r., plant, k., leonard, a.d., price, b.k., cheng, m.m.c., decuzzi, p., tour, j.m., robertson, f., ferrari, m. 2008: mesoporous silicon particles as a multistage delivery system for imaging and therapeutic applications. nature nanotechnology, 3: 151–157. teng, i.t., chang, y.j., wang, l.s., lu, h.-y., wu, l.c., yang, c.m., chiu, c.c., yang, c.h., hsu, s.l., ho, j.a. 2013: phospholipid-functionalized mesoporous silica nanocarriers for selective photodynamic therapy of cancer. biomaterials, 34: 7462–7470. teruyuki, s., hiroaki, s., atsushi, y., atsushi, n., masaki, t., hiroaki, t. 2007: matrix-free laser desorption/ionization-mass spectrometry using self-assembled germanium nanodots. analytical chemistry, 79: 4827–4832. tholey, a., zabet-moghaddam, m., heinzle, e. 2006: quantification of peptides for the monitoring of protease-catalyzed reactions by matrix-assisted laser desorption/ionization mass spectrometry using ionic liquid matrixes. analytical chemistry, 78: 291–297. thompson, d.g., enright, a., faulds, k., smith, w.e., graham, d. 2008: ultrasensitive dna detection using oligonucleotide-silver nanoparticle conjugates. analitical chemistry, 80: 2805–2810. trudeau, m.l., ying, j.y. 1996: nanocrystalline materials in catalysis and electrocatalysis: structure tailoring and surface reactivity. nanostructured materials, 7: 245–258. valcárcel, m., cárdenas, s., simonet, b.m. 2007: role of carbon nanotubes in analytical science. analytical chemistry, 79: 4788–4797. valden, m., lai, x., goodman, d.w. 1998: onset of catalytic activity of gold clusters on titania with the appearance of nonmetallic properties. science, 281: 1647–50. walker, b.n., stolee, j.a., pickel, d.l., retterer, s.t., vertes, a. 2010: tailored silicon nanopost arrays for resonant nanophotonic ion production. journal of physical chemistry c, 114: 4835– 4840. wang, t., jiang, h., wan, l., zhao, q., jiang, t., wang, b., wang, s. 2015: potential application of functional porous tio2 nanoparticles in lightcontrolled drug release and targeted drug delivery. acta biomaterialia, 13: 354–363. wang, y., song, s., liu, j., liu, d., zhang, h. 2015: zno-functionalized upconverting nanotheranostic agent: multi-modality imagingguided chemotherapy with on-demand drug release triggered by ph. angewandte chemie international edition, 54: 536–540. watanabe, t., kawasaki, h., yonezawa, t., arakawa, r. 2008: surface-assisted laser desorption/ionization mass spectrometry (saldi-ms) of low molecular weight organic compounds and synthetic polymers using zinc oxide (zno) nanoparticles. journal of mass spectrometry, 43: 1063–1071. wei, j., buriak, j.m., siuzdak, g. 1999: desorption– ionization mass spectrometry on porous silicon. nature, 399: 243–246. will, o., purkayastha, s., chan, c., athanasiou, t., darzi, a.w., gedroyc, w., tekkis, p.p. 2006: diagnostic precision of nanoparticle-enhanced mri for lymph-node metastases: a meta-analysis. the lancet oncology, 7: 52–60. wu, h.p., su, c.l., chang, h.c., tseng, w.l. 2007: sample-first preparation: a method for surfaceassisted laser desorption/ionization time-of-flight mass spectrometry analysis of cyclic oligosaccharides. analytical chemistry, 79: 6215–6221. yang, c., uertz, j., yohan, d., chithrani, b.d. 2014: peptide modified gold nanoparticles for improved cellular uptake, nuclear transport, and biologica nyssana 9 (1) ⚫ september 2018: 1-19 matijević et al. ⚫ inorganic nanoparticles in biology: drug carriers… 19 intracellular retention. nanoscale, 6: 1202612033 yin, q., shen, j., zhang, z., yu, h., li, y. 2013: reversal of multidrug resistance by stimuliresponsive drug delivery systems for therapy of tumor. advanced drug delivery reviews, 65: 1699–1715. yoon, h.y., jeon, s., you, d.g., park, j.h., kwon, i.c., koo, h., kim, k. 2017: inorganic nanoparticles for image-guided therapy. bioconjugate chemistry, 28: 124–134. yu, m., zheng, j. 2015: clearance pathways and tumor targeting of imaging nanoparticles. acs nano, 9, 6655–6674. zhang, b.t., zheng, x., li, h.f., lin, j.m. 2013: application of carbon-based nanomaterials in sample preparation: a review. analytica chimica acta, 784: 1–17. zhang, h., shan, y., dong, l. 2014: a comparison of tio2 and zno nanoparticles as photosensitizers in photodynamic therapy for cancer. journal of biomedical nanotechnology, 10: 1450–1457. zhang, h., wang, c., chen, b., wang, x. 2012: daunorubicin-tio2 nanocomposites as a “smart ” ph-responsive drug delivery system. international journal of nanomedicine, 7: 235– 242. zhang, m., qiu, h. 2015: progress in stationary phases modified with carbonaceous nanomaterials for high-performance liquid chromatography. trac trends in analytical chemistry, 65: 107–121. zhang, z., wang, z., liao, y., liu, h. 2006: applications of nanomaterials in liquid chromatography: opportunities for separation with high efficiency and selectivity. journal of separation science, 29: 1872–1878. 20 stanković et al., 2019, biologica nyssana 10(1) 10 (1) september 2019: 59-61 doi: 10.5281/zenodo.3464010 contribution to the knowledge of tachinid fauna in serbia short communication saša s. stanković university of niš, department of biology and ecology, faculty of sciences and mathematics, višegradska 33, 18000 niš, serbia sasasta@gmail.com (corresponding author) marijana ilić milošević department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, 18000 niš, serbia marijana183@gmail.com vladimir žikić department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, 18000 niš, serbia vzikic@pmf.ni.ac.rs hans-peter tschorsnig staatliches museum für naturkunde, rosenstein 1, 70191 stuttgart, germany hanspeter.tschorsnig@smns-bw.de received: august 5, 2019 revised: september 7, 2019 accepted: september 9, 2019 abstract: tachinid flies were reared from several species of lepidoptera larvae during the year 2018. eight species were recorded from two subfamilies, exoristinae and tachininae. the species ceromya bicolor is new for the serbian fauna. also, for the same territory, the genus ceromya is recorded for the first time. key words: tachinidae, ceromya bicolor, parasitoids, serbia apstract: doprinos poznavanju faune muva guseničarki u srbiji muve guseničarke su odgajene iz nekoliko vrsta larvi lepidoptera tokom 2018. godine. registrovano je osam vrsta iz dve podfamilije, exoristinae i tachininae. vrsta ceromya bicolor je nova za faunu srbije. takođe, za istu teritoriju je prvi put registrovan rod ceromya. ključne reči: tachinidae, ceromya bicolor, parazitoidi, srbija tachinid flies are well known parasitoids of other arthropods. they parasitize almost exclusively insects, especially larval lepidoptera, but very rarely spiders, scorpions and centipedes (vincent, 1985; williams et al., 1990; stireman et al., 2006). besides various parasitic hymenoptera, tachinids are the second most important natural enemies of many pest insects in both natural and agroecosystems. many tachinid species are being used in pest managements in forestry and crop production systems with notable success (greathead, 1986; grenier, 1988). the family tachinidae is one of the most diverse among diptera order, so far over 10,000 species are described worldwide from about 1,520 genera (irwin et al., 2003). the family is divided in four subfamilies: exoristinae, dexiinae, phasiinae and tachininae (herting & dely draskovits, 1993). although, the group is the most diverse in tropical regions, in europe there are registered about 877 species (according to fauna europaea, internet database). this database listed 242 species for the territory of serbia and montenegro. however, some recent faunistic papers reported several more species for the territory of serbia. for example, hubenov (2008) reported 288 species for the serbian fauna, stanković et al. (2014) reported two more species and later stanković et al. (2018) published an updated list of 295 species from which five are newly recorded and the new genus buquetia for the investigated territory. tachinids were reared from lepidoptera larvae during the year 2018 mostly from the southern part of serbia. the host larvae were grown in special plastic boxes in the laboratory until parasitoids emergence. after rearing the adults of tachinid flies, they were put in small plastic vials and preserved in 96 % ethanol. the identification of the species was done by the last author of the paper. the rearing of parasitoids and all the material is stored at the department of biology and ecology, faculty of science and mathematics, university of niš, serbia. the remarks are based on a catalogue on palaearctic tachinid-host records (tschorsnig, 2017). new species and genus is newly reported for the investigated territory and marked by an asterisk (*). © 2019 stanković, s.s. et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 59 biologica nyssana ● 10 (1) september 2019: 59-61 stanković et al. ● contribution to the knowledge of tachinid fauna in serbia species list subfamily exoristinae erycia fatua (meigen, 1824) 3 ♂, 1 ♀, serbia, vlasina lake, 27.v 2018. ex melitaea didyma (esper, 1778) (nymphalidae), on centaurea jacea l., leg. v. žikić. remark: a parasitoid of melitaea, m. didyma was already known (see tschorsnig, 2017: 181). erycia festinans (meigen, 1824) 2 ♂, 4 ♀, serbia, vlasina lake, 27.v 2018. ex melitaea phoebe (denis & schiffermüller, 1775) (nymphalidae), on centaurea jacea l., leg. s. stanković. remark: a parasitoid of melitaea, m. phoebe was already known. erycia festinans is morphologically very similar to erycia fatua (see herting, 1973; tschorsnig & herting, 1994). having the same hosts, there remains some doubt if both are really separate species. (see tschorsnig, 2017: 182). eumea mitis (meigen, 1824) 1 ♂, serbia, niš, brzi brod, 3.v 2018. unknown host, on sambucus ebulus l., leg. v. žikić. remark: a parasitoid of noctuidae and several microlepidoteran families (see tschorsnig, 2017: 187188). eurysthaea scutellaris (robineau-desvoidy, 1848) 1 ♂, serbia, sićevačka klisura (gorge), ostrovica, 1.v 2018. ex yponomeuta sp. (yponomeutidae) on euonymus japonicus thunb, leg. v. žikić. remark: common parasitoid of yponomeuta spp. (see tschorsnig, 2017: 215-216). masicera sphingivora (robineau-desvoidy, 1830) 2 ♀, serbia, vlasina lake, 27.v 2018. ex melitaea didyma (esper, 1778) (nymphalidae), on centaurea jacea l., leg. s. stanković. remark: a parasitoid with many hosts, also m. didyma was already known (see tschorsnig, 2017: 232). phryxe hirta (bigot, 1880) 1 ♂, serbia, vlasina lake, 27.v 2018. ex heterogynis zikici de freina, 2018 (heterogynidae) on chamaecytisus heuffelii (wierzb. ex griseb. and schenk) rothm, leg v. žikić. remark: typical parasitoid of heterogynis spp. (see tschorsnig, 2017: 117). phryxe vulgaris (fallén, 1810) 1 ♀, serbia, sićevačka klisura (gorge), 7.v 2018. ex brenthis daphne (bergsträsser, 1780) (nymphalidae), on rubus sp. leg v. žikić. remark: a common species which has many hosts; also b. daphne is among it (see tschorsnig, 2017: 133). subfamily tachininae *ceromya bicolor (meigen, 1824) 2 ♂, 2 ♀, serbia, paraćin, bukovik, 5.v 2018. unknown host, on unknown plant, leg n. veljković. remark: a small species which occasionally develops in numbers in large lasiocampidae (see tschorsnig, 2017: 272). here we present eight tachinid species collected in serbia from two subfamilies, seven from exoristinae and one from tachininae. the species erycia festinans is still not listed in fauna europaea for the territory of serbia although it is reported recently as a new species for this country in stanković et al. (2018). the same is with the species phryxe hirta which is reported as a new species for serbia in stanković et al. (2014). the species ceromya bicolor is the new species for the serbian fauna, as well as the genus ceromya. these observations of l. senator represent a contribution to the distribution and population census of this species, and also the need of protection of its habitat. acknowledgements. this study was supported by the ministry of education, science and technological development of the republic of serbia (iii43001). we thank to kjeld brem sørensen and boženka hric for the identification of caterpillars. references greathead, d.j. 1986: parasitoids in classical biological control. in: insect parasitoids. 13th symposium of the royal entomological society of london, 18-19 september 1985 at the department of physics lecture theatre, imperial college, london (pp. 289-318). academic press. grenier, s. 1988: applied biological control with tachinid flies (diptera, tachinidae): a review. anzeiger für schädlingskunde pflanzenschutz umweltschutz, 51: 49-56. herting, b. 1973: beiträge zur kenntnis der europäischen raupenfliegen (dipt. tachinidae) xiii. stuttgarter beiträge zur naturkunde, serie a (biologie) 254: 18 pp. herting, b., dely-draskovits, a. 1993: family tachinidae. in: soós, a., papp, l. (ed.), catalogue of palaearctic diptera 13: 118-624, hungarian natural history museum, budapest. hubenov, z. 2008: composition and zoogeographical characteristics of the family tachinidae (diptera: insecta) in serbia and bulgaria. advances in arachnology and developmental biology, 12: 375394. 60 irwin, m.e., schlinger, e.i., thompson, f.c. 2003: diptera, trueflies. in: goodman, s. m., benstead, j. p. (ed.), the natural history of madagascar, 692702, university of chicago press. chicago/london 1728 p. stanković, s.s., žikić, v., hric, b., tschorsnig, h.p. 2014: several records of tachinidae (diptera) reared from their hosts in serbia and montenegro. biologica nyssana, 5 (1): 71-73. stanković, s.s., žikić, v., ilić milošević, m., ritt, r., tschorsnig, h.p. 2018: tachinid fauna of serbia and montenegro updated with new findings (diptera: tachinidae). journal of the entomological research society, 20 (3): 53-66. stireman iii, j.o., o’hara, j.e., wood, d.m. 2006: tachinidae: evolution, behavior, and ecology. annual review of entomology, 51: 525-555. tschorsnig, h.p., herting, b. 1994: die raupenfliegen (diptera: tachinidae) mitteleuropas: bestimmungstabellen und angaben zur verbreitung und ökologie der einzelnen arten. stuttgarter beiträge zur naturkunde, serie a (biologie), 506: 170 pp. tschorsnig, h. p. 2017: preliminary host catalogue of palaearctic tachinidae (diptera). web page: www. nadsdiptera. org/tach/worldtachs/catpalhosts/ home. html) (date accessed: 15.08. 2017). vincent, l.s. 1985: the first record of a tachinid fly as an internal parasitoid of a spider (diptera: tachinidae; araneae: antrodiaetidae). panpacific entomologist, 61: 224-235. williams, s.c., arnaud, p.h., lowe, g. 1990: parasitism of anuroctonus phaiodactylus (wood) and vaejovis spinigerus (wood) (scorpiones: vaejovidae) by spilochaetosoma californicum smith (diptera: tachinidae), and a review of parasitism in scorpions. myia, 5: 11-27. 61 biologica nyssana ● 10 (1) september 2019: 59-61 stanković et al. ● contribution to the knowledge of tachinid fauna in serbia anticancer compounds from medicinal plants biologica nyssana 2 (2)  december 2011: 00-00 ljupković r.b. et al..  removal cu(ii) ions from water... 31 original article a contribution to the agromyzid leaf miners (diptera: agromyzidae) of iran zahra shahreki 1 , ehsan rakhshani* 1 , mitsohiru sasakawa 2 1 department of entomology, college of agriculture, university of zabol, 98615-538, zabol, i.r. iran 2 korigaoka, hirakata city, osaka pref., 573 japan * e-mail: rakhshani@uoz.ac.ir abstract: shahreki, z., rakshani, e., sasakawa, m.: a contribution to the agromyzid leaf miners (diptera: agromyzidae) of iran, biologica nyssana, 3 (1), september 2012: 31-36. a study was carried out during 2009 and 2010 in sistan region located in the eastern part of iran, for elucidating the agromyzid fauna. the specimens were collected by rearing of the mine-infested leaves of different cultivated and non-cultivated host plants. the leafminers of two subfamilies, agromyzinae and phytomyzinae, were studied in this paper. eight species belonging to five genera of the agromyzidae were collected. among them, three species, including chromatomyia nigra (hardy, 1849), melangromyza cunctans (meigen, 1830) and phytoliriomyza dorsata (siebke, 1863), are recorded for the first time for the territory of iran. the last species is also representative of the new genus record for iran. comments on general distribution of each species are presented here. key words: agromyzinae, eastern iran, host association, new record, phytomyzinae introduction the agromyzidae (leaf mining flies) is one of the largest dipteran families, with more than 3000 species belonging to 30 genera worldwide. about 1165 species have been recorded from the palaearctic region (civelek, 2003, 2008; çikman & sasakawa, 2008; dursun et al., 2010), one of the largest genera, the genus liriomyza mik, with over 300 species worldwide (spencer, 1973) appear common in the palaearctic region, but poorly represented in the oriental region (sasakawa, 1972). the agromyzid leaf miners including many species of economic importance, limiting production in several agroecosystems (spencer, 1973). most of species are cosmopolitan, polyphagous pests of agricultural crops and ornamental plants with an economic importance (spencer, 1973; dempewolf, 2006). they undermine the internal tissues of leaves, stems, fruits and even roots. leafminers can cause direct damage to the photosynthetic tissue of host plants caused by larval leaf mining (johnson et al., 1983) and esthetic damage caused by oviposition. adult males and females suck the exuding sap from the parenchyma cells on the punctures produced by adult females just after oviposition (spencer, 1973). extensive mining can result in premature leaf drop and leading to lack of shading (capinera, 2001). apart from direct damage caused by larval feeding on leaf tissues, the females may even act as vectors for diseases during oviposition (matteoni & broadbent, 1988; zitter & tsai, 1977). taxonomically, the agromyzids are considered one of the most difficult groups among the diptera order. this is because of high degree of uniformity between species and the small size of specimens. identification of species is an important 3 (1) • september 2012: 31-36 biologica nyssana 3 (1)  september 2012: 31-36 shahreki, z. et al.  a contribution to the agromyzid… 32 step prerequisite for establishment of effective pest control program and the quarantine measures, especially in the case of biological control (parrellaa & keil, 1984). a few attempts have been made for investigation of faunal diversity (dousti, 2010), biology (haghani et al., 2007; farrokhi et al., 2004) and the natural enemies (asadi et al., 2006; fathi, 2011) of the leafminer flies in iran. the scanty information were summarized by dousti (2010), in which the record of 26 species from iran, indicating the necessity of further investigations to extend the knowledge about the diversity and taxonomy of this poorly known group of insects. in spite of substantial efforts that have been made on systematic of agromyzidae species, there is a significant lack of knowledge in iran. furthermore, there is no record from sistan region which is located in the eastern part of the country. here, we present the data of the faunistic survey on agromyzidae from sistan region, including host associations and comments on their general distributions. materials and methods the leaves, stems and other parts of the host plants infested by the mining agromyzid larvae were collected in the various cultivated and noncultivated areas in sistan region during the period of 2009–2010. the infested plant materials were cut, preferably with representative part of the plants and then directly placed in plastic boxes (10 × 12 cm) covered with mesh on the upper side due to ventilation. the rearing boxes were then transferred to laboratory, inside the climatic chamber, under the constant conditions with the temperature of 25±1ºc and 65±5% rh. samples were kept for about 1–2 weeks expecting of emergence of the adults. adults were caught by an aspirator and gathered in the test tubes containing of 75% ethanol. illustrations were made using the nikon smz645 stereomicroscope and nikon eclips e200 microscope equipped with the canon ixus 100 is digital camera. all the samples are preserved in the collection of second author. results and disscusion eight species of agromyzidae belong to five genera and two subfamilies were identified in association with 11 different species of host plants. one genus and three species listed below are considered to be new records for iran. the newly recorded taxa are indicated by an asterisk ( ). subfamily: agromyzinae melanagromyza cunctans (meigen, 1830)* (figures 7, 15) distribution: mediterranean and western european countries. material examined: 2♀ 2♂, reared from lycopersicon esculentum – solanaceae, zabol, 11iii-2010; leg.: z.s. subfamily: phytomyzinae calycomyza humeralis (von roser, 1840) (figures 1, 9) distribution: nearctic and palaearctic region. material examined: 1♂, reared from triticum aestivum – poaceae, hamoon plain, 28-v-2009; leg.: z.s. chromatomyia horticola (gourea, 1851) (figures 2, 10) distribution: holarctic, oriental and afrotropical. expanded to central asia and europe. material examined: 30♀ 8♂, reared from gaillardia grandiflora – asteraceae, sistan dam, 20-i-2009; 2♀ 20♂, reared from helianthus annus – asteraceae, zahak, 24-iii-2009; 10♀ 8♂, reared from silybum marianum – asteraceae, zabol, 11xi-2009; 8♀ 12♂, reared from brassica rapa – brassicaceae, zahak, 06-iv-2010; 5♀ 3♂, cucumis sativus – cucurbitaceae, zahak, 02-v-2009; leg.: z.s. chromatomyia nigra (meigen, 1830)* (figures 3, 11) distribution: europe, northern asia, northern america. new record from iran. material examined: 5♀ & 3♂, reared from triticum aestivum – poaceae, hirmand, 28-v-2009; leg.: z.s. liriomyza congesta (becker, 1903) (figs. 4, 12) distribution: widespread in europe and north africa. also recorded from the eastern palaearctic and india. material examined: material: 1♀, reared from trigonella foenum-graecum – fabaceae, hamoon plain, 05-iv-2009; 2♀, reared from medicago sativa – fabaceae, zabol, 21-iv-2010; 2♂, hamoon plain, 05-xi-2009; leg.: z.s. biologica nyssana 3 (1)  september 2012: 31-36 shahreki, z. et al.  a contribution to the agromyzid… 33 figures 1-8. lateral hibatate of the adult leafminers. 1. calycomyza humeralis, 2chromatomyia horticola, 3. chromatomyia nigra, 4. liriomyza congesta, 5. liriomyza sativae, 6. liriomyza trifolii, 7. melanagromyza cunctans, 8. phytoliriomyza dorsata. biologica nyssana 3 (1)  september 2012: 31-36 shahreki, z. et al.  a contribution to the agromyzid… 34 liriomyza sativae (blanchard, 1938) (figure 5, 13) distribution: cosmopolitan. material examined: 1♀ 22♂, reared from malva sylvestris – malvaceae, zabol, 04-v-2009; leg.: z.s. liriomyza trifolii (burgess, 1880) (figure 6, 14) distribution: nearctic in origin, but a cosmopolitan species. material examined: material: 5♀ 10♂, reared from cucumis sativus – cucrubitaceae, zahak, 05-iv2009; 25♀ 6♂, reared from lactuca serriola – asteraceae, zahak, 19-iv-2009; leg.: z.s. phytoliriomyza dorsata (siebke, 1864)* (figures 8, 16) distribution: western palaearactic, north america. new record of the genus phytoliriomyza hendel from iran. material examined: 1♀, gaillardia grandiflora – asteraceae, zabol, 8-x-2009; leg.: z.s. figures 9-16. wing of agromyzid leafminers, 9. calycomyza humeralis, 10chromatomyia horticola, 11. chromatomyia nigra, 12. liriomyza congesta, 13. liriomyza sativae, 14. liriomyza trifolii, 15. melanagromyza cunctans, 16. phytoliriomyza dorsata. biologica nyssana 3 (1)  september 2012: 31-36 shahreki, z. et al.  a contribution to the agromyzid… 35 eight species belonging to five genera are known to occur in sistan region. all of them are newly recorded from eastern part of iran, too. mining larvae of different host plants are observed in cultivated or non-cultivated fields attacking crops in sistan region. most of these species have been found in warm or temperate and humid regions in the world (spencer, 1990). two species, l. trifolii and l. sativae are polyphagous leafminers known as important agricultural pests (darvas et al., 2000). among the studied agromyzids, three species, melanagromyza cunctans, phytoliriomyza dorsata and chromatomyia nigra were recorded for the first time for iran. the genus chromatomyia includes the polyphagous leafminer species, which are common agricultural pests on cosmopolitan plant species of grasses: poaceae (gramineae) (griffiths 1974, 1980). chromatomyia nigra was another newly recorded species from iran, which is an unusual species in having the densely hairy eyes and a pair of ventral processes on the distiphallus. this is an almost cosmopolitan species occurs on leaves of grasses, poaceae (gramineae) and asteraceae (compositae), aster and bellis. next species chromatomyia horticola considered also as a polyphagous species feeding on more than 30 plant families (griffiths 1974, 1980). melanagromyza hendel is the second large genus (spencer, 1966) and one of the most homogeneous taxa within the family agromyzidae (spencer, 1990). it is characterized by having a wide range of hosts undermining 23 plant families (benavent-corai et al., 2005). melanagromyza genus represents mainly internal stem-borers, but the species which has been described as melanagromyza symphyti griffiths, 1963 undermining thick-leaf stalks. the species such as melanagromyza cunctans that is newly recorded from iran, it is known as a gall-causer on lotus spp. in europe (spencer, 1976). it was collected in late winter from stems of tomato. another species of this genus, melanagromyza sativae spencer has already been recorded from the northern part of iran (spencer, 1990). it was also reared from the stems of the plants belong to the family apiaceae in fars province (dousti, 2010). the genus phytoliriomyza hendel is reported here for the first time from iran. less than 20 species of the genus phytoliriomyza have been recorded in the palaearctic region (zlobin, 2005). as a new host record, phytoliriomyza dorsata has been reared from the leaves of gaillardia grandiflora in spring. conclusion the present contribution increased our knowledge about the occurrence and the host associations of agromyzid leaf miner at a small area of eastern iran. the identified species were mostly the common and frequently encountered agricultural pests, as well as the new records, which have also economic importance in some other countries. on the other hand, further investigations are necessary to expand the data about the occurrence of other species and their host plants as an ecological refugia for the natural enemies especially the parasitoids. acknowledgements. this study was supported by the grant no. 89-9198, university of zabol, iran. references asadi, r., talebi, a.a., fathipour, y., moharramipour, s. & rakhshani, e. 2006: identification of parasitoids and seasonal parasitism of the agromyzid leafminers genus liriomyza (dip.: agromyzidae) in varamin, iran. journal of agricultural science and technology, 8: 293-303. benavent-corai, j., m. martinez & jiménez-peydró, r. 2005: catalogue of the host-plants of the world agromyzidae (diptera). bolletino di zoologia agraria e di bachicoltura. serie ii, 37: 1-97. capinera, j.l. 2001: handbook of vegetable pests. academic press, new york. 729 pp. çikman, e. & sasakawa, m. 2008: the turkish agromyzidae (diptera), with descriptions of four new species. entomological science, 11 (1): 81 86. civelek, h.s. 2003: checklist of agromyzidae (diptera) of turkey, with a new record. phytoparasitica, 31 (2): 132-138. darvas, b., skuhrav, m., & andersen, a. 2000: agricultural dipteran pests of the palaearctic region. in: papp, l. & darvas, b. (eds.), contributions to a manual of palaearctic diptera (with special reference to flies of economic importance). 1. general and applied dipterolology 565-649, science herald, bupapest. dempewolf, m. 2006: agromyzidae (diptera) of economic importance. cd-rom, institute for biodiversity and ecosystem dynamics, zoological museum amsterdam, http://www.science.uva.nl/entomol/agromyzidae .html. dousti, a. 2010: annotated list of agromyzidae (diptera) from iran, with four new records. biologica nyssana 3 (1)  september 2012: 31-36 shahreki, z. et al.  a contribution to the agromyzid… 36 journal of entomological research society, 12(3): 1-6. dursun, o., eskin, a. & atahan, t. 2010: contributions to the turkish agromyzidae (diptera) fauna with ten new records. türkiye entomology dergisi, 34 (3): 299-306. farrokhi, s., ebrahimi, e. & noori, p. 2004: study on population fluctuation of liriomyza trifolii and its parasitoids on cucumber in varamin region. proceeding of the 16 th iranian plant protection congress, vol. 1: p. 16. fathi, a.a. 2011: tritrophic interactions of nineteen canola cultivarschromatomyia horticola parasitoids in ardabil region. munis entomology and zoology, 6 (1): 449-454. griffiths, g.c.d. 1974: studies on boreal agromyzidae (diptera) v. on the genus chromatomyia hardy, with revision of caprifoliaceae-mining species. quaestiones entomologicae, 10: 15-69. griffiths, g.c.d. 1980: studies on boreal agromyzidae (diptera) xiv. chromatomyia miners on monocotyledones. entomologica scandinavica, suppl. 13: 1-61. haghani, m., fathipour, y., talebi, a.a. & baniameri, v. 2007: thermal requirement and development of liriomyza sativae (diptera: agromyzidae) on cucumber. journal of economic entomology, 100(2): 350-356. johnson, m.w., welter, c., toscano, n.c., ting, i.p. & trumble, j.t. 1983: reduction of tomato leaflet photosynthesis rates by mining activity of liriomyza sativae (diptera: agromyridae). journal of economic entomology, 76: 1061 1063. matteoni, j.a. & broadbent, a.b. 1988: wounds caused by liriomyza trifolii (diptera: agromyzidae) as sites for infection of chrysanthemum by pseudomonas cichorii. canadian journal of plant pathology, 10: 4752. parrella, m.p. & keil, c.b. 1984: insect pest management: the lesson of liriomyza. bulletin of the entomological society of america, 30: 22 25. sasakawa, m. 1972: formosan agromyzidae (diptera). the scientific reports of kyoto prefectural university, agriculture, 24: 4382. spencer, k.a. 1966: a revision of european species of the genera melanagromyza hendel and hexomyza enderlein, with a supplement on the genus ophiomyia braschnikov (diptera: agromyzidae). beiträge zur entomologie, 16: 160. spencer, k.a. 1973: agromyzidae (diptera) of economic importance. series entomologica, vol. 9. dr. w. junk b.v. the hague, the netherlands. 418 pp. spencer, k.a. 1976: the agromyzidae (diptera) of fennoscandia and denmark. fauna entomologica scandinaviaca, 5 (1): 1-304. spencer, k.a. 1990: host specialization in the world agromyzidae (diptera). bulletin of the entomological society of america, 30: 22-25. spencer, k.a. 1972: diptera, agromyzidae. handbooks for the identification of british insects, 10: 1-136. zitter, t.a. & tsai, j.h. 1977: transmission of three potyviruses by the leafminer liriomyza sativae (diptera: agromyzidae). plant disease report, 61: 1025-1029. zlobin, v.v. 2005: studies on european species of the genus phytoliriomyza hendel (diptera: agromyzidae). russian entomological journal, 14(2): 119-123. anticancer compounds from medicinal plants biologica nyssana 4 (1-2)  december 2013: 87-92 devetaković, j.,et al.  variability of morphometric caracteristic of… 87 original article variability of morphometric caracteristics of one-year seedlings of different half-sib european white elm (ulmus effusa wild.) from the great war island jovana devetaković, mirjana šijačić-nikolić, vladan ivetić faculty of forestry, university of belgrade, kneza višeslava 1, 11000 belgrade, serbia * e-mail: jovana2309@gmail.com abstract: devetaković, j., šiljačić-nikolić, m., ivetić, v.: variability of morphometric caracteristics of one-year seedlings of different half-sib european white elm (ulmus effusa wild.) from the great war island. biologica nyssana, 4 (1-2), december 2013: 87-92. european white elm is recognized as a rare and endangered species in the forest fund of the republic of serbia. during the past century massive drying of elms occurred and the indications of their extinction appeared, which consequently led to a reduction in genetic diversity and the danger of genetic drift. in the area of the great war island near belgrade we found 56 trees of european white elm that are spatially divided into three subpopulations. in order to assess the genetic potential of european white elm in the great war island and to define adequate conservation measures variability of 14 selected test trees progeny was rated. results shows that the satisfactory variability within the popoulation exists, which is a good basis for the conservation of genepool available. key words: conservation, european white elm, genepool, seedlings, ulmus effusa, variability. introduction european white elm (ulmus effusa wild., syn. u. laevis pall.) is one of the three autochthonous species of familiy ulmaceae mirb. in serbia. unlike field elm (ulmus minor miller) and mountain elm (ulmus montana with.), which occupy slightly different habitats, european white elm (ulmus effusa wild.) is a species limited to the lowlands. looking outside of serbia, european white elm grows in central and eastern europe (j o v a n o v i ć 2007). during the past century massive drying of elms occurred and the indications of their extinction appeared, both in natural populations and in urban areas, which consenquently lead to the reduction of genetic diversity and the danger of genetic drift. dutch elm disease (ded) is considered to be the main cause of the often drying of these species. however, disappearance of wetlands due to drainage for agricultural production or poplar cultivation is more often considered to be the cause of european white elm drying (c o l l i n 2003). in the forest fund of the republic of serbia, european white elm is recognized as a rare and endangered species (b a n k o v i ć et al. 2009). from the perspective of conservation and directed utilization of forest genetic resources, european white elm can be considered as priority species in sence of the available genepool, and the fact that conservation areas for in situ conservation of this species in serbia do not exist, and the methods of ex situ conservation were not performed (š i j a č i ć n i k o l i ć , m i l o v a n o v i ć 2010). the variability and growth of european white elm seedlings were studied by many authors. 11 th sfses • 13-16 june 2013, vlasina lake 4 (1-2) • december 2013: 87-92 biologica nyssana 4 (1-2)  december 2013: 87-92 devetaković, j.,et al.  variability of morphometric caracteristic of… 88 b l a c k s a m u e l s s o n et al. (2003) reported existence of a strong additive genetic variation between and within populations in growth of oneyear old seedlings as response to drought stress. w i t h e l e y et al. (2003) also reported inerand intrapopulation variability of one-year old seedlings. examining of the four-year old plants of fraxinus angustifolia and ulmus laevis, c i c e k et al. (2011), reports influence of provenance and growth density on the development of ash seedlings, but lack of significant influence on european white elm seedlings. material and methods a total of 56 trees of europen white elm were found, marked and georeferenced on great war island, near belgrade, serbia (fig. 1). the trees are spatially divided into three subpopulations: fig. 1. spatial distribution of subpopulations european white elm (ulmus effusa willd.) at great war island table 1. coordinates and basic characteristics of 14 mother trees of european white elm (ulmus effusa willd.) at great war island subpopulation mark of tree on the field coordinates height (m) average diameter (cm) age e n i 13 7456215 4965040 23,1 48.7 26 14 7456215 4965038 7,7 15.65 13 32 7456352 4964955 20,2 38.3 23 ii 18 7456050 4965266 16,7 39.5 27 19 7456054 4965270 18,4 36.5 16 21 7456046 4965285 9,5 31.5 16 33 7456138 4965226 13,7 29.35 20 34 7456170 4965254 21 31.3 20 35 7456150 4965257 20,9 30.4 20 36 7456136 4965262 13,9 37.8 23 iii 30 7455319 4965639 13,5 23.5 15 31 7455137 4965585 12 23.5 17 the first subpopulation (i) consists of 24 trees located in the inshore area, the recent alluvial deposits with characteristics of β-gley, white willow forest type (salicetum albae issl.26) on the β -gley. the second subpopulation (ii) consists of 22 marked trees inhabiting the area with a very dense canopy and impervious. it extends in the internal part of the island, close to the canal galijaš. the third subpopulation (iii) consists of 10 trees individually dispersed throughout the forest edge or in a field. the second and the third subpopulation belong to the forest of white and black poplar (populetum albo-nigra slav.52) the mosaic of alluvial soil, which is the final stage of development of floodplain forests of soft deciduous trees. european white elm (ulmus effusa willd.) is seen here in the second floor of the trees, beside black poplar (populus nigra l.) and white poplar (populus alba l) edificators (š i j a č i ć n i k o l i ć , m i l o v a n o v i ć 2012). in the spring of 2011, during may, the seeds were collected from all trees where yield was recorded (tab. 1). seeds were collected in may 2011 and after a short drying during the same month were sown in seed beds, seeding density 1200 seeds/m 2 . seed beds were covered with a thin layer of the substrate made up of sand and soil in the ratio of 1:1 (fig. 2 and fig. 3). irrigation was initially done biologica nyssana 4 (1-2)  december 2013: 87-92 devetaković, j.,et al.  variability of morphometric caracteristic of… 89 fig. 2. seed beds with seed partialy covered daily until seedlings appeared, and then less frequently (every 3-4 days) (figure 4). each of the half-sib lines samples was taken from 50 one-year plants with bare-roots, their height (h) and root collar diameter (d) were measured. roller’s sturdiness coefficient, as the ratio of the height (cm) and diameter (mm), were calculated (r o l l e r 1977, i v e t i ć 2013). fig. 3. view on the seed beds sown and covered basic statistical analysis of the data collected included the calculation of mean values, standard deviations (sd) and width variation. clustering of the obtained mean values and the homogenity of groups were tested by tukey hsd test. the origin of variability was examined by analysis of variance (one-way anova). the interdependence of the morphological parameters was tested by calculating the pearson correlation coefficient. closeness (distance) of mother trees based on the above parameters was tested by cluster analysis. fig. 4. appearance of seedlings after germination fig. 5. the layout of rows of seedlings biologica nyssana 4 (1-2)  december 2013: 87-92 devetaković, j.,et al.  variability of morphometric caracteristic of… 90 table 2. mean values of height (h), diameter (d) and roller’s sturdiness coefficient (sq) (standard deviation (sd in parentheses), the limits for h, d, sq and hsd test of 14 tested maternal line (ml) n=700 h d sq ml mean values (sd) xmиn-xmax mean values (sd) xmиn-xmax mean values (sd) xmиn-xmax 13 20.55 (5.92) abc 12.3-40.2 3.25 (1.07) abc 1.47-7.05 6.52 (1.35) a 3.73-10.1 14 19.34 (5.64) ab 10.4-36.5 3.13 (1.17) abc 1.43-6.49 6.45 (1.29) a 3.98-9.5 18 23.34 (6.44) abc 12.2-41.2 3.46 (1.31) abc 1.67-6.57 7.18 (1.81) abc 3.81-10.6 19 23.83 (8.13) bc 9.3-46.8 3.42 (1.59) abc 1.30-7.69 7.52 (1.97) abc 4.24-15.8 21 18.87 (7.55) a 7.9-45.0 2.80 (1.35) a 0.88-7.33 7.12 (1.48) ab 4.40-10.4 29 21.93 (7.29) abc 11.0-43.4 3.52 (1.35) abc 1.55-6.97 6.51 (1.44) a 3.25-10.3 30 29.89 (9.33) d 13.4-55.7 3.91 (1.66) c 1.79-8.86 8.29 (2.80) c 2.84-21.8 31 21.67 (8.87) abc 8.9-47.1 2.91(1.35) ab 1.17-6.97 7.71 (1.53) bc 4.44-11.5 32 20.33 (5.88) abc 10.6-35.8 2.89 (0.96) ab 1.48-5.97 7.25 (1.50) abc 4.03-11.1 33 24.92 (7.94) c 7.0-43.7 3.73 (1.32) bc 1.31-7.84 6.87 (1.53) ab 4.52-10.9 34 24.73 (7.54) c 10.5-43.5 3.45 (1.34) abc 1.11-7.96 7.53 (1.64) abc 3.68-11.1 35 23.41 (6.27) abc 7.8-38.7 3.31 (1.04) abc 1.73-6.00 7.36 (1.98) abc 3.50-12.1 36 21.84 (5.94) abc 11.1-35.0 3.29 (0.98) abc 1.36-5.24 6.88 (1.45) ab 4.18-9.9 37 20.48 (7.56) abc 6.5-35.4 2.80 (1.35) a 0.97-6.96 7.86 (1.91) bc 3.46-11.6 all lines 22.5 (7.70) 6.5-55.7 3.28 (1.32) 0.88-8.86 7.22 (1.79) 2.84-21.8 results and discussion the highest average h (29.89 cm) and d (3.91 mm) was observed in half-sib seedlings line 30 of the parent tree (table 2). the maximum values of 55.7 cm h and d of 8.86 mm were recorded in the same line of seedlings at the same time. mean values in the same column followed by different letters are statistically different for p <0.05. according to the observed parameters seedlings from 30 lines segregate into a separate group. they also show the highest variability of observed parameters. thus, for h sd = 9.33 with variational width of 42.30 cm for d sd = 1.66 with the variation width of up to 7.07 mm, and for sq sd = 2.8 the variation width of 18.96. minimum value of 6.5 cm h and d of 0.97 mm were measured in seedlings of maternal line 37, while the minimum mean values of both measured parameters were recorded in the line 21. analysis of variance indicated that the variability of physiological parameters is the consequence of a mother tree, not a coincidence, and that the variability between seedlings of maternal lines (p = 0.00) is greater than the variability of plants within a maternal line (table 3). d and h showed a significant and strong positive correlation. sq shows a relatively strong, negative correlation with d, and a weak, but significant correlation with h. table 3. analysis of variance (one-way anova) height (h) and diameter (d) of 14 half-sib lines of european white elm between maternal lines својст во mean square f-ratio pvalue h 40721.00 7.74 0.00 d 1.67 3.48 0.00 table 4. correlations between the morphological parameters: the diameter (d), the height (h) and roller’s sturdiness coefficient (sq) of 14 half-sib lines of european white elm n=700 (casewise deletion of missing data) d h sq d 1.00000 h 0.79095* 1.00000 sq -0.48323* 0.10136* 1.00000* figure 6 shows a constant growth of sq with increasing of h. on the other hand, the value of sq decreases with growth of d, to certain, approximately mean values. after that, with the increase of d, sq begins to grow. biologica nyssana 4 (1-2)  december 2013: 87-92 devetaković, j.,et al.  variability of morphometric caracteristic of… 91 cluster analysis (fig. 7) shows clustering of examined maternal lines into three groups. the first group consists of the maternal lines 18, 19, 33, 34 and 35. the second group consists of lines 13, 14, 21, 29, 31, 32, 36 and 37. the third group consists of 30 lines, which are groups at the greatest distance with other maternal lines. the density of growth, as a result of planting density has a strong effect on the growth of seedlings. applied seeding density of 1,200 seeds per m 2 significantly exceeds the growth of 75 plants per m 2 , where c i c e k et al. (2011) found the highest values of h and d of one-year old seedlings of european white elm with bare-root. however, it did not significantly affect the sq, which is satisfactory and which is close to the results got by the listed authors. sq change with increasing d should be explored, especially in the second year of growth of seedlings. variability within the population of parameters observed is significant, as shown by the analysis of variance, and does not depend on the spatial distribution of mother trees. considering the number of trees in the reference population as an appropriate method may be recommended the in situ conservation of genetic resources. in endangered populations of european white elm, with a significantly reduced number of trees, may be recommended the ex situ conservation (a l e k s i c , o r l o v i c 2004). for the purpose of ex situ conservation, the level of variation within the population should be compared with the close spatial populations of european white elm of coastal areas of the danube and sava rivers, during their work, w h i t e l e y et al. (2003) found that studied populations differ in the level of genetic variation within the population. also, exploring the genetic variability of the border populations of european white elm, v a k k a r i et al. (2009) suggest that differences between populations follow the structure of isolation caused by distance (isolation by distance). conclusion the vulnerability of european white elm (ulmus effusa wild.) population on the great war island, is sufficiently explained by the record that only 56 trees of this species exist, and their age structure indicates the existence of different generations of trees in the population. according to the research of variability of morphometric caracteristics of one-year old seedlings, it can be concluded that the satisfactory variability within the popoulation exists, which is a good basis for the conservation of genepool available. special attention should be paid to the half-sib lines whose characteristics (height, diameter and roller’s sturdiness coefficient) provide the best quality seedlings, which in this case is the line halfsib seedlings of mother tree 30. but from the aspect of conservation and directed utilization the specific genotypes which manifested the minimum dimensions such as tree seedlings of lines 21 and 37 should not be ignored. for effective protection of genetic resources of european white elm on the great war island, in situ and ex situ methods should be combinated for a long time. in situ conservation is already partially realized, given that it is a protected nature reserve. to apply the ex situ methods it is necessary to continue monitoring of half-sib lines and establish progeny tests, in order to select appropriate trees for the production of plants and to expand the species population and conservation of her genetic variability in this area. 3d surface: h vs. d vs. sq (casewise deletion of missing data) z = distance weighted least squares 40 30 20 10 0 fig. 6. area of a combination of height (h) and diameter (d) as a predictor, as opposed to roller’s sturdiness coefficient (sq) as the dependent variable biologica nyssana 4 (1-2)  december 2013: 87-92 devetaković, j.,et al.  variability of morphometric caracteristic of… 92 t r e e d ia g r a m f o r 1 4 c a s e s s in g le l in k a g e e u c lid e a n d is t a n c e s 0 1 0 2 0 3 0 4 0 5 0 l in k a g e d is t a n c e 3 0 3 4 3 3 1 9 3 5 1 8 3 1 3 6 2 9 2 1 1 4 3 2 3 7 1 3 fig. 7. dendrogram of the cluster analysis was done on the basis of height and root collar diameter of onehalf-sib seedlings of european white elm references aleksić, j., orlović, s. 2004: ex situ conservation of genetic resources of field elm (ulmus minor mill.) and european white elm (ulmus laevis pall.). – genetika, vol. 36, no. 3, 221-227. banković, s., medarević, m., pantić, d., petrović, n., obradović, s. 2009: šumski fond republike srbije-stanje i problemi, glasnik šumarskog fakulteta, beograd, br. 100, (7-30). collin, e. 2003. euforgen technical guidelines for genetic conservation and use for european white elm (ulmus laevis). international plant genetic resources,institute, rome, italy. 6 p. black-samuelsson s., whiteley r. e., junzhan g. 2003: growth and leaf morphology response to drought stress in the riparian broadleaved tree, ulmus laevis (pall.). silvae genetica 52, 5-6. cicek e., cicek n., tilki f. 2011: four-year field performance of fraxinus angustifolia vahl. and ulmus laevis pall. seedlings grown at different nursery seedbed densities. research journal of forestry, 5: 89-98. jovanović, b. 2007: dendrologija. šumarski fakultet univerziteta u beogradu. str. 1-536ultet, beograd: 1-77. ivetić, v. 2013: praktikum iz semenarstva rasadničarstva i pošumljavanja. univerzitet u beogradu, šumarski fakultet, 213 p.. šijačić-nikolić, m., milovanović, j. 2010: konzervacija i usmereno korišćenje šumskih genetičkih resursa, šumarski fakultet univerziteta u beogradu, beograd, 1-200. šijačić-nikolić, m., milovanović, j. 2012: conservation and sustainable use of foerst genetic resources throught an example of watland ecosystems, international conference: role of research in sustainable development of agriculture and rural areas, may 23-26, podgorica, montenegro, book of abstracts, 128. whiteley, r.e., black-samuelsson, s., jansson, g. 2003: within and between population variation in adaptive traits in ulmus laevis pall., the european white elm. forest genetics 10(4):313323. stojković piperac, m. et al.  the application of the abundance/biomas... biologica nyssana 6 (1)  september 2015: 25-32 stojković-piperac, m. et al.  the application of the abundance/biomas... 25 original article received: 24 june 2015 revised: 28 june 2015 accepted: 01 july 2015 the application of the abundance/biomass comparison method on riverine fish assemblages: limits of use in lotic systems milica stojković piperac1*, djuradj milošević1, vladica simić2 1university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia 2university of kragujevac, faculty of sciences and mathematics, department of biology and ecology, radoja domanovića 12, 34000 kragujevac, serbia * e-mail: milicas@pmf.ni.ac.rs abstract: stojković piperac, m., milošević, dj., simić, v.: the application of the abundance/biomass comparison method on riverine fish assemblages: limits of use in lotic systems. biologica nyssana, 6 (1), september 2015: 25-32. fish assemblages have been widely used as ecological indicators for assessing the level of environmental degradation and ecosystem health. environmental disturbances affect the aquatic community structure in terms of abundance and biomass. therefore, we tested the utility of abundance/biomass comparison (abc) method, originally developed for marine ecosystems, to detect the anthropogenic disturbance in lotic systems using fish community data. electrofishing was conducted in the period between 2003 and 2011 at 35 sites along the southern morava river basin. the results indicated that species richness strongly influences the utility of abc method to detect the anthropogenic disturbance in lotic systems. the warwick (w) statistic showed the positive correlation and the expected direction of response with some factors defining environmental quality, applying it on the samples with greater species richness. this approach has significant power for detecting environmental quality disturbance but may be limited due to effects of habitat variability in riverine environments. key words: abundance/biomass comparison method, lotic systems, fish community, pca analysis, southern morava river basin apstrakt: stojković piperac, m., milošević, đ., simić, v.: primena metode poređenja abundance i biomase na zajednicu riba tekućih voda: ograničenja prilikom upotrebe u lotičkim ekosistemima. biologica nyssana, 6 (1), septembar 2015: 25-32. ribe predstavljaju dobro poznate ekološke indikatore u proceni kvaliteta životne sredine. poznato je da narušavanje sredine utiče na strukturu akvatičnih zajednica u smislu abundance i biomase. zbog toga, u ovom radu testirana je korisnost metode poređenja abudance i biomase (abc method, eng. abundance/biomass comparison method), inicijalno predviđene za primenu nad marinskim ekosistema, u detekciji antropogeno izazvanih promena u lotičkim ekosistemima korišćenjem podataka o zajednici riba. procedura elektroribolova sprovedena je u periodu od 2003 do 2011 godine na 35 lokaliteta raspoređenih duž sliva južne morave. rezultati ove studije ukazuju da bogatstvo vrsta u značajnoj meri utiče na korisnost 6 (1) • september 2015: 25-32 biologica nyssana 6 (1)  september 2015: 25-32 stojković piperac, m. et al.  the application of the abundance/biomas... 26 ispitivane metode u proceni intenziteta antropogenog delovanja na lotičke ekosisteme. warwick-ova (w) statistika pokazuje pozitivnu korelaciju sa faktorima koji definišu kvalitet sredine, kao i očekivani odgovor na stres, samo prilikom primene nad lokalitetima sa većim brojem vrsta. pokazano je da ova metoda ima značajnu moć u detekciji narušenja životne sredine ali sa izvesnim ograničenjima izazvanim prirodnom varijabilnošću u lotičkim ekosistemima. key words: metoda poređenja abundance i biomase, lotički sistemi, zajednice riba, pca analiza, sliv južne morave introduction anthropogenic modifications of natural hydrology have been altering freshwater ecosystems worldwide, threatening their ecological integrity (r e h a g e & t r e x l e r , 2006). anthropogenic alteration may change the hydrological patterns of the river basin (w a r d , 1998) and modify the physical characteristics of the aquatic habitat (k a r r , 1981), strongly influencing the structure and composition of the aquatic biota. the major human activities with negative impacts on aquatic communities include: human impact on habitat morphology, intensive agriculture and urbanization, construction of channels and dams, removal of snags, and pollution (j o h n s o n et al., 1995; s p a r k s , 1995; r i c h t e r et al., 1997; e i t z m a n n & p a u k e r t , 2010). considering the fish fauna, it is also necessary to emphasise the possible changes caused by over-fishing by both anglers and poachers as an important factor that may dramatically diminish fish populations (p e n c z a k & k r u k , 1999; a n t i c a m a r a et al., 2010). however, the major threat to freshwater fish is modification of aquatic environment, which may cause decline in many species (c o l l a r e s p e r e i r a & c o w x , 2004). assessing fish community structure is one of the efficient ways of evaluating the biotic integrity in rivers in different parts of the world ( k a r r , 1981; o b e r d o r f f & h u g h e s , 1992; h u g u e n y et al., 1996; g a n a s a n & h u g h e s , 1998; b r e i n e et al., 2004; s t o j k o v i ć et al, 2014). changes in fish community composition are an important factor used to characterize environmental quality, as fishes respond to changes in the aquatic environment with great sensitivity. they play an important role in aquatic ecosystems due to their dependence on both, the physical features of their environment and the other forms of aquatic life. therefore, the health of each fish assemblage reveals conditions present in the entire aquatic community (f o l t z , 1982). fig. 1. map of the sampling sites under investigation. site codes for studied streams are the same as in table 1. biologica nyssana 6 (1)  september 2015: 25-32 stojković piperac, m. et al.  the application of the abundance/biomas... 27 environmental disturbances also affect the aquatic community structure in terms of abundance and biomass, both measured by the abundance/biomass comparison (abc) method. the abc method was initially applied on marine macrobenthic communities to detect influence of the anthropogenic activities such as pollution (w a r w i c k , 1986). in addition, many studies conducted by now, stressed that this method could be also applicable to detect the effect of anthropogenic changes on freshwater biota caused by pollution (c o e c k et al., 1993), industrial plant impact (pinto et al., 2006), over-fishing (p e n c z a k & k r u k , 1999) and water and habitat disturbance (c a s a t t i et al., 2006). however, p e n c z a k & k r u k (1999) proposed a threshold regarding a minimal number of species caught in the sample when applying abc method on riverine fish assemblages. bearing all this in mind, we here aimed to test the performance of the abc method in assessing the changes in the fish community as a response to environmental degradation in lotic systems. furthermore, we tested how species richness influences the utility of abc method to detect the anthropogenic disturbance in riverine environments. material and methods study area the source of southern morava river, also known as binačka morava, is in the skopska crna gora mountains, macedonia. this river flows in the roughly northerly direction. at the 49th kilometer it coalesces with the preševska moravica and at 295th kilometer it discharges in the morava, tributary of danube and therefore part of the black sea catchment area. the southern morava river has a catchment area of 15,469 km2, of which 14,372 km2 (92.91%) are in serbia and 1,097 km2 (7.09%) in bulgaria through its right-hand tributary the river nišava. it has 157 tributaries but most of them dry out during summer. larger, permanent left-hand tributaries are jablanica, veternica, toplica and pusta reka rivers. right-hand tributaries include vlasina, nišava (the longest) and sokobanjska moravica rivers (g a v r i l o v i c & d u k i c , 2002). sampling fish fauna was sampled along the southern morava river basin in the period between 2003 and 2011. during the investigated period, out of total number of 35 sampling sites (fig. 1), 12 were sampled ones, 18 twice, 2 three and 3 four times. since each sampling occasion was considered as separate entity in data processing, the final data matrix was consisted of 66 samples. the electrofishing procedure was conducted using the dc electrofisher “aquatech” ig 1300 (2.6 kw, 80– 470 v). a more detailed sampling procedure is described in s t o j k o v i c et al. (2013). together with the fish data collection, water and habitat quality variables were measured for each sample in order to characterize the extent of stress. water quality was expressed by five variables strongly dependent on anthropogenic disturbance (dissolved oxygen (do), electro-conductivity (ec), concentrations of ammonia nitrogen (nh4), nitrate nitrogen (no3) and orthophosphates (po4)). dissolved oxygen and electro-conductivity were estimated by a wtw multi 340i probe, while the concentrations of ammonia nitrogen, nitrate nitrogen and orthophosphates were measured using the spectrophotometer shimatzu uv–vis. habitat quality was presented by three disturbance variables: hydrological alteration (ha), channel alteration (ca) and land use intensity (lu). each site was given a score of 1, 3, or 5 if slight, moderate, or severe alteration for each habitat disturbance variable was observed (s t o j k o v i ć et al., 2014). data analysis to test how species richness influences the output of the abc method, two data matrix have been constructed. the first data matrix (a) contained all samples collected during the sampling period, which finally counts 66 samples, regardless of the number of species caught. in contrast, the second data matrix (b) was constructed to contain only samples where species richness was greater than 5, as suggested by p e n c z a k & k r u k (1999), finally presenting 28 samples. according to w a r w i c k (1986), condition of an aquatic community can be illustrated by using combined k-dominance plots of abundance and biomass, where species are ranked in the order of importance on the x-axis (logarithmic scale) with percentage dominance on the y-scale (cumulative scale). the area between the two curves is called the warwick or w statistic, calculated according to the following formula (c l a r k e , 1990). w =       150 1 11     s ajbj s j i j i j , where w is the standardized sum of the differences between each pair of species’ cumulative biomass and cumulative abundance value ranked in decreasing order. the curves    i j bj 1    i j aj 1 biologica nyssana 6 (1)  september 2015: 25-32 stojković piperac, m. et al.  the application of the abundance/biomas... 28 classify the quality of the environment (m a g u r r a n , 2004) either as undisturbed (biomass curve overlaps that of abundance, w > 0) or disturbed (abundance curve overlaps that of biomass, w < 0). w ranges from -1 to +1. w statistic was calculated using the primer v6 (c l a r k e & g o r l e y , 2006), a multivariate statistical package developed at plymouth marine laboratory. since some variables in this study were presented as ordinal, categorical principal components analysis (catpca) was used to reveal the relationship between w statistic and disturbance variables. the data matrix were consisted of 9 variables, out of with 6 were numerical and 3 categorical (given in columns), measured along the 66 and 28 samples (given in rows), depending on the data matrix used in the analysis. the catpca analysis was performed using software spss version 15.0 (spss inc, chicago, il, usa). results dominance of abundance or biomass was calculated for all 66 samples. the w statistics values, observed at the majority of sites, were positive, with an exception of a few sites, mainly situated on upper river courses (tab. 1). according to this, the abc method indicated that the majority of sites were undisturbed, as represented by positive values of w statistic. nevertheless, there were several sites having the positive values the w statistic but close to zero, where both curves roughly coincided, indicating a moderately disturbed condition. table 1. description of code, stream order and w statistic value for each sample collected along the southern morava river basin. after the each site code, abbreviation for year of sampling is included river code stream order w statistic river code stream order w statistic southern morava *1-03 2 0.428 visočica ntv1-08 1 0.109 southern morava *1-10 2 -0.031 visočica ntv1-10 1 0.536 southern morava *2-03 2 0.247 visočica ntv1-11 1 0.501 southern morava *2-08 2 0.094 visočica ntv2-06 2 0.308 southern morava *3-03 3 -0.124 visočica ntv2-10 2 -0.126 southern morava *4-03 3 0.111 dojkinačka reka ntvd-08 1 0.000 southern morava *4-08 3 0.122 jelovička reka ntvj-08 1 0.280 southern morava *5-03 4 0.090 jerma nj1-10 1 0.109 southern morava *5-08 4 0.176 jerma nj1-11 1 -0.158 southern morava *6-10 4 0.200 jerma nj2-10 1 0.380 jablanica jb1-03 2 0.217 jerma nj2-11 1 0.143 jablanica jb2-03 2 0.088 pusta reka p1-10 2 0.153 sokobanjska moravica m1-10 1 -0.020 pusta reka p2-03 2 0.232 sokobanjska moravica m2-08 1 0.053 pusta reka p2-10 2 0.634 sokobanjska moravica m3-08 1 0.162 toplica t1-03 1 0.175 sokobanjska moravica m3-10 1 0.334 toplica t1-10 1 -0.205 nišava n1-10 2 0.120 toplica t2-08 2 0.180 nišava n1-11 2 0.081 toplica t2-10 2 0.256 nišava n2-10 3 0.019 toplica t3-03 2 0.144 nišava n2-11 3 0.139 toplica t3-08 2 0.284 nišava n3-03 3 0.197 toplica t4-03 2 0.201 nišava n3-08 3 0.180 toplica t4-10 2 0.257 nišava n3-10 3 0.299 banjska reka tb-08 1 0.223 nišava n3-11 3 0.242 lukovska reka tl-08 1 -0.133 nišava n4-08 3 0.022 lukovska reka tl-10 1 0.007 nišava n4-10 3 0.160 veternica ve1-10 2 0.070 nišava n4-11 2 0.253 vlasina vl1-03 1 0.158 temska nt1-03 1 -0.160 vlasina vl1-04 1 0.000 temska nt1-08 1 -0.109 vlasina vl1-08 1 0.000 temska nt1-10 1 -0.433 vlasina vl1-10 1 -0.739 temska nt1-11 1 -0.068 vlasina vl2-10 1 0.165 temska nt2-10 2 0.264 vlasina vl3-03 2 0.291 temska nt2-11 2 -0.128 vlasina vl3-08 2 0.030 biologica nyssana 6 (1)  september 2015: 25-32 stojković-piperac, m. et al.  the application of the abundance/biomas... 29 table 2. contributions of the w statistic and water and habitat quality variables to the two fist axis of the catpca and the total variance accounted for (vaf) for a) data matrix a, b) data matrix b component loadings total vaf component loadings total vaf a) axis b) axis 1 2 1 2 w -0.431 0.230 0.189 w -0.397 0.490 0.288 no3 -0.860 -0.146 0.433 no3 0.842 0.219 0.486 po4 -0.827 -0.152 0.430 po4 0.822 0.391 0.509 nh4 -0.840 0.085 0.379 nh4 0.757 -0.193 0.380 ec -0.722 -0.335 0.913 ec 0.518 0.633 1.000 do 0.893 0.064 0.462 do 0.875 -0.064 0.478 ha -0.030 0.949 0.468 ha -0.004 -0.790 0.323 ca -0.857 0.256 0.436 ca 0.953 -0.328 0.466 lu -0.844 0.157 0.377 lu 0.764 -0.352 0.449 fig. 2. catpca plots showing relationships between w statistic and stressor gradient based on a) data matrix a and b) data matrix b the catpca analysis, conducted under the data matrix a, extracted the first and the second dimensions, which explained 56.45% and 13.46% of the total variance between 66 samples, respectively. the first axis was associated with all factors, excluding factor ha. also, the first axis had high loading for all factors except for w (tab. 2, fig. 2a). the catpca plot a indicated that w statistic is positively correlated with some factors associated with the first axis that define water and habitat quality and negatively correlated with do. according to this result, positive and high values of w statistic occur when the water and habitat quality parameters indicate stress condition (fig. 2a). on the other side, in the catpca result of the data set b, the first and second axis accounted for 49.82% and 19.31% of the observed variation, respectively (fig. 2b). the catpca plot b showed that values of w statistic increase when high values of do and the decrease of water and habitat quality variables were observed. discussion the w statistics values, according to the catpca plot a, showed correlation with factors defining stress condition but in the opposite than expected direction (fig.2a). such a result could be explained as a consequence of a low number of species found at some particular sites situated on upstream reaches (p e n c z a k & k r u k , 1999) which caused unexpected negative values of w statistic. despite the premise that the undisturbed water courses should be characterized by greater species richness and diversity indices (a r g e n t et al., 2003; g a f n y et al., 2000), fish richness and biologica nyssana 6 (1)  september 2015: 25-32 stojković piperac, m. et al.  the application of the abundance/biomas... 30 diversity are strongly influenced by stream order and longitudinal heterogeneity (p l a t t s , 1979; b a r i l a et al., 1981). longitudinal zonation in stream fish assemblages revealed that species diversity increases from upstream to downstream areas due to the greater variety of habitat diversity (g o r m a n & k a r r , 1978; f o l t z , 1982; b a i n et al., 1988). likewise, as stream order increases, the richness and total number of fish specimens also increase (p l a t t s , 1979; t h o m a s & h a y e s , 2006). similar problem was detected when using the taxonomic distinctness index applied on freshwater fish (c l a r k e & w a r w i c k , 1998; b h a t & m a g u r r a n , 2006). b h a t & m a g u r r a n (2006) found that the sites on the first and second stream order fell below the confidence limits, indicating false environmental disturbance. explanation provided in that study suggested that this may be caused by the fact that total species richness and taxonomic distinctness increase with stream order (b h a t , 2004), as downstream areas contained several upstream species as well as species unique to downstream area, whereas upstream sites tended to include closely related species. another factor considered necessary to emphasize is that the total fish biomass decrease in the upstream direction (s c h l o s s e r , 1991; t h i e l et al., 1995; h i c k s , 2003) which was also reflected on negative values of w statistic at the upper river courses. in contrast, considering the second catpca plot, where upper river courses were excluded from analysis, w statistic showed the expected direction of response to the extent of human alteration. this result unambiguously confirms presumed obstacles in applying abc method on riverine fish assemblages, which are mainly caused by natural longitudinal heterogeneity in lotic systems. consequently, we believe that abc method may be a promising tool for characterization of lotic systems under stress conditions, but may be limited to the sites with greater species richness. conclusion the results of this study have shown limited effectiveness of the abc method in pinpointing effects of the disturbance at upstream sites, since the resulting values of w statistic were highly confusing and not aligned with the real picture of the fish fauna. in some instances, the real situation in the fish community was presumably hidden by influences of longitudinal heterogeneity (b h a t & m a g u r r a n , 2006). however, we stressed that this approach has significant power for detecting environmental quality disturbance but may be limited due to effects of habitat variability in riverine ecosystems. we feel that further research should be undertaken to test the sensitivity of abc method to recognize the influence of some socioeconomic factors on riverine fish assemblages, such as effects of commercial and recreational fisheries. acknowledgements. this study was supported by funding from the ministry of education and science, republic of serbia (grant #43002; biosensing technologies and global system for long-term research and integrated management of ecosystems). references anticamara, j.a., zeller, d., vincent, a.c.j. 2010: spatial and temporal variation of abundance, biomass and diversity within marine reserves in the philippines. diversity and distributions, 16: 529-536. argent, d.g., bishop, j.a., stauffer, j.r. 2003: predicting freshwater fish distributions using landscape-level variables. fisheries research, 60: 17-32. bain, m.b., finn, j.t., booke, h.e. 1988: streamflow regulation and fish community structure. ecology, 69: 382-392. barila, t.y., williams, r.d., stauffer, j.r. 1981: the influence of stream order and selected stream bed parameters on fish diversity in raystown branch, susquehanna river drainage, pennsylvania. journal of applied ecology, 18: 125-131. bhat, a. 2004: patterns in the distribution of freshwater fishes in rivers of central western ghats, india and their associations with environmental gradients. hydrobiologia, 529: 83-97. bhat, a., magurran, a.e. 2006: taxonomic distinctness in a linear system: a test using a tropical freshwater fish assemblage. ecography, 29: 104-110. breine, j., simoens, i., goethals, p., quataert, p., ercken, d., van liefferinghe, c., belpaire, c. 2004: a fish-based index of biotic integrity for upstream brooks in flanders (belgium). hydrobiologia, 522: 133-148. casatti, l., langeani, f., ferreira, c.p. 2006: effects of physical habitat degradation on the stream fish assemblage structure in a pasture region. environmental management, 38: 974982. clarke, k., gorley, r., 2006: primer v6. user manual/tutorial. plymouth routine in mulitvariate ecological research. plymouth marine laboratory. biologica nyssana 6 (1)  september 2015: 25-32 stojković-piperac, m. et al.  the application of the abundance/biomas... 31 clarke, k., warwick, r. 1998: a taxonomic distinctness index and its statistical properties. journal of applied ecology, 35: 523-531. clarke, k.r. 1990: comparisons of dominance curves* 1. journal of experimental marine biology and ecology, 138: 143-157. coeck, j., vandelannoote, a., yseboodt, r., verheyen, r. 1993: use of the abundance/biomass method for comparison of fish communities in regulated and unregulated lowland rivers in belgium. regulated rivers: research & management, 8: 73-82. collares pereira, m., cowx, i. 2004: the role of catchment scale environmental management in freshwater fish conservation. fisheries management and ecology, 11: 303-312. dash, m.c. 2001: fundamentals of ecology. tata mcgraw-hill, new delhi. 557 p. dias, a.m., tejerina-garro, f.l. 2010: changes in the structure of fish assemblages in streams along an undisturbed-impacted gradient, upper paraná river basin, central brazil. neotropical ichthyology, 8: 587-598. eitzmann, j.l., paukert, c.p. 2010: longitudinal differences in habitat complexity and fish assemblage structure of a great plains river. american midland naturalist, 163: 14-32. foltz, j.w. 1982: fish species diversity and abundance in relation to stream habitat characteristics. in: sweeney, j.r. (ed.), proceedings of the thirty-sixth annual conference of the southeastern association of fish and wildlife agencies, seafwa, jacksonville. 305-311 p. gafny, s., goren, m., gasith, a. 2000: habitat condition and fish assemblage structure in a coastal mediterranean stream (yarqon, israel) receiving domestic effluent. hydrobiologia, 422: 319-330. ganasan, v., hughes, r.m. 1998: application of an index of biological integrity (ibi) to fish assemblages of the rivers khan and kshipra (madhya pradesh), india. freshwater biology, 40: 367-383. gavrilovic, l., dukic, d. 2002: reke srbije, zavod za udzbenike i nastavna sredstva, beograd. 218 p. gorman, o.t., karr, j.r. 1978: habitat structure and stream fish communities. ecology, 59: 507515. hicks, b.j. 2003: distribution and abundance of fish and crayfish in a waikato stream in relation to basin area. new zealand journal of zoology, 30: 149-160. hugueny, b., camara, s., samoura, b., magassouba, m. 1996: applying an index of biotic integrity based on fish assemblages in a west african river. hydrobiologia, 331: 71-78. johnson, b.l., richardson, w.b., naimo, t.j. 1995: past, present, and future concepts in large river ecology. bioscience, 45: 134-141. karr, j.r. 1981: assessment of biotic integrity using fish communities. fisheries, 6: 21-27. magurran, a.e. 2004: measuring biological diversity, wiley-blackwell, oxford. 256 p. oberdorff, t., hughes, r. 1992: modification of an index of biotic integrity based on fish assemblages to characterize rivers of the seine basin, france. hydrobiologia, 228: 117-130. penczak, t., kruk, a. 1999: applicability of the abundance/biomass comparison method for detecting human impacts on fish populations in the pilica river, poland. fisheries research, 39: 229-240. pinto, b.c.t., peixoto, m.g., araújo, f.g. 2006: effects of the proximity from an industrial plant on fish assemblages in the rio paraíba do sul, southeastern brazil. neotropical ichthyology, 4: 269-278. platts, w.s. 1979: relationships among stream order, fish populations, and aquatic geomorphology in an idaho river drainage. fisheries, 4: 5-9. rehage, j.s., trexler, j.c. 2006: assessing the net effect of anthropogenic disturbance on aquatic communities in wetlands: community structure relative to distance from canals. hydrobiologia, 569: 359-373. richter, b.d., braun, d.p., mendelson, m.a., master, l.l. 1997: threats to imperiled freshwater fauna. conservation biology, 11: 1081-1093. schlosser, i.j. 1991: stream fish ecology: a landscape perspective. bioscience, 41: 704-712. sparks, r.e. 1995: need for ecosystem management of large rivers and their floodplains. bioscience, 45: 168-182. stojkovic m., simic v., milosevic dj., mancev d., penczak t. 2013: visualization of fish community distribution patterns using the selforganizing ap: a case study of the great morava river system (serbia). ecological modeling, 248: 20-29. stojković m., milošević dj., simić s., simić v. 2014: using a fish-based model to assess the ecological status of lotic systems in serbia. water resources management, 28: 4615-4629. thiel, r., sepulveda, a., kafemann, r., nellen, w. 1995: environmental factors as forces structuring the fish community of the elbe estuary. journal of fish biology, 46: 47-69. thomas, d.a., hayes, d.b. 2006: a comparison of biologica nyssana 6 (1)  september 2015: 25-32 stojković piperac, m. et al.  the application of the abundance/biomas... 32 fish community composition of headwater and adventitious streams in a coldwater river system. journal of freshwater ecology, 21: 265-275. ward, j. 1998: riverine landscapes: biodiversity patterns, disturbance regimes, and aquatic conservation. biological conservation, 83: 269278. warwick, r. 1986: a new method for detecting pollution effects on marine macrobenthic communities. marine biology, 92: 557-562. anticancer compounds from medicinal plants biologica nyssana 4 (1-2)  december 2013: 81-85 stamenković, j. et al.  optimization of hplc method for the… 81 original article optimization of hplc method for the isolation of hypericum perforatum l. methanol extract jelena stamenković, ivana radojković, aleksandra đorđević, olga jovanović, goran petrović, gordana stojanović department of chemistry, faculty of science and mathematics, university of niš, serbia * e-mail: jelena.stamenkovic@pmf.edu.rs abstract: stamenković, j., radojković, i., đorđević, a., jovanović, o., petrović, g., stojanović, g.: optimization of hplc method for the isolation of hypericum perforatum l. methanol extract. biologica nyssana, 4 (12), december 2013: 81-85. st. john's wort (hypericum perforatum l.) is one of the most studied plant species in the family hypericaceae. the aim of this study was the identification of the constituents of methanol extract of h. perforatum and optimization of conditions for their isolation. the main components of the methanol extract were isolated on preparative zorbax eclipse xdb c18 column with solvent system consisting of methanol and 1x10 -2 m ammonium acetate in water. constituents of the extract were identified by comparing their retention times with the retention times of the standards, with the literature data and the uv spectra. by varying the conditions of chromatography, the optimal conditions for isolation of the methanol extract constituents were determined: mobile phase consisting of methanol and 1x10 -2 m ammonium acetate in water in ratio 1 : 1, sample concentration 100 mg/ml, sample volume 30 µl, flow 2 ml/min. under these conditions 7 components of the methanol extract were isolated. key words: hypericum perforatum l., methanol extract, isolation, preparative hplc . introduction st. john’s wort (hypericum perforatum) is a herbaceous perennial plant of the hypericaceae family, which is distributed in europe, asia, and northern africa, and naturalized in the us. the plant grows approximately one metre high with opposite and paired branches. the leaves are opposite and sessile, up to 2 cm long, oblong and contain numerous translucent glandular dots, which are visible against light. the yellow flowers contain 5 petals with many stamens protruding. these flowers contain a group of reddish fluorescent dianthrone pigments with biological activity (v a t t i k u t i & c i d d i , 2005). hypericum species are medicinal plants known as healing herbs due to their various medicinal properties for the last two hundred years. oily hypericum preparations may be applied externally to treat minor burns, wounds, inflammation of the skin, and nerve pain (b l u m e n t h a l et al., 1998). internally, the herbal preparation is indicated for the treatment of anxiety and depression. st john’s wort (hypericum perforatum l.) has a chemical composition well studied: anthraquinone derivatives naphthodianthrones – hypericin, pseudohypericin and isohypericin, protohypericin and protopseudohypericin (biosynthetic precursors of hypericin and pseudohypericin, respectively); flavonoids – flavonols (kaempferol, quercetin), flavones (luteolin) and glycosides (hyperoside, isoquercitrin, quercitrin, and rutin), biflavonoids including biapigenin (a flavone) and amentoflavone (a biapigenin derivative) and catechins (flavonoids 11 th sfses • 13-16 june 2013, vlasina lake 4 (1-2) • december 2013: 81-85 biologica nyssana 4 (1-2)  december 2013: 81-85 stamenković, j. et al.  optimization of hplc method for the… 82 often associated with condensed tannins); prenylated phloroglucinols – hyperforin and adhyperforin; tannins; procyanidins (condensed type); other phenols – caffeic, chlorogenic, pcoumaric, ferulic, p-hydroxybenzoic and vanillic acids; volatile oils, carotenoids, choline, β-sitosterol etc. (c r e t u et al., 2011). naphthodianthrones, namely hypericin and pseudohypericin, are found in the flowering portions of the plant. until recently, these two compounds were considered responsible for the purported antidepressant effect of hypericum. numerous flavonoid compounds, including hyperoside, quercitrin, isoquercitrin, rutin, quercetin, campferol, luteolin, and myricetin, as well as biflavonoids i3,ii8-biapigenin and i3′,ii8-biapigenin (amentoflavone) are found in the aboveground portions of the plant, including the leaves, stalk, flowers, and buds. phloroglucinol compounds, including hyperforin and adhyperforin, are present in the flowers and buds (g r e e s o n et al., 2001). several methods for the analysis of h. perforatum by tlc, photometry, hplc, hplc-ms and hplc-nmr have been reported, but most of these focus on the determination of naphthodianthrones and/or hyperforin only (gray et al., 2000; liu et al., 2000; mulinacci et al., 1999). the aim of this study was to develop the new simple and efficient method for isolation and identification of the hypericum perforatum l. methanol extract main constituents. for this purpose, the following methods were used: maceration of dried plant material with methanol to obtain the extract; high-pressure chromatography (hplc) method for the isolation, identification and determination of purity of isolated compounds. materials and methods plant material was collected in the vicinity of leskovac in july 2011. voucher specimen is deposited at the herbarium of the department of biology and ecology, faculty of sciences and mathematics, university of niš. naphthodianthrones and hyperforin are known to be thermolabile and especially photosensitive; thus, the extraction process was performed at room temperature and protected from the light (w i r z et al., 2001; o r t h et al., 1999). dry aerial part of the plant was chopped and powdered. 60 g of dry material was transferred in a dark bottle and submeged in 600 ml (1:10, v/v) methanol (sigma aldrich, p.a.), and left in the dark for 48 hours. the methanol extract was filtered through filter paper (white bar) into the premeasured flask. methanol was evaporated under reduced pressure in a rotary evaporator and 12.10 g of dry extract was obtained. re-extraction of the previously treated material was performed with another 500 ml of methanol. after 5 days, the methanol extract was again filtered through filter paper (white bar) into the same flask. the extract was evaporated and 16.28 g of dry extract was obtained in whole after repeated extraction. the dry extract was extracted with 100 ml of hexane (sigma-aldrich, p.a.) in the ultrasonic bath for 30 min. the crude hexane extract was evaporated to dryness at reduced pressure by rotoevaporation and 0.28 g of dry material was obtained. the respective yields were calculated as percentage using the formula: (crude extract weight/plant material weight) x 100 and were: 20.16%, 27.13% and 1.72% respectively. for hplc analysis a weighed amount of extract was dissolved in hplc grade methanol. all samples were filtered through a 0.45 μm filter before undertaking hplc analysis. each sample solution was injected in triplicate and standard deviations were below 2.2 % for all experiments, indicating that the method is precise and reproducible. hplc analysis was performed on hplc chromatography with dad detector (hplc system 1200 series, agilent technologies, usa). separation and purification of the extract components was performed on zorbax eclipse xdb c18 column with dimensions (5 μm, 150 mm × 4.6 mm, analytical and 5 μm, 250 mm x 9.4 mm, semipreparative). solvent system consisting of methanol and 1x10 -2 m ammonium acetate in water was used as the mobile phase in isocratic and gradient mode. the ratio of the solvents was varied in order to determine the optimum conditions for separation. all experiments were carried out at room temperature (25±2°c). chromatograms were recorded at different wavelengths. the constituents of the extract were identified by comparing their retention times with the retention times of standards, based on literature data and comparing the uv spectra. fractions were isolated and purified using the fraction collector. results and discussion there are numerous literature data on the hplc analysis of different extracts h. perforatum l. (b r o l i s et al., 1998; r o e m p p et al., 2004). the fact common to all of the researched methods is that thay were developed primarily for analytical biologica nyssana 4 (1-2)  december 2013: 81-85 stamenković, j. et al.  optimization of hplc method for the… 83 purposes in order to achieve a better separation of the constituents of the extract (l i & f i t z l o f f , 2001). described methods are based on the usage of the system with different solvents in gradient and isocratic mode. therefore they are not suitable to be applied in preparative purposes primarily due to long duration of analysis. in order to determine the optimal conditions for isolating components, preliminary investigations were carried out on the analytical column, varying the amount of injected sample, eluent polarity and flow rate of mobile phase. work on the analytical column was carried out in isocratic and gradient mode and chromatograms were recorded at different wavelengths. the methanol extract first was eluated on the analytical column under isocratic conditions. the mobile phase was run at flow rate of 0.5 ml/min and consisted of methanol : water (1 % hcooh) = 80 : 20.5 µl of sample (concentration 5 mg/ml) was injected in all experiments performed on analytical column. since the separation in isocratic mode was not obtained in satisfactory manner, gradient elution was performed by using the following solvent gradient: 0-5 min – 50 % meoh; 5-10 min – 70 % meoh; 10-30 min – 90 % meoh. the flow-rate was kept at 0.5 ml/min. as gradient conditions did not provide good results, the possibility of using isocratic method with the changed polarity of the mobile phase was tested. a mixture of methanol : water (1x10 -2 m ch3coonh4) = 50 : 50 was used as mobile phase. the resulting chromatogram is shown in fig. 1. fig. 1. hplc chromatogram obtained in isocratic conditions, methanol : water (1x10 -2 m ch3coonh4) = 50 : 50. fig. 2. hplc chromatogram with the sample volume of 20 µl, at a flow rate of 2 ml/min. this system was the basis for the transition from the analytical to semipreparative column, by varying the flow rate and injected sample volume, in order to obtain optimal results. all samples that were injected to semipreparative column were significantly more concentrated (100 mg/ml), aimed to increase method efficiency. fig. 2 shows the chromatogram of hplc analysis that was performed on semipreparative column with the optimal composition of mobile biologica nyssana 4 (1-2)  december 2013: 81-85 stamenković, j. et al.  optimization of hplc method for the… 84 phase (methanol and 1x10 -2 m ammonium acetate in water, ratio (1:1). this system was chosen because of good separation and short runtime. injection volume of the sample was 20 µl, with a flow rate of 2 ml/min. the peaks in the hplc chromatogram were identified based on literature data (hansen et al.,1999), by comparing the retention time and uv spectra (hillwing et al., 2008) with reference standards. fig. 3. hplc chromatogram with the sample volume of 30 µl, at a flow rate of 2 ml/min. table 1. retention time of identified and isolated compounds, mass of isolated compounds and their purity. no. compound rt (min) isolated mass (mg) purity % 1. chlorogenic acid 4.033 / / 2. gallic acid 4.213 / / 3. transp-coumaric acid 4.535 / / 4. unidentified compound 1 5.600 1.4 87.65 5. rutin 7.425 / / 6. hyperoside 7.425 / / 7. isoquercitrin 7.631 16.8 79.84 8. quercitrin 8.467 / / 9. quercetin 9.444 5.5 92.71 10. pseudohypericin 10.634 0.8 96.66 11. hypericin 12.860 1.0 97.12 12. unidentified compound 2 16.464 0.9 97.43 13. hyperforin 20.974 1.4 98.26 table 1 shows the retention times of identified and isolated components, mass of isolated components and their purity, based on the integral of the peak areas. the purity was determined by the integration of hplc spectra of the isolated compounds and it was directly proportional to the achieved resolution. in order to increase method efficiency, 40 μl of sample was injected, but this led to the reduced resolution. as a compromise solution, 30 μl of sample volume was injected at the same flow rate of 2 ml/min (fig. 3). based on obtained chromatograms, it was concluded that the best results for the separation and isolation of constituents of h. perforatum l. methanol extract on the semipreparative column, were obteined by injecting the volume of 30 µl of the sample (concentration 100 mg/ml) at a flow rate of 2 ml/min. biologica nyssana 4 (1-2)  december 2013: 81-85 stamenković, j. et al.  optimization of hplc method for the… 85 conclusion the results showed that the optimal conditions for isolation of the constituents of hypericum perforatum l. methanol extract on the semipreparative column were: mobile phase methanol : water (1x10 -2 m ch3coonh4) = 50 : 50, sample concentration 100 mg/ml, injection sample volume 30 µl and the flow rate of 2 ml/min. seven compounds were isolated from the extract with purity : unidentified compound (peak 4) 87.65 %, isoquercitrin (peak 7) 79.84 %, quercetin (peak 9) 92.71 %, pseudohypericin (peak 10) 96.66 %, hypericin (peak 11), 97.12 %, unidentified compound (peak 12) 97.43 %, hyperforin (peak 13) 98.26 %. according to obtained results a new simple isocratic hplc method was developed, allowing the baseline separation of thirteen main hypericum perforatum standard compounds in less than 25 min, which means an improved separation 25-50 min less than other published systems. acknowledgements. the results obtained in this study are part of the research project 172047, funded by ministry of education, science and technological development of serbia. references blumenthal, m., busse, w.r., goldberg, a., grüenwald, j., hall, t., riggins, c.w., rister, r.s.(eds), 1998: the complete german commission e monographs: therapeutic guide to herbal medicines. american botanical council, boston. brolis, m., gabetta, b., fuzzati, n., pace, r., panzeri, f., peterlongo, f. 1998: identification by high-performance liquid chromatographydiode array detection-mass spectrometry and quantification by high-performance liquidchromatography-uv absorbance detection of active constituents of hypericum perforatum. journal of chromatography a, 825: 9–16. cretu, r., mihailescu, r., mitroi, g., iacob, e., verdes, r., tebrencu, c. 2011: phytochemical investigation of crataegi folium cum flos (hawthorn leaves and flowers) and hyperici herba (st. johns wort aerial parts) hydroalcoholic extracts., annals of the ''alexandru ioan cuza'' university sect.ii a. genetics and molecular biology, 12 (4): 133-138. gray, d.e., rottinghaus, g.e., garrett, h.e., pallardy, s.g. 2000: simultaneous determination of the predominant hyperforins and hypericins in st. john's wort (hypericum perforatum l.) by liquid chromatography. journal of aoac international, 83 (4): 944-949. greeson, j. m., sanford, b., monti, d.a. 2001: st. john's wort (hypericum perforatum): a review of the current pharmacological, toxicological, and clinical literature. psychopharmacology, 153 (4): 402-414. hansen, s.h.,. jensen, a., cornett, c., bjørnsdottir, i., taylor, s., wright, b., wilson, i.d. 1999: high-performance liquid chromatography online coupled to high-field nmr and mass spectrometry for structure elucidation of constituents of hypericum perforatum l. analytical chemistry, 71 (22): 5235–5241. hillwig, m.l., hammer, k.d.p., birt, d.f., wurtele, e.s. 2008: characterizing the metabolic fingerprint and anti-inflammatory activity of hypericum gentianoides. journal of agricultural and food chemistry, 56 (12): 4359–4366. li, w., fitzloff, j.f. 2001: high performance liquid chromatographic analysis of st. john’s wort with photodiode array detection. journal of chromatography, 765 (1): 99–105. liu, f.f., ang, c.y., heinze, t.m., rankin, j.d., beger, r.d., freeman, j.p., lay, j.o. 2000: evaluation of major active components in st. john’s wort dietary supplements by highperformance liquid chromatography with photodiode array detection and electrospray mass spectrometric confirmation. journal of chromatography a, 888 (1–2): 85–92. mulinacci, n., bardazzi, c., romani, a., pinelli, p., vincieri, f.f., costantini, a. 1999: hplc-dad and tlc-densitometry for quantification of hypericin in hypericum perforatum l. extracts. chromatographia, 49 (3-4): 197-201. orth, h.c.j., rentel, c., schmidt, p.c. 1999: isolation, purity analysis and stability of hyperforin as a standard material from hypericum perforatum l. journal of pharmacy and phamacology, 51: 193-200. römpp h., seger c., kaiser c.s., haslinger e., schmidt p.c. 2004: enrichment of hyperforin from st. john’s wort (hypericum perforatum) by pilot-scale supercritical carbon dioxide extraction. european journal of pharmaceutical sciences, 21: 443-451. vattikuti u.m.r., ciddi, v. 2005: an overview on hypericum perforatum linn. natural product radiance, 4 (5): 368-381. http://www.ncbi.nlm.nih.gov/pubmed?term=gray%20de%5bauthor%5d&cauthor=true&cauthor_uid=10995119 http://www.ncbi.nlm.nih.gov/pubmed?term=rottinghaus%20ge%5bauthor%5d&cauthor=true&cauthor_uid=10995119 http://www.ncbi.nlm.nih.gov/pubmed?term=garrett%20he%5bauthor%5d&cauthor=true&cauthor_uid=10995119 http://www.ncbi.nlm.nih.gov/pubmed?term=pallardy%20sg%5bauthor%5d&cauthor=true&cauthor_uid=10995119 dimitrov, d.s.: floristic and habitat diversity of dospat dere in the dabrash mountain (west rhodopes), bulgaria. biologica nyssana, 8 (1), september 2017 biologica nyssana 8 (1)  september 2017: 39-45 dimitrov, d.  floristic and habitat diversity of dospat dere… 39 original article received: 09 april 2017 revised: 17 july 2017 accepted: 02 september 2017 floristic and habitat diversity of dospat dere in the dabrash mountain (west rhodopes), bulgaria dimitar s. dimitrov national natural history museum, 1 tsar osvoboditel blvd., sofia 1000, bulgaria * e-mail: dimitrov.npm@gmail.com abstract: dimitrov, d.s.: floristic and habitat diversity of dospat dere in the dabrash mountain (west rhodopes), bulgaria. biologica nyssana, 8 (1), september 2017: 39-45. a research has been done of the flora and natural habitats of the dospat dere area, which is located on the right bank of the dospat river, between the tuhovitsa and zhizhevo villages, next to the border with greece. the systematic specter of the flora contains 197 species of vascular plants (excluding the moss species). these vascular plants are referred to 143 genera and 57 families. as a result of this research, 5 habitats were established: 10 е1 pseudo-steppe with grasses and annual of the thero-brachypodietea, 08h3 calcareous rocky slopes with chasmophytic vegetation, 21g1 supra mediterranean hop-hornbeam woods, 02g1 southern helleno-balkanic swamp alder woods and 07g1 helleno-balkanic riparian plane forests. key words: floral analysis, floristic elements, natural habitats apstrakt: dimitrov, d.s.: floristički i stanišni diverzitet dospat derea na planini dabraš (zapadni rodopi), bugarska. biologica nyssana, 8 (1), septembar 2017: 39-45. izvršeno je istraživanje flore i prirodnih staništa oblasti dospat dere, koja se nalazi na desnoj obali reke dospat, između sela tuhovica i žiževo, pored granice sa grčkom. sistematski spektar flore sadrži 197 vrsta vaskularnih biljaka (sa izuzećem mahovina). ove vaskularne biljke se svrstavaju u 143 roda i 57 familija. kao rezultat ovog istraživanja, utvrđeno je 5 staništa: 10 e1 psedostepe sa travama i jednogodišnjim biljkama therobrachypodietea, 08h3 krečnjačke stene sa hazmofitskom vegetacijom, 21g1 supramediteranske šume crnog graba, 02g1 južne grčko-balkanske močvarne šume jove i 07g1 grčko-balkanske riparijalne ravničarske šume. ključne reči: floristička analiza, florni elementi, prirodna staništa introduction the dabrash mountain is the westernmost part of the rhodopes mountain range, which happens to be the oldest piece of land on the balkan peninsula. its highest peak is located in the northern part of this area: beslet peak (1937 m height). its geological makeup is rather variable. there are predominantly silicate rocks: granite and granite gneiss. however, there are also basic and ultrabasic rocks, especially in the southern part, where the investigated area is located. the rocks are marble dating back to the 8 (1) • september 2017: 39-45 doi: 10.5281/zenodo.963435 biologica nyssana 8 (1)  september 2017: 39-45 dimitrov, d.  floristic and habitat diversity of dospat dere… 40 proterozoic eon (s t e f a n o v , 1981). the highest peaks here are pobit kamak 1086 m and chukata 1010 m (fig. 1). there are several caves in this area: mecha dupka and gargina dupka. the climate is transitional continental, since the valley of the dospat river follows a meridional direction and thus allows for strong mediterranean climate influence. the average annual temperature here is 10 °с. the average temperature in january is between 0 °с and 7 °с. there are predominantly autumn-winter and spring-summer rainfalls. the composition of the rocks is similar to the one in northern and southern pirin, stargach, slavyanka and trigradsko zhdrelo, which of course has an impact on the flora in this area. until now, the flora of the dabrash mountain has not been researched specifically in details. there are some brief notifications about some new and rare species for the bulgarian flora (k i t a n o v , 1936) and also about some new localities of macedonian pine (j o r d a n o v , 1939, к i t a n o v , 1939b). there are new chorological data for 25 new species of the floristic area in the west rhodopes (dabrash) which are reported by d i m i t r o v & v u t o v (2016). material and methods the researched area is 1.35 кm2. the species determination was done according to: j o r d a n o v (1963-1979), v e l c h e v (1982-1989), k o z u h a r o v (1992), d e l i p a v l o v & c h e s h m e d i j e v (2004), p e e v (2012), t u t i n et al. (1964-1980). the transect and trial sites methods were used for the determination of the vegetation in this case. the habitats were determined according to b i s s e r k o v et al. (2015). the geoelemental makeup was analyzed according to a s s y o v & p e t r o v a (2012). the species` category with conservation status was determined according to p e t r o v a & v l a d i m i r o v (2009). the protected species` status is based on the law for biological diversity of the republic of bulgaria. the herbarium specimens have been deposited in the herbarium of the institute of biodiversity and eco systematic research (som). results and discussion the systematic specter of the flora of dospat dere is comprised mainly with specimens from phylum magnoliophyta – 52 families (91.2%), 137 genera (90.6%) and 188 species (95.4%). from class dicotyledonae – 45 families (78.8%), 109 genera (76.2%), and 152 species (77.1%). from class monocotyledonae – 7 families (12.2%), 28 genera (19.5%) and 36 species (18.2%). from phylum gymnospermae there are 2 families (3.5%), 2 genera (1.3%) and 4 species (2.03%). from phylum pteridophyta – 3 families (5.2%), 4 genera (2.7%) and 5 species (2.5%). out of the examined families, the ones with the biggest number of species are: fabaceae (23 species; 11.6% of the total number of species), poaceae (17; 8.6%), asteraceae (15; 7.6%), caryophyllaceae (14; 7.1%) and lamiaceae (11; 5.5%). other families contain less than 10 species: apiaceae, rosaceae, scrophulariaceae (7; 3.5%), orchidaceae (6; 3.0%), liliaceae, brassicaceae, cyperaceae (5; 2.5%), campanulaceae, dipsacaceae, linaceae (4; 2.03%), polygalaceae, ranunculaceae, aspleniaceae, betulaceae (3; 1.5%), juniperaceae, pinaceae, boraginaceae, cornaceae, cistaceae, crassulaceae, euphorbiaceae, geraniaceae, hyperica fig. 1 – a geographical position of the investigated territory in western rhodopes (tourist map, domino, stara zagora, isbn 978-954-651-2055, m= 1:100 000) and on the balkan peninsula (down left) biologica nyssana 8 (1)  september 2017: 39-45 dimitrov, d.  floristic and habitat diversity of dospat dere… 41 ceae, loranthaceae, oleaceae, orobanchaceae, rubiaceae (2; 1.02%), polypodiaceae, equisetaceae, aceraceae, amarillidaceae, anacardiaceae, asclepiadaceae, cannabaceae, convolvulaceae, dioscoreaceae, fagaceae, globulariaceae, iridaceae, plantaginaceae, platanaceae, plumbaginaceae, polygonaceae, rutaceae, salicaceae, santalaceae, thymeleaceae, ulmaceae, urticaceae, valerianaceae, violaceae (1; 0.5%). according to biological type, there are predominately perennial herbaceous species (120 species; 60.8% of the total number of species), followed by annual species (29; 10.1%), arboreal species (10; 5.07%), bushes (10; 5.07%), annual to biennial species (9; 4.51%), semi-bushes (7; 3.5%), biennial (4; 2.02%), biennial to perennial (4; 2.07%) and annual to perennial species (4; 2.07%). based on the conducted research, the following floristic elements were determined: there are predominantly mediterranean elements (34 species; 17.7% of the total number of species), followed by euro-asiatic (23; 11.6%), euromediterranean (19; 9.6%), pontic-mediterranean (16; 8.1%), mediterranean (14; 7.1%), balkan endemics (10; 5.07%), euro-siberian (9; 4.5%), boreal, european (8; 4.5%), subboreal (6; 3.4%), euro-submediterranean elements (5; 2.53%), subpontic and pontic elements (4; 2.03%), mediterano-central asiatic, apennino-balcanic and balcano-anatolian (3; 1.5%), submediterranoasiatic, euro-central asiatic, mediterrano-asiatic, euro-orientalo turanian, subbalcanic, kosmopolitian (2; 1.01%), mediterano-orientalo turanian, ponto-submediterranean, ponto-balcanic, alpo-mediterranean, south siberian, bulgarian, carpato-balcanic and ponto-submediterranean elements (1; 0.5%). the genera represented by the biggest number of species are: trifolium, silene: (5 species; 2.5% of the total number of species), carex, scabiosa, vicia, linum (4; 2.03%), scorzonera, dianthus, astragalus, coronilla, ajuga, festuca, polygala, veronica (3; 1.5%). the floristic complex of the investigated flora includes 1 bulgarian endemite cerastium velenovskyi and 13 balkan endemites: anthemis macedonica, chondrilla urumoffii, onosma thracica, trachelium rumelianum, dianthus cruentus, dianthus drenovskyanus, scabiosa triniifolia, hypericum umbellatum, micromeria dalmatica, linum thracicum, orobanche esulae, festuca penzesii and silene frivaldszkyana. there are also 20 tertiary relics, some of them being: trachelium rumelianum, chondrilla urumoffii, onosma thracica, festuca penzesii. the research has found 15 species with conservation status: galanthus elwesii hook f. en b1ab(ii,iii,iv,v)+2ab(ii); c2a(i) (e v s t a t i e v a , 2009), bda, annex 2, 3. chondrilla urumoffii degen vu b2ab(ii,iii) (d i m i t r o v a , 2009), bda, annex 3 trachelium rumelianum hampe vu b1ab(ii,iii,iv) (g o r a n o v a & a n c h e v , 2009), bda, annex 2, 3. romulea bulbocodium (l.) sebast. & mauri vu b2ab(ii,iv); c2a(i) (m e s h i n e v , 2009), bda, annex 2, 3. limodorum abortivum (l.) schwartz vu b2b(iv)c(iv) (p e t r o v a , 2009), bda, annex 2, 3. ophrys cornuta steven vu b2b(ii,iv)c(iv) (p e t r o v a 2009), bda, annex 2, 3. lotus aegaeus (griseb.) boiss. lc (s o p o t l i e v a , 2009) arabis collina ten. bda, annex 2, 3. trinia glauca (l.) dumort. ssp. carniolica (a. kerner ex janchen) h. wolff. bda, annex 2. veronica multifida l. bda, annex 2, 3. dianthus drenowskyanus rech. f. bda, annex 3. lilium martagon l. bda, annex 4 asparagus tenuifolius lam., bda, annex 4. stipa tirsa steven, bda, annex 4. pulsatilla montana (hoppe) rchb. bda, annex 4. out of these species, 7 are protected by the law for biological diversity of the republic of bulgaria (annex 2, 3): galanthus elwesii hook. f., trachelium rumelianum hampe, romulea bulbocodium (l.) sebast. & mauri, limodorum abortivum (l.) schwartz, ophrys cornuta steven, arabis collina ten. and veronica multifida l. one species trinia glauca (l.) dumort. ssp. carniolica (a.kerner ex janchen) h.wolff is reported in annex 2. two species chondrilla urumoffii degen and dianthus drenowskyanus rech. f. are in annex 3 and 4 species lilium martagon l., asparagus tenuifolius lam., stipa tirsa steven and pulsatilla montana (hoppe) rchb. are in annex 4. three species from the researched flora are reported in the red data book of the republic of bulgaria, v.1 plants and fungi (p e e v et al., 2015): dianthus drenovskyanus rech. f. cr b2b(ii,v)c(ii,iv); d (d e n c h e v & a s s y o v , 2015), chondrilla urumoffii degen vu b2ab(ii,iii) (d i m i t r o v a , 2015) and trachelium rumelianum hampe vu b1ab (ii,iii,iv) (g o r a n o v a & a n c h e v , 2015). six species are also under the protection of the cites convention: cephalanthera rubra (l.) rich., epipactis atrorubens (hoffm.) besser, limodorum biologica nyssana 8 (1)  september 2017: 39-45 dimitrov, d.  floristic and habitat diversity of dospat dere… 42 abortivum (l.) schwarz, ophrys cornuta steven, orchis coriophora l. and platanthera chlorantha (custer) rchb. out of all the determined species in this research, there are 75 (38% of the total number) medicinal plants. regarding the natural habitats here, there are: 10 е1 pseudo-steppe with grasses and annual of the thero-brachypodietea, 08h3 calcareous rocky slopes with chasmophytic vegetation, 21g1 supra mediterranean hop-hornbeam woods, 02g1 southern helleno-balkanic swamp alder woods and 07g1 helleno-balkanic riparian plane forests. in general, the habitat 10 е1 pseudo-steppe with grasses and annual of the thero-brachypodietea has a conservation status: vulnerable [vua1,2 b1 c1 d2 e2 f2 g2 h2 i l2] (g u s s e v , 2015). however, this habitat in the west rhodopes has not been reported in the literature (b i s e r k o v et al., 2015). at the moment, the habitat is being used as a grazing land for domestic sheep and goats. the habitat 08н3 calcareous rocky slopes with chasmophytic vegetation has a conservation status: vulnerable [vu – a1, 2 b2 d2 h2 i j] (g u s s e v & r u s s a k o v a , 2015). the habitat 21g1 supra mediterranean hophornbeam woods has a conservation status: vulnerable [vua1 b2 c1 d2 e2 f1 g1 h2i l2] (r u s s a k o v a & t a s h e v , 2015). it is located on the steep and vertical slopes above the right bank of the dospat river. this habitat has not been reported either according to b i s e r k o v et al. (2015). the habitat 02g1 southern helleno-balcanic swamp alder woods has a conservation status: endangered [en – a1, 2 c2 d2 e2 f2 g2 h2 i j l2] (d i m i t r o v & t a s h e v , 2015). this habitat also has not been reported according to b i s e r k o v et al. (2015). the habitat 07g1 helleno-balcanic riparian plane forest has a conservation status: endangered [en – a1, 2 c1 d1 e2 f2 g2 h2 l3] (g o g o u s h e v , 2015). this habitat has not been reported according to b i s e r k o v et al. (2015) either. conclusion the existence of this rich and interesting local flora brings up an important point – its preservation in the future. when the following facts are taken into consideration: the presence of 1 species with endangered conservation status, 5 vascular plant species with vulnerable conservation status, 1 species with a least concern status, the high percentage of endemites (7,1%) and tertiary relicts (10,6%), the presence of two habitats with endangered status, 3 habitats with vulnerable status, as well as 15 species protected according to the law for biological diversity of the republic of bulgaria – are more than enough for this area of the west rhodopes to be officially classified as a natural protected one. references anonimous 2002: law for biological diversity of the republic of bulgaria (official gazette, copy 77 of 9 august 2002). assyov, b., petrova, a. 2012: conspectus of bulgarian vascular flora. distribution maps and floristic elements. 4th edition. bulgarian biodiversity foundation, s. evstatieva, l. 2009: galanthus elwesii. in: petrova, a., vladimirov, v. (eds.). red list of bulgarian vascular plants. phytologia balcanica, 15 (1):76. delipavlov, d., cheshmedjiev, i. 2003: a key of plants in bulgaria. agrarian university. plovdiv, 591 pp (in bulgarian). denchev, c., assyov, b. 2015: dianthus drenovskyanus. in: peev, d. et al. (eds.). red data book of r. bulgaria, v. 1. plants & fungi. bas & mew, s.: 233. dimitrov, d., vutov, v. 2016: reports 79-109. in: vladimirov, v. et al. (eds.). new floristic records in the balkans: 31. phytologia balcanica, 22 (3): 440-442. dimitrov, m., tashev, a. 2015: southern hellenobalkanic swamp alder woods. in: bisserkov, v. et al. (eds.). red book of r. bulgaria. v. 3. natural habitats, bas & mew, s.: 83. dimitrova, d. 2009: chondrilla urumoffii. in: petrova, a., vladimirov, v. (eds.). red list of bulgarian vascular plants. phytologia balcanica, 15 (1): 83. dimitrova, d. 2015: chondrilla urumoffii. in: peev, d. et al. (eds.). red data book of r. bulgaria, v. 1. plants & fungi. bas & mew, s.: 668. gogoushev, g. 2015: helleno-balcanic riparian plane forests. in: bisserkov, v. et al. (eds.). red book of r. bulgaria. v. 3. natural habitats, bas & mew, s.: 287-288. goranova, v., anchev, m. 2009: trachelium rumelianum. in: petrova, a. & vladimirov, v. (eds.). red list of bulgarian vascular plants. phytologia balcanica, 15 (1): 87. goranova, v., anchev, m. 2015: trachelium rumelianum.in: peev, d. et al. (eds.). red data book of r. bulgaria, v. 1. plants & fungi. bas & mew, s.: 691. gussev, ch. 2015: helleno-balkanic short grass and therophytic communities. in: bisserkov, v. et al. (eds.). red book of r. bulgaria. v. 3. natural habitats, bas & mew, s: 152-154. biologica nyssana 8 (1)  september 2017: 39-45 dimitrov, d.  floristic and habitat diversity of dospat dere… 43 gussev, ch., roussakova, v. 2015: rhodopide calcicolous chasmophyte communities. in: bisserkov, v. et al. (eds.). red book of r. bulgaria. v. 3. natural habitats, bas & mew, s.: 381-384. jordanov, d. (ed.). 1963-1979: flora of r. p. bulgaria. v.1-7. bas, s. jordanov, l. 1936: locality of macedonian pine round peak beslet, west rhodope. forestry ideas, 1: 31-32. kitanov, b. 1936: new and rare plants for the bulgarian flora. bulletin of the bulgarian botanical society, 7: 116-123. kitanov, b. 1939b: some new localities of macedonian pine in west rhodope. forestry ideas, 2: 121-124. kozuharov, s. (ed.). 1992: guidebook to the vascular plants in bulgaria. sofia. nauka i izkustvo. peev, d. (ed.). 2012: flora of r. bulgaria. v. 11. bas, s. 523 pp. peev, d. et al. 2015: red data book of r. bulgaria, v. 1. plants & fungi. bas & mew, s.: 887 pp. petrova, a. 2009: limodorum abortivum, ophrys cornuta.-in: petrova, . & vladimirov, v. (eds.). red list of bulgarian vascular plants. phytologia balcanica, 15 (1): 85. petrova, a., vladimirov, v. (eds.). 2009: red list of bulgarian vascular plants. phytologia balcanica, 15 (1): 63-94. sopotlieva, d. 2009: lotus aegaeus. in: petrova, a. & vladimirov, v. (eds.). red list of bulgarian vascular plants. phytologia balcanica, 15 (1): 92. stefanov, p. 1981: peculiarities of karst in dabrash. in: national congress of geographs in bulgaria. 4. collection of summary and reports. tutin, t. g., heywood, v. h., burges, n. a., moore, d. m., valentine, d. h., walters, s. m., webb, d. a. (eds.). 1964-1980: flora europaea, 1-5. cambridge, university press. london. appendix list of determined plants from the researched area pteridophyta aspleniaceae 1. asplenium ruta-muraria l. 2. a. trichomanes l. 3. ceterach officinarum dc. polypodiaceae 4. polypodium vulgare l. equisetophyta equisetaceae 5. equisetum palustre l. pinophyta juniperaceae 6. juniperus communis l. 7. j. oxycedrusl. pinaceae 8. pinus nigra arnold 9. p. sylvestris l. magnoliophyta aceraceae 10. acer campestre l. ssp. campestre amaryllidaceae 11. galanthus elwesii hook.f. en apiaceae 12. eryngium campestre l. 13. ferulago sylvatica (besser) rchb. 14. orlaja daucoides (l.) greuter 15. o. grandiflora (l.) hoffm. 16. peucedanum alsaticum l. 17. torilis japonica (houtt.) dc. 18. trinia glauca (l.) dumort. anacardiaceae 19. pistacia terebinthus l. asclepiadaceae 20. vincetoxicum hirundinaria medicus ssp. nivale (boiss. et heldr.) markgr. asteraceae 21. achillea setacea waldst. & kit. 22. anthemis macedonica boiss. ssp. orbelica (balkan endemic) 23. carlina acanthifolia all. 24. chondrilla urumoffii degen balcan end. vu, bda 25. crepis sancta (l.) babc. 26. inula aschersoniana janka (balkan endemic) 27. leontodon crispus vill. 28. i. ensifolia l. 29. jurinea mollis (l.) rchb. ssp. anatolica (boiss.) stoj.et stef. 30. scorzonera hispanica l. 31. s. laciniata l. 32. s. purpurea l. 33. senecio vernalis waldst.& kit. 34. tanacetum vulgare l. 35. tragopogon dubius scop. betulaceae 36. alnus glutinosa (l.) gaertn. 37. carpinus orientalis mill. 38. ostrya carpinifolia scop. boraginaceae 39. buglossoides purpurocaerulea (l.) i.m.johnst. 40. onosma thracica velen. balcan.end. brassicaceae biologica nyssana 8 (1)  september 2017: 00-00 dimitrov, d.  floristic and habitat diversity of dospat dere… 44 41. aethionema saxatile (l.) r.br. 42. alyssum alyssoides (l.) l. 43. arabis collina ten. 44. a. sagittata (bertol.) dc. 45. erysimum diffusum ehrh. campanulaceae 46. asyneuma canescens (waldst. & kit.) griseb. et schenk 47. campanula lingulata waldst. & kit. 48. c. persicifolia l. 49. trachelium rumelianum hampe (balkan endemic) vu, bda cannabaceae 50. humulus lupulus l. caryophyllaceae 51. cerastium luridum guss. 52. c. velenovskyi hayek bul. end. 53. dianthus cruentus griseb. (balkan endemic) 54. d. drenowskyanus rech. f. (balkan endemic) 55. d. petraeus waldst. & kit. 56. herniaria hirsuta l. 57. lychnis coronaria (l.) desr. 58. minuartia mesogitana (boiss.) hand.-mazz. 59. petrorhagia prolifera (l.) p.w.ball & heywood 60. silene conica l. 61. s. flavescens waldst. & kit. 62. s. frivaldszkyana hampe (balkan endemic) 63. s. italica (l.) pers. 64. s. radicosa boiss. & heldr. convolvulaceae 65. convolvulus cantabrica l. cornaceae 66. cornus mas l. 67. cornus sanguinea l. cistaceae 68. fumana procumbens (dunal) gren. & godr. 69. rhodax alpestris (jacq.) fuss. crassulaceae 70. sedum alpestre vill. 71. s. anopetalum dc. cyperaceae 72. blysmus compressus (l.) panz. ex link 73. carex distans l. 74. c. divisa huds. 75. c. hirta l. 76. c. liparocarpos gaudin dipsacaceae 77. scabiosa argentea l. 78. s. columbaria l. 79. s. ochroleuca l. var. ochroleuca 80. s. triniifolia friv. (balkan endemic) dioscoreaceae 81. tamus communis l. euphorbiaceae 82. euphorbia myrsinites l. 83. e. niciciana borbas f. niciciana fabaceae 84. anthyllis vulneraria l. ssp. pulchella (vis.) bornm. 85. astragalus glycyphyllos l. 86. a. monspessulanus l. 87. a. onobrychis l. 88. coronilla emerus l. 89. c. scorpioides (l.) c. koch 90. c. varia l 91. dorycnium herbaceum vill. 92. genista lydia boiss. 93. lotus aegaeus (griseb.) boiss. lc 94. medicago minima (l.) bartal 95. onobrychis gracilis besser 96. ononis adenotricha boiss. 97. o. pusilla l. 98. trifolium alpestre l. 99. t. arvense l. 100. t. campestre schreb. var. campestre 101. t. incarnatum l. 102. t. scabrum l. ssp. scabrum 103. vicia cracca l. 104. v. grandiflora scop. 105. v. varia host 106. v. villosa roth fagaceae 107. quercus pubescens willd. geraniaceae 108. geranium robertianum l. 109. g. sanguineum l. globulariaceae 110. globularia aphyllanthes crantz hypericaceae 111. hypericum rumeliacum boiss. 112. h. umbellatum a. kern. (balkan endemic) iridaceae 113. romulea bulbocodium (l.) sebast. & mauri, vu, bda juglandaceae 114. juglans regia l. lamiaceae 115. ajuga chamaepitys (l.) schreb. 116. a. genevensis l. 117. a. laxmannii (l.) benth. 118. calamintha sylvatica bromf. 119. micromeria dalmatica benth. (balkan endemic) 120. salvia glutinosa l 121. sideritis montana l. 122. stachys recta l. 123. teucrium polium l. 124. thymus callieri borb.ex velen. 125. th. striatus vahl. liliaceae 126. allium moschatum l. 127. anthericum liliago l. biologica nyssana 8 (1)  september 2017: 39-45 dimitrov, d.  floristic and habitat diversity of dospat dere… 45 128. asparagus tenuifolius lam. 129. lilium martagon l. 130. muscari comosum (l.) mill. linaceae 131. linum austriacum l. 132. l. tenuifolium l. 133. l. nervosum waldst. & kit. 134. l. thracicum (griseb.) degen (balkan endemic) loranthaceae 135. arceutobium oxycedri (dc.) m.bieb. 136. viscum laxum boiss. & reuter ssp. laxum oleaceae 137. fraxinus ornus l. 138. ligustrum vulgare l. orchidaceae 139. cephalanthera rubra (l.) rich. 140. epipactis atrorubens (hoffm.) besser 141. limodorum abortivum (l.) schwarz, vu, bda 142. ophrys cornuta steven vu, bda 143. orchis coriophora l. 144. platanthera chlorantha (custer) rchb. orobanchaceae 145. orobanche elatior sutton 146. o. esulae pancic plantaginaceae 147. plantago lanceolata l. platanaceae 148. platanus orientalis l. plumbaginaceae 149. armeria rumelica boiss. (balkan endemic) poaceae 150. aegilops neglecta req. ex bertol. 151. bromus intermedius guss. 152. b. sterilis l. 153. chrysopogon gryllus (l.) trin. 154. festuca nigrescens lam. 155. f. penzesii(acht. )markgr.-dann. (balkan endemic) 156. f. spectabilis jan. ssp. affinis (boiss. & heldr. ex hack.) hack. 157. koeleria nitidula velen. 158. melica ciliata l. 159. molinia coerulea (l.) moench 160. phleum pratense l. 161. sclerochloa dura (l.) p.beauv. 162. sesleria alba sm. 163. stipa tirsa steven 164. trachynia distachya (l.) link 165. vulpia ciliata dumort. 166. v. myurus (l.) c.c.gmel. polygalaceae 167. polygala comosa schkuhr 168. p. major jacq. 169. p. rhodopea (velen.) janch. (balkan endemic) polygonaceae 170. rumex acetosella l. ranunculaceae 171. pulsatilla montana (hoppe) rchb. 172. ranunculus sardous crantz 173. thalictrum aquilegifolium l. rosaceae 174. agrimonia eupatoria l. 175. crataegus monogyna jacq. 176. filipendula vulgaris moench 177. potentilla argentea l. 178. prunus spinosa l. 179. rosa micrantha borrer ex sm. 180. sanguisorba minor scop. ssp. muricatum briq. rubiaceae 181. asperula tenella heuff. ex deg. in kerner 182. cruciata laevipes opiz rutaceae 183. dictamnus albus l. salicaceae 184. salix alba l. santalaceae 185. thesium arvense horv. scrophulariaceae 186. euphrasia rostkoviana hayne 187. melampyrum sylvaticum l. 188. parentucellia latifolia (l.) caruel 189. rhinanthus wagneri degen ssp. wagneri 190. veronica annagalis-aquatica l. 191. v. chamaedrys l. 192. v. multifida l. thymeleaceae 193. thymelaea passerina (l.) coss. & germ. ulmaceae 194. ulmus minor mill. urticaceae 195. urtica dioica l. valerianaceae 196. valerianella rimosa bast. violaceae 197. viola alba besser ssp. scotophylla (jordan) nyman anticancer compounds from medicinal plants biologica nyssana 2 (2)  december 2011: 00-00 ljupković r.b. et al..  removal cu(ii) ions from water... 11 original article preliminary investigation of free radical scavenging activity and total phenolic content of three cultivated thymus chemotypes from bulgaria milena nikolova 1* genka zhekova 2 anatoli dzhurmanski 2 1 institute of biodiversity and ecosystem research, 23, acad.g. bonchev st., 1113 sofia, bulgaria 2 institute of roses, essential and medicinal cultures, bul. osvobozhdenie 49, 6100 kazanlak, bulgaria * e-mail: milena_n@bio.bas.bg abstract: nikolova, m., jekova, g., dzhurmanski, a.: preliminary investigation of free radical scavenging activity and total phenolic content of three cultivated thymus chemotypes from bulgaria , biologica nyssana, 3 (1), september 2012: 11-15. methanol extracts of three varieties of thymus “slava”, “german winter” and “pagane”, representatives respectively of citral, thymol and geraniol chemotype were evaluated for their antioxidant potential and phenolic content. total content of phenols was determined by spectrophotometric method using folinciocalteu reagent. the antioxidant potential of methanol extracts was estimated by 1,1-diphenyl-2picrylhydrazyl radical (dpph) free radical assay. all of the examined extracts exhibited significant free radical scavenging activity and their ic50 values were below 50 μg/ml. the highest antioxidant activity was determined for the extract of thymol chemotype “german winter” with ic50 value – 19.34 µg/ml. the extract of this variety had the highest amount of phenols 75.61 mg/g extract. the other two varieties “slava” and “pagane” have a lower content of phenols and antiradical activity. influence of three stages of flower development on antiradical activity and total phenolic content was examined for variety “pagane”. the highest antioxidant activity was determined on the extract prepared from plant material collected at the stage “end of flowering”. for the first time, provides data on the phenolic content and antiradical activity of varieties “slava” and “pagane”. these results are the basis for selection of varieties with high antioxidant potential for the preparation of cosmetic and herbal products. key words: thymus vulgaris, thymus marschalianus, dpph, citral, thymol, geraniol introduction the genus thymus l. (lamiaceae) comprises about 300 species of perennial aromatic, herbaceous plants with many subspecies, varieties, subvarieties and forms. the plants are extensively used in phytotherapy, cosmetic and food industries (s t a n e v , 1974; n i k o l o v , 2006). essential oils, flavonoids (highly methylated flavonoids, luteolin derivatives), biphenils and phenolic compounds have been determined as main constituents of the thyme with antioxidant, antiseptic, antiinflamatory and antimicrobial properties (j o v a n o v i ć et al., 1995; h a r a g u c h i et al., 1996; f e c k a & t u r e k , 2008; b o r o s a et al., 2010). the thyme shows a great species diversity which reflects to the composition of the essential oil content. based on the dominant monoterpene in the essential oils of different thymus chemotypes, compounds such as geraniol, thymol, α-terpineol, linalool, carvacrol can be distinguished (t h o m p s o n et al., 2003; c h i z z o l a et al., 3 (1) • september 2012: 11-15 biologica nyssana 3 (1)  september 2012: 11-15 nikolova, m. et al.  preliminary investigation of free radical… 12 2008). in recent years, several reports have been published concerning the antioxidant activity on the essential oils on different thyme chemotype (j u k i et al., 2005; b o u n a t i r o u et al., 2007; m i g u e l et al., 2007; s t o i l o v a et al., 2008; g r i g o r e et al., 2010; t e p e et al., 2011) while the studies on antioxidant activity of methanol extracts from different chemotypes and varieties are insufficient (j o v a n o v i ć et al., 1995; k u l i š i ć et al., 2006). a positive correlation between antioxidant activity and phenolic content in plant extracts has been well documented (m i l i a u s k a s a et al., 2004; k i s e l o v a et al., 2006). widely used methods for assessing the total content of phenols and antioxidant properties of plant extracts are based on folin-ciocalteu reagent and 1-diphenyl-2picrylhydrazyl (dpph) radicals (k i s e l o v a et al., 2006, g i o r g i et al., 2009; n i ć i f o r o v i ć et al., 2010; m a r i n o v a & b a t c h v a r o v , 2011). the purpose of the present work is to examine antioxidant activity and total phenolic content varieties created and cultivated in bulgaria: thyme “slava” (thymus vulgaris, citral chemotype) and “pagane” (thymus marschalianus wild., geraniol chemotype) as well as of the introduced variety “german winter” (thymus vulgaris, thymol chemotype). this study contributes to the knowledge of the antioxidant properties of the thymus chemotypes. materials and methods plant material the plants used for the present study were cultivated in the experimental field of institute of roses, essential and medicinal cultures, kazanlak. aerial parts of two varieties “slava” and “german winter” were collected at full flowering stage. aerial parts of the variety “pagane”, collected in three stages of flowering – beginning of flowering, full flowering and end of flowering were analyzed. preparations of extracts air-dried, powdered plant material (1 g) was extracted with 80% methanol in an ultrasonic bath. after evaporation of the solvent the crude extract was subjected to subsequent analyses. determination of total phenolic content total phenolic content of the methanol extracts was determined by employing the methods given in the literature including folin–ciocalteu reagent and gallic acid as standard (g i o r g i et al., 2009; n i ć i f o r o v i ć et al., 2010). plant extracts were diluted to the concentration of 1 mg/ml, and aliquots of 0.25 ml were mixed with 2.5 ml of folin–ciocalteu reagent (previously diluted 10-fold with distilled water) and 2 ml of na2co3 (6%). after 1 h of staying at the room temperature, the absorbances of the samples were measured at 765 nm on spectrophotometer versus blank sample. total phenols were determined as gallic acid equivalents (mg ga/g extract) by the following formula: c = c x v / m where c total content of phenolic compounds, mg/g plant extract, in gae; c the concentration of gallic acid established from the calibration curve, mg/ml; v the volume of extract, ml; m the weight of pure plant methanolic extract, g. free radical scavenging activity free radical scavenging activity of plant extracts was evaluated using a 1,1-diphenyl-2picrylhydrazyl (dpph) assay (c h o i et al., 2002; s t a n o j e v i ć et al., 2009). different concentrations of plant extract (10, 20, 50, 100 and 200 μg/ml, in methanol) were added at an equal volume (2.5 ml) to methanol solution of dpph (0.3 mm, 1 ml). after 30 min at room temperature, the ab (absorbance) values were measured at 517 nm on a spectrophotometer (jenway 6320d) and converted into the percentage antioxidant activity using the following equation: dpph antiradical scavenging capacity (%) = [ 1( absample – abblank ) / abcontrol ] x 100 plant extract solution (2.5 ml) in methanol (1.0 ml) was used as a blank, while dpph solution in methanol was used as a control. the ic50 values were calculated by sigmoid non-linear regression model using plots, where the abscissa represented the concentration of tested plant extracts and the ordinate the average percent of scavenging capacity (software prizm 3.00). ic50 values denote the concentration of sample required to scavenge 50% of dpph radical. statistical analysis statistical analysis was carried out using excel. all experiments were performed in triplicate. results are presented as a value ± standard deviation (sd). significant levels were defined at p<0.05. biologica nyssana 3 (1)  september 2012: 11-15 nikolova, m. et al.  preliminary investigation of free radical… 13 results and discussion methanol extracts of three chemotypes of thymus (citral, thymol and geraniol) were examined for their antiradical potential using a dpph assay and expressed as ic50 valueextract concentration providing 50% inhibition of the dpph solution (table 1). the all examined extracts exhibited considerable free radical scavenging activity and their ic50 values were below 50 μg/ml. the extract of variety “german winter” (thymol chemotype) showed the strongest antioxidant activity with ic50 value 19.34 µg/ml, followed by citral chemotype “slava” (33.18µg/ml) and geraniol chemotype “pagane” (47.72 µg/ml). the established differences in the antioxidant potential of studied varieties can be explained by different chemotype – different composition of essential oils. the thyme with the highest content of thymol (“german winter”) has the strongest antioxidant activity. this is in accordance with results reported by chizzola et al. (2008) which prove that the essential oil with high content of thymol and/or carvacrol have the highest antioxidant activity. these data allow us to assume antioxidant potential of thyme based on the essential oil composition and individual components (geraniol, linalool, thymol, etc.). a total phenolic content of studied varieties of thymus “slava”, “german winter” and “pagane” was determined (table 1). the extracts of variety “german winter” contain the highest amount of phenols (75.61 mg/g), followed by “slava” (56.6 mg/g) and “pagane” (45.42 mg/g). the greater amount of phenolic compounds positively correlated to more potent radical scavenging effect. carried out correlation analyses shows strong positive mutual dependence between total phenolic content and antioxidant (dpph scavenging) activity of investigated extracts (r=0, 9812). this is in accordance of several reports for such positive correlation between phenols and antioxidant activity of plant extracts (m i l i a u s k a s a et al., 2004; c a i et al., 2004; s h a n et al., 2005; k i s e l o v a et al., 2006; v i l l a ñ o et al., 2007). the antioxidant potential and total phenolic content of extracts obtained from material collected at differing stages of flower development of the variety “pagane” was examined. it was found that extracts of plant material gathered at stage “end of flowering” had the highest antioxidant activity with ic50 value of 34.28 µg/ml (table 2). the extracts prepared from plant material collected at the stages “beginning of flowering” and “full flowering” showed ic50 values 47.72 and 67.00 µg/ml, respectively. thyme is recommended to be picked up at the “end of flowering” stage, when their antioxidant activity is 40% higher than in a “full flowering” stage and 95% higher than in stage “beginning of flowering”. opposite to the results on antioxidant activity, it was found that there are no statistical significant differences (p>0,05) in the phenolic content of the examined extracts during flowering period. total phenols ranges from 44.08 to 46.82 mg/g. this fact suggests that radical scavenging activity of the studied extracts is not only due to phenol compounds. table 1. total phenolic content and free radical scavenging activity of methanol extracts of three thymus varieties thymus varieties total phenols mg gae/g extract* free radical scavenging activity ic50 (µg/ml)* pagane 46.82±1.22 47.72±5.41 slava 56.6±1.25 33.18±1.89 german winter 75.61±2.71 19.34±1.76 * values represent mean ±sd, n=3 table 2. total phenolic and antiradical activity of extracts of variety “pagane” collected at three stage of flowering stage of flowering total phenols mg gae/g extract* free radical scavenging activity ic50 (µg/ml)* beginning of flowering 44.08±1,80 67.00±7.63 full flowering 46.82±1.22 47.72±5.41 end of flowering 45.42±2.62 34.28±1.98 * values represent mean ±sd, n=3 biologica nyssana 3 (1)  september 2012: 11-15 nikolova, m. et al.  preliminary investigation of free radical… 14 conclusion in the present study, the total phenolic content and antioxidant capacity of three varieties of thymus “slava”, “german winter” and “pagane” were determined. high positive correlation was found between investigated indexes. bulgarian varieties thyme – “slava” and “pagane” was studied for the first time for their total phenolic content and the antioxidant activity. the methanol extracts of all examined samples showed high radical scavenging activity (ic50 <50 µg/ml). extracts of thymol chemotype “german winter” exhibited the strongest antiradical activity and highest content of phenols among studied varieties. therefore, thymol chemotype can be priority for utilization in phytoterapy and cosmetics. the recommended concentration of thyme solution for lipid stabilizing in cosmetics and food products is 50 µg/ml, when it provides 95% free radical inhibition whilst for the other types of thyme, it is twice higher. thyme is recommended to be picked up at the “end of flowering” stage, because then the content of antioxidants is the highest. obtained data will be employed as a basis for selection of varieties with high antioxidant potential for the preparation of cosmetic and herbal products, as well as for the optimization of harvesting time. references borosa, b., jakabová, s., dörnyei, a., horváth, g., pluhár z., kilár, f., felingera, a. 2010: determination of polyphenolic compounds by liquid chromatography–mass spectrometry in thymus species. journal of chromatography a, 1217: 7972–7980. bounatirou, s., smiti, s., miguel, m.g., faleiro, l., rejeb, m.n., neffati, m., costa, m.m., figueiredo, a.c., barroso, j.g., pedro, l.g. 2007: chemical composition, antioxidant and antibacterial activities of the essential oils isolated from tunisian thymus capitatus hoff. et link. food chemistry, 105: 146–155 cai, y., luo, q., sun, m., corke, h. 2004: antioxidant activity and phenolic compounds of 112 chinese medicinal plants associated with anticancer. life science, 74: 2157–2184. chizzola, r., michitsch, h., franz, c. 2008: antioxidative properties of thymus vulgaris leaves: comparison of different extracts and essential oil chemotypes, journal of agricultural and food chemistry, 56: 6897–6904. choi, c.w., kim, s.c., hwang, s.s., choi, b.k., ahn, h.j., lee, m.y. 2002: antioxidant activity and free radical scavenging capacity between korean medicinal plants and flavonoids by assay – guided comparsion. plant science, 163: 11611168. nikolov, s. (ed.), 2006: specialized encyclopedia of medicinal plants. trud publishing house, sofia. 566 p. (in bulgarian) fecka, i., turek, s. 2008: determination of polyphenolic compounds in commercial herbal drugs and spices from lamiaceae: thyme, wild thyme and sweet marjoram by chromatographic techniques. food chemistry, 108: 1039-1053. giorgi, a., mingozzi, m., madeo, m., speranza, g., cocucci, m. 2009: effect of nitrogen starvation on the phenolic metabolism and antioxidant properties of yarrow (achillea collina becker ex rchb.). food chemistry, 114: 204-211. grigore, a., ina, p., colceru-mihul, s., bubueanu, c., draghici e., ichim, m. 2010: chemical composition and antioxidant chemical composition and antioxidant activity of thymus vulgaris l. volatile oil obtained by two different methods. romanian biotechnological letters, 15: 5436–5443. haraguchi, h., saito, t., ishikawa, h., date, h., kataoka, s., tamura, y., mizutani, k. 1996: antiperoxidative components in thymus vulgaris. planta medica, 62: 217-221. juki, m., milo, m. 2005: catalytic oxidation and antioxidant properties of thyme essential oils (thymus vulgarae l.). croatica chemica acta, 78: 105-110. kiselova, y., ivanova, d., chervenkov, t., gerova, d., galunska, b., yankova, t. 2006: correlation between the in vitro antioxidant activity and polyphenol content of aqueous extracts from bulgarian herbs. phytotherapy research, 20: 961-965. kulišić, t., dragović-uzelac v., miloš, m. 2006: antioxidant activity of aqueous tea infusions prepared from oregano, thyme and wild thyme. food technology and biotechnology, 44: 485– 492. marinova, g., batchvarov, v. 2011: evaluation of the methods for determination of the free radical scavenging activity by dpph. bulgarian journal of agricultural science, 17: 11-24. miguel, m.g., costa, l.a., figueiredo, a.c., barroso, j.g., pedro, l.g. 2007: assessment of the antioxidant ability of thymus albicans, th. mastichina, th. camphoratus and th. carnosus essential oils by tbars and micellar model systems, natural product communications, 2: 399-406. miliauskasa, g., venskutonisa, p.r., van beek, t.a. 2004: screening of radical scavenging activity of biologica nyssana 3 (1)  september 2012: 11-15 nikolova, m. et al.  preliminary investigation of free radical… 15 some medicinal and aromatic plant extracts. food chemistry, 85: 231–237. nićiforović, n., mihailović, v., masković, p., solujić, s., stojković, a., muratspahić, d.p. 2010: antioxidant activity of selected plant species; potential new sources of natural antioxidants. food and chemical toxicology, 48: 3125-3130. picuric jovanovic, k., milovanovic, m., vrbaski, z. 1995: thymus vulgaris as a sourse of natural lipid antioxidant. review of research work at the faculty of agriculture, 40: 141-146. thompson, j.d., chalchat, j.c., michet, a., linhart, y.b., ehlers, b. 2003: qualitative and quantitative variation in monoterpene cooccurrence and composition in the essential oil of thymus vulgaris chemotype. journal of chemical ecology, 29: 859-880. tepe, b., sarikurkcu, c., berk, s., alim, a., akpulat, h.a. 2011: chemical composition, radical scavenging and antimicrobial activity of the essential oils of thymus boveii and thymus hyemalis. records of natural products, 5: 208220. shan, b., cai, y.z., sun, m., corke, h. 2005: antioxidant capacity of 26 spice extracts and characterization of their phenolic constituents. journal of agricultural and food chemistry, 53: 7749–7759. stanev, d. 1974: study of thymus vulgaris in bulgaria. plant science, 2: 29-33. stanojević, l., stanković, m., nikolić, v., nikolić, l., ristić, d., čanadanovic-brunet, j., tumbas, v. 2009: antioxidant activity and total phenolic and flavonoid contents of hieracium pilosella l. extracts. sensors, 9: 5702-5714. stoilova, i., bail, s., buchbauer, g., krastanov, a., stoyanova, a., schmidt, e., jirovetz, l. 2008: chemical composition, olfactory evaluation and antioxidant effects of an essential oil of thymus vulgaris l. from germany. natural product communications, 3: 1047-1050. villaño, d., fernández-pachón, m.s., moyá, m.l., troncoso, a.m., garcía-parrilla, m.c. 2007: radical scavenging ability of polyphenolic compounds towards dpph free radical. talanta, 71: 230–235. 16 anticancer compounds from medicinal plants biologica nyssana 4 (1-2)  december 2013: 9-14 nikolić, lj. et al.  analysis of weed flora in conventional… 9 original article analysis of weed flora in conventional and organic potato production ljiljana nikolić, olivera ilić, dejana džigurski, branka ljevnaić-mašić faculty of agriculture, university of novi sad, trg dositeja obradovića 8, 21 000 novi sad, serbia * e-mail: ljnik@polj.uns.ac.rs abstract: nikolić, lj., ilić, o., džigurski, d., ljevnaić-mašić, b.: analysis of weed flora in conventional and organic potato production. biologica nyssana, 4 (1-2), december 2013: 9-14. composition of weed flora is highly dynamic and depends upon great number of factors, of which cultural practices that are applied by humans in certain crops are the most important. one of the most frequently grown plants in the world and in our country is potato (solanum tuberosum l., solanaceae), due to its high biological and nutritive value. therefore, in the paper was presented taxonomic analysis of weed flora in potato grown conventionally and according to the principles of organic agricultural production, with the intention to point out to eventual differences between present weeds. of the total number of identified species, from phylum equisetophyta and class equisetopsida, in organic potato crop, was determined only one, equisetum arvense. of remaining 38 weeds from phylum magnoliophyta., classified into two classes, magnoliopsida and liliopsida. on both of potato growing systems, 39 weed species were found, classified into 16 families and 32 genus. of the total number, 31 species was identified in conventional potato crop, and only 23 species in potato crop grown according to organic principles, which is for about quarter less. biological spectrum of weed flora in both potato growing systems is pronouncedly of terrophytic – geophytic type. in the spectrum of area types were recorded differences, i.e. in the conventional potato crop represented are only widely distributed species, while in the organic crop, beside species of wide distribution are also present elements of pontic group. key words: taxonomic analysis, weeds, potato introduction floristic diversity, as well as overall biodiversity of natural and anthropogenic ecosystems, is a subject of numerous studies. fundamental research comprising of direct verification and systematization of biodiversity forms the foundations for biodiversity conservation in general. biodiversity changes due to anthropogenic factors pose numerous risks, while yielding paucity of immediate benefits (s t e v a n o v i ć & v a s i ć 1995). hence, research focusing on studying and preserving the biodiversity of agroecosystems that are under direct human influence is of particular importance, as reflected in the development of a european-level action plan (the 2001 biodiversity action plan for agriculture /com/2001/0162). since the early nineties, interest in organic farming has significantly increased in europe, with particular attention to agroecosystems and their biodiversity, emphasizing the differences between conventional crops and those grown according to organic principles (r o s c h e w i t z et al., 2005; w e i b u l l et al., 2003; h y v ö n e n et al., 2003; h y v ö n e n , 2007). the main difference between conventional and organic farming principles is reflected in the fact that the latter completely exclude the use of chemicals and fertilizers. it is this specificity that, 11 th sfses • 13-16 june 2013, vlasina lake 4 (1-2) • december 2013: 9-14 mailto:ljnik@polj.uns.ac.rs biologica nyssana 4 (1-2)  december 2013: 9-14 nikolić, lj. et al.  analysis of weed flora in conventional… 10 on one hand, raises the question of whether organic farming could contribute to the restoration and preservation of biodiversity (h y v ö n e n , 2007). however, on the other hand, control of weed proliferation—which, as a regular crop companion, has a significant negative impact on the quality and quantity of agricultural products—is a major problem in organic production, as it almost completely excludes chemical weed control measures (b à r b e r , 2002; k r i s t i a n s e n , 2003). to address these issues, this paper presents the analysis of the weed flora accompanying potato (solanum tuberosum l.), one of the most widely cultivated plants in the world and in our country, grown both conventionally and according to the organic farming principles, in order to identify potential differences between the outcomes of the two systems. the reported results provide a foundation for long-term monitoring and study of weed flora as an important component of agroecosystem biodiversity. material and methods floristic weed analysis in conventional potato production was carried out during the 2008-2010 vegetative period at the experimental plots in the vicinity of bečej. the two-factor experiment was carried out, whereby conventional potato production with and without herbicide application was conducted in the varying cultivation conditions (number of inter-row potato earthing repetitions). this paper reports only on the weed flora found in the control part of the experiment, which included cultivation and planting, with no other crop care or weed protection measures. floristic weed analysis in organic potato production was carried out during 2012, on the localities registered for organic farming—family "biofarm" in kelebija and biofarm "mamudžić" in ljutovo—where weed proliferation is suppressed by mechanical means only. determination of plant material was performed according to josifović (1970-1977), whereas taxonomy conformed to takhtajan (2009), life-form categorization followed ujvárosi (1973), and floral elements were determined according to gajić (1980). results and discussion the following sections present the analysis of weed flora in potato crops grown conventionally and according to organic principles. in both potato cultivation systems, 39 weed species were identified, grouped into 32 genera, 16 families, 12 orders, 5 subclasses, 3 classes and 2 divisions. more specifically, the control variant of conventional potato crops showed the presence of 31 weed species, classified into 25 genera, 11 families, 7 orders, 5 subclasses, 2 classes and 1 division. on the other hand, in the organic potato crops, only 23 weed species were recorded, which were classified in 22 genera, 15 families, 12 orders, 5 subclasses, 3 classes and 2 divisions (table 1 and 2). of the total number of identified species, equisetum arvense l. (2.56%) from the equisetophyta division and equisetopsida class was found in organic potato crops only. the remaining 38 weeds from the magnoliophyta division were noted in varying numbers in both conventional and organic potato crops. these were divided into two classes, whereby the magnoliopsida (82.05%) class was represented by four subclasses (caryophyllidae, dillenidae, lamiidae and asteridae) and the liliopsida class (15.38%) was represented by the commelinidae subclass only (table 1, 2). as noted above, greater floristic diversity (31 species) and more significant weed proliferation was found in the control variant of the conventionally grown potatoes, while nearly 25% (23 species) reduction in floristic diversity is evident in organically grown potato crops. the analysis revealed presence of 15 common species, as well as 8 specific to organic and 16 to conventional potato crops. in the conventionally grown crops, the following were found in significant numbers: panicum crus-galli l., cirsium arvense (l.) scop., amaranthus retroflexus l., sorghum halepense l. and polygonum lapathifolium l., while only individual weeds were generally recorded in organic potato crops, except for equisetum arvense l. and polygonum convolvulus l. in ljutovo, both of which were more abundant. although the findings reported by some authors (hald, 1999; menalled et al., 2001; boguzas et al., 2004) indicate greater species wealth in organic crops compared to conventional cultivars, data obtained in this work point to the greater weed floristic diversity in conventional potato crops. this discrepancy can be explained by the fact that our analyses were conducted on the control variant of the experiment, which did not permit any herbicide application. however, it should be noted that, as the soil on which the experiment was conducted had been previously used for conventional agricultural production, the previously applied agrotechnical practices certainly affected the obtained results. biologica nyssana 4 (1-2)  december 2013: 9-14 nikolić, lj. et al.  analysis of weed flora in conventional… 11 table 1. taxonomic review of weed flora in potato crops p h y lu m c la ss s u b c la ss order family genus plant species life form floristic elements c o e q u is e to p h y ta e q u is e to p si d a equisetales equisetaceae equisetum e. arvense l. g1 cirk. + m a g n o li o p h y ta m a g n o li o p si d a c a r y o p h y ll id a e caryophyllales portulacaceae portulaca p. oleracea l. t4 cosm.. + + caryophyllaceae stellaria s. media l. t1 cosm.. + amaranthaceae amaranthus a. retroflexus l. t4 adv. + + chenopodiaceae chenopodium c. album l. t4 cosm.. + + c. hybridum l. t4 subcirk. + + polygonales polygonaceae polygonum p. convolvulus l. t4 subeur. + + p. lapathifolium l. t4 subcirk. + p. persicaria l. t4 eur. + p. aviculare l. t4 cosm.. + d il le n id a e capparales brassicaceae capsella c. bursa-pastoris (l.) med. t1 cosm.. + + sinapis s. arvensis l. t3 subeur. + malvales malvaceae hibiscus h. trionum l. t4 pont.ea.subm. + malva m. silvestris l. ht-h4 eur. + euphorbiales euphorbiaceae euphorbia e. helioscopia l. t4 subeur. + l a m ii d a e solanales solanaceae datura d. stramonium l. t4 cosm.. + + solanum s. nigrum l. t4 cosm.. + + convolvulales convolvulaceae convolvulus c. arvensis l. g3 cosm.. + + cuscutaceae cuscuta c. epithymum l. t4 subeur. + c. europea l. t4 eur. + boraginales boraginaceae heliotropium h. europaeum l. t4 pont.-subm. + lamiales lamiaceae stachys s. annua l. t4 subpont. subm. + lamium l. amplexicaule l. t1 subeur. + a st e r id a e asterales asteraceae ambrosia a. artemisiifolia l. t4 adv. + + cirsium c. arvense (l.) scop. g3 subeur. + + galinsoga g. parviflora cav. t4 adv. + erigeron e. canadensis l t4 adv. + matricaria m. chamomilla l. t2 eur. + m. inodora l. t4 eur. + senecio s. vulgaris l. t1 eur. + sonchus s. arvensis l. g3 eur. + + s. oleraceus l. t4 subeur. + xanthium x. strumarium l. t4 adv. + l il io p si d a c o m m e li n id a e poales poaceae agropyrum a. repens beauv. g1 eur. + cynodon c. dactylon pers. g1 cosm.. + digitaria d. sanguinalis scop. t4 cosm.. + panicum p. crus-galli l. t4-t4(h) cosm.. + + setaria s. glauca p.b. t4 cosm.. + + sorghum s. halepense l. g1 cosm.. + + 2 3 5 12 16 32 39 31 23 biologica nyssana 4 (1-2)  december 2013: 9-14 nikolić, lj. et al.  analysis of weed flora in conventional… 12 table 2. the number of weed species of the higher taxonomic categories in potato crop division class subclass family genus species no % equisetophyta equisetopsida 1 1 1 2,56 magnoliophyta magnoliopsida 4 14 25 32 82,05 liliopsida 1 1 6 6 15,38 ukupno 2 3 5 16 32 39 100 the 25% reduction in weed variety in the organic potato crops, when compared to the conventional production, could be potentially explained by the still not fully balanced ecological conditions in these plots, their partial isolation, as well as regular mechanical weed control. still, some authors (weibull et al., 2003) maintain that the cultivation (farming) system is yet to be established as a factor affecting the floristic diversity, while roschewitz et al. (2005) reported that the floristic diversity of conventional crops, especially their peripheral parts, is significantly influenced by dissemination of various species from the surrounding area. as can be seen in table 3, the total floristic spectrum of weed flora found in potato crops is dominated by the asteraceae family with 10 species (25.65%), and poaceae family with 6 species (15.39%). this relationship is not surprising, given that these vascular plant families are already very rich in species, among which the representatives from the weed category are very numerous. table 3. floristic range of families according to species number familija broj vrsta % asteraceae 10 25,65 poaceae 6 15,39 polygonaceae 4 10,26 chenopodiaceae 2 5,13 brassicaceae 2 5,13 malvaceae 2 5,13 solanaceae 2 5,13 cuscutaceae 2 5,13 lamiaceae 2 5,13 equisetaceae 1 2,56 portulacaceae 1 2,56 caryophyllaceae 1 2,56 amaranthaceae 1 2,56 euphorbiaceae 1 2,56 convolvulaceae 1 2,56 boraginaceae 1 2,56 ukupno 39 100 polygonaceae family is represented by 4 species (10.26%), whereas chenopodiaceae, brassicaceae, malvaceae, solanaceae, cuscutaceae and lamiaceae families are represented by 2 species (each with 5.13% participation). finally, only one species (2.56%) each was found for equisetaceae, portulacaceae, caryophyllaceae, amaranthaceae, euphorbiaceae, convolvulaceae and boraginaceae families. life-form analysis of the identified potato crop weed flora indicated its terophytic-geophytic character (fig. 1). in conventional potato crops, terophytes comprised 80.64% (25 species), and were dominated by t4 terophytes—annual weeds whose seeds germinate in spring and bear fruit in summer—with 64.52% (20 species). the remaining 19.36% (6 species) were of geophytic character, of which three species (9.68%) belonged to the g1 category, with developed rhizomes, and the remaining three (9.68%) to the g3 category, characterized by adventitious root buds. in the organic potato crops, the ratio is similar, as the terophytes predominate with 73.91% (17 species), albeit with a slightly lower share compared to the conventional crops. the participation of geophytes is also similar, with 21.74% (5 species), of which three (13.05%) are in g3 and two (8.69%) in the g1 category. only one species (malva silvestris) belongs to the hemiterophyte-hemicryptophyte category (fig. 1). this life-form ratio is characteristic of agroecosystems, where due to intensive agricultural practices, the conditions are most favorable for development of terophytes (kojić et al., 1998; knežević & baketa, 1990; gabriel & tscharntke, 2007). the spectrum of areal types, as is characteristic of agroecosystems, is dominated by species capable of wide spatial distribution, due to their wide ecological valence, and adaptation to intense anthropogenic impacts (kojić et al., 1998). although they cover relatively small surface and comprise only a few species, we note a few differences. namely, in the conventional potato crops, only highly proliferative species are present, among which, the cosmopolitan floral elements predominate with 41.94% (13 species). in contrast, biologica nyssana 4 (1-2)  december 2013: 9-14 nikolić, lj. et al.  analysis of weed flora in conventional… 13 in the organic crops, in addition to proliferative species, the conditions are favorable for the development of markedly continental representatives of pontic groups (ponticsubmediterranean – heliotropium europaeum l., pontic-eastern submediterranean – hibiscus trionum l., and subpontic-submediterranean – stachys annua l.) (fig. 2). fig. 1. biological spectrum of weed flora in potato crop, a – conventional, and b organic these results point to slight differences in the structure of the weed flora found in potato crops grown conventionally and according to organic principles, probably due to differences in applied agrotechnical practices. it should be noted that, although the weed flora plays a significant role in the overall biodiversity and the functioning of these anthropogenic ecosystems, their optimal operation still requires maintaining a correct balance between cultivated and wild plants, as weeds contribute to better regulation of environmental conditions (bound & grundy, 2001; boguzas et al., 2004). protection and conservation of sensitive weed species can be achieved by different means, and given their increasing endangerment, the focus should be on the intensification of organic agriculture, with reduced use of herbicides and fertilizers. however, conservation of these species in botanical gardens is also one of the possible measures (znaor, 1996; moss et al., 2004; hulina, 2005). fig. 2. area type spectrum of weed flora in potato crop, a – conventional, and b – organic conclusion in both organic and conventional potato crops, the presence of 39 weed species was noted, which were grouped into 16 families and 32 genera. greater floristic diversity (31 species) and greater weed infestation was found in the control plots of conventionally grown potatoes, while nearly 25% (23 species) reduction in floristic diversity was established in organically grown crops. the two cultivation methods had 15 species in common, while 8 were specific to organic and 16 to conventional potato crops. in the total floristic spectrum of potato crops weed flora, asteraceae family predominates with 10 species, followed by poaceae family with 6 species. biological spectrum of weed flora in both potato production systems is of predominantly terophytic-geophytic character, with a slightly lower participation of terophytes in organic potato crops. biologica nyssana 4 (1-2)  december 2013: 9-14 nikolić, lj. et al.  analysis of weed flora in conventional… 14 in terms of the spectrum of areal types, only species capable of wide distribution are present in conventional potato crops, while in organic crops, elements of pontic group are also found. the results reveal small differences in the weed flora structure between conventionally and organically grown potato crops, most likely due to agrotechnical practices. acknowledgements. this study is part of the project tr – 31027 «organic agriculture: improvement of production by use of fertilizers, biopreparations and biological measures« funded by subsidized by the ministry for science and technological development of the republic of serbia. references bàrberi, p. 2002: weed management in organic agriculture: are we addressing the right issues? weed research, 42(3): 177-193. boguzas, v., marcinkeviciene, a., kairyte, a. 2004: quantitative and qualitative evaluation of weed seed bank in organic farming. agronomy research, 2(1): 13-22. bound, w., grundy, a.c. 2001: non-chemical weed management in organic farming systems. agronomy research, 41: 383-405. gabriel, d., tscharntke,t. 2007: insect pollinated plants benefit from organic farming. agriculture, ecosystems and environment, 118: 43-48. gajić. m. 1980: pregled vrsta flore sr srbije sa biljnogeografskim oznakama. glasnik šumarskog fakulteta, 54(a): 111-141. hald, a.b. 1999: weed vegetation (wild flora) of long estabilished organic versus conventional cereal fields in denmark. annals of applied biology, 134: 307-314. http://europa.eu/legislation_summaries/agriculture/e nvironment/l28024_en.htm the 2001 biodiversity action plan for agriculture /com/2001/0162). hulina, n. 2005: list of threatened weeds in continental part of croatia and their possible conservation. agriculturae conspectus scientificus, 70 (2): 37-42. hyvönen, t., ketoja, e., salonen, j., jalli, h., tiainen, j. 2003: weed species diversity and community composition in organic and conventional cropping of spring cereals. agriculture, ecosystems and environment, 97: 131-149. hyvönen, t. 2007: can conversion to organic farming restorethe species composition of arable weed communities? biological conservation, 137: 382-390. josifović, m. (ed.), 1970-1977: flora sr srbije, iix, sanu, beograd. kojić, m., popović, r., karadžić, b. 1998: sintaksonomski pregled vegetacije srbije. institut za biološka istraživanja »siniša stanković«, beograd, 1-218. knežević, m., baketa, e. 1990: phytocenological characteristics of the buckwheat weed community in north-eastern croatia. fagopyrum, 11: 15-18 kristiansen, p. e. 2003: sustainable weed management in organic herb and vegetable production. monash, new england, australia, university of new england, phd thesis, archived at http://orgprints.org/4829 menalled, f.d., gross, k.l., hammond, m. 2001: weed aboveground and seedbank community response to agricultural management systems. ecological applications, 9: 634-641. moss, r.s., storkey, j., cussans, w.j., perryman, a.m.s., hewitt, v.m. (2004): simposium the broadbalk long-term experiment at rothamsted: what has it told us about weeds? weed science, 52: 864-873. roschewitz, i., gabriel, d., tscharntke, t., this c. 2005: the effects of landscape complexity on arable weed species diversity in organic and conventional farming. journal of applied ecology, 42: 873-882. stevanović, v. , vasić, v. 1995. o biodiverzitetu. in: stevanović, v. & vasić, v. (eds.). biodiverzitet jugoslavije sa pregledom vrsta od međunarodnog značaja, 1-9, ecolibri, beograd, biološki fakultet, beograd. weibull, a.c., östman, ö., granqvist, a. 2003: species richnes in agroecosystems; the effect of landscape, habitat and farm management. biodiversity and conservation, 12: 1335-1355. takhtajan, a. 2009: flowering plants. second edition. springer. ujvárosi, m. 1973: gymnövények. mezőgazdasági kiado, budapest. znaor, d. 1996: ekološka poljoprivreda. nakladni zavod globus, zagreb, 469pp. http://europa.eu/legislation_summaries/agriculture/environment/l28024_en.htm http://europa.eu/legislation_summaries/agriculture/environment/l28024_en.htm http://orgprints.org/4829 vasić et al., 2019, biologica nyssana 10(1) 10 (1) september 2019: 17-21 doi: 10.5281/zenodo.3463990 phytopathogenic fungi causers fungal diseases of the faba bean (vicia faba l.) in serbia original article tanja vasić faculty of agriculture, university of niš, kosančićeva 4, 37000 kruševac, serbia tanjavasic82@gmail.com (corresponding author) sanja živković faculty of agriculture, university of niš, kosančićeva 4, 37000 kruševac, serbia gajicsanja43@gmail.com jordan marković institute for forage crops, 37251 kruševac, serbia jordan.markovic@ikbks.com ivana stanojević faculty of agriculture, university of niš, kosančićeva 4, 37000 kruševac, serbia stanojevic.ivana85@gmail.com sonja filipović faculty of agriculture, university of niš, kosančićeva 4, 37000 kruševac, serbia sonjafilipovic86@yahoo.com dragan terzić institute for forage crops, 37251 kruševac, serbia dragan.terzic.agro@gmail.com received: june 24, 2019 revised: september 8, 2019 accepted: september 9, 2019 abstract: faba bean (vicia faba l.) is one of the oldest legume crops mainly grown as a valuable protein-rich food, both for human and animal consumption, where, in latter case, it provides an alternative to soybean meals in the temperate regions. there has not been systematic research of faba bean mycoflora in serbia. this paper aims to present the results of preliminary investigation of mycopopulation of 10 different genotypes of faba bean. total of 400 plant parts were examined, and 6 genera of fungi were isolated: fusarium, rhizoctonia, phoma, sclerotinia, alternaria and ascochyta. the results indicate that faba bean is vulnerable to a large number of phytopathogenic fungi that can have a significant impact on reducing its yield and quality. key words: faba bean, mycopopulation apstract: fitopatogene gljive izazivači gljivičnih oboljenja boba (vicia faba l.) u srbiji bob (vicia faba l.) je najstarija kultura koja se uzgaja kao hrana bogata proteinima, kako za ljudsku, tako i za stočnu ishranu, predstavljajući alternativu sojinim obrocima u umerenim područjima. s obzirom da nije bilo sistematskog istraživanja mikoflore boba u srbiji, cilj ovog rada je da se predstave preliminarni rezultati mikopopulacije 10 različitih genotipova boba. ispitano je ukupno 400 biljnih delova i izolovano je 6 rodova gljiva: fusarium, rhizoctonia, phoma, sclerotinia, alternaria i ascochyta. dobijeni rezultati ukazuju da je bob osetljiv na veliki broj fitopatogenih gljiva koje mogu imati značajan uticaj na smanjenje prinosa i kvaliteta boba. ključne reči: bob, mikopopulacija introduction faba bean is an annual plant from the genus of vetches (vicia), the family of legumes (fabaceae). it is originally of african-asian origin, and it is grown all over the world. faba bean was known as an annual field legume since ancient times. faba bean was cultivated in ancient egypt, greece, and rome, as confirmed by numerous records. it was used for human nutrition as well as for green fertilisation. today, faba beans, both garden and fodder, are grown in many countries of the world. in europe it is cultivated in england, belgium, scandinavian countries, germany, russia and italy (mišković, 1986). in our country faba bean is grown in a low capacity, especially as a fodder. faba bean is a versatile fodder crop. the content of nutrients in the grain of the faba bean and the plant as a whole is significant. the grain contains about 85% dry matter, 25.1% of crude proteins, 46.8% of bem, 1.6% of crude fat, 9.4% of raw cellulose and about 3.5% of crude ash. because of this composition, faba bean serves as an excellent concentrate feed for most species of livestock. also, fabae bean grain is used in human nutrition, like stew, as it has a high nutritional value (vučković, 1999; aleksić et al., 2015). vicia faba l. can fix nitrogen through symbiosis with rhizobium leguminosarum in its root nodules (stoddard et al., 2010). the most important and widespread fungal diseases observed at all locations in the world are: rust (uromyces fabae), chocolate spot (botrytis fabae and b. cinerea), ascochyta blight (ascochyta fabae), leaf © 2019 vasić et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 17 spots (alternaria alternata and cercospora fabae) and wilt/root rot complex (fusarium oxysporum and macrophomina phaseolina dominating). other diseases of apparent minor importance recorded at low incidence levels at most locations in the world are sclerotinia blight (sclerotinia sclerotiorum), downy mildew (peronospora viciae) (akem and bellar, 1999; sillero et al., 2010; stoddard et al., 2010). in ethiopia chocolate spot (botrytis fabae and b. cinerea) and rust (uromyces fabae) are the major diseases which can reduce yield by about 61 and 21%, respectively (tegen, 2017). in croatia two fusarium species were determined on the seed vetch, f. verticillioides and f. proliferatum (miličević et al., 2013). since faba bean has become very important in recent years as a fodder crop in serbia, the aim of this paper was to determine the phytopathogenic fungi disease causative agents in faba bean for more precise understanding of the problems (plant die-off, reduction of yield and quality, etc.) that arise as a result of the presence of phytopathogenic fungi in faba bean. materials and methods the samples were collected between march and june 2016-2017 at the location of the institute for forage crops in globoder. the samples of faba bean seed for the study of mycoflora were taken from different sites: the rasina region (kruševac 1, kruševac 2, gaglovo 2), zaječar region (crni kao), nišava region (praskovče, rujevica, šarbanovac, sokobanja) and pomoravlje region (jagodina 3, oparić). parts of plants are carefully washed under running water. after washing, the parts of stem and roots were cut to pieces of 0.5-1 cm in size. prepared samples of roots and stems were disinfected with 96% ethanol for 10 seconds and with 1% sodium hypochlorite (naocl) for 1 minute and then washed three times in sterile distilled water. they were then dried on sterile filter paper and placed on potato dextrose agar (pda) with streptomycin. five pieces of the plant parts (root and stem) were each placed in petri dish in four replications. they were kept in a thermostat at 25°c in 12 h light / 12 h dark regime. the observations were performed every 3 days, and the majority of mycelium samples were developed up to 14 days. developed mycelia were screened to a new pda substrate and, after an initial grow, the peak part of the mycelium was reseeded on pda again. microscopic examination was performed using microscopes olympus cx31. morphological identification of fungi to the genus was carried out using a standard key. the frequency of isolation was calculated in percents according to the formula by vrandečić et al. (2011): results and discussion in these studies, the mycopopulation of faba bean genotypes was examined in a total of 400 plant parts. the isolation frequency of the pathogens from diseased samples largely confirmed the field diagnosis based on visual symptoms. there were clearly expressed symptoms on the leaves in the form of necrotic spots and lesions in all the plants where fungi were isolated. fungi of genera ascochyta and alternaria were isolated from these plants (tab. 1). likewise, necrosis with the presence of white, aerial mycelium was observed on stems in a large number of plants. fungi from the genus sclerotinia were isolated from the lower third of the stems of these (tab. 1). symptoms in the form of light to dark brown necrosis were present at the plant root system, and from these plants were isolated fungi from the genera fusarium, phoma and rhizoctonia (tab. 1). in these studies, there was a difference in the isolation frequency of some genera of phytopathogenic fungi in faba bean genotypes originating from different regions of serbia. in the samples from the rasina region (krusevac 1, krusevac 2, gaglovo 2), on the leaves, the infection by genus alternaria was 10-40%, and by genus ascochyta was represented in all tested samples at 30% (tab. 1). fungi from genera fusarium with 66.67% and phoma with 40% were dominant in the crowns of the root in the faba bean plants from rasina region (tab. 1). in the zaječar region (crni kao), the most represented fungi were ascochyta with 40% in leaves, and sclerotinia with 43.33% it roots (tab. 1). in the nišava region (praskovče, rujevica, šarbanovac, sokobanja), the prevalent species were from the genus alternaria with 80% in leaves, while in the root system, fungi from genus rhizoctonia were the most abundant with 46.67%. genera alternaria with 80% and sclerotinia with 60% were the most prevalent in pomoravlje region (jagodina 3, oparić) (tab. 1). the genera fusarium, phythophthora, rhizoctonia, phoma, verticillium, alternaria and sclerotinia were dominant in annual and perennial legumes in the world (tivoli et al., 2006; villegas-fernández and rubiales, 2011; salam et. al., 2011; sillero et al., 2014; o’sullivan and angra, 2016). miličević et al. (2013) determined two fusarium species, f. verticillioides and f. proliferatum in vetch seed in croatia. rhizoctonia solani kühn is a soil parasite that can 18 biologica nyssana ● 10 (1) september 2019: 17-21 vasić et al. ● phytopathogenic fungi causers fungal diseases of the faba bean (vicia faba l.) in serbia (%) isolation frequency number of segments containing the fungal species total number of segments used in the isolation x 100= cause serious problems on many legumes, especially on faba bean (assunção, 2011). in canada, 304 faba bean genotypes were tested for resistance to r. solani and only five were identified with high resistance (rashid and bernier, 1993). sclerotinia stem rot, a fungal disease caused by sclerotinia trifoliorum, is often a serious problem in faba beans (vicia faba) in greece (lithourgidis et al., 2005). chocolate spot is caused by botrytis cinerea pers. and b. fabae sard., the latter being the most important since its action can result in serious plant damage, which is not usually the case with b. cinerea. chocolate spot is especially severe in humid areas, having been reported to be the cause of heavy reductions in yields in places such as the maghreb, southern china, egypt, uk or france (tivoli et al., 2006). the type of alternaria tenuissima was detected on the broad bean in japan. the disease was found in all surveyed fields. the initial lesion was brown, water-soaked, circular to slightly irregular. then the lesion enlarged and became concentric. mature 19 leaves had coalescing necrosis surrounded by yellowing. older leaves of the plant were particularly affected. in a later stage of the disease, the leaves became blighted from the margin to the centre and most of the diseased plants defoliated (rahman et al., 2002). ascochyta blight, caused by ascochyta fabae speg., is a common and destructive disease of faba bean (vicia faba l.) in the middle east, europe, canada, new zealand (díaz-ruiz et al., 2009). in syria ascochyta blight were frequently isolated from infected samples showing typical chocolate spot symptoms (akem and bellar, 1999). symptoms occur on leaves, stems and pods of infected plants, and can be confused with the early stages of chocolate spot (botrytis fabae). on leaves, small, circular, dark-brown spots appear first. as the disease develops, lesions enlarge and turn light and then change to dark grey. they become irregular in shape, often zonate, and may coalesce to cover most of the leaf surface. leaf tissue next to the lesions may become black and necrotic. within the lesions, numerous pinhead-sized black fruiting bodies (pycnidia) of genotypes number of samples (plant part) fungi species (leaf) (%) isolation frequency fungi species (root) (%) isolation frequencyleaf root 3/ii jagodina 3 10 30 alternaria sp. 80 sclerotinia sp. 60 19 red rujevicasokobanja 10 30 alternaria sp. 80 sclerotinia sp. rhizoctonia sp. 26.67 20 41 red kruševac 1 10 30 alternaria sp. ascochyta sp. 30 30 fusarium sp. 66.67 7/ii oparić 10 30 alternaria sp. ascochyta sp. 30 10 fusarium sp. sclerotinia sp. 23.33 6.67 15 red šarbanovacsokobanja 10 30 alternaria sp. ascochyta sp. 60 10 fusarium sp. sclerotinia sp. 43.33 16.67 20 red crni kao 10 30 alternaria sp. ascochyta sp. 20 40 sclerotinia sp. 43.33 kruševac 2 10 30 alternaria sp. ascochyta sp. 10 30 phoma sp. rhizoctonia sp. 40 23.33 10 red gaglovo 2 10 30 alternaria sp. ascochyta sp. 40 30 rhizoctonia sp. fusarium sp. 33.33 26.67 4 red sokobanja 10 30 alternaria sp. ascochyta sp. 50 20 rhizoctonia sp. 46.67 13 red praskovče 10 30 alternaria sp. ascochyta sp. 30 30 fusarium sp. sclerotinia sp. rhizoctonia sp. 16.67 13.33 13.33 table 1. frequency of fungal isolation on vicia faba l. biologica nyssana ● 10 (1) september 2019: 17-21 vasić et al. ● phytopathogenic fungi causers fungal diseases of the faba bean (vicia faba l.) in serbia 20 the fungus develop. these appear only under moist conditions and are often concentrically arranged (elkomy, 2014). ascochyta blight is a common disease that causes up to 90% yield losses in susceptible cultivars when environmental conditions are favourable for disease development (díaz-ruiz et al., 2009). conclusion this paper presents preliminary results of mycopopulation of 10 experimental faba bean genotypes. the obtained results indicate that faba bean is susceptible to the attack by a large number of phytopathogenic fungi that can significantly reduce its yield and affect its quality. faba bean has more and more significance in our country, becoming an important fodder crop as livestock feed. this paper is the beginning of a more comprehensive study of phytopathogenic fungi in faba bean. so far, there has been no significant research in this field in serbia, so future investigations related to the selection of genotypes with increased tolerance to the disease-causing agents will be conducted. selection to disease resistance can be used as part of the integral plant protection program (ipp), which can include biological and agronomic measures, as well as cultivation of resistant cultivars and hybrids, and which aims to prevent economically significant damage and preserve the environment. principles and practice of ipp include the monitoring or other detection methods, accurate identification of the target pest (phytopathogenic fungi), population monitoring, rotation of products with different mechanisms of action, and the use of plant protection products when the target pest population (phytopathogenic fungi) threshold at the local level reaches economically significant level. references akem, c., bellar, m. 1999: survey of faba bean (vicia faba l.) diseases in the main faba bean-growing regions of syria. arab journal of plant protection, 17 (2): 113-116. aleksić, j., banović, b., miljuš-đukić, j., jovanović, ž., mikić, a., ćupina, b., zlatković, b., anđelković, s., spanu, i., jelić, m., maksimović, v. 2015: a rapid and cost-effective procedure for delineation and utilization of genomic microsatellites for paralleled genotyping in vicia faba. czech journal of genetics and plant breeding, 51 (1): 36–39. assunçăo, i.p., nascimento, l.d., ferreira, m.f., oolivira, f.j., michereff, s.j., lima, g.s.a. 2011: reaction of faba bean genotypes to rhizoctonia solani and resistance stability. horticultura brasileira 29: 492-497. díaz-ruiz, r., satovic, z., ávila, c.m., alfaro, c.m., gutierrez, m.v., torres, a.m., román, b. 2009: confirmation of qtls controlling ascochyta fabae resistance in different generations of faba bean (vicia faba l.). crop & pasture science, 60: 353–361 el-komy, mahmoud h. 2014: comparative analysis of defense responses in chocolate spot-resistant and -susceptible faba bean (vicia faba) cultivars following infection by the necro-trophic fungus botrytis fabae. the plant pathology journal, 30 (4): 355366 lithourgidis, a.s., roupakias, d.g., damalas, c.a. 2005: inheritance of resistance to sclerotinia stem rot (sclerotinia trifoliorum) in faba beans (vicia faba l.). field crops research, 91 (2–3): 125–130. miličević, t., kaliterna, j., ivić, d., stričak, a. 2013: identification and occurrence of fusarium species on seeds of common vetch, whithe lupine and some wild legumes. agriculture, 19 (1): 25-32. mišković, b. 1986: krmno bilje. naučna knjiga, beograd. 503. o’sullivan, d.m., angra, d. 2016: advances in faba bean genetics and genomics. frontiers in genetics, 7 (150): 1-12. rashid, k.y., bernier, c.c. 1993: genetic diversity among isolates of rhizoctonia solani and sources of resistance in vicia faba. canadian journal of plant pathology, 15: 23-28. rahman, m.z., honda, y., islam, s.z., muroguchi, n., arase, s. 2002: leaf spot disease of broad bean (vicia faba l.) caused by alternaria tenuissima a new disease in japan. journal of general plant pathology, 68 (1): 31-37. salam, m.u., davidson, j.a., thomas, g.j., ford, r., jones, r.a.c., lindbeck, k.d., macleod, w.j., kimber, r.b.e., galloway, j., mantri, n., van leur, j.a.g., coutts, b.a., freeman, a.j., richardson, h., aftab, m., moore, k.j., knights, e.j., sillero, j.c., rojas-molina, m.m., emeran, a.a., rubiales, d. 2011: rust resistance in faba bean. grain legumes, 56: 27-28. sillero, j.c., villegas-fernàndez, a.m., thomas, j., rojas-molina, m.m., emeran, a.a., fernàndez-aparicio, m., rubiales, d. 2010: faba bean breeding for disease resistance. field crops research, 115 (3): 297–307. sillero, j.c., rubiales d. 2014: response of vicia species to ascochyta fabae and uromyces viciaefabae. czech journal of genetics and plant breeding, 50: 109–115. biologica nyssana ● 10 (1) september 2019: 17-21 vasić et al. ● phytopathogenic fungi causers fungal diseases of the faba bean (vicia faba l.) in serbia 21 stoddard, f.l., nicholas, a.h., rubialesc, d., thomas, j., villegas-fernàndez, a.m. 2010: integrated pest management in faba bean. field crops research, 115: 308–318. tegegn, a. 2017: effect of aerated and non-aerated compost seepages on the severity and incidence of major fungal diseases of faba bean; botrytis fabae, uromyces vicia fabae and ascochyta fabae. plant, 5 (6): 85-92. tivoli, b., baranger, a., avila, c.m., banniza, s., barbetti m., chen w.d., davidson j., lindeck, k., kharrat, m., rubiales, d., sadiki, m., sillero, j.c., sweetingham, m., muehlbauer, f.j. 2006: screening techniques and sources of resistance to foliar diseases caused by major necrotrophic fungi in grain legumes. euphytica, 147: 223–253. villegas-fernández, a.m., rubiales, d. 2011: chocolate spot resistance in faba bean. grain legumes, 56: 29-30. vrandečić, k., ćosić, j., jurković, d., poštić, j. 2011: mycopopulation of medicinal plants in croatia . agriculture, 17 (2): 18-21. vučković, s. 1999: krmno bilje. institut za istraživanja u poljoprivredi srbija, beograd; bonart, stara pazova. 553. biologica nyssana ● 10 (1) september 2019: 17-21 vasić et al. ● phytopathogenic fungi causers fungal diseases of the faba bean (vicia faba l.) in serbia air quality lichen monitoring at three selected urban areas biologica nyssana 7 (1)  september 2016: 19-29 stamenković, s. et al.  air quality lichen monitoring at three… 19 original article received: 09 mart 2016 revised: 21 mart 2016 accepted: 01 june 2016 air quality lichen monitoring at three selected urban areas in the southern serbia slaviša stamenković1*, tatjana djekić2, svetlana ristić1, vladica novković1, tatjana mitrović1, marija marković1 1university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia 2university of niš, faculty of sciences and mathematics, department of geography, višegradska 33, 18000 niš, serbia * e-mail: sslavisa@pmf.ni.ac.rs abstract: stamenković, s., djekić, t., ristić, s., novković, v., mitrović, t., marković, m.: air quality lichen monitoring at three selected urban areas in the southern serbia. biologica nyssana, 7 (1), september 2016: 19-29. the results of a study using epiphytic lichens as bioindicators, on teritory of three different urban areas in the southern serbia (leskovac, vlasotince, lebane) are reported. the analysis of samples from 142 investigated points indicates presence of 53 lichens taxa. the aim of the study is to present the difference in air quality over a certain time interval. using the index of atmospheric purityiap values, it has been found that there are different air pollution zones: „normal“ zone, „struggle“ zone and „lichen desert“ zone. the comparison of the results obtained in 2002 and 2013 in vlasotince indicates the presence of „normal“ zone in 2002 but, in 2013, the „normal zone“ area was completely replaced with the „struggle“ zone. in leskovac, unlike the previous studies, a slightly narrowing “lichen desert" zone is noticed. in lebane, there is a presence of all lichen zones. key words: biomonitoring, serbia, leskovac, vlasotince, lebane apstrakt: stamenković, s., djekić, t., ristić, s., novković, v., mitrović, t., marković, m.: lišajski monitoring kvaliteta vazduha određenih urbanih područja južne srbije. biologica nyssana, 7 (1), septembar 2016: 1929. istraživanja kvaliteta vazduha korišćenjem lišaja kao bioindikatora sprovedena su na teritoriji tri različita urbana područja južne srbijeleskovac, vlasotince i lebane. analiza uzoraka sa 142 istraživane tačke, ukazuje na prisustvo 53 lišajskih taksona. cilj ovog istraživanja je da prikaže promene kvaliteta vazduha koje su nastale u određenom vremenskom periodu. korišćenjem vrednosti indeksa atmosferske čistoće, konstantovano je prisustvo različitih zona zagađenja vazduha: „normalna“ zona, zona „borbe“ i zona „lišajske pustinje“. poređenje rezultata dobijenih 2002. i 2013. godine u vlasotincu, ukazuju na prisustvo „normalne“ zone u 2002. godini, međutim 2013. godine oblast „normalne“ zone je u potpunosti zamenjena zonom „borbe“. na području leskovca, razlika se ogleda u blagom suženju zone „lišajske pustinje“. u lebanu je uočeno prisustvo sve tri zone lišajske indikacije. key words: biomonitoring, srbija, leskovac, vlasotince, lebane 7 (1) • september 2016: 19-29 doi: 10.5281/zenodo.159100 biologica nyssana 7 (1)  september 2016: 19-29 stamenković, s. et al.  air quality lichen monitoring at three… 20 introduction the air quality is getting worse all over the world. the strongest effects are felt in urban areas. air pollution continuously increases, suggesting the need for timely detection, quantification and determination of levels of atmospheric pollution and introduction of monitoring system. biological monitoring is based on the assumption that changes in the environment affect the indicator organisms and these changes can be effectively used as an early warning signal for detecting of environmental changes (g a r t y , 2001). lichens are used in the biomonitoring studies because they are one of the most sensitive and valuable biomonitors of atmospheric pollution (s u j e t o v i e n e , 2015). lichens are symbiotic organisms composed of a fungus and an alga, usually either a cyanobacterium or green algae. it is estimated that there are approximately 25000 species of lichens (c h a p m a n , 2009). these unique organisms have a number of specific properties and therefore can be used for different purposes. changes in epiphytic lichen diversity are used as indicators of environmental conditions and have been widely applied in air quality assessments and monitoring programs around the world (l l o p et al., 2012). they are very good indicators of air quality. in the last decade of the twentieth century, a more intense use of epiphytic lichens as bioindicators on the territory of the republic of serbia began (m i l i ć , 1980; s a v i ć , 1991; 1997; 1998; s t a m e n k o v i ć , 1992; m i l i ć & b l a ž e n č i ć , 1993; t o d o r o v i ć , 1995; c v i j a n et al., 1995; 1997; 2008; s t a m e n k o v i ć , 1995; 1996; 1997; 1998; 2002; c v i j a n & s t a m e n k o v i ć , 1996a; 1996b; s t a n k o v i ć et al., 1999; p e j č i n o v i ć , 2000; s t a m e n k o v i ć & c v i j a n , 2002; 2003; 2004; 2010; s t a m e n k o v i ć et al., 2010; 2012; s t a m e n k o v i ć et al., 2013a; 2013b; j e v t i ć , 2013; n o v k o v i ć , 2013). this research is intended to present the results of the air quality on selected urban ecosystems and contribute the knowledge in biogeography and diversity of lichens on the territory of the republic of serbia and create the basis for the evaluation of lichens as indicators of the investigated urban territory. this is of great importance because there are no continuous physical and chemical measurements of air quality in these areas and this kind of monitoring is the only indicator of the air quality in urban conditions in the selected territory. material and methods sample location the southern serbia includes jablanica district. the administrative centre of the district is the town of leskovac, the biggest settlement in the southern serbia. this is the town with around 65289 inhabitants. the mean annual temperature is 10.7 °c and the mean annual rainfall is 591.6 mm. the most frequent winds are north, northwest and south (republic hydrometeorological service of serbia). leskovac has got a developed textile, chemical and pharmaceutical industry. the town of vlasotince is located in the southeastern part of serbia, in the area of middle and bottom basin of the river vlasina. the town has a population of 15882 people. the mean annual temperature is 11.8 °c and the mean annual rainfall is 724.3 mm, which is much higher than in the surrounding towns. east – west is the most common direction of movement of the winds (republic hydrometeorological service of serbia). fig. 1. geographical position of investigated region investigation region is framed. the town of lebane is an area which is characterized by extremely poor infrastructure, financial, economic and human resources. this is a small town having about 9272 inhabitants. the climate is moderate continental. the mean annual temperature is 10.2 °c and the mean annual rainfall in the vegetation period is 349 mm. the highest mean biologica nyssana 7 (1)  september 2016: 19-29 stamenković, s. et al.  air quality lichen monitoring at three… 21 wind speed from the north-northwest is 3.5 m/s, and the least is 1 m/s, from the northeast direction (republic hydrometeorological service of serbia). bioindication of air quality using lichens, as well as the physical and chemical measuring of air pollution have not been done in lebane until now. bioindication in vlasotince was made in 2002 and now. the institute of public health in leskovac controls physicochemical measuring of the level of air pollution since 2004. bioindication of air pollution in leskovac was made in 2002 and 2008. sample collection epiphytic lichens are determined and collected from the bark of various species of trees at the level of 1.52 m above the ground, exclusively from the trunks angled no higher than 5º. lichens were collected from 142 investigated points on the territory of the southern serbia. lichen materials were collected since 2002 to 2013. points of investigation are located in urban part of three different towns in the southern serbia. lichen samples were collected from the bark of various species of trees (tab. 1). the voucher specimens of the lichens were determined and deposited in the lichenological herbarium (under designation l; 1-9 hmn) of the department of biology and ecology, faculty of sciences and mathematics, university of niš, serbia. determination of collected lichen species was done by using a few sources (d o b s o n , 2005; w i r t h , 1995). determination of plant species was done by using “flora of serbia” (j o s i f o v i ć , ed., 1970-1977). the nomenclature of plant species follows euro+med plant base (http://www.emplantbase.org). method description the method of lichen indication of air quality means identifying, collecting, detecting and mapping the lichens and calculation of the index of atmospheric purity (iap) and it is based on determination of the zones of different degrees of air quality. investigation points have been noted and iap values for each point calculated. in this research, we were using the numeric method known as the index of atmospheric purity iap (k r i c k e & l o p p i , 2002): table 1. substrates in the three different surveys (the cells are filled with serial number of the investigated points) l o c a t i o n a n d t i m e o f c o l l e c t i o n supstrate (plant species) vlasotince 2002 vlasotince 2013 leskovac 2002 lebane 2013 acer campestre ͞ ͞ 1 ͞ carpinus betulus ͞ 2 ͞ ͞ fraxinus excelsior ͞ ͞ 6, 29, 35 ͞ fraxinus sp. ͞ ͞ ͞ 7 juglans regia 2, 5, 8, 10, 11 7 3, 4, 11, 21, 22 6, 9 malus domestica ͞ 10, 11, 13 15, 23 ͞ morus alba ͞ ͞ 18, 34 17 platanus acerifolia ͞ ͞ 33 ͞ populus alba 1 ͞ ͞ ͞ populus deltoides 4, 6, 7, 9, 13, 16 1, 4 5, 10, 19, 25, 30, 32, 36, 39 ͞ prunus armeniaca ͞ ͞ 8, 13 ͞ prunus avium ͞ 3, 12 20, 24 10 prunus domestica 3 8, 14 ͞ 4, 12, 16, 18, 19, 22 pyrus communis ͞ ͞ 12, 28, 31 ͞ quercus ceris ͞ ͞ ͞ 1, 2, 3, 8, 11, 14, 15, 24 robiania pseudoacacia ͞ ͞ 7, 27 13, 20 tilia argentea ͞ ͞ ͞ ͞ tilia plathyphyllos 12, 15 5, 6, 9 ͞ ͞ tilia sp. ̶ ̶ ͞ 5, 21, 23 biologica nyssana 7 (1)  september 2016: 19-29 stamenković, s. et al.  air quality lichen monitoring at three… 22 table 2. epiphytic lichen flora with values of frequency for each investigated point in the southern serbia taxon place/ time investigated point frequency (%) amandinea sp. leskovac/ 2002 28 2.56 amandinea punctata (hoffm.) coppins & scheid. leskovac / 2002 1, 8, 29, 30, 32, 33, 36 20.48 leskovac / 2008 25, 26, 28, 40, 41, 45, 46, 48, 49 18.37 vlasotince / 2002 7, 10, 12, 16 25.00 lebane / 2013 2, 11 8.33 arthonia punctiformis ach. lebane / 2013 19 4.16 caloplaca cerina (hedw.) th. fr. vlasotince / 2002 6, 10 12.50 caloplaca sp. leskovac / 2002 9, 14, 26 7.68 vlasotince / 2002 3, 16 12.50 candelariella aurella (hoffm.) zahlbr. leskovac / 2002 11, 14, 19, 20, 32 12.80 candelariella vitellina (hoffm.) müll. arg. vlasotince / 2013 7 7.14 candelariella xanthostigma (pers. ex ach.) lettau leskovac / 2002 1, 4, 9, 19, 26, 28, 29 12.80 leskovac / 2008 2, 9, 15, 22, 25, 26, 27, 28, 31, 36, 40, 41, 45, 46, 47, 48, 49 34.69 vlasotince / 2002 2, 13, 14, 16 25.00 vlasotince / 2013 1, 9, 12, 13 28.57 lebane / 2013 1, 3, 4, 5, 9, 11, 12, 14, 16, 17, 19, 21, 23, 24 58.33 candelariella sp. vlasotince / 2002 2 6.25 evernia prunastri (l.) ach. leskovac / 2002 1, 29, 32 12.80 leskovac / 2008 27, 28, 29, 45, 46, 47, 48, 49 16.33 vlasotince / 2002 5, 9 12.50 vlasotince / 2013 2 7.14 lebane / 2013 1, 2, 8, 11, 12, 16, 19, 21, 22, 24 41.66 flavoparmelia caperata (l.) hale leskovac / 2002 37 2.56 leskovac / 2008 47, 48 4.08 lebane / 2013 1, 2, 8, 11, 12, 14, 16, 19, 21, 22 41.66 gyalecta sp. vlasotince / 2002 16 6.25 vlasotince / 2013 10 7.14 graphis sp. vlasotince / 2002 3 6.25 hypogymnia physodes (l.) nyl. leskovac / 2002 37, 38 5.12 leskovac / 2008 36, 48 4.08 lebane / 2013 4, 6, 12 12.50 lecanora allophana (ach.) nyl. vlasotince / 2002 2, 5, 7, 8, 9, 10, 12 43.75 lecanora argentata (ach.) röhl. leskovac / 2002 9, 34, 36 10.24 leskovac / 2008 28 2.04 vlasotince / 2002 2, 4, 9, 10 25.00 lecanora carpinea (l.) vain. vlasotince / 2002 4, 9, 10, 12 25.0 lecanora glabrata (ach.) malme vlasotince / 2002 5, 10, 12 18.75 lecanora intumescens (rebent.) rabenh. leskovac / 2002 3, 9, 10, 14, 30, 34 17.92 vlasotince / 2002 2, 4, 5, 7, 8, 9, 10, 12, 13 56.25 lecanora muralis (schreb.) rabenh. vlasotince / 2002 2, 8, 9 18.75 lecanora pulicaris (pers.) ach. leskovac / 2002 22 5.12 leskovac / 2008 28 2.04 vlasotince / 2002 2, 4, 5, 8, 9 31.25 lecanora sp. leskovac / 2002 1, 8, 11, 14, 18, 19, 26, 28, 29, 32, 35, 36, 37, 39 42.18 leskovac / 2008 22, 28, 39, 45, 46 10.20 vlasotince / 2002 3, 11 6.25 vlasotince / 2013 2, 7, 9 21.43 lecidea sp. leskovac / 2008 47, 48 4.08 lecidella elaeochroma (ach.) m. choisy leskovac / 2002 1, 2, 6, 8, 16, 17, 24, 29, 30, 31 30.72 vlasotince / 2002 12, 13 12.50 lecidella sp. leskovac / 2008 47 2.04 lepraria aeruginosa (ach.) turner lebane / 2013 11 4.16 lepraria incana (l.) ach. leskovac / 2008 28 2.04 lebane / 2013 10, 16, 22 12.50 melanohalea elegantula (zahlbr.) o. blanco et al. leskovac / 2002 37, 38 5.12 melanohalea exasperata (de not.) o.blanco et al. leskovac / 2002 38, 39 5.12 vlasotince / 2002 1, 2, 8 18.75 melanohalea exasperatula (nyl.) o. blanco, a. crespo, divakar, essl., d. hawksw. & lumbsch leskovac / 2002 1, 29 7.68 melanelixia glabra (schaer.) o. blanco et al. leskovac / 2002 1 5.12 leskovac / 2008 36 2.04 melanelixia glabratula (lamy) sandler berlin & arup leskovac / 2002 1, 2, 29 7.68 leskovac / 2008 27, 28, 47, 49 8.16 vlasotince / 2002 1 6.25 biologica nyssana 7 (1)  september 2016: 19-29 stamenković, s. et al.  air quality lichen monitoring at three… 23 continuation of table 2 taxon place/ time investigated point freque ncy (%) melanelixia subargentifera (nyl.) o. blanco, a. crespo, divakar, essl., d. hawksw. & lumbsch lebane / 2013 18, 19, 21 12.50 melanelixia subaurifera (nyl.) o.blanco et al. leskovac / 2002 1 5.12 leskovac / 2008 47, 48 4.08 lebane / 2013 1, 2, 8, 11, 12, 14, 16, 24 33.33 melanelia sp. leskovac / 2002 2, 20, 28 7.68 leskovac / 2008 4 2.04 vlasotince / 2002 1, 2 12.50 vlasotince / 2013 2, 6 14.29 ochrolechia sp. vlasotince / 2013 11 7.14 parmelia sulcata taylor leskovac / 2002 1, 2, 7, 8, 16, 20, 24, 27, 28, 29, 33 33.28 leskovac / 2008 15, 36, 39, 47, 48 10.20 vlasotince / 2002 1, 3, 5, 8, 9, 10, 16 43.75 vlasotince / 2013 2, 3, 4, 5, 13, 14 42.86 lebane / 2013 1, 4, 6, 17, 18, 19, 20, 21, 22, 23, 24 45.83 parmelia tiliacea (hoffm.) ach. leskovac / 2002 1, 3, 32 7.68 leskovac / 2008 4, 14, 27, 28, 37, 48 12.24 lebane / 2013 5, 21, 23 12.50 parmelina pastillifera (harm.) hale leskovac / 2002 37, 38, 39 7.68 vlasotince / 2002 8 6.25 parmelina quercina (willd.) hale vlasotince / 2002 3, 13 12.50 lebane / 2013 3, 8, 14, 15 16.66 phaeophyscia orbicularis (neck.) moberg leskovac / 2002 1, 2, 3, 4, 5, 6, 8, 10, 11, 12, 13, 15, 16, 18, 19, 20, 21, 28, 29, 30, 35 56.32 leskovac / 2008 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 14, 15, 16, 17, 19, 21, 22, 23, 24, 25, 26, 27, 28, 31, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 79.59 vlasotince / 2002 1, 2, 3, 4, 6, 7, 8, 9, 10, 11, 12, 14, 15 81.25 vlasotince / 2013 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 100.00 lebane / 2013 1, 2, 3, 5, 8, 9, 10, 14, 16, 19, 22 45.83 physcia adscendens (fr.) h. olivier leskovac / 2002 1, 3, 8, 10, 11, 12, 19, 20, 28, 29, 30, 32, 33 40.96 leskovac / 2008 1, 2, 3, 4, 5, 6, 7, 8, 9, 12, 13, 14, 15, 16, 18, 19, 20, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 81.63 vlasotince / 2002 1, 4, 6, 8, 9, 10, 11, 13, 16 56.25 vlasotince / 2013 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 100.00 lebane / 2013 2, 5, 6, 9, 10, 11, 12, 13, 15, 17, 19, 22, 24 54.16 physcia aipolia (ehrh. ex humb.) fürnr. leskovac / 2002 29 5.12 vlasotince / 2002 8, 11 12.50 lebane / 2013 3 4.16 physcia stellaris (l.) nyl. leskovac / 2002 1, 3, 14, 20, 32 10.24 leskovac / 2008 28, 29, 48, 49 8.16 vlasotince / 2002 6, 8, 13 18.75 physcia tenella (scop.) dc. leskovac / 2002 1, 7, 20, 28, 29 17.92 leskovac / 2008 48 2.04 vlasotince / 2002 4, 8, 11 18.75 physconia enteroxanta (nyl.) poelt leskovac / 2002 1 7.68 leskovac / 2008 48 2.04 vlasotince / 2013 6 7.14 lebane / 2013 2, 4, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 19, 21, 23 62.50 physconia grisea (lam.) poelt leskovac / 2002 1, 25 10.24 leskovac / 2008 27, 28 4.08 vlasotince / 2002 1, 4, 5, 6, 10 31.25 lebane / 2013 2 4.16 pleurosticta acetabulum (neck.) elix & lumbsch leskovac / 2002 39 2.56 leskovac / 2008 28 2.04 lebane / 2013 7, 24 8.33 ramalina calicaris (l.) röhl. lebane / 2013 12, 19, 22 12.50 ramalina farinacea (l.) ach. leskovac / 2002 38 2.56 ramalina sp. leskovac / 2002 38, 39 5.12 usnea hirta (l.) weber ex f.h. wigg. leskovac / 2002 37 2.56 xanthoria parietina (l.) th. fr. leskovac / 2002 1, 2, 5, 8, 10, 14, 16, 19, 20, 25, 29, 36 35.84 leskovac / 2008 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 18, 19, 20, 22, 23, 24, 25, 26, 27, 28, 30, 32, 33, 34, 36, 37, 38, 39, 40, 41, 43, 45, 46, 47, 48, 49 83.67 vlasotince / 2002 1, 4, 5, 6, 7, 8, 10, 12, 14, 16 62.50 vlasotince / 2013 1, 3, 4, 5, 6, 8, 9, 10, 13, 14 71.43 lebane / 2013 1, 2, 3, 4, 5, 6, 8, 9, 10, 12, 13, 14, 15, 17, 18, 20, 21, 22, 23, 24 83.33 biologica nyssana 7 (1)  september 2016: 19-29 stamenković, s. et al.  air quality lichen monitoring at three… 24 𝐼𝐴𝑃 = 1 10 ∑(𝑄𝑖 × 𝑓𝑖) 𝑛 𝑖=1 where n = a number of species on the spot, f = coefficient which represents the frequency and cover of each species, and q = the ecological index of each species, i.e. the mean value of other lichen species growing with the species under study within the surveyed area. results and discussion the analysis of samples from the described localities indicate the presence of 53 lichen taxa on 142 investigated points from the territory of the southern serbia (tab. 2). the most frequent species were: phaeophyscia orbicularis, xanthoria parietina, physcia adscendens and parmelia sulcata. the highest frequency of these lichen species were found in all three investigated urban ecosystems. the least frequent species were: amandinea sp., ramalina farinacea and usnea hirta. these lichen species were found on only one of all investigated localities, in leskovac in 2002, having a low frequency 2.56%. the calculated iap values are very variable and they are between 0 and 47 (tab. 3). according to the calculated iap values, we got borders of the lichen zones of different air pollution levels in lebane (fig. 2). also, we got a picture which presents a comparison of moving the lichen zones in relation to the time of the survey in leskovac (fig. 3) and vlasotince (fig. 4). on the basis of these results and according to schulze (s c h u l z e et al., 2005), we can see that the investigated points in central part of urban ecosystems are included in “lichen desert” zone, characterized by the presence of species that are good indicators of air pollutiont. this indicates a high level of air pollution. the presence of this zone is recorded in most parts of urban areas of the southern serbia. in leskovac, we found presence of this zone in 2002 and 2008 (fig. 3). in lebane, three points belong to the “lichen desert” zone. in vlasotince, this zone did not exist in the year of 2002, but it is present in the year of 2013. (fig. 4). many investigated points belong to the “struggle” zone. in this zone, the air quality is slightly better than in the previous one. the maps clearly show that this zone usually includes peripheral parts of the city. smaller parts of the towns belong to “normal zone” which is detected in lebane and leskovac in the years of 2002 and 2008. we can also notice an interesting situation in vlasotince, where we detected the “normal zone” in the year of 2002 but, after eleven years, this zone does not exist anymore. such a picture of the different air pollution levels in the investigated areas is a logical consequence of air pollution and the mutual influence of microclimate, substrate and geophysical features, as well as the distribution of areas and objects. composition of lichens community shows certain deviations. the urban area of lebane includes all air quality zones. there is a relatively high level of air pollution, caused by the main road intersecting the center of the town and the former textile factory. agricultural land occupies 53% of the total area of the municipality of lebane, so, in this area, the presence of lichen is less probable. in 2002 and 2008, the bioindication of air quality by using lichens, in the area of leskovac was analyzed and compared. unlike in 2002, the boundaries of the “lichen desert” moved in 2008. there was a slight reduction of “lichen desert” zone. however, comparing to the year of 2002, in 2008, we can see a reduced number of lichen species of all types of the thallus morphology. other zones did not show significant changes in their limits, but the composition of the lichen species shows discrepancies. in leskovac concentrations of sulfur dioxide (so2), nitrogen oxides (nox), aero sediments and heavy metals (cd, pb, zn) in the suspended particles have been decreasing (institute of public health leskovac), which might have to do with the closing down of many industrial facilities. the reduction of the “lichen desert” zone might be correlated with a decrease of pollutants (sulfur dioxide, nitrogen oxides, aero sediments and heavy metals in suspended particles), while the reduced number of registered taxa might be linked to an increasing concentration of soot and heavy metals in the sediments. by comparing the results of bioindication of air quality by using lichens, in the vlasotince area in 2002 and in 2013, we found that the borders of the zones with different levels of air pollution using lichens as bioindicators in vlasotince are different in 2002 and 2013. investigation conducted in vlasotince in 2002 showed the presence of “struggle zone” and “normal zone”. eleven years later, in the investigated area, there aren't any records of the presence of “normal zone”. "normal" zone from 2002 is commuted into a “struggle zone" in 2013, and the "struggle zone" area has changed into "lichen desert" zone (fig. 4). compared to 2002, the absence of a number of taxa in 2013 provides the evidence for a considerably reduced diversity, which is supported by the altered frequency of some species’ presence. biologica nyssana 7 (1)  september 2016: 19-29 stamenković, s. et al.  air quality lichen monitoring at three… 25 this indicates that, in relation to the resistant species, coverage of the sensitive ones is reduced. domination of the more resistant species and a reduced total diversity of the lichen flora are indicating an increased air pollution ratio in vlasotince within the last 11 years. tabele 3. values of iap for every investigated point in the southern serbia l/t ip iap l/t ip iap l/t ip iap l/t ip iap l e sk o v a c /2 0 0 2 1 38 l e b a n e /2 0 1 3 1 6 v la so ti n c e /2 0 0 2 1 17 v la so ti n c e /2 0 1 3 1 4 2 4 2 6 2 19 2 5 3 3 3 6 3 7 3 3 4 1 4 7 4 19 4 4 5 3 5 7 5 14 5 5 6 1 6 8 6 14 6 3 7 2 7 3 7 7 7 2 8 4 8 10 8 24 8 2 9 4 9 9 9 17 9 4 10 8 10 5 10 28 10 3 11 6 11 7 11 3 11 2 12 1 12 7 12 11 12 3 13 0 13 4 13 8 13 3 14 9 14 7 14 3 14 3 15 0 15 5 15 1 16 2 16 6 16 11 17 1 17 7 18 3 18 4 19 7 19 8 20 6 20 2 21 0 21 18 22 0 22 5 23 / 23 5 24 1 24 9 25 2 26 3 27 1 28 21 29 39 30 3 31 1 32 12 33 6 34 1 35 1 36 4 37 35 38 23 39 47 biologica nyssana 7 (1)  september 2016: 19-29 stamenković, s. et al.  air quality lichen monitoring at three… 26 fig. 2. zones of different levels of air pollution in lebane using lichens as bioindicators (novković, 2013). fig. 3. borders of the zones with different levels of air pollution using lichens as bioindicators in 2002 (stamenković, 2002) and 2008 (nikolić, 2008), with "wind rose" in leskovac. biologica nyssana 7 (1)  september 2016: 19-29 stamenković, s. et al.  air quality lichen monitoring at three… 27 fig. 4. the "wind rose" with the delimitation of different lichen indication zones in vlasotince from investigations in 2002 (stamenković, 2002) and 2013 (jevtić, 2013). *delimitation of different lichen zones of indication in 2002; **delimitation of different lichen zones of indication in 2013 conclusion the method used in this research on relatively simple and quick way provides an insight into the quality of the air on investigated area by calculating the index of atmospheric purity (iap) based on the lichen diversity and abundance. according to the calculated iap values, we created a map of the zones of different air pollution levels in lebane and maps which present comparison of lichen zones movement in different time periods in vlasotince and leskovac. due to the fact that during the last 11 years the level of air pollution has increased so, if we do not take measures of protection, it can be assumed that the trend of the further growth of pollution will continue. this work is a mere contribution to the biomonitoring of air pollution i.e. air quality on the territory of the southern serbia. acknowledgements. this research was supported by the ministry of education and science of the republic of serbia, project oi 171025. authors express gratitude to marina jevtić for the ceded data from its master's thesis. references chapman, a.d. 2009: numbers of living species in australia and the world. australian biological resources study (abrs). canberra. 80 p. cvijan, m., todorović, b., joksimović, v., 1995: lichens as bioindicators of air pollution in the towns of mali zvornik and aranđelovac, glasnik instituta za botaniku i botaničke bašte univerziteta u beogradu, 29: 175-186, beograd. cvijan, m., stamenković, s., 1996a: bioindication of air pollution in the niš area by use of lichens. ekologija 31 (1), 151-157. cvijan, m., stamenković, s., 1996b: lignicolous lichens in the urban area of niš town. archive of biological sciences, 48 (3-4): 115-118, belgrade. cvijan, m., savić, s., szabados, k., 1997: lichens as bioindicators of air pollution in belgrade area. ekologija, 32 (1): 99-106. cvijan, m., subakov-simić, g., krizmanić, j. 2008: monitoring of the «lichen desert» in the belgrade area (1980/81, 1991 and 2007). archive of biological sciences, 60 (2), 215-222. dobson, f. s. 2005: lichens. richmond, richmond publishing co. ltd. biologica nyssana 7 (1)  september 2016: 19-29 stamenković, s. et al.  air quality lichen monitoring at three… 28 garty, j., tamir, o., hassid, i., eshel, a., cohen, y., karnieli, a., orlovsky, l. 2001: photosynthesis, chlorophyll integrity and spectral reflectance in lichens exposed to air pollution. journal of environmental quality, 30: 884-893. gombert, s., asta, j., seaward, m. r. d. 2004: assessment of lichen diversity by index of atmospheric purity (iap), index of human impact (ihi) and other environmental factors in an urban area (grenoble, southeast france). science of the total environment, 324: 183-199. hawksworth, d. l. and rose, f. 1970: qualitative scale for estimation sulfur dioxide air pollution in england and wales using epiphytic lichens. nature, 227: 145-148. institute of public health leskovac. http://www.zzjzle.org.rs/ jevtić, m., 2013: promene lišajskih zona biološke indikacije kvaliteta vazduha u vlasotincu u periodu 2002 – 2013. god. master thesis ( in serbian). prirodno-matematički fakultet. niš. kricke, r., loppi, s. 2002: bioindication: the iap approach. in: nimis, p., scheidegger, c. & wolseley, p. (eds.) monitoring with lichensmonitoring lichens. dordrecht, kluwer academic. llop, e., pinho, p., matos, p., pereira, m.j., branquinho, c. 2012: the use of lichen functional groups as indicators of air quality in a mediterranean urban environment. ecological indicators, 13: 215221. milić, m. 1980: bsc thesis lichens and air pollution in belgrade. senior thesis (in serbian), faculty of biology, university of belgrade. milić, m. and blaženčić, j. 1993: the epiphytic lichenes in the city of belgrade. glasnik instituta za botaniku i botaničke bašte univerziteta u beogradu, 24-25: 83-96. nikolić, m. 2009: monitoring životne sredine na urbanoj teritoriji grada leskovca u periodu od 2002 2008, diplomski rad. prirodnomatematički fakultet. niš. novković, v. 2013: lišaji kao pokazatelji kvaliteta vazduha u lebanu. master thesis (in serbian). prirodno-matematički fakultet. niš. pejčinović, d. 2000: lišajna flora vranja i uže okoline, 6. simpozijum o flori jugoistočne srbije i susednih područja, univerzitet u prištini, sokobanja, 3941 str. republic hydrometeorological service of serbia. http://www.hidmet.gov.rs/ savić, s. 1991: promene u sastavu flore lišajeva kao bioindikatora aerozagadjenja na području beograda za poslednjih deset godina. senior thesis (in serbian), faculty of biology, university of belgrade, 87 pp. savić, s. 1997: epifitski lišajevi kao bioindikatori aerozagadjenja na području beograda. master thesis (in serbian), faculty of biology, university of belgrade, 60 pp. savić, s. 1998: epiphytic lichens as bioindicators of air pollution in the area of belgrade. sauteria 9, ial 3 proceedings 331-340. schulze ed, beck e, muller-hohenstein k. 2005. plant ecology, berlin⁄heidelberg, germany: springer stamenković, s. 1992: bioindikacija aerozagađenja lignikolnim lišajevima na području grada niša. master thesis (in serbian). biološki fakultet. beograd. stamenković, s. 1995: lignikolna lišajska flora prokuplja. ekologija, 30(1-2): 41-46, beograd. stamenković, s., 1996: biological indication of air pollution in vlasotince using lignicolous lichens. acta biologica jugoslavica – serija d: ekologija, 33 (1–2): 71–74. belgrade. stamenković, s. 1997: biological indication of air pollution in prokuplje by means of lignicolous lichens. ekologija, 32: 107-110. belgrade. stamenković, s. 1998: biological indication of air pollution in vlasotince using lignicolous lichens, ekologija, 33 (1-2): 71-74, belgrade. stanković, s., stanković, a., pantelić, g. 1999: zagađenost lišaja i mahovina istočne srbije prirodnim i veštačkim radionuklidima, ekološka istina vii naučno-stručni skup o privrednim vrednostima i zaštiti životne sredine, zaječar, 16. zbornik radova. stamenković, s. 2002a: indikacija aerozagađenja u urbanim centrima južne i jugoistočne srbije korišćenjem lišajeva kao bioindikatora, phd disseratation (in serbian), biološki fakultet, univerzitet u beogradu. stamenković, s. 2002b: bioindication of air pollution in pirot by use of lichens. ekologija, 37 (1-2), 3340, belgrade. stamenković, s., cvijan, m. 2002: epiphytic lichens as bioindicator of air quality in leskovac (southern serbia). ekologija, 37(1-2), 41-46, belgrade. stamenković, s. and cvijan, m. 2003: bioindication of air pollution in niš by using epiphytic lichens. archive of biological sciences, 55: 133-140. stamenković, s., cvijan, m. 2004: using of epiphytic lichens for bioindication of air pollution in vranje. archive of biological sciences, 56 (3-4): 139-143. belgrade. stamenković, s., cvijan, m., 2010: determination of air pollution zones in knjaževac (southeastern serbia) by using epiphytic lichens, biotechnology and biotechnological equipment, special editionbiologica nyssana 7 (1)  september 2016: 19-29 stamenković, s. et al.  air quality lichen monitoring at three… 29 online (2nd balkan conference on biology, plovdiv, bulgaria, 2010), 278-283, bulgaria stamenković, s., cvijan, m., aranđelović, m. 2010: lichen as bioindicators of air quality in dimitrovgrad (southeastern serbia), archive of biological sciences, 62 (3): 643-648, belgrade, serbia. stamenkovic, s., djekić, t., mitrović, t., stojičić, d., cvetković, v., nikolić, m. 2012: monitoring of air quality and „lichen desert“ in the city of leskovac (southeastern serbia) in the period 2000-2011, abstract book, iv congress of ecologists of the republic of macedonia with international participation, 106p, skoplje, makedonija (fyrm) stamenković, s., mitrović, t., cvetković, v., krstić, n., baošić, r., marković, m., nikolić, n., marković, v., cvijan, m. 2013: biological indication of heavy metal pollution in the areas of donje vlase and cerje (southeastern serbia) using epiphytic lichens. archive of biological sciences, 65(1): 151-159, belgrade, serbia. stamenković, s., ristić, s., đekić, t., mitrović, t., baošić, r. 2013a: air quality indication in blace (southeastern serbia) using lichens as bioindicators. archive of biological sciences, 65 (3): 893-897. sujetoviene, g. 2015: monitoring lichen as indicators of atmospheric quality. recent advances in lichenology, 4: 87118. todorović, b. 1995: the lichens as bioindicators of air pollution in the towns mali zvornik and aranđelovac. glasnik instituta za botaniku i botaničke bašte univerziteta u beogradu, 29: 175186. vantova, i., backor, m., klejdus, b., backorova, m., kovacik, j. 2013: copper uptake and copperinduced physiological changes in the epiphytic lichen evernia prunastri. plant growth regulation, 69: 1-9. wirth, v. 1995: die flechten baden-wurtembergs. verbreitungsatlas, 1&2. wolterbeek, b. 2002: biomonitoring of trace element air pollution: principles, possibilities and perspectives. environmental pollution, 120: 1121. the ecological and floristic characteristics of natural population of micromeria juliana (l.) benth. ex rchb. in bulgaria biologica nyssana 8 (1)  september 2017: 105-111 lazarević, m. et al. morphological discrimination of the genera… 105 original article received: 25 january 2017 revised: 11 july 2017 accepted: 15 july 2017 morphological discrimination of the genera binodoxys mackauer and trioxys haliday (hymenoptera: braconidae: aphidiinae) based on the general shape of forewings maja lazarević*, marijana ilić milošević, saša s. stanković, vladimir žikić university of niš, faculty of science and mathematics, department of biology and ecology, višegradska 33, niš, serbia * e-mail: majalazarevic9@gmail.com abstract: lazarević, m., ilić milošević, m., stanković, s.s., žikić, v.: morphological discrimination of the genera binodoxys mackauer and trioxys haliday (hymenoptera: braconidae: aphidiinae) based on the general shape of forewings. biologica nyssana, 8 (1), september 2017: 105-111. the genera binodoxys and trioxys, members of the aphidiinae subfamily, are classified in the same subtribe trioxina within the tribe trioxini. these two genera are morphologically very similar, but differ in a large degree in their ecology. generally, binodoxys species are specialized in parasitizing aphids on herbaceous plants, unlike trioxys species, which have co-evolved with the tree-attacking aphids. for this study, we analyzed the right forewings of four binodoxys and two trioxys species, using the method of geometric morphometrics. sixteen specific landmarks were digitalized for each forewing of 80 parasitoid individuals. the results of two statistical methods anova and manova demonstrated statistically significant differences of the wing shapes, especially between the genera. visualizing the results of the dispersion of specimens in the morphospace via multivariate tests pca and cva, we found that the most variable character in wing structure was the radial sector, differentiating binodoxys from trioxys. key words: trioxina, geometric morphometrics, variability, parasitoids apstrakt: lazarević, m., ilić milošević, m., stanković, s.s., žikić, v.: morfološko razdvajanje rodova binodoxys mackauer i trioxys haliday (hymenoptera: braconidae: aphidiinae) na osnovu oblika krila. biologica nyssana, 8 (1), septembar 2017: 105-111. rodovi binodoxys i trioxys, članovi podfamilije aphidiinae, su klasifikovani u isti podtribus trioxina u okviru tribusa trioxini. pomenuta dva roda su morfološki veoma slična, ali se ekološki dosta razlikuju. generalno posmatrajući, vrste roda binodoxys su se specijalizovale za parazitiranje biljnih vaši koje se hrane zeljastim biljkama, za razliku od vrsta roda trioxys, koje su koevoluirale sa biljnim vašima koje žive na drvenastim biljkama. za ovu studiju, analizirana su desna krila četiri vrste roda binodoxys i dve trioxys vrste, koristeći metod geometrijske morfometrije. za svako krilo, od ukupno 80 analiziranih jedinki parazitoida, izabrano je šesnaest tačaka. rezultati dve statističke metode, anova i manova, pokazali su postojanje statistički značajnih razlika u obliku krila, naročito između rodova. vizuelizacijom rezultata multivarijantnim testovima, 8 (1) • september 2017: 105-111 doi: 10.5281/zenodo.964447 biologica nyssana 8 (1)  september 2017: 105-111 lazarević, m. et al. morphological discrimination of the genera… 106 pca i cva, uočeno je da je najvarijabilnija struktura radijalni sektor, na osnovu koga se rodovi binodoxys i trioxys razdvajaju. ključne reči: trioxina, geometrijska morfometrija, varijabilnost, parazitoidi introduction the aphidiinae subfamily can be distinguished from other members of the braconidae family for being the exclusive parasitoids of aphids (hemiptera: aphididae). they are solitary koinobiont endoparasitoids. uniquely parasitizing aphids was one of the reasons why s t a r ý (1981) recognized this group as a separate entity, raising it to the family level – aphidiidae, which can be found in older literature. nowadays, this group of parasitoids is usually considered as one of the subfamilies of braconids (q u i c k e & v a n a c h t e r b e r g , 1990; w h a r t o n et al., 1992). this is a moderately large subfamily with about 500 described species worldwide (y u et al., 2012). the subfamily is divided into five tribes, where the tribe trioxini in europe comprises nine genera: betuloxys mackauer 1960, binodoxys mackauer 1960, calaphidius mackauer 1961, falciconus mackauer 1959, harkeria cameron 1900, lipolexis föerster 1862, monoctonia starý 1962, monoctonus haliday 1833, and trioxys haliday 1833 (m a c k a u e r & s t a r ý , 1967; m a c k a u e r et al., 1968). the tribe trioxini consists of two subtribes, trioxina and monoctonina. subfamily aphidiinae have a diverse forewing venation, which is often quite a useful trait for determination of parasitoids on the generic level. among the subfamily, several members of the subtribe trioxina possess highly reduced forewing venation, with only the stigma, radial vein and radial sector vein. beside wings, which are the most useful trait, other morphological structures are important for determination of aphidiines on lower taxonomic levels, such as species (w h a r t o n et al., 1997). several genera of the subtribe trioxina contain paired prongs, which are extensions of the female’s seventh metasomal sternite. in european fauna, only the species of the genera betuloxys, binodoxys and trioxys possess those abdominal excrescences. these structures are very important for the identification of specimens on the generic and also species level; especially the shape and the curvature of prongs, the number and disposition of setae on them (s t a r ý , 1981). furthermore, european genera which have prongs can be differentiated from one another by the morphology of the second metasomal segment, petiole. while the genera betuloxys and trioxys have one pair of tubercles with spiracles, the genus binodoxys possesses two pairs, primary and secondary. in this genus, the primary tubercles are with spiracles, while the secondary are without them. the position of the secondary tubercles on petiole is variable and highly important for the identification of binodoxys at the species level (m a c k a u e r , 1959, 1960; f u l b r i g h t et al., 2007). knowing that the variability of the forewings in shape and size, and also in the wing venation can be a significant, often not conspicuous, characteristic to separate two morphologically similar species, there were a lot of studies where the geometric shape of the wing was used to accent that variability and those differences. in contrast to monoctonina, trioxina can be easily recognized by the forewing venation which is significantly reduced, but all minute differences between genera and among species of the same genus can be explained using the method of geometric morphometrics (z e l d i t c h et al., 2012). this particular method has already been successfully applied in many previous studies on aphidiinae wings (ž i k i ć et al., 2009, 2014; m i t r o v s k i -b o g d a n o v i ć et al., 2009, 2014; k o s et al., 2011; t o m a n o v i ć et al., 2014; i l i ć m i l o š e v i ć et al., 2015; s t a n k o v i ć et al., 2015), sometimes revealing a new species, such as ephedrus lonicerae tomanović, kavallieratos & starý 2009 in ž i k i ć et al. (2009). beside the morphological differences, there is also a biological dissimilarity between these genera, as their hosts feed on different plants. the species of the genus binodoxys attack aphids on herbaceous plants, while betuloxys and trioxys species are known mostly from the hosts feeding on trees and shrubs (m e s c h e l o f f & r o s e n , 1993; p i k e et al., 1996, 2000; a k h t a r et al., 2011; ž i k i ć et al., 2012). the aim of this study was to explore the variability of the shape and size of forewings, comparing the wing venation using the method of geometric morphometrics, and to determine whether there was a significant difference that would help in the further application of geometric morphometrics on the species of both investigated genera binodoxys and trioxys. material and methods sampled material the specimens for this study were collected in the period between 2010 and 2013 on the territory of serbia and montenegro (tab. 1). plant parts infested biologica nyssana 8 (1)  september 2017: 105-111 lazarević, m. et al. morphological discrimination of the genera… 107 with aphids were cut and placed into plastic containers and covered with a muslin cloth. according to the instructions given in k a v a l l i e r a t o s et al. (2010), these containers were placed in a growing cabinet and kept at the following conditions: 22.5 °c, 65% relative humidity and photo period of 16l:8d for 3-4 weeks until parasitoid emergence. thus collected parasitoids were placed in vials containing 96% ethanol. several wingless adult aphids were selected, put in 70% ethanol and sent to a specialist for identification. wing preparation, selection of landmarks and geometric morphometrics the method of geometric morphometrics was applied to explore the morphological differences in wing size and shape between the genera binodoxys and trioxys (z e l d i t c h et al., 2012). we examined 80 right forewings of females in total; four binodoxys species: b. acalephae (marshall 1896), b. angelicae (haliday 1833), b. brevicornis (haliday 1833) and b. heraclei (haliday 1833), and two species of trioxys: t. complanatus quilis perez 1931 and t. pallidus (haliday 1833) (see tab. 1). the right forewings of each specimen were detached, mounted on microscopic slides in berlese solution and photographed using leica system dm2500 microscope with leica dfc490 digital camera (leica microsystems©, wetzlar, germany). the specific landmarks from 1 to 9, together with the landmarks 11, 12 and 14, represent true landmarks (lm), while the other four (10, 13, 15 and 16) were set as semilandmarks (s-lm). these semilandmarks are used to better describe the distal part of the wing which lacks wing venation (fig. 1). the landmarks 1, 4 and 6 define the length and width of the stigma; lm 4 and 5 represent the metacarpal (r1) vein; lm 2, 6 and 7 describe the radial sector, while lm 8 represents the projection of the radial sector to the edge of the wing. the landmarks 3, 9, 10, 11 and 12, together with the s-lm 13, delineate the proximal part of the wing, while lm 5, 8, 14, and s-lm 15 and 16 define the distal part of the wing. the constellation of selected landmarks and the semi-landmarks was positioned and processed using the software makefun6 (s h e e t , 2003) and tpsdig2 (r o h l f , 2005). the general procrustes analysis was applied to eliminate variation which can be caused due to the differences in the wing size, orientation and/or positioning during the digitalization (b o o k s t e i n , 1991) and for each wing to compute the centroid size (cs) as a geometric measure of size. based on the cs, the differences in the wing size were analyzed using the analysis of variance (anova), while the shape variability was computed using the multivariate analysis of variance (manova). all statistical methods were done in statistica software package (stat soft inc. 7.0). to visualize the differences in the wing shape and size, the principal component analysis (pca) and canonical variate analysis (cva) were done using the software morphoj (k l i n g e n b e r g , 2011). table 1. the list of sampled material used in the analysis; abbreviations: srb – serbia, mne – montenegro; mj – m. janković, ss – s. stanković, zk – z. kojičić, vž – v. žikić parasitoid species no. of individuals country, locality, date, legator b. acalephae (marshall 1896) 14 srb, niš, popovac, 04.07.2010. vž b. angelicae (haliday 1833) 15 srb, sićevačka klisura (gorge) 12.05.2013. ss b. brevicornis (haliday 1833) 10 mne, durmitor mt., crno jezero (lake), 25.07.2012. vž b. heraclei (haliday 1833) 11 srb, dukat mt, 29.06.2012. ss t. complanatus quilis perez 1931 16 srb, smederevo, malo orašje, 10.06.2012. mj t. pallidus (haliday 1833) 14 srb, kruševac, 19.05.2013. zk fig. 1 set of specific landmarks and semi-landmarks on the forewing of binodoxys angelicae biologica nyssana 8 (1)  september 2017: 105-111 lazarević, m. et al. morphological discrimination of the genera… 108 results and discussion for the variability among and between the selected groups, anova test showed statistically significant differences in wing size (f(45216.25) = 3.62; p ˂ 0.006). the wing shape variability which was tested by manova also displayed statistically significant differences: wilks`λ = 0.000444; f(113631211) = 6; p ˂ 0.001. delineation of the genera in morphospace is presented over principal component analysis defined by the first two pc axes (fig. 2). the discrimination between bionodoxys and trioxys in the analysis of the forewing shape is evident, with a very small overlapping between the two genera (fig. 2), where the first two pc axes accounted for 64.76% of the total variability. the analyzed species are separated along pc1 = 34.08%, according to the length of the radial sector (fig. 1, 2) which was calculated as the most variable part of the wing. placing all binodoxys species in the negative part of pc1, this structure points a shorter radial sector than in trioxys. to a lesser extent, all other parts of the forewing contribute to the separation of the analyzed genera. as a consequence of all shape changing in the wing along the positive and the negative senses of the pc1, the wings in binodoxys species are generally shorter and broader in their distal part, lm (5, 7, 8, 14, 15 and 16), unlike trioxys species, which wings are elongated. changes in wing morphology illustrated by pc2 = 30.68% are reflected not so much in the length of the wings as much as in the width. thus, these kinds of changes explained by pc2 are more related to intragroup variability than the differences between the two genera. although there is a strong trend of delimitation of binodoxys and trioxys applying pca, to obtain a clearer view of the discrimination of these two genera, we used canonical variate analysis (cva). after applying cva, the ordination of analyzed specimens in the morphospace, which was defined by the first two canonical axes, has pointed out the interpreted morphological differences in wings much better (fig. 3). the first canonical axis (cv1) explained 53.57% of the total variability of the wing shape, thus splitting binodoxys and trioxys species in the two distinct groups. both analyzed species of the genus trioxys, t. complanatus and t. pallidus, are positioned in the negative part of the cv1, while the four species of binodoxys are distributed along the positive part of the cv1 axis. as it can be seen from the cva graph, most of the differences are caused by the variability of the distal part of the wing, where the lm 7 contributes most to the differences of the wing shape with respect to all other landmarks in this region. unlike both species of trioxys, the species of fig. 2 ordination of the binodoxys and trioxys species in the morphospace defined by the first two principal axes, for an interval of confidence of 0.8. the thin-plate transformation grids illustrate forewing changes along pc1 and pc2 biologica nyssana 8 (1)  september 2017: 105-111 lazarević, m. et al. morphological discrimination of the genera… 109 the genus binodoxys have a much shorter radial sector and rather wider wings. also, the wings of binodoxys are little larger (in average) than in trioxys. changes in wing size and shape are presented via transformation grids. the best discrimination was shown in b. acalephae, where this species is abstracted in the positive part of cv1, and the negative cv2 = 23.26% (fig. 3). the other species, b. angelicae, is clearly separated from the other three binodoxys species, being positioned in the positive part of the cv2. the other two species of binodoxys, b. brevicornis and b. heraclei, mostly overlap in the center of the morphospace defined by cv1 x cv2, sharing more or less the similar shape of the wings. in this analysis, both trioxys species are well discriminated. the reason of the partial overlapping lies in the morphology of the forewing, especially in the variation of the radial sector (f u l b r i g h t et al., 2007). since the genus trioxys is very large, comprising about 60 species (ž i k i ć et al., 2017), the radial sector can be variable. for example, it can be extremely short like in t. bonnevillensis (smith 1944), medium-sized in t. ibis (mackauer 1961) and very long in t. curvicaudus (mackauer 1967). having this in mind, it should be noted that the use of geometric morphometrics is suitable only for the species with similar wing armature. contrariwise, the majority of binodoxys species have a less variable radial sector on the forewings, and in comparison to the trioxys species listed above, we can say that it is of a medium size. therefore, we can see much greater overlapping among the binodoxys species. conclusion for a very long time, the taxonomic status of the genus binodoxys was considered as a subgenus within the genus trioxys. since m a c k a u e r (1959) separated these two genera, there was a need for the revision of both genera. most keys for determination of aphidiines are based on the morphology of petiole and shape of ovipositor and accessory prongs. adding the geometric shape of the wings to the characters listed above, it is confirmed that binodoxys and trioxys are two genera. acknowledgements. we express our gratitude to dr. željko tomanović and dr. andjeljko petrović (faculty of biology, university of belgrade, serbia) for useful comments concerning the manuscript. we would like to thank dr. olivera petrovićobradović (faculty of agronomy, university of belgrade, serbia) for the identification of the aphids. this study was funded by the ministry of education, science and technological development of the republic of serbia (iii43001). fig. 3 distribution of the species of binodoxys and trioxys in the morphospace defined by the cv1 and cv2 axes, for an interval of confidence of 0.85 biologica nyssana 8 (1)  september 2017: 105-111 lazarević, m. et al. morphological discrimination of the genera… 110 references akhtar, m.s., dey, d., usmani, m.k. 2011: a catalogue of aphid parasitoids (hymenoptera: braconidae: aphidiinae) from india. insecta mundi, 151: 1-31. bookstein, f.l. 1991: morphometric tools for landmark data: geometry and biology. cambridge university press, new york, 435 pp. fulbright, j.k., pike, k.s., starý, p. 2007: a key to north american species of trioxys haliday (hymenoptera: braconidae: aphidiinae), with a summary of the geographic distribution, hosts, and species diagnostic features. proceeding of the entomological society of washington, 109: 779790. ilić milošević, m., petrović, a., stanković, s.s., čkrkić, j., starý, p., žikić, v., tomanović, ž. 2015: taxonomic position and phylogenetic relationships of the genera and species euaphidius and remaudierea (hymenoptera: braconidae: aphidiinae) analyzed using molecular markers and geometric morphometrics. annals of entomological society of america, 108: 435-445. kavallieratos, n.g., tomanović, ž., starý, p., žikić, v., petrović-obradović, o. 2010: parasitoids (hymenoptera: braconidae: aphidiinae) attacking aphids feeding on solanaceae and cucurbitaceae crops in southeastern europe: aphidiine aphidplant associations and key. annals of entomological society of america,103: 153–164. klingenberg, c.p. 2011: morphoj: an integrated software package for geometric morphometrics. molecular ecology resources, 11: 353-357. kos, k., petrović, a., starý, p., kavallieratos, n.g., ivanović, a., toševski, i., jakše, j., trdan, s., tomanović, ž. 2011: on the identity of cereal aphid parasitoid wasps aphidius uzbekistanicus, aphidius rhopalosiphi, and aphidius avenaphis (hymenoptera: braconidae: aphidiinae) by examination of coi mitochondrial gene, geometric morphometrics, and morphology. annals of the entomological society of america, 104: 1221-1232. mackauer, m. 1959: die mittel-, westund nordeuropäischen arten der gattung trioxys haliday (hymenoptera: braconidae: aphidiinae). beiträgezur entomologie, 9: 144-179. mackauer, m. 1960: zur systematik der gattung trioxys haliday (hymenoptera: braconidae: aphidiinae). beiträgezur entomologie, 10: 137160. mackauer, m., starý, p. 1967: hymenoptera. ichneumonoidea. world aphidiidae. in index of entomophagous insects (v. delluchi and g. remaudière eds.). le fransois, paris, pp. 1-95. mackauer, m. ferriere, c., van der vecht, j. 1968: hymenopterorum catalogus. pars 3. aphidiidae. dr w. junk, the hague. 103 pp. mescheloff, e., rosen, d. 1993: biosystematic studies of the aphidiidae of israel (hymenoptera: ichneumonoidea). 5. the genera trioxys and binodoxys. israel journal of entomology, 27: 3147. mitrovski-bogdanović, a., ivanović, a., tomanović, ž., žikić, v., starý, p., kavallieratos, n.g. 2009: sexual dimorphism in ephedrus persicae (hymenoptera: braconidae: aphidiinae): intraspecific variation in size and shape. the canadian entomologist, 141: 550-560. mitrovski-bogdanović, a., tomanović, ž., mitrović, m., petrović, a., ivanović, a., žikić, v., starý, p., vorburger, c. 2014: the praon dorsale – yomenae s.str. complex (hymenoptera, braconidae, aphidiinae): species discrimination using morphometrics and molecular makers with description of a new species. zoologischer anzeiger, 253: 270-282. pike, k.s., starý, p., miller, r., allison, d., boydston, l., graf, g., miller, t. 1996: new species and host records of aphid parasitoids (hymenoptera: braconidae: aphidiinae) from the pacific northwest, u.s.a. proceeding of entomological society of washington, 98: 570591. pike, k.s., starý, p., miller, t., graf, g., allison, d., boydston, l., miller, r. 2000: aphid parasitoid (hymenoptera: braconidae: aphidiinae) of northwest usa. proceeding of entomological society of washington, 102: 688-740. quicke, d., van achterberg, c., 1990: phylogeny of the subfamilies of the family braconidae (hymenoptera: ichneumonoidea). zoologische verhandelingen, 258: 1–95. rohlf, f.j. 2005: tpsdig, digitize landmarks and outlines, version 2.05. department of ecology and evolution, state university of new york at stony brook (http://life.bio.sunysb.edu/morph/). sheets, h.d. 2003: ipm suite(integrated morphometrics package). canisius college, buffalo, ny. stanković, s.s., petrović, a., ilić milošević, m., starý, p., kavalllieratos, n.g., žikić, v., tomanović, ž. 2015: morphological and molecular characterization of common european species of adialytus (hymenoptera: braconidae: aphidiinae) based on the mtcoi barcoding gene and geometric morphometrics of forewings. european journal of entomology, 112: 165-174. biologica nyssana 8 (1)  september 2017: 105-111 lazarević, m. et al. morphological discrimination of the genera… 111 starý, p. 1981: biosystematical clasification of trioxys haliday and related genera (hymenoptera, aphidiidae). bollettino del laboratorio di entomologia agraria portici, 38: 85-93. statsoft inc. 2004: statistica (data analysis software system), version 7, www.statsoft.com. tomanović, ž., petrović, a., mitrović, m., kavallieratos, n.g., starý, p., rakhshani, e., rakhshanipour, m., popović, a. shukshuk, a.h., ivanović, a. 2014: molecular and morphological variability within the aphidius colemani group with redescription of aphidius platensis brethes (hymenoptera: braconidae: aphidiinae). bulletin of entomological research, 104: 552-565. yu, d.s., van achterberg, c., horstmann, k. 2012: world ichneumonoidea 2011. taxonomy, biology, morphology and distribution. taxapad (scie ntific names for information management), interactive catalogue, ottawa available on: www.taxapad.com. wharton, r., shaw, s., sharkey, m., wahl, d., woolley, j., whitfield, j., marsh, p., johnson, w., 1992: phylogeny of the subfamilies of the family braconidae (hymenoptera: ichneumonoidea): a reassessment. cladistics,8: 199-235. zelditch, m.l., swiderski, d.l., sheets, h.d. 2012: geometric morphometrics for biologist: a primer. academic press, london, uk, 488 pp. žikić, v., tomanović, ž., ivanović, a., kavallieratos, n.g., starý, p., stanisavljević, lj.ž., rakhshani, e. 2009: morphological characterization of ephedrus persicae biotypes (hymenoptera: braconidae: aphidiinae) in the palaearctic. annals of the entomological society of america, 102: 1-11. žikić, v., ilić milošević, m., stanković, s., petrović, a., petrović-obradović, o., kavallieratos, n. g., starý, p., tomanović, ž., 2012: aphidiinae (braconidae: hymenoptera) of serbia and montenegro – tritrophic interactions. acta entomologica serbica, 17: 83-105. žikić, v., petrović, a., ivanović, a. 2014: allometric shape changes indicate significant divergence in the wing shape between asexual and sexual lineages of lysiphlebus fabarum (marshall) (hymenoptera: braconidae: aphidiinae). acta entomologica serbica, 19: 53-62. žikić, v., lazarević, m., milošević, d. 2017: host range patterning of parasitoid wasps aphidiinae (hymenoptera: braconidae). zoologischer anzeiger – a journal of comparative zoology, 268: 75-83.doi: 10.1016/j.jcz.2016.10.001 mitrović et al., 2019, biologica nyssana 10(2) 10 (2) december 2019: 65-75 doi: 10.5281/zenodo.3600172 serbian spruce (picea omorika (pančić) purkyné) endemicity and advantages review article aleksandra lj. mitrović institute for multidisciplinary research, university of belgrade, kneza višeslava 1, 11000 belgrade, serbia mita@imsi.rs (corresponding author) jelena bogdanović pristov institute for multidisciplinary research, university of belgrade, kneza višeslava 1, 11000 belgrade, serbia mala@imsi.rs jasna simonović radosavljević institute for multidisciplinary research, university of belgrade, kneza višeslava 1, 11000 belgrade, serbia jasna@imsi.rs lloyd donaldson scion, private bag 3020, rotorua 3010, new zealand lloyd.donaldson@scionresearch.com ksenija radotić institute for multidisciplinary research, university of belgrade, kneza višeslava 1, 11000 belgrade, serbia xenia@imsi.rs received: july 09, 2019 revised: october 12, 2019 accepted: december 06, 2019 abstract: conifers, as a response to mechanical stress, such as wind and stem lean, form reaction wood called compression wood (cw). cw occurs in a range of gradations from near normal wood (nw) to severe cw (scw). as the severity of cw affects the mechanical and chemical properties of wood, and as cw has limited value in the forest products industry, it is desirable to be able to measure cw severity. picea omorika belong to slow-growing conifer species in which cw typically occurs in a severe form. we developed different morphometric and non-morphometric methods for estimation of cw severity tested on wood samples of p. omorika juvenile trees exposed to long term static bending. this specific review is aimed at presenting p. omorika as one of the most adaptable spruces, and as a good model for testing of methods for estimation of compression wood severity. first, we summarize main knowledge about p. omorika, features of cw, and methods for assessment of wood quality. then, we present breifly our recently published methods for estimation of compression wood severity tested on p. omorika juvenile wood samples. key words: cell wall, cellulose fibrils, compression wood, fluorescence-detected linear dichroism microscopy, double wall thickness apstract: omorika (picea omorika (pančić) purkiné) endemičnost i perspektive konifere kao odgovor na mehanički stres (vetar, savijanje) formiraju reakciono drvo koje se naziva kompresiono drvo (cw). cw se javlja u nizu gradacija od skoro normalnog drveta (nw) do jako izraženog cw (scw). s obzirom da stepen izraženosti osobina cw ima značajan uticaj na mehaničke i hemijske osobine drveta i da cw ima ograničenu vrednost za drvnu industriju, poželjno je moći odrediti stepen izraženosti osobina cw u uzorku. picea omorika spada u sporo rastuće četinarske vrste kod kojih se cw tipočno javlja u jako izraženoj formi. mi smo razvili nekoliko morfometrijskih i ne-morfometrijskih metoda za procenu izraženosti osobina cw u uzorku, testiranih na uzorcima drveta juvenilnih stabala p. omorika koja su bila izložena dugotajnom statičkom savijanju. ovaj revijski rad ima za cilj da predstavi pančićevu omoriku kao jednu od najadaptabilnijih smrča i kao dobar model za testiranje metoda za procenu izraženosti osobina cw u uzorku. u prvom delu sumiramo znanja o pančićevoj omorici, osobinama cw i metodama za procenu kvaliteta drveta, a u drugom ukratko predstavljamo naše nedavno objavljene metode za za procenu izraženosti osobina cw u uzorku, testirane na uzorcima drveta juvenilnih stabala p. omorika. ključne reči: ćelijski zid, celulozni fibrili, kompresiono drvo, fluorescentna konfokalna mikroskopija, debljina ćelijskog zida introduction picea omorika (pančić) purkynĕ is a a rare and endangered tertiary relict and endemic species (jovanović, 1970). despite its endemism, p. omorika is considered as one of the most adaptable spruces (sevill et al., 2017). conifers, as a response to mechanical stress, form reaction wood called compression wood (cw) (timell, 1986). cw occurs in a range of gradations from near normal wood (nw) to severe cw (scw). the degree of development of particular features of cw does not necessarily change in parallel to each other, so the severity of a given tracheid is represented as a function of the degrees of development of individual features, mainly lignification, helical cavi© 2019 mitrović et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 65 13th symposium on the flora of southeastern serbia and neighboring regions ties and cell wall thickness (yumoto et al., 1983). as cw has limited value in the forest products industry it is of great importance to be able to measure cw severity (altaner et al., 2009). in recent years we worked on the development of different morphometric and non-morphometric methods for distinguishing wood samples on the compression severity scale. they are based on tracheid double wall thickness (nedzved et al., 2018), cellulose microfibrils order (savić et al., 2016), or variation in lignin structure (mitrović et al., 2015). we used confocal fluorescence microscopy and spectroscopy, combined with development of additional equipment, new algorithms and statistical analysis. we tested our methods on stem samples of p. omorika juvenile trees exposed to long term static bending. picea omorika belongs to slow-growing conifer species, its wood is characterized by small, densely packed tracheids, while cw typically occurs in a severe form (timell 1986; donaldson et al., 2004). juvenile conifer wood is characterized by randomly distributed mild compression wood (mcw), nw often being absent (donaldson et al., 2004). these are the features that suggest p. omorika juvenile wood as a good choice of samples for evaluation of the precision of methods suggested for estimation of compression wood severity. our methods for distinguishing wood samples on a compression severity scale provide a fine gradation from nw to the severest form of cw, compression severity scales being partially different. these methods, alone or in combination with each other, could be a useful tool for fine gradation of wood samples on the compression severity scale, either in the estimation of wood quality or environmental influences during growth and developmental process. they confirm juvenile p. omorika stem samples as a good choice of samples for evaluation of the methods suggested for compression wood severity estimation. picea omorika: endemicity, natural range, habitat, planting outside its natural range picea omorika (pančić) purkynĕ is a slow growing endemic coniferous species and tertiary relict of the european flora. its natural habitat is fragmented and reduced to the middle and upper courses of the drina river, in western serbia and eastern bosnia and herzegovina (jovanović, 1970, sevill et al., 2017). the species was widespread in europe and asia, but after the pleistocene glaciations, this region represents species long-term, cryptic and last refugium (aleksić & geburek, 2014). an asian origin of serbian spruce has been recently confirmed (lockwood et al., 2013), grouping p. omorika with the caucasian p. orientalis, and the two japanese endemics p. alcoquiana and p. maximowiczii. serbian spruce ancestors appeared in asia at the end of the neogene, but the increasing seasonality and aridity during the late miocene led to the extinction of picea in the mid latitudes of eurasia. until the middle of the 19th century, the natural range of p. omorika was more continuous and less fragmented than it is today (sevill et al., 2017). it has been legally protected since 1964. its current distribution is on one side the result of anthropogenic factors such as general forest clearance and harvesting for timber, pastoralism and wildfires (jovanović, 1986; sevill et al., 2017). fire has perhaps been the biggest threat, and logging has been a subsidiary one (sevill et al., 2017). on the other side the limited natural range of serbian spruce is the result of the species poor competing ability. it retreats to areas less inhabitable by its competitors, predominantly picea abies and fagus orientalis (johnson, 1993; jovanović, 2000). it inhabits open habitats comprising cliffs and forest clearings, characterized by a strong northerly wind, snow, and rockfalls. the climate in its natural range is characterized by very high humidity, high precipitation, regularly distributed over the year, deep snow cover which lasts 4-5 months, and low winter temperatures. picea omorika is drought tolerant, its cold hardiness limit is between -28 °c and -23 °c, it tolerates wide soil ph range and polluted urban conditions (sevill et al., 2017). in short, it is adapted to extreme environmental conditions. planting serbian spruce outside its natural range has a long tradition in europe since the late 19th century. in addition to the initial use as ornamental plant species in parks, serbian spruce has a long tradition of use in forestry (ivetić & aleksić, 2016). it is grown to a small extent for christmas trees, timber and paper production, particularly in northern europe, although its slow growth makes it less important than sitka spruce or norway spruce (sevill et al., 2017). however, in britain p. omorika and p. orientalis are among the alternative species to sitka spruce and norway spruces particularly in areas where they might be subject to damage due to drought as the impacts of climate change (sevill et al., 2017). the great value of this species appears to be successful planting in places where other spruces are susceptible to injury by drought or spring frosts. at present, serbian spruce is of major importance only as an ornamental tree, mainly in northern europe and north america. it is regarded as one of the most attractive spruces because of its elegant form and the ability to grow on a wide range of soils (sevill et al., 2017). despite its endemism, p. omorika is considered as one of the most adaptable spruces. 66 biologica nyssana ● 10 (2) december 2019: 65-75 mitrović et al. ● serbian spruce (picea omorika (pančić) purkyné endemicity and adventages wood properties in the forest products industry and methods for estimation of wood quality forest products industry is based on wood properties, while wood properties are directly determined by cell arrangement, cell size and shape, and cell wall structure and thickness. in softwood species differences in wood structure result from genetic and abiotic factors: 1) plant age (zobel & sprague, 1998; burdon et al., 2004) juvenile wood and mature wood; 2) season of maturation within the growth ring (uggla et al., 2001) – early wood (ew) and late wood (lw); or 3) mechanical stress as a consequence of wind and stem lean (timell, 1986) compression wood, opposite wood and normal wood. in the forest products industry, juvenile wood, early wood and compression wood, generally have limited value, and therefore determination of their amounts is of great importance. in this regard, different morphometric and non-morphometric methods were developed for the evaluation of wood quality (tab. 1). the number of developed methods speak in favor of their significance for the forest products industry. compression wood reaction wood in conifers: formation, occurrence, range of gradations the resistance of trees to mechanical perturbation depends on structural modifications for mechanical biologica nyssana ● 10 (2) december 2019: 65-75 mitrović et al. ● serbian spruce (picea omorika (pančić) purkyné endemicity and adventages 67 strength. the formation of reaction wood in the stem is a reaction of the tree to leaning, as part of the geotropic response. in conifers, reaction wood is known as compression wood (timell, 1986). its formation occurs on the lower side of the leaning stem (fig. 1), resulting in eccentric growth (timell, 1986; donaldson & singh, 2013). inclination at the high angle results in severe compression wood (scw) formation (yumoto et al., 1983). cw occurs in a range of gradations from near nw to scw, mild cw (mcw) forming a continuum between nw and scw (fig. 1). wood opposite to the cw in the same growth ring is termed opposite wood (ow) (fig. 1), while wood from growth rings that do not contain any cw is termed normal wood (nw). also, in the leaning stem, compression wood severity declines from the stem base to the top of the stem (fig. 1). wood tracheid cell walls are composed of several layers containing an ordered array of cellulose microfibrils, embedded in a matrix of polysaccharides such as pectin, hemicellulose, and lignin (harris, 2006). cw is characterized by (fig. 2): increased tracheid wall thickness, reduced lumen diameter, rounder cell cross-sectional profile, presence of intercellular spaces, absence of the s3 cell wall layer and presence of helical cavities in the s2 layer, compared to nw (donaldson et al., 2004; donaldson & singh, 2013). cw is highly lignified, with the changed composition of lignin, increased amounts of p-hydroxyphenyl monomers and increased conmethods parameter for estimation of m or ph om et ri c m et ho ds manual or automated measurements on micrographs (mork, 1928; brown et al., 1949; gofas & tsoumis, 1975; klisz, 2009; selig et al., 2012) automated measurements from distance maps reconstructed from digital images (travis et al., 1996; lorbach et al., 2012; nedzved et al., 2018) cell wall area radial cell wall width double wall thickness cell lumen ew/lw ratio (mork, 1928) differences between juvenile and mature wood (mitchell and denne, 1997; loustarinen, 2012) compression wood severity (andersson & walter, 1995; nyström & hagman, 1999; moëll & fujita, 2004; duncker & spiecker, 2009; nedzved et al., 2018) n on -m or ph om et ri c m et ho ds fluorescence spectroscopy (donaldson et al., 2010) chemical analysis (nanayakkara et al., 2009) scanning fourier transform infrared microspectroscopy and immunolabeling (altaner et al., 2009) fluorescence-detected linear dichroism (fdld) microscopy (savić et al., 2016) confocal microscopy (donaldson et al., 2004) lignin and carbohydrate content/structure cellulose and noncellulosic polysaccharides composition and organization microfibrillar angle compression wood severity table 1. some of the methods for estimation of wood quality 68 densation of monomer units in the polymer (timell, 1986). consequently, cw contains less cellulose, with greatly increased amounts of galactan, and slightly lower amounts of mannan and xylan, together with a higher angle of cellulose microfibrils in the s2 layer of the cell wall, compared to nw (nanayakkara et al., 2009; donaldson & knox, 2012; donaldson & singh, 2013). the degree of development of particular features of cw does not necessarily change in parallel to each other, so the severity of a given tracheid is represented as a function of the degrees of development of individual features, mainly lignification, helical cavities and cell wall thickness (yumoto et al., 1983). visual detection of compression wood severity, more precisely the determination of mcw, is difficult. as the severity of cw affects mechanical and chemical properties of wood in the forest products industry, it is desirable to be able to measure cw severity (altaner et al., 2009). fig. 1. scheme of compression wood formation; compression wood occurs in a range of gradations from near normal wood to severe compression wood, mild compression wood forming a continuum between normal wood and severe compression wood; compression wood severity declines from stem base to the top of the stem; nw – normal wood, cw – compression wood. mcw mild compression wood, scw severe compression wood, ow – opposite wood fig. 2. scheme for changes in degree of development of tracheid cell wall features characterizing the transition from normal wood (nw) to severe compression wood (scw), mild compression wood (mcw) forming a continuum between nw and scw; on the left – field emission scanning electron microscopy (fesem) images of nw, on the right – fesem images of scw; nw – normal wood, mcw mild compression wood, scw severe compression wood, s1, s2, s3 – layers of secondary cell wall biologica nyssana ● 10 (2) december 2019: 65-75 mitrović et al. ● serbian spruce (picea omorika (pančić) purkyné endemicity and adventages 69 our morphometric and nonmorphometric methods for distinguishing conifer wood samples on a compression severity scale in recent years we worked on the development of different morphometric and non-morphometric methods for distinguishing wood samples on a compression severity scale. the first method is based on tracheid double wall thickness analysis (nedzved et al., 2018). the second method is based on cellulose microfibrils order (distribution and alignment of cellulose microfibrils) analysis in tracheid (double) walls (savić et al., 2016). the third one we are still developing, based on the analysis of structural modifications of lignin, and it is related to our results published a few years ago (mitrović et al., 2015). these 3 methods cover the main features of cw, related to changes in tracheid cell wall shape, lignin and cellulose organization. hence, alone or in combination with each other, they could be suggested for use in fine gradation of wood samples on the compression severity scale, either in the estimation of wood quality or in the estimation of environmental influences during growth and developmental process. we present here, in more detail, 2 methods, one morphometric (nedzved et al., 2018) and one non-morphometric (savić et al., 2016), for distinguishing wood samples on a compression severity scale. in tab. 2, the main characteristics, similarities, differences, and advantages of these methods are summarized. it is known that radial and tangential walls can vary significantly regarding different features of wood cell walls. so far, most investigations, regarding cellulose microfibrils (donaldson, 2008), or other features of wood cell walls such as cell wall thickness (mork, 1928), have been carried out on radial cell walls. accordingly, our methods confirm the selection of radial walls for the analysis as an excellent choice of tracheid cell wall region for the determination of mcw. fig. 3. reprinted by permission from: springer nature, trees 32, 1347–1356; nedzved et al. (2018) automatic image processing morphometric method for the analysis of tracheid double wall thickness tested on juvenile picea omorika trees exposed to static bending. trees 32, 1347–1356.: the clsm images (first column), corresponding binary images (second column) and distance maps (third column) of p. omorika stem samples; s1 and s2 – nw samples; s3 and s4 mcw samples; s5 and s6 – scw samples biologica nyssana ● 10 (2) december 2019: 65-75 mitrović et al. ● serbian spruce (picea omorika (pančić) purkyné endemicity and adventages 70 compression wood – reaction wood in conifers: formation, occurrence, range of gradations measurements of various anatomical characteristics of wood cells are of great importance in the research of wood structure. tracheid double wall thickness, as an important wood anatomical feature, besides being known as an indicator of transition from ew to lw within an annual ring in gymnosperms (the ratio between radial double wall thickness and cell lumen diameter, mork, 1928), also characterizes the differences between juvenile and mature wood (mitchell & denne, 1997; loustarinen, 2012), as well as between normal and compression wood (timell, 1986; plomion et al., 2001). hence, the determination of tracheid double wall thickness is of great importance in estimation of wood quality. image-processing techniques are used for estimation of the ew and lw ratio as a significant feature for forest products industry (mork, 1928; jagels & dyer, 1983; diao et al., 1999). however, such techniques for estimation of compression wood severity were more (duncker & spiecker, 2009) or less (andersson & walter, 1995; nyström & hagman, 1999; moëll & fujita, 2004) successful in recognizing mcw. for testing of our automatic image processing morphometric method for the analysis of tracheid double wall thickness (nedzved et al., 2018) we used confocal laser scanning microscopy (clsm) images of stem cross sections of juvenile p. omorika trees exposed to static bending. our algorithm (nedzved et al., 2018) was developed using imagewarp a&b software company (usa) and software for image analysis qtip developed in united institute of informatics problems (uiip), national academy of sciences (belarus). it consists of the extraction of cell patterns from the original micrograph via binarization using otsu’s threshold method (otsu, 1979) and reconstruction of the distance maps (kimmel et al., 1996). the use of euclidian distance maps for calculating the thickness of the wood cell wall was suggested earlier in estimations of pulp or paper quality (travis et al., 1996; koskenhely & paulapuro, 2005; selig et al., 2012; lorbach et al., 2012). the novelty of our fig. 4. reprinted by permission from: springer nature, trees 32, 1347–1356; nedzved et al. (2018) automatic image processing morphometric method for the analysis of tracheid double wall thickness tested on juvenile picea omorika trees exposed to static bending. trees 32, 1347–1356.: distribution of double wall thickness of a) entire tracheid walls, b) tracheid at cell corners, c) tangential walls and d) radial walls, determined by morphological processing of structural elements with different orientation from distance maps obtained from the clsm images of (s1 – s6) samples (fig. 3); a, b, c, d, e, f significant difference at 5% level of significance of the maxima positions of gaussian curves between samples s1 – s6; µ mean values, σ – standard deviation biologica nyssana ● 10 (2) december 2019: 65-75 mitrović et al. ● serbian spruce (picea omorika (pančić) purkyné endemicity and adventages 71 method is the use of morphological image processing of structural elements with different orientation (dougherty, 1992) on euclidian distance maps reconstructed from microscopic images, allowing determination of the distribution of tracheid double wall thickness separately for tangential walls, radial walls, and cell corners (it yields valuable information on circularity/rectangularity of cross-sectional profile of tracheid cell wall). we applied ibm spss software, nonlinear curve fit function, for fitting the gaussian curves on the data (distribution of double wall thickness in pixels) to show the overall variation in tracheid double wall thickness in a response to mechanical stress. for comparison of the maxima positions of gaussian curves between samples, one-way anova and duncan test were used. as a result, our nonmorphometric image processing method provides a fine gradation of p. omorika juvenile wood samples on the compression severity scale from nw to scw (fig. 4 a-d), suggesting that it could be used as a tool for estimation of compression wood severity. in addition, as different regions of p. omorika tracheid cell wall show somewhat different cell wall thickening in response to mechanical stress (fig. 4), compression severity scales based on double wall thickness distribution of entire tracheids, tracheids at cell corners, tangential and radial walls, differ to some degree. radial wallsdouble wall thickness distribution (fig. 4d) shows the additional advantage and specificity over a number of methods for estimation of compression wood severity: it groups, and consequently sharply distinguishes mcw samples from scw samples. our non-morphometric method for estimation of compression wood severity based on cellulose microfibrils order (savić et al., 2016) short report the distribution and orientation of cellulose microfibrils (mfs) in wood cell walls is determined by both, genetic and abiotic factors. genetic factors include: cell wall layer (primary wall, s1, s2, s3 layer of secondary cell wall), position (radial or tangential cell wall), plant age (juvenile or mature wood), season of maturation within the growth ring (early and late wood), while abiotic factors include wind and stem lean (compression wood, normal wood). since cellulose fibrils, as reinforcing material in conifer wood cell walls, determine tracheid cell wall properties and wood quality, one of the most frequently measured ultrastructural variables in wood cell wall is microfibrilar angle (mfa) (donaldson, 2008). various microscopic techniques have been used to study the orientation of cellulose microfibrils in the wood cell wall, using variations in polarised light techniques or directly visualizing orientation of the microfibrils (donaldson, 2008). in our method for estimation of compression wood severity (savić et al., 2016) we observed the relative order of cellulose fibrils in cell walls. fluorescence detected linear dichroism (fdld) imaging was performed using confocal laser scanning microscope (clsm) additionally equipped with constructed differential polarization extension (dplsm). fdld microscopy exploits fluorescence originating from cellulose fibrils stained specifically by congo red, enabling screening of cellulose microfibrils order in the x–y plane of the cross section, which means separately in tangential and radial walls. the method was tested on stem cross sections of juvenile p. omorika trees exposed to static bending. blue colour represents dipoles (cellulose fibers) predominantly parallel with x-axis (tangential walls), yellow colour indicates dipoles (cellulose fibers) oriented predominantly parallel with the yaxis (radial walls) (fig. 5), while the grey colour represents fibers orientated at about 45°. image processing was performed using imagej program with macros developed for this analysis. fdld images were quantified and presented as histograms (fig. 5). the decrease in cellulose fibrils order from cw to nw samples is obvious, in both radial and tangential tracheid walls (fig. 5). this is in line with xu et al. (2011) work; they showed that the characteristics of cellulose fibrils reinforcements in s2 layer of severe compression juvenile wood include lower number, abundant dislocation segments and shorter length of cellulose mf, compared to nw. we additionally showed (savić et al., 2016) that radial and tangential tracheid cell walls in p. omorika juvenile wood differ considerably regarding cellulose fibril order, and that fdld of radial walls (fig. 5 u), showing fine gradation from cw to nw, could be suggested as an easily applicable technique for estimation of cw severity. picea omorika juvenile wood samples – a model for testing of methods for estimation of compression wood severity. we tested our methods on stem samples of p. omorika juvenile trees exposed to long term static bending (bending procedure described in detail in mitrović et al., 2015). p. omorika belong to slow-growing conifer species in which cw typically occurs in a severe form (scw) (donaldson et al., 2004). juvenile conifer wood is characterized by randomly distributed mcw, nw often being absent (donaldson et al., 2004). these are the features that suggest p. omorika juvenile wood a good choice of samples for evaluabiologica nyssana ● 10 (2) december 2019: 65-75 mitrović et al. ● serbian spruce (picea omorika (pančić) purkyné endemicity and adventages 72 fig. 5. reprinted by permission from: cambridge university press, microscоpy and microanalysis 22, 361–367.; savić et al. (2016) fluorescence-detected linear dichroism of wood cell walls in juvenile serbian spruce: estimation of compression wood severity. microscоpy and microanalysis 22, 361–367.: fluorescence-detected linear dichroism (fdld) images of p. omorika sections and corresponding anisotropy histograms. a: normal wood (nw) samples; (e) mild compression wood (mcw) samples; (i,m) severe compression wood (scw) samples; (b,f,j,o) pixel values were collected in the marked areas, in tangential walls (blue boxes) and in radial walls (yellow boxes), and used to obtain anisotropy distributions; (c,g,k,p) tangential walls fdld distributions; (d,h,l,q) radial walls fdld distributions; (t) overlaid distributions (c,g,k,p) with black lines representing corresponding gaussian fits (white arrow represents gradual shifts toward gray—increasing number of disorientated fibrils); (u) overlaid distributions (d,h,l,q) with black lines representing corresponding gaussian fits (white arrows represent gradual shifts toward gray—increasing number of disorientated fibrils); (r,s) schemes of nw and cw tracheid sections, respectively. excitation at 488 nm, emission above 560 nm; image size is 64 × 64 μm biologica nyssana ● 10 (2) december 2019: 65-75 mitrović et al. ● serbian spruce (picea omorika (pančić) purkyné endemicity and adventages tion of the precision of methods suggested for estimation of compression wood severity. after testing of our methods (savić et al., 2016; nedzved et al., 2018) on stem cross sections of juvenile p. omorika trees exposed to long term static bending, we can confirm p. omorika juvenile wood samples as a good model for testing of methods suggested for estimation of compression wood severity. conclusion picea omorika, despite its endemism, and thanks to her adaptability and long tradition of planting, nowadays is present widely outside its natural range. we confirmed picea omorika juvenile wood samples as a good model for testing of methods suggested for estimation of compression wood severity. our methods, based on the analysis of tracheid double wall thickness, cellulose fibril order and structural modifications of lignin, using confocal fluorescence microscopy and spectroscopy, cover all the main features of cw. therefore, alone or in combination with each other, they could be a useful tool for fine gradation of wood samples on compression severity scale, as a valuable advantage over many other methods for the estimation of compression wood severity, as the determination of mild compression wood is difficult. hence they can be of great benefit either for estimation of wood quality in forest products industry, or for estimation of environmental influences during growth and developmental process in tree physiology. acknowledgements. this study was supported by grant 173017 of the ministry of education, science and technological development of the republic of serbia. previous version of this manuscript has been presented at 13th symposium on the flora of southeastern serbia and neighboring regions, 20 23 june 2019., stara planina, serbia, in the form of introductory lecture. references aleksić, m.j., geburek, t. 2014: quaternary population dynamics of an endemic conifer, picea omorika, and their conservation implications. conservation genetics, 15: 87-107. altaner, c.m., tokareva, e.n., wong, j.c., hapca, a.i., mclean, j.p., jarvis, m.c. 2009: measuring compression wood severity in spruce. wood science and technology, 43: 279-290. andersson, c., walter, f. 1995: classification of compression wood using digital image analysis. forest products journal, 45: 87-92. brown, h.p., panshin, a.j., forsaith, c.c. 1949: textbook of wood technology. mcgraw hill book company inc, new york, ny, usa. burdon, r.d., kibblewhite, r.p., walker, j.c.f., megraw, r.a., evans, r., cown, d.j. 2004: juvenile versus mature wood: a new concept, orthogonal to corewood versus outerwood, with special reference to pinus radiata and p. taeda. forest science, 50: 399-415. diao, x.m., furuno, t., fujita, m. 1999: digital image analysis of cross-sectional tracheid shapes in japanese softwoods using the circularity index and aspect ratio. journal of wood science, 45: 98-105. donaldson, l.a., grace, j.c., downes, g. 2004: within tree variation in anatomical properties of compression wood in radiata pine. iawa journal, 25: 253–271. donaldson, l.a. 2008: microfibril angle: measurement, variation and relationships – a review. iawa journal, 29: 345–386. donaldson, l.a., knox, j.p. 2012: localisation of cell wall polysaccharide sin normal and compression wood of radiata pine relationships with lignification and microfibril orientation. plant physiology, 158: 642–653. donaldson, l.a., singh, a.p. 2013: structure and formation of compression wood. in: fromm j (ed) cellular aspects of wood formation, plant cell monographs. springer, berlin, pp 225–256. donaldson, l.a., radotić, k., kalauzi, a., djikanović, d., jeremić, m. 2010: quantification of compression wood severity in tracheids of pinus radiata d. don using confocal fluorescence imaging and spectral deconvolution. journal of structural biology, 169: 106–115. dougherty, e.r. 1992: an introduction to morphological image processing. spie optical engineering press, washington usa. duncker, p., spiecker, h. 2009: detection and classification of norway spruce compression wood in reflected light by means of hyperspectral image analysis. iawa journal, 30: 59–70. gofos, a, tsoumis, g. 1975: a method for measuring characteristics of wood. wood science and technology, 9: 145-152. harris, p.j. 2006: primary and secondary plant cell walls: a comparative overview. new zealand journal of forestry science, 36: 36–53. ivetić, v., aleksić, j. 2016: response of rare and endangered species picea omorika to climate change the need for speed. reforesta, 2: 81-99. jagels, r., dyer, m. 1983: morphometric analysis 73 biologica nyssana ● 10 (2) december 2019: 65-75 mitrović et al. ● serbian spruce (picea omorika (pančić) purkyné endemicity and adventages applied to wood structure. i. cross-sectional cell shape and area change in red spruce. wood and fiber science, 15: 376-386. johnson, h. 1993: the international book of trees. london: mitchell beazley. jovanović, b. 1970: gymnospermae. in: josifović, m. (ed.), flora sr srbije, 1. sanu, beograd. jovanović, b. 1986: red coniferales – četinari, 168-234. in: sarić, m. (ed.), flora srbije, 1, 2nd edition. sanu, beograd. 429 str. kimmel, r., kiryati, n., bruckstein, a.m. 1996: distance maps and weighted distance transforms. journal of mathematical imaging and vision, special issue on topology and geometry in computer vision, 6: 223-233. klisz, m. 2009: wincell – an image analysis tool for wood cell measurements. forest research papers, 70: 303–306. koskenhely, k., paulapuro, h. 2005: effect of refining intensity on pressure screen fractionated softwood kraft. nordic pulp & paper research journal, 20: 169-175. lockwood, j.d., aleksić, j.m., zou, j., wang, j., liu, j., renner, s.s. 2013: a new phylogeny for the genus picea from plastid, mitochondrial, and nuclear sequences. molecular phylogenetics and evolution, 69: 717-727. lorbach, c., hirn, u., kritzinger, j., bauer, w. 2012: automated 3d measurement of fiber cross section morphology in handsheets. nordic pulp & paper research journal, 27: 264-269. luostarinen, k. 2012: tracheid wall thickness and lumen diameter in different axial and radial locations in cultivated larix sibirica trunks. silva fennica, 46: 707–716. mitchell, m.d., denne, m.p. 1997: variation in density of picea sitchensis in relation to withintree trendsin tracheid diameter and wall thickness. forestry, 70: 47–60. mitrović, a., donaldson, l.a., djikanović, d., bogdanović pristov, j., simonović, j., mutavdžić, d., kalauzi, a., maksimović, v., nanayakkara, b., radotić, k. 2015: analysis of static bendinginduced compression wood formation in juvenile picea omorika (pančić) purkynĕ. trees structure and function, 5: 1533-1543. moëll, m.k., fujita, m. 2004: fourier transform methods in image analysis of compression wood at the cellular level. iawa journal, 25: 311 – 324. mork, e. 1928: die qualität des fichtenholzes unter besonderer rücksichtnahme auf schleif– und papierholz. der papier-fabrikant, 26: 741–747. nyström, j., hagman, o.j. 1999: real-time spectral classification of compression wood in picea abies. wood science, 45: 30-37. nanayakkara, b., manley-harris, m., suckling, i.d., donaldson, l.a. 2009: quantitative chemical indicators to assess the gradation of compression wood. holzforschung, 63: 431-439. nedzved, a., mitrović, a.lj, savić, a., mutavdžić, d., radosavljević simonović, j., bogdanović pristov, j., steinbach, g., garab, g., starovoytov, v., radotić, k. 2018: automatic image processing morphometric method for the analysis of tracheid double wall thickness tested on juvenile picea omorika trees exposed to static bending. treesstructure and function, 32: 1347-1356. otsu, n. 1979: a threshold selection method from gray-level histograms. ieee transactions on systems, man, and cybernetics, 9: 62–66. plomion, c., le provost, g., stokes, a. 2001: wood formation in trees. plant physiology, 127: 1513–1523. savić, a., mitrović, a., donaldson, l., simonović radosavljević, j., bogdanović pristov, j., steinbach, g., garab, g., radotić, k. 2016: fluorescence-detected linear dichroism of wood cell walls in juvenile serbian spruce: estimation of compression wood severity. microscopy and microanalysis, 22: 361-367. savill, p., s. wilson, b. mason, r. jinks, v. stokes, christian, t. 2017: alternative spruces. part 1 serbian spruce (picea omorika). quarterly journal of forestry, 111: 32-39. selig, b., luengo hendriks, c.l., bardage, s., daniel, g., borgefors, g. 2012: automatic measurement of compression wood cell attributes in fluorescence microscopy images. journal of microscopy, 246: 298-308. timell, t.e. 1986: compression wood in gymnosperms. springer-verlag, heidelberg. travis, a.j., hirst, d.j., chesson, a. 1996: automatic classification of plant cells according to tissue type using anatomical features obtained by the distance transform. annals of botany, 78: 325-331. uggla, c., magel, e., moritz, t., sundberg, b. 2001: function and dynamics of auxin and carbohydrates during earlywood/latewood transition in scots pine. plant physiology, 125: 2029-2039. xu, p., huawu liu, h., donaldson, l., zhang, 74 biologica nyssana ● 10 (2) december 2019: 65-75 mitrović et al. ● serbian spruce (picea omorika (pančić) purkyné endemicity and adventages 75 y. 2011: mechanical performance and cellulose microfibrils in wood with high s2 microfibril angles. journal of materials science, 46: 534–540. yumoto, m., ishida, s., fukazawa, k. 1983: studies on the formation and structure of compression wood cells induced by artificial inclination in young trees of picea glauca. iv. gradation of the severity of compression wood tracheids. research bulletins of the college experiment forests hokkaido university, 40: 409– 454. zobel b.j., sprague j.r. 1998: juvenile wood in forest trees. springer series in wood science. springer, berlin, heidelberg. 300 p. biologica nyssana ● 10 (2) december 2019: 65-75 mitrović et al. ● serbian spruce (picea omorika (pančić) purkyné endemicity and adventages anticancer compounds from medicinal plants biologica nyssana 4 (1-2)  december 2013: 93-96 petrović, s.  a contribution to the knowledge of the neuroptera … 93 original article a contribution to the knowledge of the neuroptera (insecta) fauna of serbia sonja petrović university of nis, faculty of science and mathematics, department of biology and ecology, višegradska str. 33, 18000 niš, serbia * e-mail: sonjichka@gmail.com abstract: petrović, s.: a contribution to the knowledge of the neuroptera (insecta) fauna of serbia. biologica nyssana, 4 (1-2), december 2013: 93-96. we present a record of a rare species libelloides lacteus brullè, 1832 (ascalaphidae, ascalaphinae) inhabiting jelašnica gorge, formerly known to be present only in one single site located inside serbia's province kosovo and metohija. existing literature data regarding balkan peninsula distribution of this species is listed here. jelašnica gorge's adult and larval stage specimen findings are illustrated. morphoanatomical parameters essential for species determination are listed. species habitat conditions presented here, have a closer determination according to vegetation units and according to eunis classification system. it is concluded that a jelašnica gorge population has the most continental distribution on balkan peninsula. further species investigation is recommended, especially the one regarding it's ecology and it's relationship to other populations. key words: libelloides lacteus brullè, 1832, jelašnica gorge, serbia. introduction serbian neuroptera fauna is not researched sufficiently enough, and it's study is rather fragmented. first written data is to be found in p o n g r a c z (1923) work that refers to fauna belonging to this group in albania which states 12 more species in kosovo and metohija. researching an insect fauna on university land near majdanpek, ž i v o j i n o v i ć (1950) affirmed the existence of 7 more neuroptera species among large number of others. in a contribution about kosovo and metohija's neuroptera species, d e v e t a k and j a k š i ć (2003) are listing findings of a 51 species of this group. j o n e s and d e v e t a k (2009) gave new fauna data on nevrorthus apatelios species. as part of an ecological study on macro-invertebrates in ivanštica river, had been noted a finding of neuroptera osmylus fulvicephalus (scopoli, 1763) (đ u k n i ć et al., 2010). beside this faunistic works, there is existent literature data where neuroptera is not the object of primary faunistic research, but mentioned and regarded to as a predator of other groups of insects (g r a o r a , 2009; j e r i n i ć -p r o d a n o v i ć , 2010; t o m a n o v i ć , 2008, and others). summing the results of data in this literature, we are free to conclude that there are 54 species of neuroptera described in serbia. in balkan peninsula neuroptera fauna, there are 244 species known (p o p o v & l e t a r d i , 2010). genus libelloides is occurring in europe by the number of 9 species. looking into an overview of a literature quoted here, we can conclude that serbian fauna recognizes only two species of libelloides: libelloides macaronius (scopoli, 1763) (syn.: ascalaphus macaronius) and libelloides lacteus brullè, 1832 [syn.: ascalaphus ottomanus (germar, 1817)]. however, this work present a newly discovered finding of libelloides lacteus brullè, 1832 species. 11 th sfses • 13-16 june 2013, vlasina lake 4 (1-2) • december 2013: 93-96 biologica nyssana 4 (1-2)  december 2013: 93-96 petrović, s.  a contribution to the knowledge of the neuroptera … 94 material and methods field research on jelašnica gorge domain, had been done during 2011 and 2012. material concerning this species had been registered on the site, habitat and species pictures photographed using a digital camera. due to population preservation concerns, only a few of evidential specimens had been collected. morphologic and anatomical parameter analysis used for determination, had been done in a standard procedure in a form of classic microscopic slide preparation. photographs of specimen in situ had been taken using “olympus” sp-510uz (7.1 megapixel and 10x optical zoom), photographs of mandible had been taken using "leica dm 1000" microscope with "camera leica dec 290”. all of the material (specimens and mandible slides) are deposited in the author's collection. results and discussion in a few recent years of fieldwork in jelašnica gorge area, species l. macaronius had been found rather regularly. in early may of 2012, however, we had found a few adult specimens of a. lacteus species, clearly discerned by it's charachteristic habitus of white base colored wings with blue tones heading toward an apex area, as opposed to l. macaronius whose wings are dominated by a yelow. we have sampled evidential specimens and made our snapshots in situ (fig.1). fig. 1. libelloides lacteus in jelašnica gorge (photo s. petrović) one month later and according to our expectations, during a careful field inspection we have noticed a number of characteristic sand funnels (fig. 2), on bottoms of which we had found and sampled a few adult larvae. fig. 2. characteristic habitat (sand funnels) of the larvae of libelloides lacteus (photo s. petrović) conserved larvae have been photographed previously (fig. 3), followed by dissection of a mandible under a binocular and preserved in a form of a permanent microscopic slide preparation (fig. 4). comparison of larvae habitus and morphological details of mandible to the same parameters found on other related species, led to an unambiguous conclusion of the matter being a larval species listed (g e p p, 1984). mandible is characterised by three pronounced teeth on the inside, with presence of an accompanying hairs over a whole surface. fig. 3. larva of libelloides lacteus (photo s. petrović) ecological conditions of the habitat, and the species that exist on it, fully correspond to those in which the species had been found in bulgaria (b u r e s c h , 1936) or on the adriatic side of balkan peninsula (d e v e t a k , 1998). we may charachterise them as being an exeptionally arid climate habitats, exposed to a south wind, and being poor in vegetation. in jelašnica gorge we have established the species exactly on such kind of a site, inscribed in a geographical literature by the toponym ploča (fig. 5). this site's vegetation is represented by a plant associations of species poterio-festucetum valesiacae danon 1969, and biologica nyssana 4 (1-2)  december 2013: 93-96 petrović, s.  a contribution to the knowledge of the neuroptera … 95 sedo-potentilletum arenariae ružić 1978. according to eunis habitat classification system, habitats being registered on this site are : e1.224 (perennial calcareous grassland and basic steppes) dominated by festuca valsiaca s. lat. and e1.5 (mediterranean-montane grassland). fig. 4. mandubula of adult larva of libelloides lacteus (photo i. gnjatović) summary review shown on a map (fig.6), displays a distribution of this species on a balkan peninsula according to a i s t l e i t n e r (2007), supplemented with our own finding in jelašnica gorge. it is shown that the find of this anatolian ponto mediterranean species, is being the most continental one. b u r e s c h ' s (1936) findings are comming from thrace part of european turkey and bulgaria (patlejna monastery near preslav, st. todor monastery near bodom, dede agač) . place of discovery in jelašnica gorge, is located 350 km further away from that teritory. it is certain that a communication to populations on the east of bulgaria is achived over a valley of nišava river. due to non existent, inadequate habitats and mountain barriers (kukavica mt., radan mt., jastrebac mt., kopaonik mt.) which do not permit populations mixing, a communication with populations from kosovo and metohija is a less certain one. bearing these facts in mind, we may infer that further ecological and molecular-genetic research would complement a comprehensive biologycal knowledge on this species. fig. 5. site pleče in the jelašnica gorge fig. 6. distribution of libelloides lacteus on balkan penninsula, italy and turkey (dashed line is the boundary of the distribution of species on the balkan peninsula according to aistleitner, 2007) conclusion on a basis of morphological parameters in adults and on a basis of a larvae structure details, a pressence of a libelloides lacteus brullè, 1832 (neuroptera, ascalaphidae) in jelašnica gorge, near niš, has been proven. this is by far the most continental finding of this species on balkan peninsula. references aistleitner, e., 2007. zur taxonomie und chorologie des schmetterlingshaftes libelloides biologica nyssana 4 (1-2)  december 2013: 93-96 petrović, s.  a contribution to the knowledge of the neuroptera … 96 lacteus (brullè, 1832). entomofauna, 28(26): 357-368. buresch, i., 1936. prinos' k'm' izučvaneto na mrežokrilnata fauna na b'lgaria. izvestija na b'lgarskoto entomologično družestvo, ix: 135150. devetak, d,.1998. libelloides ottomanus (germar, 1817) in the northwestern part of the balkan peninsula (neuroptera, ascalaphidae). ent.croat., (1997) 1998. 3(1-2): 45-48. devetak, d. and jakšić, p. 2003. neuroptera of kosovo and metohija (serbia). z. arb. germ. ost. ent.öst.ent., 55: 45-53. đuknić, j., bjelanović, k., durutović, a. and jovanović, j. 2010. ecological survey of macroinvertebrate communities in the vrelska padina and the ivanštica rivers (eastern serbia). balwois 2010 ohrid, republic of macedonia. pp.: 1-12. davies, c., moss, d., hill, o.m., 2004. eunis habitat classification revised 2004. http://www.searchmesh.com/pdf/gmhm1%20e unis_habitat_classification_revised_2004.pdf gepp, j., 1984. erforschungsstand der neuropterenlarven der erde. progress in world's neuropterology. gepp j., h. aspöck & h. hölzel ed, 265 pp 1984, pp.: 183-239, graz. graora, d. 2009. parazitoidi i predatori štitastih vašiju iz familije diaspididae u nekim voćnjacima u srbiji. pestic. fitomed. (beograd), 24(4), 2009, 295-301. jerinić-prodanović, d., 2010. predatori i parazitoidi cacopsylla pyri (l.) (hemiptera: psyllidae) u srbiji. pestic. fitomed. (beograd), 25(1), 2010, 29-42 jones, r. and devetak, d. 2009. first record of nevrorthidae from slovenia. acta entomologica slovenica 17(2): 99-106. pongracz, s. 1923. recesszarnyuak. neuropteroiden. – in: csiki erno allattani kutatasai albaniaban. explorationes zoologicae ab e. csiki in albania perectae. ix. a. magyar tudomanyos akademia balkan-kutatasainak tudomanyos eredmenyei. budapest, 1(1): 143166. popov, a., letardi, a., 2010. comparative zoogeographical analysis of neuropterida of the apennine and balkan peninsulas. proceedings of the tenth international symposium on neuropterology. piran, slovenia, 2008. devetak, d., lipovšek, s. & arnett, a.e. (eds). maribor, slovenia, 2010. pp. 239–256. tomanović, ž., kavallieratos, n.g., stary, p., petrović-obradović, o., athanassiou, c.g., stanisavljević, lj.ž., 2008: cereal aphids (hemiptera: aphidoidea) in serbia: seasonal dynamics and natural enemies. eur. j. entomol. 105: 495–501. živojinović, s. 1950. fauna insekata šumske domene majdanpek. san, knjiga clx (2), beograd. anticancer compounds from medicinal plants biologica nyssana 4 (1-2)  december 2013: 1-7 stešević, d. caković, d.  contribution to the alien flora of montenegro … 1 original article contribution to the alien flora of montenegro and supplementum to the preliminary list of plant invaders danijela stešević, danka caković faculty of natural sciences and mathematic, university of montenegro, džordža vašingtona bb, 81000 podgorica, montenegro * e-mail: danijela.stesevic@ac.me abstract: stešević, d., caković, d.: contribution to the alien flora of montenegro and supplementum to the preliminary list of plant invaders. biologica nyssana, 4 (1-2), december 2013: 1-7. this contribution is based on the field observations from 2011 to 2013. besides new data about distribution of some known plant invaders, one new alien species for the flora of montenegro is reportedsolidago gigantea. this plant was recorded in 2011, on two distinct localities near the road side in peri-urban area of nikšić and mojkovac, in the vicinity of gardens, were it has been grown as ornamental. in 2012 survey, species was again reported for mojkovac, but it disappeared from nikšić, due to environmental changes caused by road construction. remaining locality is placed near the tara river bank, so considering ecological preferences (roadsides, disturbed river banks and moist soils), this species might become more frequent in the area. it is included into the eppo list of invasive alien plants. in addition, alien plant tagetes minuta is added to the preliminary list of plant invaders in montenegro. key words: alien flora, invasive flora, montenegro, solidago gigantea, tagetes minuta introduction since our last publication from 2010 (s t e š e v i ć & p e t r o v i ć , 2010) situation with funding of the research and control of ias (invasive alien species) didn’t change much, but some progress in the legislative has been made. pursuant to the requirements of the convention on biological diversity, on 29 july 2010 th , the first national biodiversity strategy and action plan for 2010 2015th was adopted (www.gov.me/.../filedownload.aspx?rid=121033). this document defined the long-term goals and a number of measures for the protection of biodiversity and protected natural resources, as well as its implementation according to overall economic and social conditions in the country. the strategy has identified the main threats to biodiversity and ecosystems. introduction of alien and invasive species was considered as so far poorly understood threat, with comment that its greater significance among the threats to biodiversity can be expected soon. in the meantime, on 14 march 2013 th , the government of montenegro on the proposal agency for environmental protection adopted a regulation on the national list of indicators in environmental protection, by which alien and invasive species are an indicator (b05) of pressure on dpsir (driving forces pressures state-impact-response) model. therefore, in coming period they will be systematically monitored in annually dynamics, while summary data will be published each 10 years (official gazette no 29/2013). according to recommendations of l a m b d o n et al. (2008), national check list of alien and invasive flora need to be further assessed within respect to the invasion status and residence time of the species included. for future in-depth analyses it is crucial that especially the naturalized/casual status is evaluated. furthermore, 11 th sfses • 13-16 june 2013, vlasina lake 4 (1-2) • december 2013: 1-7 biologica nyssana 4 (1-2)  december 2013: 1-7 stešević, d. caković, d.  contribution to the alien flora of montenegro … 2 more rigorous approach is also needed at the national level in terms of recording of habitat (c h y t r ỳ et al., 2008) and abundance data, and according to recognized standards which are comparable across the continent. this paper is a contribution to the alien and invasive flora of montenegro and results given in it presents our field observation, gathered during different floristic and habitat investigations. material and methods data on invasive flora presented in this paper were gathered during different floristic and habitat investigations of central and coastal part of the country, undertaken in period late summer 2011 to spring 2013. each location where alien plants were recorded were geo-coded by using a gps device garmin e-trex vista c. field data about the habitats were also collected. collected specimens were deposited in the herbarium collection of the faculty of natural sciences in podgorica (tgu!), while identification of materials was conducted according to m c n e i l (1976) and p i g n a t t i (1982). results and discussion solidago gigantea aiton, giant goldenrod/late goldenrod (fam. asteraceae) during the floristic survey in augustseptember 2011,on two distinct localities near the road side in peri-urban area of nikšić (n42º 47’21'‘e 18º 55’42'') and mojkovac (n42º 57’28'‘e 19º 34’23''), several individuals of ornamental plant solidago gigantea aiton, were recorded. the plant belongs to the solidago canadensis l. complex within the subgenus triplinervae (w e b e r & j a c o b s , 2005). plant is known as a giant goldenrod/late goldenrod. it is up to 280 cm tall, erect rhizomatous perennial with annual aboveground shoots 5-11mm in diameter. shots are branched only in the inflorescence. they are mainly glabrous, or weakly hairy in the inflorescence. leaves are alternate, oblong to lanceolate, often acuminate, with margins mostly serrate, occasionally entire, triple-nerved, glabrous or sometimes pubescent beneath. they are largest in the middle of shoots and become smaller towards the apex and within inflorescence. inflorescences form broad pyramidal panicles with recurving branches and a central axis. bracts of the involucre are linear, obtuse or somewhat acute, 3.5-5mm. ray florets are golden yellow, female and fertile, disc florets are bisexual and fertile, capitula 3-5 mm long. achenes are pubescent, 1-2 mm long, with a pappus of 1 mm long (m c n e i l , 1976; w e b e r & j a c o b s , 2005). giant goldenrod originates from north america (m c n e i l , 1976). it was introduced into europe as an ornamental plant in the mid-1700s, with naturalized populations first observed in the mid-1800s (w a g e n i t z , 1979). continuous spread was noted between 1850 and 1950, by which time most of the present range was occupied (w e b e r , 1998). according to (euro+med, 2006-), plant is naturalized in west, central and part of south europe. it is included into the eppo list of invasive alien plants (http://www.eppo.int/invasive plants/ias_lists.htm#a1a2lists). it grows in a wide range of different soil conditions but is not shade tolerant (e l l e n b e r g et al., 1992). initially it occupied ruderal sites, such as roads, railway lines and rivers, it has also established in semi-natural and natural habitat since the late 19 th century (g u z i k o w a & m a y c o c k 1986; l o h m e y e r & s u k o p p , 1992). in the peri-urban area of nikšić and mojkovac, plant was reported near the roadside, in the vicinity of gardens, were it has been grown as ornamental. in 2012 survey, species was again fig. 1. solidago gigantea, plant habitus and detail of glabrous shoot (photo a. haines) http://www.eppo.int/invasive%20plants/ias_lists.htm#a1a2lists http://www.eppo.int/invasive%20plants/ias_lists.htm#a1a2lists biologica nyssana 4 (1-2)  december 2013: 1-7 stešević, d. caković, d.  contribution to the alien flora of montenegro … 3 reported for mojkovac, but it disappeared from nikšić, due to environmental changes caused by the road construction. the remaining locality in mojkovac is placed near the tara river bank, so considering habitat preferences: disturbed river banks and moist soils (j a c o b s et al., 2004; s t a n č i ć , 2010; k o u t i k a et al., 2011), this species might become more frequent in the area. besides giant goldenrod (solidago gigantea), canada goldenrod (s. canadensis l.) is even more frequently planted in central and northern part of montenegro. both species are known as honey bee plants. according to (w a l t e r , 1987) planting of goldenrods as feeding plant for bees, may also have facilitated its invasion into new habitats. these closely related species are distinguished by following features: s. gigantea have glabrous and glaucous shoots, while shoots of s. canadensis are at least weakly pubescent (fig. 1, 2). involucre in s. gigantea is 3.5-5mm, while in s.canadensis is up to 3mm. flowers of s. gigantean are bright yellow and the pappus is brownish-white, whereas the flowers of s. canadensis are somewhat lemon-yellow and the pappus silvery whitish (mcneil, 1976; pignatti, 1982). canada goldenrod is also on the eppo list of invasive alien plants (http://www.eppo.int/invasive_plants/ias_list s.htm#a1a2lists). considering bad habits of gardeners, who cut down the fruiting shoots or dump rhizome fragments on rubbish heaps outside the gardens, on riversides, brooks or waste places near the roadside, it is possible to generate subspontaneously growing individuals/populations of this species, too. in the field survey at the long ulcinj beach and buljarice bay, undertaken in late autumn 2012 to spring 2013, eleven natura 2000 habitats have been mapped: 1210 annual vegetation of drift lines, 1410 mediterranean salt meadows (juncetalia maritimi), 2110 embryonic shifting dune, 2120 shifting dunes along the shoreline with ammophila arenaria (white dunes), 2190 humid dune slack, 2220 dunes with euphorbia terracina, 2240 brachypodietalia dune grasslands with annuals, 2270 * wooded dunes with pinus pinea and/or pinus pinaster, 3170 * mediterranean temporary ponds, 92a0 salix alba and populus alba galleries sand 91aa eastern white-oak wood. almost each habitat type has an alien component of the flora (tab. 1). as it is shown in table 1, in the field survey of long ulcinj beach and buljarice bay, 28 alien species were recorded. the richest alien flora has the embryonic shifting dune and brachypodietalia dune grasslands with annuals (10 species each), while the poorest with alien species are mediterranean salty meadows (only one). number of aliens would definitely be much higher if the survey included whole hinterland of both localities and all floristic aspects. among them twenty are considered as invasive (stešević & petrović, 2010), while tagetes minuta is proposed to be included in the list. our own field investigation of the long ulcinj beach dates from the beginning of 2000, when species was noticed sporadically. in the meantime it occupied larger area in the first line of the beachs’hinterland, just near the sandy road that parallel goes with the sea-line, distant 250-600m from the sea. the most abundant population was recorded at ada bojana. as a new alien species for montenegro this plant has been recorded at the beginning of 1970es in boka kotroska bay: dobrota, kostanjica, kamenjari, igalo. actually, it has been reported as a noxious weed of whole mediterranean and submediterranean part of former yugoslavia, starting from sw istria to s macedonia (šilić, 1973). the plant is worldwide known as invasive south-american species that establishes readily in disturbed sites in the coastal areas and can form dense populations. the large size and dense growth makes it highly competitive to native plant species (weber 2003). considering the fact that plant is recorded at 3 distant localities: boka kotorska bay (šilić, 1973; obradović, 1980), ulcinj area and buljarice bay, therefore, it would be interesting to fig. 2. solidago canadensis, plant habitus (photo d. stešević) and detail of pubescent shoot (photo a. haines) http://www.eppo.int/invasive_plants/ias_lists.htm#a1a2lists http://www.eppo.int/invasive_plants/ias_lists.htm#a1a2lists biologica nyssana 4 (1-2)  december 2013: 1-7 stešević, d. caković, d.  contribution to the alien flora of montenegro … 4 make a survey and see if it is established at some “in between” places. general remarks of the survey are that the most aggressive and widespread aliens at surveyed sites at the long ulcinj beach and its hinterland are xanthium strumarium subsp. italicum, oenothera glazioviana, o. fallax, conyza canadensis, c. albida, aser squamatus, sporobolus poirettii, and tagetes minuta, while in buljarice bay are: paspalum dilatatum, sorghum halepense, conyza canadansis, c.albida and aster squamatus. alike other species, that can invade different kind of devastated habitats with small plant-cover value, dallis grasspaspalum dilatatum prefere the grassy one, on which it soon begins to dominate. it is often accompanied by sporobolus poiretti. conclusion i) alien flora of montenegro is richer for one species: giant goldenrodsolidago gigantea. besides it, another goldenrod species is frequently planted in central and northern part of montenegro canada goldenrod (s. canadensis). considering bad habits of gardeners, who cut down the fruiting shoots or dump rhizome fragments on rubbish heaps outside the gardens, on riversides, brooks or waste places near the roadside, it is possible to generate subspontaneously growing individuals/populations of this species, too. ii) the list of plant invaders in montenegro is enlarged for one species: tagetes minuta. the plant is recorded at 3 distant localities: boka kotorska bay, ulcinj area and buljarice bay, therefore it would be interesting to make a survey and see if it is established at some “in between” places. iii) the most aggressive and widespread aliens at surveyed sites at the long ulcinj beach and its hinterland are xanthium strumarium subsp. italicum, oenothera glazioviana, o. fallax, conyza canadensis, c. albida, aser squamatus, sporobolus poirettii, and tagetes minuta, while in buljarice bay are: paspalum dilatatum, sorghum halepense, conyza canadansis, c.albida and aster squamatus. alike other species, that can invade different kind of devastated habitats with small plant-cover value, dallis grasspaspalum dilatatum prefer the grassy one, on which it soon begins to dominate. it is often accompanied by sporobolus poiretti. table 1. alien and invasive species on different natura 2000 and anthropogenized habitats at the long ulcinj beach and buljarice bay species long ulcinj beach, habitat type/cooridnates buljarice bay, habitat type/cooridnates ailanthus altissima (mill.) swingle graveyard (n 42°11’18.1" e 18°59’25.7"). agave americana l. 2110 embryonic shifting dune (n 41°54'31.2" e 19°14'55.2"). amaranthus hybridus l. agricultural habitat (n 41°54'48.6" e 19°15'12.4", n 41°54'45.0" e 19°15'32.1"). amorpha fruticosa l. 92a0 salix alba and populus alba galleries (n 41°53’32.0" e 19°18’29.1"), 2270* wooded dunes with pinus pinea and/or pinus pinaster (n 41°54’37" e 19°15’02.0"), 2190 humide dune slack (n 41°54’19.4"e 19°16’00.8", n 41°54’09.8" e 19°16’42.3"), 2240 brachypodietalia dune grasslands with annuals (n 41°51’51.6" e 19°20’27"). aster squamatus (spreng.) hieron 1410 mediterranean salt meadows (juncetalia maritimi) (n 41°52’52.9 e 19°19’48.5), 2190 humide dune slack (n 41°54’19.4" e 19°16’00.8", n 41°53’19.0" e 19°18’42.6"), 2270* wooded dunes with pinus pinea and/or pinus pinaster (n 41°54’15.18" e 19°15’58"), ruderal habitat (n 41°53’14.9" e 19°18’29.2"). graveyard (n 42°11’18.1" e 18°59’25.7"). bidens subalternans dc. graveyard (n 42°11’18.1" e 18°59’25.7"). biologica nyssana 4 (1-2)  december 2013: 1-7 stešević, d. caković, d.  contribution to the alien flora of montenegro … 5 species long ulcinj beach, habitat type/cooridnates buljarice bay, habitat type/cooridnates conyza albida willd. 2110 embryonic shifting dune (n 41°54’39" e 19°14’34.7"), 2270* wooded dunes with pinus pinea and/or pinus pinaster (n 41°54’15.18" e 19°15’58"). agricultural habitat (n 41°54’45.0” e 19°15’32.1”), ruderal habitat (n 41°54’31.7“ e 19°16’21.1“,n 41°53’52.2“ e 19°17’58.2“, n 41°53’14.9“ e 19°18’29.2“). mesophilic meadows (n 42°11’02.7” e 18°58’05.5”), graveyard (n 42°11’18.1” e 18°59’25.7”). conyza canadensis (l.) cronq. 2110 embryonic shifting dune (n 41°54’ 31.2” e 19°14’ 55.2”), 2190 humide dune slack (n 41°54’19.4” e 19°16’00.8”, n 41°53’19.0” e 19°18’42.6”), 2240 brachypodietalia dune grasslands with annuals (n 41°54’08.1” e 19°16’59.8”, n 41°53’55.3” e 19°18’04.9”), 2270* wooded dunes with pinus pinea and/or pinus pinaster (n 41°54’33.0” e 19°15’23.3”, n 41°54’15.18” e 19°15’58”), agricultural habitat (n 41°54’48.6” e 19°15’12.4”), ruderal habitat (n 41°54’31.7” e 19°16’21.1”, n 41°53’52.2” e 19°17’58.2”n 41°53’14.9” e 19°18’29.2”, n 41°53’27.9” e 19°18’26.8”), agricultural land (n 41°54’02.7” e 19°186’00.2”). mesophilic meadows (n 42°11’02.7” e 18°58’05.5”), graveyard (n 42°11’18.1” e 18°59’25.7”). datura stramonium l. 2270* wooded dunes with pinus pinea and/or pinus pinaster (n 41°54’15.18” e 19°15’58”), agrucultural habitat (n 41°53’52.2” e 19°17’58.2”). chenopodium multiflorum moq. ruderal habitat (n 41°53’52.2“ e 19°17’58.2“). cuscuta caesattiana bertol. 2270* wooded dunes with pinus pinea and/or pinus pinaster (n 41°54’15.18” e 19°15’58”). datura stramonium l. agricultural habitat (n 41°54’45.0” e 19°15’32.1”), ruderal habitat (n 41°53’52.2” e 19°17’58.2”). eleusine indica (l.) gaertn. ruderal habitat (n 41°54’37.0" e 19°14’27.7", n 41°53’14.9" e 19°18’29.2"). euphorbia maculata l. ruderal habitat (n 41°53’14.9" e 19°18’29.2"). lepidium virginiacum l. graveyard (n 42°11’18.1" e 18°59’25.7"). melia azedarach l. 2110 embryonic shifting dune (n 41°54’ 31.2" e 19°14’ 55.2") roadside vegetation (n 42°11’32.0" e 18°58’20.6"). mentha × piperita ruderal habitat (n 41°53’52.2" e 19°17’58.2"). oenothera fallax renner & o. glazioviana micheli 2110 embryonic shifting dune (n 41°54’31" e 19°14’55.2", n 41°54’39" e 19°14’34.7", n 41°54’28.0" e 19°15’19.7", n 41°54’19.2" e 19°15’50.3"), 2120 shifting dunes along the shoreline with ammophila arenaria (white dunes) (n 41°53’47.4" e 19°17’21.0", n 41°53’32.0" e 19°18’01.0") ruderal habitat (n 41°53’27.9" e 19°18’26.8"). oenothera suaveolens desf. 2110 embryonic shifting dune (n 41°53’18.9" e 19°18’33.3"). biologica nyssana 4 (1-2)  december 2013: 1-7 stešević, d. caković, d.  contribution to the alien flora of montenegro … 6 species long ulcinj beach, habitat type/cooridnates buljarice bay, habitat type/cooridnates robinia pseudoacacia l. 2110 embryonic shifting dune (n 41°54’ 31.2" e 19°14’ 55.2"), 2270* wooded dunes with pinus pinea and/or pinus pinaster (n 41°54’37" e 19°15’02.0", n 41°54’15.18" e 19°15’58") ruderal habitat (n 41°54’40.2" e 19°14’10.4", n 41°54’40.2" e 19°14’10.4"), agricultural habitat (n 41°54’48.6" e 19°15’12.4"). parthenocissus quinquefolia (l.) planch. ruderal habitat (n 41°54’40.2" e 19°14’10.4"). paspalum dilatatum poir. 92a0 salix alba and populus alba galleries (n 41°54’31.5" e 19°15’53.9"). mesophilic meadows (n 42°11’42.2" e 18°58’10.3", n 42°11’36.1" e 18°58’17.1", n 42°11’02.7" e 18°58’05.5"). paspalum paspaloides (michx.) schribn. 2270* wooded dunes with pinus pinea and/or pinus pinaster (n 41°54’33.0" e 19°15’23.3") ruderal habitat (n 41°54’31.7" e 19°16’21.1", n 41°53’27.9" e 19°18’26.8"). phytolacca americana l. agricultural habitat (n 41°54’48.6" e 19°15’12.4"), ruderal habitat (n 41°54’40.2" e 19°14’10.4", n 41°54’31.7" e 19°16’21.1", n 41°53’52.2" e 19°17’58.2"). sorghum halepense (l.) pers. agricultural habitat (n 41°54’48.6" e 19°15’12.4", n 41°54’45.0" e 19°15’32.1"). mesophilic meadows (n 42°11’00.2" e 18°58’50.4", n 42°11’02.7" e 18°58’05.5"). sporobolus poirettii (r.et s.) hitche 2240 brachypodietalia dune grasslands with annuals (n 41°54’08.1" e 19°16’59.8", n 41°53’55.3" e 19°18’04.9"), 2270* wooded dunes with pinus pinea and/or pinus pinaster (n 41°54’15.18" e 19°15’58") 92a0 salix alba and populus alba galleries (n 41°54’31.5" e 19°15’53.9"9), agricultural habitat (n 41°54’48.6" e 19°15’12.4", n 41°54’45.0" e 19°15’32.1"), ruderal habitat (n 41°54’31.7" e 19°16’21.1", n 41°53’14.9" e 19°18’29.2", n 41°53’27.9" e 19°18’26.8"). mesophilic meadows (n 42°11’42.2" e 18°58’10.3"), graveyard (n 42°11’18.1" e 18°59’25.7"). tagetes minuta l. 2240 brachypodietalia dune grasslands with annuals, and ruderal habitat in line transect: (n 41°53’52.2" e 19°17’58.2" to n 41°51’51.6" e 19°20’27"). graveyard (n 42°11’18.1" e 18°59’25.7"). xanthium strumarium l. italicum (moretti) d.löve 2110 embryonic shifting dune (n 41°54’31" e 19°14’55.2", n 41°54’39" e 19°14’34.7", n 41°54’19.2" e 19°15’50.3", n 41°53’22.6" e 19°18’22.7"), 2270* wooded dunes with pinus pinea and/or pinus pinaster (n 41°54’15.18" e 19°15’58"), 2120 shifting dunes along the shoreline with ammophila arenaria (white dunes) (n 41°54’09.7" e 19°16’19.5", n 41°53’47.4" e 19°17’21.0", n 41°53’32.0" e 19°18’01.0", n 41°53’19.0" e 19°18’42.6"), ruderal habitat (n 41°54’40.2" e 19°14’10.4", n 41°54’31.7" e 19°16’21.1", n 41°53’14.9" e 19°18’29.2"), agricultural land (n 41°54’02.7" e 19°186’00.2", n 41°53’27.9" e 19°18’26.8"). 1210annual vegetation of drift lines (n 42°11’36.4" e 18°578’54.7"). yucca gloriosa l. 2110 embryonicshifting dune (n 41°54’ 31.2" e 19°14’ 55.2"). biologica nyssana 4 (1-2)  december 2013: 1-7 stešević, d. caković, d.  contribution to the alien flora of montenegro … 7 acknowledgements. authors would like to thank arthur haines for his valuable photo contribution. the field investigation was partly funded by undp, office in montenegro (project number mne 12-057) and by the montenegrin academy of sciences and arts. references chytrý, m., maskell, l., pino, j., pyšek, p., vilà, m., font, x., smart, s. 2008: habitat invasions by alien plants: a quantitative comparison between mediterranean, subcontinental and oceanic regions of europe. journal of applied ecology, 45: 448–458. ellenberg, h., weber, h.e., düll, r., wirth, v., werner, w., paulissen, d. 1992: zeigerwerte von pflanzen in mitteleuropa. scripta geobotanica, 18:1-258. guzikowa, m., maycock, p. f. 1986: the invasion and expansion of three north american species of goldenrod (solidago canadensis l. sensu lato, s. gigantea aid. and s. graminifolia (l.) salisb.) in poland, acta societatis botanicorum poloniae, 55 (3): 367-384. jacobs, g., weber, e., edwards, p.j. 2004: introduced plants of the invasive solidago gigantea (asteraceae) are larger and grow denser than conspecifics in the native range. diversity & distributions, 10: 11-19. koutika, l.s., rainey, h.j., dassonville, n. (2011): impacts of solidago gigantea, prunus serotina, heracleum mantegazzianum and fallopia japonica invasions, applied ecology and environmental research, 9(1): 73-83 lambdon, p.w.et al. 2008: alien flora of europe: species diversity, patterns and research needs. preslia, 80: 101-149. lohmeyer, w. and sukopp, h. 1992: agriophyten in der vegetation mitteleuropas. schriftenreihe für vegetationskunde, 25: 185 p. mcneil, j. 1976: solidago l. in: tutin tg, heywood vh, burges na, moore dm, valentine dh, walters sm, and weeb da [eds.], flora europaea, vol. 4. 110-111, cambridge university press. obradović, m. 1980: prilog poznavanju adventivne flore herceg-novog, boka 12: 203-212. official gazette no 29/2013 pignatti, s. 1982: flora d' italia. 3, edagricole, bologna. stančić, z. 2010: marshland vegetation of the class phragmito-magnocaricetea in northwest croatia (krapina river valley). biologia 65(1): 39-53. stešević, d., petrović, d. 2010: preliminary list of plant invaders in montenegro. biologica nyssana, 1 (1-2): 35-42. šilić, č. 1973: tagetes minuta l. sve masovniji i opasniji korov na poljoprivrednim površinama dalmacije, hercegovine, crnogorskog primorja i južne makedonije. jugoslavenski simpozijum o borbi protiv korova u brdsko-planinskim područjima (sarajevo), 27–34. wagenitz, g., 1979: solidagol. in: hegi, g. ed. illustrierte flora von mitteleuropa, 16-29, carl hanser verlag, münchen. walter, e. 1987: zur verbreitung und zum verhalten nordamerikanischer goldruten (solidago canadensis und s. gigantea) in oberfranken, berieht der naturforschenden gesellschaft bamberg, 62: 2768. weber, e. 1998: the dynamics of plant invasions: a case study of three exotic goldenrod species (solidago l.) in europe. journal of biogeography, 25(1):147-154. weber, w., jacobs, g. 2005: bilogical flora of central europe, solidago gigantea. flora, 200: 109-118. weber, e. 2003: invasive plants of the world. cabi publishing, cab international, wallingford, uk. 548 pp. internet sorces: euro+med 2006-: euro+med plantbase the information resource for euro-mediterranean plant diversity. http://ww2.bgbm.org/europlusmed/ [accessed may 2013]. ministarstvo turizma i održivog razvoja 2013: drugi izvještaj o implementaciji nacionalne strategije biodiverziteta sa akcionim planom. http://www.gov.me/.../filedownload.aspx?rid=1210 33 [accessed semptember 2013]. http://www.eppo.int/invasive_plants/ias_lists. htm#a1a2lists, [accessed may 2103] rajković, j., joković, n.  probiotic properties and safety assesment… biologica nyssana 6 (2)  december 2015: 81-89 rajković, j., joković, n.  probiotic properties and safety assesment… 81 original article received: 02 december 2015 revised: 14 december 2015 accepted: 20 december 2015 probiotic properties and safety assessment of lactic acid bacteria isolated from kajmak jelena rajković, nataša jokovića* university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia * e-mail: jole35@mts.rs abstract: rajković, j., joković, n.: probiotic properties and safety assessment of lactic acid bacteria isolated from kajmak. biologica nyssana, 6 (2), december 2015: 81-89. one hundred forty-one strains of lactic acid bacteria (lab) were tested for probiotic features such as antimicrobial activities, autoaggregation ability, hydrophobicity of bacterial cell walls and ability to grow on and hydrolyze bile salts. additionally, resistance to antibiotics and production of biogenic amines were performed as safety assessment of lab strains. five lc. lactis ssp. lactis and six lc. raffinolactis strains showed antibacterial activities against used indicator strains. protein nature of antibacterial compounds was proven by the addition of pronase. enterococcus durans strains had the highest degree of autoaggregation and hydrophobicity while the rest of the strains showed low degree for both characteristics. lb. paracasei, lb. plantarum, ln. mesenteroides, en. faecium, en. faecalis and en. durans strains could growth on bile salts and some of them were able to hydrolyse them. safety assessment of lab indicates that many strains of enterococci were resistant to chloramphenicol, tetracycline, cephalosporin and to the lowest used concentration of erythromycin. the strains from other lab genera were resistant to antibiotics used in lower concentration and in smaller numbers when compared to enterococci. key words: lactic acid bacteria, probiotic properties, safety assessment apstrakt: rajković, j., joković, n.: probiotska svojstva i bezbednost upotrebe mlečno kiselinskih bakterija izolovanih i kajmaka. biologica nyssana, 6 (2), december 2015: 81-89. probiotska svojstva mlečno kiselinskih bakterija kao što su antimikrobna aktivnost, autoagregacija, hidrofobnost i sposobnost da rastu na žučnim solima i vrše njihovu hidrolizu ispitivana su na sto četrdeset jednom bakterijskom izolatu. pored toga, ispitivana je rezistentnost ovih bakterijih izolata na antibiotike i sposobnost produkcije biogenih amina u okviru procene bezbednosti izolata za upotrebu u starter kulturama. pet lc. lactis ssp. lactis i šest lc. raffinolactis izolata pokazala su antimikrobno delovanje prema korišćenim indikatorskim sojevima. proteinska priroda antimikrobnih jedinjenja je potvrdjena dodavanjem pronaze. najveći stepen hidrofobnosti i autoagregacije imali su en. durans izolati, dok su kod ostalih izolata ova probiotska svojstava bila izražena u manjem stepenu. lb. paracasei, lb. plantarum, ln. mesenteroides, en. faecium, en. faecalis i en. durans izolati su pokazali rast na podlogama sa žučnim solima, dok su neki od njih imali sposobnost da vrše njihovu hidrolizu. procenom sigurnosti upotrebe izolata utvrđeno je da su mnogi izolati enterokoka bili rezistentni na hloramfenikol, tetraciklin, cefalosporin i na najniže korišćene koncentracije eritromicina. izolati iz ostalih rodova mlečno kiselinskih bakterija su bili rezistentni na niske koncentracije antibiotika, ali u manjem broju u poređenju sa enterokokama. key words: mlečno kiselinske bakterije, probiotska svojstva, procena bezbednosti 6 (2) • december 2015: 81-89 biologica nyssana 6 (2)  december 2015: 81-89 rajković, j., joković, n.  probiotic properties and safety assesment… 82 introduction lactic acid bacteria (lab) are a heterogeneous group of bacteria, including gram-positive cocci and bacilli, with common feature to produce lactic acid as the end-product of metabolism. they are mainly isolated from the various fermented foods produced from milk, meat or vegetables but also present a normal microflora of the nasopharyngеal, intestinal and vaginal mucosa of humans and animals (s t i l e s & h o l z a p f e l , 1997). various strains of lab are widely used in the industry as a part of starter cultures for improving the quality and shelf life of fermented foods in different food processes. in recent years, lab has been intensively studied as probiotic bacteria. probiotics are defined as "live microorganisms which when administered in adequate amounts confer a health benefit to the host'' (f a o / w h o , 2002). in dairy products bifidobacteria and lactobacilli are commonly used as probiotic bacteria, but other lab, such as enterococci, lactococci or streptococci, also may possess probiotic properties (p r i n g s u l a k a et al., 2015). intake of probiotic bacteria could lead to the establishment of intestinal microbial balance, prevention of allergies and alleviation of certain intolerances. antimutagenic, anticarcinogenic, hypocholesterolemic, antihypertensive, antiosteoporosis, and immunomodulatory effects on the host have been also reported for some probiotic bacteria (c h i a n g & p a n , 2012). considering the fact that beneficial effects of lab are strain-dependent, screening of lab isolated from different sources for probiotic properties is a common way for searching new probiotic bacteria. synthesis of antimicrobial compounds, adhesion potential to the intestinal epithelium and ability to survive conditions in the gastrointestinal tract are the characteristics that have been studied for preliminary selection of potential probiotics (b a u t i s t a g a l l e g o et al., 2013; p r i n g s u l a k a et al., 2015; a n g m o et al., 2016). the strains of lab which have capacity to produce different antimicrobial compounds could inhibit the pathogenic bacteria in gastrointestinal tract, thus reducing the risk of gastrointestinal diseases. in order to achieve the effect in intestine, probiotic bacteria must have ability to pass through upper parts of gastrointestinal tract and adhere to intestinal epithelial cells. conditions in the upper gastrointestinal tract, such as ph values and bile salts, may adversely affect the probiotic bacteria. therefore, it is necessary to examine the possibility of survival of the probiotic bacteria in the presence of bile salts. aggregation ability and hydrophobicity of bacterial cell walls are correlated to adherence properties of probiotic bacteria (d u a r y et al., 2011; j a n k o v i ć et al., 2012). required property of potential probiotic bacteria is the safety for human consumption. resistance to different antibiotics, the presence of virulence factors and synthesis of biogenic amines are commonly tested undesirable characteristics of probiotic bacteria (c h a m b a & j a m e t , 2008). traditional fermented products are the source of the new probiotic bacteria that can be used in the production of various types of functional foods. the aim of current study was screening of lab isolated from serbian traditional dairy product, kajmak, for some probiotics features such as antimicrobial activities, autoaggregation ability, hydrophobicity of bacterial cell walls and the ability to grow on and to hydrolyze bile salts. additionally, lab strains were tested for resistance to antibiotics and production of biogenic amines as safety assessment of strains. material and methods bacterial strains, media, and growth conditions one hundred forty-one strains of lab isolated from different samples of kajmak were used in this study. all strains were previously identified by molecular methods to the species level as lactococcus lactis ssp. lactis (19), lactococcus raffinolactis (6), streptococcus thermophilus (4), enterococcus faecalis (9), enterococcus faecium (19), enterococcus durans (10), leuconostoc mesenteroides (50), lactobacillus plantarum (15), lactobacillus paracasei (6), lactobacillus satsumensis (1), lactobacillus kefiri (1) and lactobacillus kefiranofaciens (1) (j o k o v i c et al., 2008; j o ko vi ć et al., 2014). the strains were stored at -20 c and activated by triple streaking on appropriate media. mrs agar (merck, gmbh, darmstadt, germany) was used for growth of leuconostoc and lactobacillus strains whereas lactococcus, streptococcus and enterococcus strains were cultured on m17 agar (merck, gmbh, darmstadt, germany) supplemented with glucose (0.5%, w/v; gm17 broth). the inoculated media were incubated overnight at 30 c for mesophilic and 37 c for thermophilic strains. tryptic soy agar (himedia, india) was used for the growth of non-lab strains (escherichia coli, bacillus subtilis and listeria innocua) which were incubated at 37 c for 24 h. yeast (candida albicans) was cultured on sabouraud maltose agar (torlak, serbia) at 30 c for 48 h. biologica nyssana 6 (2)  december 2015: 81-89 rajković, j., joković, n.  probiotic properties and safety assesment… 83 antimicrobial activity of strains the lab strains were screened for antimicrobial activity by the agar-well diffusion method (t a g g & m c g i v e n , 1971) using various indicator strains that are listed in table 1. asssay was performed by overlaying solid mrs or gm17 medium in petri dishes with 5 ml of soft (0.7% agar) mrs, gm17, tryptic soy and sabouraud maltose media inoculated with 105-106 cells of indicator strain/ml medium. the wells, 5 mm in diameter, were made in soft agar and filled with 100 l of overnight strain culture with a potential of bacteriocin production. the presence of antimicrobial substances was detected by the appearance of clear zones around wells as a result of growth inhibition of sensitive bacterial strains. the protein nature of bacteriocins was confirmed by adding a cristal of pronase e near to the edge of the well. absence of a zone of inhibition in the place where pronase e crystals were added indicates the protein nature of bacteriocins. adhesive properties of strains bacterial adhesion to hydrocarbons was determined by the method described by f o r t i n a et al. (2008). cells from overnight culture were struck down by centrifugation at 5000 rpm for 15 min, washed twice in pbs buffer and resuspended in 0.1 m kno3 (ph 6.2). the initial absorbance of the cell suspension was adjusted to 0.5-0.6 at 600 nm (a0). then, 3 ml of cell suspension was mixed with 1 ml of nhexadecane. the mixture was incubated at room temperature for 10 min and mixed well on vortex for 2 min. after 20 min of incubation at room temperature, the aqueous phase was carefully separated and the optical density was measured at 600 nm (at). the percentage of bacterial adhesion to n-hexadecane was expressed as: % hydrophobicity = 1(at/a0) x100, where a0 and at represent absorbance values of aqueous phase before and after contact with n-hexadecane. aggregation of strains was monitored in phosphate buffered saline (pbs) according to f o r t i n a et al. (2008). bacterial cells from overnight cultures were centrifuged at 5000 rpm for 15 min, washed twice in pbs and resuspended in 4.0 ml of pbs to give an od600nm of 1.0. the cell suspensions were further well mixed on a vortex for 10 s and autoaggregation was determined during 5 h of incubation at room temperature. every hour, 100 l from the surface of the suspension was transferred to new microtube containing 900 l pbs buffer and od600nm was measured. the percentage of aggregated cells was calculated as 1-(at/a0) x100 where a0 represents absorbance values at time t=0, while at represents absorbance values at time t= 5 h. the growth of strains on bile salts the growth of strains in the presence of bile salts was monitored on laptg medium consisted of: 15 g/l peptone, 10 g/l tryptone, 10 g/l yeast extract, 10 g/l glucose, 15 g/l agar, 0.01 ml/l of tween 80 and 0.3 g/l bovine bile (f o r t i n a et al., 2008). the ability of strains to hydrolyse bile salts was detected by precipitation of deconjugated bile acids around the colony that grew on the laptg medium without bovine bile and with the addition of 0.05 g/l bile salts no. 3 (torlak, belgrade, serbia) and 0.2 g/l cacl2. a positive reaction for the presence of hydrolase is the appearance of a precipitate around the colonies. safety assessment the resistance of strains to antibiotics was tested by spot agar test on antibiotic-containing gm17 and mrs agar media optimal for lab growth. tests were done for the following antibiotics and concentration: chloramphenicol (10 g/ml, 20 g/ml), tetracycline (10 g/ml, 20 g/ml, 30 g/ml) and erythromycin (5 g/ml, 10 g/ml). table 1. list of strains used in antimicrobial activity assay bacterial strains source or reference lactococcus lactis ssp. lactis biovar. diacetylactis, s50 (kojic et al., 2005) lactococcus lactis ssp.cremoris, ns1 (kojic et al., 1991) lactococcus lactis ssp. lactis, bgmn1-596 (gajic et al., 1999) lactococcus lactis ssp. lactis, np45 lab. kolekcija lactobacillus paracasei ssp. paracasei, bgbuk2-16/k4 (lozo et al., 2004) lactobacillus plantarum, a112 (vujcic & topisirovic, 1993) escherichia coli, atcc 25923 atcc bacilus subtilis, atcc 6633 atcc listeria innocua, atcc 33090 atcc candida albicans, atcc 10231 atcc biologica nyssana 6 (2)  december 2015: 81-89 rajković, j., joković, n.  probiotic properties and safety assesment… 84 the ability of lab isolates to produce biogenic amines was determined according to the method described by b o v e r -c i d and h o l z a p f e l (1999). histidine, tyrosine, ornithine and lysine were used as precursors for the biogenic amines synthesis. the color change of colonies to purple or occurrence of precipitates around the colonies in case of tyramine synthesis indicated the decarboxylation activities of isolates. results and discussion antimicrobial activity lab produce different substances that have antimicrobial activity, such as an organic acid (lactic, formic, acetic acid etc.), co2, h2o2, diacetyl, ethanol, and bacteriocins. among these products, bacteriocins are the most important because of their possible application in the food industry as biopreservatives (d e v u y s t & l e r o y , 2007). since bacteriocins have the highest inhibitory effect against related gram positive bacteria, bacteria from genus lactococcus, lactobacillus, bacillus and listeria were used as indicator strains (tab. 1). additionally, gram negative bacteria e. coli and yeast c. albicans were also used in antimicrobial assays. results of antimicrobial assays showed that a small number of strains could synthesize bacteriocins (tab. 2). five lc. lactis ssp. lactis, and six lc. raffinolactis strains produced bacteriocins with inhibitory effect on the growth of lactococcal indicator strains lc. lactis ssp. cremoris ns1, lc. lactis ssp. lactis bgmn1-596 and lc. lactis ssp. lactis np45 (tab. 2). lactococcal strains formed table 2. antimicrobial effect of tested strains shown through the zones of inhibition species n o . o f is o la te s indicator strains lactococcus lactis ssp. lactis bgmn1-596 lactococcusl actis ssp. cremoris ns1 lactococcus lactis ssp. lactis np45 lactococcus lactis ssp. lactis biovar. diacetylactis s50 escherichia coli atcc2592 listeria innocua atcc 33090 lc. lactis ssp. lactis 3 1 mm s 1 mm s 1 mm s lc. lactis ssp. lactis biovar. diacetylactis 4 7 mm s 7 mm s 3 mm s lc. raffinolactis 6 5 mm s 5 mm s 3 mm s 2 mm s 1 mm s 2 mm s en. faecium 1 2 mm s 5 mm s en. faecalis 1 4 mm s 2 mm s 0.5 mm s mm – zone width; s – clear zone. table 3. autoaggregation, hydrophobicity, growth on bile salts and hydrolysis of bile salts of tested lab strains l c . la c ti s s sp . la c ti s (1 9 )1 l c . ra ff in o la c ti s (6 ) s t. t h e rm o p h il u s (4 ) e n . fa e c a li s (9 ) e n . fa e c iu m ( 1 9 ) e n . d u ra n s (1 0 ) l n . m e se n te ro id e s (5 0 ) l b . p a rc a se i ( 6 ) l b . p la n ta ru m ( 1 5 ) l b . sa ts u m e n si s (1 ) l b . k e fi ri ( 1 ) l b . k e fi ra n o fa c ie n s (1 ) hydrophobicity (%)2 19 2 10 14 28 45 17 6 6 27 2 3 autoaggregation (%)2 39 31 36 31 44 71 36 50 27 36 8 37 growth in the presence of bile salts3 4 10 10 3 3 9 hydrolysis of bile salts 3 1 4 5 3 3 8 1 -numbers in parenthesis represent the number of isolates 2 -results are presented as mean value 3 -results are shown as the number of isolates that has a certain property biologica nyssana 6 (2)  december 2015: 81-89 rajković, j., joković, n.  probiotic properties and safety assesment… 85 fig. 1. inhibitory effects of lc. lactis ssp. lactis and lc. raffinolactis strains on lc. lactis ssp. cremoris ns1 as indicator strain clear zones of inhibition, which lacked on the points where pronase e was added (fig. 1). lc. raffinolactis strains also inhibited the growth of lc. lactis ssp. lactis biovar. diacetylactis s50, e. coli atcc 2592 and l. inoccua atcc 33090 but zones of inhibition were smaller in these cases (tab. 2). one strain of en. faecium formed clear zones of inhibition against lc. lactis ssp. lactis biovar. diacetylactis s50, and l. innocua atcc 33090 while en. faecalis strain had inhibitory effects on lc. lactis ssp. cremoris ns1, lc. lactis ssp. lactis bgmn1-596 and l. inoccua atcc 33090 (tab. 2). zones of inhibition formed by enterococci were not sensitive to pronase e, so it can be concluded that the antimicrobial substance doesn’t have protein nature. strains which have been found to have antimicrobial activity against any of the above mention indicator strains, did not act inhibitory against lb. plantarum a112, lb. paracasei ssp. paracasei bgbuk2-16 / k4, b. subtilis atcc 6633 and c. albicans atcc 10231 indicator strains. synthesis of bacteriocins is significant characteristics of probiotic bacteria because bacteriocin producing strains could inhibit pathogenic bacteria in the gastrointestinal tract. except probiotic effects that strains with antimicrobial activities could have, bacteriocin producing lab strains are a part of starter cultures for improving the microbiological quality and safety of dairy products, or for accelerating the ripening of fermented foods (a y a d et al., 2004; m o h a m m e d et al., 2009). adhesive properties of strains autoaggregation and hydrophobicity, as surface characteristics of the lab strains, indicate potential adhesion ability of probiotic lab strains to epithelial cells of human intestine (f o r t i n a et al., 2008). the high degree of autoaggregation indicates better adhesive capability of isolates, while physicochemical properties of the cell wall of lab may probably affect autoaggregation and adhesion (f o r t i n a et al., 2008). according to m a l d o n a d o et al. (2012), three different groups of lab strains can be distinguished on the basis of autoaggregation and hydrophobicity degrees: high (60–100%), medium (30–60%) and low (0–30%). the average values of these characteristics of tested lab species are summarized in table 3. enterococcus durans strains showed the highest autoaggregation and hydrophobicity degrees with the average values of 71% and 45%, respectively. among six en. durans strains, three of them aggregated quickly after 15 min, forming a precipitate and clear solution. these strains also expressed hydrophobicity values higher than 60%. lc. lactis, lc. raffinolactis, st. thermophilus, en. faecalis, en. faecium, ln. mesenteroides, lb. paracasei, lb. satsumensis and lb. kefiranofaciens strains showed a medium degree of autoaggregation. en. faecium and lb. paracasei strains had the highest degree of autoaggregation in this group (44% and 50%, respectively). all strains in this group had very low level of binding to n-hexadecane and only en. faecium strains had average degree of hydrophobicity higher than 20%. lb. plantarum strains had low degree of autoaggregation and hydrophobicity (27% and 6%, respectively) while one lb. kefiranofaciens strain had the lowest degree of both properties among tested strains. high intraspecies variability in autoaggregation and hydrophobicity degrees was observed for lab strains evidencing that these properties seems to be strain specific. similar findings have been reported by other authors (t o d o r o v et al., 2008; g i a o u r i s et al., 2009; i y e r et al., 2010). growth in the presence of bile salts the growth of the lab strains in the presence of bile salts is a significant characteristic of probiotic lab strains, which indicates the possibility of their survival in gastrointestinal tract (d u n n e et al., 2001). biologica nyssana 6 (2)  december 2015: 81-89 rajković, j., joković, n.  probiotic properties and safety assesment… 86 table 4. resistance of lab strains to different antibiotics antibiotic chloramphenicol, g/ml tetracycline,g/ml erythromycin, g/ml cephalosporin,g/ml lab species 10 20 10 20 30 5 10 10 20 30 lc. lactis ssp. lactis (19)1,2 5 19 18 8 19 18 18 lc. raffinolactis (6) st. thermophilus (4) 4 2 1 4 2 2 en. faecalis (9) 9 6 9 9 3 8 9 9 9 en. faecium (19) 19 13 19 19 5 19 19 19 19 en. durans (10) 8 10 10 7 10 10 10 ln. mesenteroides(50) 50 45 4 40 50 40 9 lb. parcasei (6) 6 6 4 6 6 4 lb. plantarum (15) 15 14 14 4 15 14 12 lb. satsumensis (1) 1 1 1 1 1 1 lb. kefiri (1) 1 1 1 1 1 lb. kefiranofaciens (1) 1 -numbers in parenthesis represent the number of isolates of the certain strain 2 -the results are presented as number of isolates which are resistant to a particular antibiotic the ability of bile salts hydrolysis is desirable probiotic characteristics of lab strains, because the bile salts deconjugation contributes to the intestinal microflora balance (m a t h a r a et al., 2008). selection of probiotic strains based on their deconjugation activities is also important, because of the correlation between this activity and the ability of lab strains to decrease cholesterol level (p e r e i r a et al., 2003). in our study, both characteristics were detected only in strains of genus enterococcus, leuconostoc and lactobacillus (tab. 3). the most of the strains that could grow in the presence of bile salts belonged to species en. faecium, en. durans and lb. plantarum while half of the lb. plantarum strains could hydrolyse bile salts. the growth of lb. plantarum, lb. paracasei and en. faecium strains on the media with the addition of bile salts was also found in earlier studies (x a n t h o p o u l o s et al., 2000; s a a v e d r a et al., 2003; t o d o r o v et al., 2008). certain lb. plantarum, lb. paracasei and en. faecium strains were found to be able to deconjugate bile salts (s a a v e d r a et al., 2003; m a r a g k o u d a k i s et al., 2006; n g u y e n et al., 2007). safety assessment although lab has gras (generally recognized as safe) status, the strains isolated from fermented foods that can be used as starter culture should be tested for their potentially adverse effects on human health. antibiotic resistance and the synthesis of biogenic amines are mostly analyzed characteristics of lab strains important for their safe use in starter cultures (f r a n c i o s i et al., 2009). antibiotic resistance of lab strains is a major problem in the food industry after the discovery that certain strains of lactococci, lactobacilli and enterococci have genes for antibiotics resistance located on plasmids and transposons. thus, antibiotic resistance can be transferred to other bacterial species (k a s t n e r et al., 2006). in our work, resistance to antibiotics was determined by growing the isolates on the appropriate medium with various antibiotics (chloramphenicol, tetracycline, erythromycin, and cephalosporin) added in certain concentrations (tab. 4). cephalosporin belongs to the group of antibiotics which inhibit the synthesis of the cell wall, while tetracycline, erythromycin, chloramphenicol affect the protein synthesis in the bacterial cell. results from the present study showed that the most of the tested lab strains were resistant to cephalosporin (tab. 4). all enterococci, lb. satsumensis strain, lb. kefiri strain and the most of lc. lactis ssp. lactis, st. thermophilus, lb. paracasei and lb. plantarum strains grew well on the media where cephalosporin was added in the concentration of 30 µg/ml. strains belonging to species ln. mesenteroides showed the highest sensitivity to cephalosporin with only 9 strains resistant to the highest concentration of cephalosporin used (tab. 4). biologica nyssana 6 (2)  december 2015: 81-89 rajković, j., joković, n.  probiotic properties and safety assesment… 87 the most of the lab strains were resistant to tetracycline when it was added in media in low concentration (10 g/ml and 20 g/ml). increasing of tetracycline concentration in media reduced the number of resistant strains for all lab species but some strains of enterococci were resistant to this high concentration of tetracycline. when chloramphenicol was added in the concentration of 10 g/ml, all enterococcal strains and a small number of lactococcal strains were resistant. increasing concentrations of chloramphenicol at 30 g/ml did not affect growth of the most of en. faecium and en. faecalis strains. addition of erythromycin in a concentration of 5 µg/ml in media did not inhibit the growth of en. faecium, en. faecalis, ln. mesenteroides, lb. paracasei, lb. plantarum and lb. satsumensis strains, but some of lc. lactis, all st. thermophilus and en. durans strains and one lb. kefiri strain were sensitive to addition of this antibiotic in low concentration. only four lb. plantarum strains were resistant to the higher concentration of erythromycin (10 µg/ml). the most sensitive to the tested antibiotics were lc. raffinolactis and lb. kefiranofaciens strains that showed no growth on any of the medium with the addition of antibiotics. antibiotic resistant strains from dairy environment were also found by other researchers (f l ó r e z et al., 2005; d e v i r g i l i i s et al., 2010). data on proposed minimum inhibitory concentrations of antibiotics used for lab isolates from dairy products are different in the literature due to the differences between selected methods (a m m o r et al., 2007). overall, lab isolates from dairy products are the most sensitive to erythromycin, while their resistance is most pronounced to cephalosporins (k a t l a et al., 2001; t e m m e r m a n et al., 2003). resistance to chloramphenicol and tetracycline is distinguished among lab isolates from different genus and it is usual to obtain strains with a much higher resistance compared to the proposed minimal inhibitory concentration for a particular species (d a n i e l s e n & w i n d , 2003; f l ó r e z et al., 2005; m a r a g k o u d a k i s et al., 2006). in fermented food, biogenic amines formed by bacterial decarboxylation of amino acids histidine, tyrosine, lysine or ornithine, lead to the undesirable flavor of final product and may have negative impact on human health (l a n d e t e et al., 2007). among analyzed lab strains, all en. faecalis strains, 12 strains of en. faecium and 5 strains of en. durans synthesized tyramine from tyrosine. production of tyramine is commonly detected characteristics of enterococci from dairy products (m a r t í n p l a t e r o et al., 2009). conclusion traditional fermented products, such kajmak is, are new sources of lab strains with interesting characteristics. in recent years, lab strains that have probiotic properties are particularly popular because they can be used for the production of new types of functional foods. the results of the present research indicate that among analyzed lab strains isolated from kajmak, a few strains had interesting probiotic characteristics. five lc. lactis ssp. lactis and six lc. raffinolactis strains synthesized bacteriocins, so they could be used for a control of pathogenic bacteria. enterococcus durans strains with high degree of autoaggregation and hydrophobicity should be further tested for adhesion to human cell lines. strains of lb. paracasei, lb. plantarum and ln. mesenteroides that hydrolyze bile salts could have cholesterol lowering effects. all the abovementioned strains were low resistance to used antibiotics, and they didn’t have the ability to synthesize biogenic amines. on the other hand, all enterococcal strains that hydrolyze bile salts showed high resistance to used antibiotics and could decarboxylate amino acids. acknowledgements. the authors are grateful to the ministry of education, science and technological development for financial support through the grant within frame of basic research, no tr 31032. references ammor, ms., flórez, ab., mayo, b. 2007: antibiotic resistance in non-enterococcal lactic acid bacteria and bifidobacteria. food microbiology, 24 (6): 559-570. angmo, k., kumari, a., bhalla, tc. 2016: probiotic characterization of lactic acid bacteria isolated from fermented foods and beverage of ladakh. lwt-food science and technology, 66: 428-435. ayad, e., nashat, s., el-sadek, n., metwaly, h., elsoda, m. 2004: selection of wild lactic acid bacteria isolated from traditional egyptian dairy products according to production and technological criteria. food microbiology, 21 (6): 715-725. bautista-gallego, j., arroyo-lópez, f., rantsiou, k., jiménez-díaz, r., garrido-fernández, a., cocolin, l. 2013: screening of lactic acid bacteria isolated from fermented table olives with probiotic potential. food research international, 50 (1): 135-142. biologica nyssana 6 (2)  december 2015: 81-89 rajković, j., joković, n.  probiotic properties and safety assesment… 88 bover-cid, s., holzapfel, wh. 1999: improved screening procedure for biogenic amine production by lactic acid bacteria. international journal of food microbiology, 53 (1): 33-41. chamba, jf., jamet, e. 2008: contribution to the safety assessment of technological microflora found in fermented dairy products. international journal of food microbiology, 126 (3): 263-266. chiang, s-s., pan, t-m. 2012: beneficial effects of lactobacillus paracasei subsp. paracasei ntu 101 and its fermented products. applied microbiology and biotechnology, 93 (3): 903916. danielsen, m., wind, a. 2003: susceptibility of lactobacillus spp. to antimicrobial agents. international journal of food microbiology, 82 (1): 1-11. de vuyst, l., leroy, f. 2007: bacteriocins from lactic acid bacteria: production, purification, and food applications. journal of molecular microbiology and biotechnology, 13 (4): 194199. devirgiliis, c., barile, s., caravelli, a., coppola, d., perozzi, g. 2010: identification of tetracycline‐ and erythromycin‐resistant gram‐positive cocci within the fermenting microflora of an italian dairy food product. journal of applied microbiology, 109 (1): 313-323. duary, rk., rajput, ys., batish, vk., grover, s. 2011: assessing the adhesion of putative indigenous probiotic lactobacilli to human colonic epithelial cells. the indian journal of medical research, 134 (5): 664-671. dunne, c., o'mahony, l., murphy, l., thornton, g., morrissey, d., o'halloran, s., feeney, m., flynn, s., fitzgerald, g., daly, c. 2001: in vitro selection criteria for probiotic bacteria of human origin: correlation with in vivo findings. the american journal of clinical nutrition, 73 (2): 386s-392s. fao/who, 2002: guidelines for the evaluation of probiotics in food. report of a joint fao/who working groupon drafting guidelines for the evaluation of probiotics in food.ontario, canada, april 30 and may 1, 2002, 1–11. flórez, ab., delgado, s., mayo, b. 2005: antimicrobial susceptibility of lactic acid bacteria isolated from a cheese environment. canadian journal of microbiology, 51 (1): 51-58. fortina, m., ricci, g., borgo, f., manachini, p., arends, k., schiwon, k., abajy, m., grohmann, e. 2008: a survey on biotechnological potential and safety of the novel enterococcus species of dairy origin, e. italicus. international journal of food microbiology, 123 (3): 204-211. franciosi, e., settanni, l., cavazza, a., poznanski, e. 2009: biodiversity and technological potential of wild lactic acid bacteria from raw cows' milk. international dairy journal, 19 (1): 3-11. gajic, o., kojic, m., banina, a., topisirovic, l. 1999: characterization of natural isolate lactococcus lactis subsp. lactis bgmn1-5, a strain producing two bacteriocins, cell wallassociated proteinase and showing clumping phenotype. archives of biological sciences, 51: 69-78. giaouris, e., chapot-chartier, m-p., briandet, r. 2009: surface physicochemical analysis of natural lactococcus lactis strains reveals the existence of hydrophobic and low charged strains with altered adhesive properties. international journal of food microbiology, 131 (1): 2-9. iyer, r., tomar, s., kapila, s., mani, j., singh, r. 2010: probiotic properties of folate producing streptococcus thermophilus strains. food research international, 43 (1): 103-110. janković, t., frece, j., abram, m., gobin, i. 2012: aggregation ability of potential probiotic lactobacillus plantarum strains. international journal of sanitary engineering research, 6 (1): 17-22. jokovic, n., nikolic, m., begovic, j., jovcic, b., savic, d., topisirovic, l. 2008: a survey of the lactic acid bacteria isolated from serbian artisanal dairy product kajmak. international journal of food microbiology, 127 (3): 305-311. joković, n., rajković, j., veljović, k., tolinački, m., topisirović, l. 2014: screening of lactic acid bacteria isolated from serbian kajmak for use in starter cultures. biologica nyssana, 5 (1): 37-46. kastner, s., perreten, v., bleuler, h., hugenschmidt, g., lacroix, c., meile, l. 2006: antibiotic susceptibility patterns and resistance genes of starter cultures and probiotic bacteria used in food. systematic and applied microbiology, 29 (2): 145-155. katla, a-k., kruse, h., johnsen, g., herikstad, h. 2001: antimicrobial susceptibility of starter culture bacteria used in norwegian dairy products. international journal of food microbiology, 67 (1): 147-152. kojic, m., svircevic, j., banina, a., topisirovic, l. 1991: bacteriocin-producing strain of lactococcus lactis subsp. diacitilactis s50. applied and environmental microbiology, 57 (6): 1835-1837. kojic, m., strahinic, i., topisirovic, l. 2005: proteinase pi and lactococcin a genes are located on the largest plasmid in lactococcus lactis subsp. lactis bv. diacetylactis s50. canadian journal of microbiology, 51 (4): 305-314. landete, jm., de las rivas, b., marcobal, a., muñoz, r. 2007: molecular methods for the detection of biologica nyssana 6 (2)  december 2015: 81-89 rajković, j., joković, n.  probiotic properties and safety assesment… 89 biogenic amine-producing bacteria on foods. international journal of food microbiology, 117 (3): 258-269. lozo, j., vukasinovic, m., strahinic, i., topisirovic, a. 2004: characterization and antimicrobial activity of bacteriocin 217 produced by natural isolate lactobacillus paracasei subsp. paracasei bgbuk2-16. journal of food protection, 67 (12): 2727-2734. maldonado, nc., de ruiz, cs., otero, mc., sesma, f., nader-macías, me. 2012: lactic acid bacteria isolated from young calves–characterization and potential as probiotics. research in veterinary science, 92 (2): 342-349. maragkoudakis, pa., zoumpopoulou, g., miaris, c., kalantzopoulos, g., pot, b., tsakalidou, e. 2006: probiotic potential of lactobacillus strains isolated from dairy products. international dairy journal, 16 (3): 189-199. martín-platero, am., valdivia, e., maqueda, m., martínez-bueno, m. 2009: characterization and safety evaluation of enterococci isolated from spanish goats' milk cheeses. international journal of food microbiology, 132 (1): 24-32. mathara, jm., schillinger, u., guigas, c., franz, c., kutima, pm., mbugua, sk., shin, h-k., holzapfel, wh. 2008: functional characteristics of lactobacillus spp. from traditional maasai fermented milk products in kenya. international journal of food microbiology, 126 (1): 57-64. mohammed, m., el-aziz, ha., omran, n., anwar, s., awad, s., el-soda, m. 2009: rep-pcr characterization and biochemical selection of lactic acid bacteria isolated from the delta area of egypt. international journal of food microbiology, 128 (3): 417-423. nguyen, t., kang, j., lee, m. 2007: characterization of lactobacillus plantarum ph04, a potential probiotic bacterium with cholesterol-lowering effects. international journal of food microbiology, 113 (3): 358-361. pereira, di., mccartney, al., gibson, gr. 2003: an in vitro study of the probiotic potential of a bilesalt-hydrolyzing lactobacillus fermentum strain, and determination of its cholesterol-lowering properties. applied and environmental microbiology, 69 (8): 4743-4752. pringsulaka, o., rueangyotchanthana, k., suwannasai, n., watanapokasin, r., amnueysit, p., sunthornthummas, s., sukkhum, s., sarawaneeyaruk, s., rangsiruji, a. 2015: in vitro screening of lactic acid bacteria for multi-strain probiotics. livestock science, 174 :66-73. saavedra, l., sesma, f., de valdez, gf. 2003: homemade traditional cheeses for the isolation of probiotic enterococcus faecium strains. international journal of food microbiology, 88 (2): 241-245. stiles, me., holzapfel, wh. 1997: lactic acid bacteria of foods and their current taxonomy. international journal of food microbiology, 36 (1): 1-29. tagg, j., mcgiven, a. 1971: assay system for bacteriocins. applied microbiology, 21 (5): 943. temmerman, r., pot, b., huys, g., swings, j. 2003: identification and antibiotic susceptibility of bacterial isolates from probiotic products. international journal of food microbiology, 81 (1): 1-10. todorov, s., botes, m., guigas, c., schillinger, u., wiid, i., wachsman, m., holzapfel, w., dicks, l. 2008: boza, a natural source of probiotic lactic acid bacteria. journal of applied microbiology, 104 (2): 465-477. vujcic, m., topisirovic, l. 1993: molecular analysis of the rolling-circle replicating plasmid pa1 of lactobacillus plantarum a112. applied and environmental microbiology, 59 (1): 274-280. xanthopoulos, v., hatzikamari, m., adamidis, t., tsakalidou, e., tzanetakis, n., litopouloutzanetaki, e. 2000: heterogeneity of lactobacillus plantarum isolates from feta cheese throughout ripening. journal of applied microbiology, 88 (6): 1056-1064. biologica nyssana 6 (2)  december 2015: 81-89 rajković, j., joković, n.  probiotic properties and safety assesment… 90 ćušterevska, r.: armerio rumelicae-potentillion micevski 1978 in south-central balkan with emphasis on galičica mountain vegetation. biologica nyssana, 8 (1) biologica nyssana 8 (1)  september 2017: 00-00 ćušterevska, r.  armerio rumelicae-potentillion micevski 1978… 61 original article received: 09 august 2017 revised: 17 august 2017 accepted: 13 september 2017 armerio rumelicae-potentillion micevski 1978 in south-central balkan with emphasis on galičica mountain vegetation renata ćušterevska institute of biology, faculty of natural sciences and mathematics, university of ss. cyril and methodius, arhimedova 5, mk-1000 skopje, republic of macedonia * e-mail: renatapmf@yahoo.com abstract: ćušterevska, r.: armerio rumelicae-potentillion micevski 1978 in south-central balkan with emphasis on galičica mountain vegetation. biologica nyssana, 8 (1), september 2017: 61-72. the article provides an overview of current knowledge about distribution of perennial grasslands supported by nutrient-poor soils on siliceous bedrocks at elevations characterized by the submediterranean climate of southcentral balkan peninsula (armerio rumelicae-potentillion). we used our own field data from galičica mountain and literature sources to compile a set of vegetation plots that have been stored in a vegetation database, data-mined and analyzed using numerical-analytical tools such as cluster analysis and ordination. five already known associations, were confirmed as well-defined floristic complexes, while the floristic composition of our data enabled recognition of ass. euphrasio-plantaginetum holostei micevski 1994 subass. festucetosum hirtovaginatae subass. nova. key words: balkan peninsula, silicicolous perennial grasslands, syntaxonomy, vegetation classification apstrakt: ćušterevska, r.: armerio rumelicae-potentillion micevski 1978 u južnom delu centralnog balkana sa posebnim osvrtom na vegetaciju planine galičice. biologica nyssana, 8 (1), septembar 2017: 61-72. članak obezbeđuje pregled postojećeg znanja o distribuciji višegodišnjih travnjaka na silikatnim zemljištima siromašnim nutrijentima i nadmorskim visinama okarakterisanim submediteranskom klimom južnocentralnog dela balkanskog poluostrva (armerio rumelicae-potentillion). korišćeni su autorski terenski podaci sa planine galičice i literaturni izvori kako bi se sastavio skup vegetacijskih plotova, koji su zatim sačuvani u vegetacijskoj bazi podataka i analizirani korišćenjem numeričko-analitičkih alata poput klaster analize i ordinacije. pet poznatih asocijacija potvrđene su kao dobro definisani floristički kompleksi, dok je iz florističkog sadržaja naših podataka prepoznata ass. euphrasio-plantaginetum holostei micevski 1994 subass. festucetosum hirtovaginatae subass. nova. ključne reči: balkansko poluostrvo, višegodišnji travnjaci na silikatima, sintaksonomija, klasifikacija vegetacije 8 (1) • september 2017: 61-72 doi: 10.5281/zenodo.964015 biologica nyssana 8 (1)  september 2017: 61-72 ćušterevska, r.  armerio rumelicae-potentillion micevski 1978… 62 introduction in the framework of a broader research of the grassland vegetation on galičica mountain, among other it was investigated the vegetation which is developing to deep decalcified soil on carbonate substrate distributed in the area between 1000-1500 m a.s.l.. whereby there was a need to define its affiliation to higher sintaxonomic categories. communities that develop on silicate surface up to 1000 m a.s.l. in south-central balkan belong to the alliance trifolion cherleri micevski 1970 (festuco-brometea), while communities that develop over this belt belong to the alliance armeriopotentillion micevski 1978. according to the original concept (m i c e v s k i , 1970, 1971, 1978), armerio-potentillion was subordinated to the order astragalo-potentilletalia. in the classification scheme, as adopted by the eurovegchecklist (m u c i n a et al., 2016), the astragalo-potentilletalia (festuco-brometea) comprises only open calcicolous grasslands of the saturejo-thymion. the trifolion cherleri represents submediterranean therophyte communities in the southern balkans, classified in the helianthemetalia guttati br.-bl. in br.-bl. & al. 1940 (helianthemetea guttati rivas goday et rivas-mart. 1963). the armerio-potentillon cannot be classified into either the astragalo-potentilletalia or in the helianthemetalia guttati. this vegetation is perennial grassland supported by nutrient-poor soils on siliceous bedrocks at elevations characterized by the submediterranean climate of macedonia, southern serbia and bulgaria. the following associations have so far been classified in the armerion-potentillon: genisto-agrostietum byzanthinae micevski 1978, koelerio-festucetum stojanovii micevski 1978, diantho-armerietum rumeliacae n. ranđelović 1978, euphrasio-plantaginetum holostei micevski 1994 and plantagini subulatae-agrostietum capillaris pedashenko et al. 2013. similar vegetation was described or reported from southeastern serbia by n. r a n đ e l o v i ć (1978), where ass. dianthoarmerietum rumelicae n. ranđelović 1978 in describing is arranged in alliance chrysopogonodanthonion kojić 1957 (order brometalia erecti br.bl. 1936 class festuco-brometea br.-bl. et tx. ex klika et hadac (1944). later, m i l o s a v l j e v i c et al. (2008), r a n đ e l o v i ć et al. (2008) and v. r a n đ e l o v i ć & z l a t k o v i ć (2010) classified this vegetation in the armerio-potentillon. v. r a n đ e l o v i ć & z l a t k o v i ć (2010) proposed classification of the armerio-potentillon into a new order – the armerietalia rumelicae, however since they failed to designate the holotypus in expressis verbis (as required by the icpn after 01.01.2002), the order name remained invalid. mucina et čarni (d i p i e t r o et al. 2015) agreed with the syntaxonomic concept of the new order suggested by r a n đ e l o v i ć & z l a t k o v i ć (2010) and therefore formally validated the armerietalia rumelicae v. randelovic et n. randelovic in v. randelovic et zlatkovic ex mucina et čarni in d i p i e t r o et al. 2015. material and methods the studied area comprised the central and southern regions of the balkan peninsula, namely territory of southeastern serbia, eastern and western macedonia and central bulgaria (fig. 1). we focused on the collection of vegetation plot data of supratemperate and submediterranean, silicicolous perennial grasslands. original vegetation plot data were collected according to the standard braun-blanquet method of field-plot sampling (b r a u n -b l a n q u e t , 1964). the sampling was done in optimal phenological period of the year – from the second half of june to mid-july during 2009 and 2010 on the galičica mountain, were investigated perennial grasslands supported by nutrient-poor soils which usually occur on silicate substrate and into this case on deep soil where carbonates are washed. the size of the sampled plots was 100 m². the choice of this sample size was motivated by the relatively homogenous vegetation at this scale and the preponderance of vegetation plot data collected at that scale in the past. fig. 1. locations (relevés numbered with ordinal numbers) of the studied area 6 3 table 1. association euphrasio-plantaginetum holostei micevski 1994 subass. festucetosum hirtovaginatae subass. nova (e-ph-fh) (g-ab genisto-agrostidetum byzanthinae; k-fs koelerio-festucetum stojanovii; e-ph euphrasio-plantaginetum holostei (bistra mt.); ps-ac plantagini subulatae-agrostietum capillaris; d-ar diantho-armerietum rumelicae) number of releve 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 e -p h -f h g -a b k -f s e -p h p sa c d -a r euphrasia pectinata + + + + 1 . 1 1 1 1 1 1 + 1 1 1 1 + + 1 + 1 1 + . + + + 1 1 1 + v v ii v plantago holosteum . . . . 1 . . 1 1 . 1 4 3 4 3 3 3 + 1 1 + 1 . . . . . 1 1 1 1 + iv v v v arenaria serpyllifolia . . + . . 1 + . . . . + . . . . . . . . . . . + . . . . . . . . i iv i subass. festucetosum hirtovaginatae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . achillea seidlii 1 + + 1 + + 1 2 1 + 1 + 1 + + + + 1 + 1 + 1 + + + 1 1 . . . . . v centaurea deusta . . . . . + + 2 1 1 1 1 2 1 + + + + 1 1 1 1 1 + + + . 1 1 1 . + iv hypericum barbatum 1 1 + 1 1 + + 1 + + + . + + . . . 1 + 1 + . + . + + 1 + . + + + iv i verbascum speciosum . . . . . . . + + 1 1 + + + 1 + + + + + + + + + + + + . . . . . iv festuca hirtovaginata . . . . 1 . . . . 1 2 + 2 2 1 2 + 4 5 4 3 4 4 4 4 4 1 . . . . . iii bupleurum falcatum subsp. cernuum + . . + . . . . . . . . . . . . . + 1 . 1 + 1 + 1 + + 1 2 2 1 1 iii armerio-potentillion agrostis castellana . . . . . . . 2 1 4 3 . + + + + + + + 1 + . 1 . . + 1 1 1 + + + iv v i leontodon hispidus + + + + . . + 1 1 1 + + + . . . . + . + + . . . . . 1 1 1 1 1 + iv iv i festuca valesiaca 3 4 4 3 4 4 4 . . . . . . . . . . . . . . . . . . . . 1 1 1 + . ii iii iv helianthemum nummularium . . . . . . . . . . . . . . . . . . . . . . . . . . + + . + . + i iii v i iii veronica austriaca subsp. austriaca . + . . . + . 1 + . + . . . . . . . . . . . . . . . . . . . . . i iii i armerietalia rumelicae , stipo giganteae-agrostietea castellanae anthoxanthum odoratum 2 1 + + 1 + 1 1 1 1 + 1 + 1 + 1 + + + 1 + + + + + + 1 1 1 1 + . v iv iii iv iii i plantago lanceolata 1 1 + + + + + 1 1 + + . + + + + + 1 . + . + + + . . + + + + + . v v v v ii v cruciata pedemontana 1 1 + + + 1 + . + . . . . . . . . 1 + 1 + + 1 1 + 1 1 . . . . . iii i i i poa bulbosa . . . . . . . + 1 1 1 1 1 1 1 1 + . . . . + + + + . . . . . . . iii v iii v ii trifolium campestre . . + . 2 2 + + 1 1 1 1 + + + + + . . . . . . . . . . . . . + . iii v i iii iii i dianthus deltoides 1 + + + . . . . . . . . . . . . . + . . . + . . . . 1 1 1 + . . ii i trifolium repens + 2 + + 2 . . + + . . . . . . . . . . . . . . . . . . + . . . . ii iv v iv ii iv veronica arvensis + . + . 2 + + . . . . . . + + + + . . . + . . + . . . . . . . . ii iii ii i i cynosurus echinatus + + + + + . . . + . . + . . . . . . . . . . . . . . . . . . . . ii ii anthemis cretica subsp. carpatica . . . . . . . 1 + . . + + + . + . . . . . . . . . . . . . . . . i arabidopsis thaliana . . . . . . . . . . . 1 . . . . . . . . . . . . . . . . . . . . i erodium cicutarium . . + + . . . . . . . . . . . . . . . . . . . . . . . . . . . . i filago arvensis . . . . . . . . . . . . . . . . . . . . + . . . . . . . . . . . i filago minima . . + + 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . i myosotis stricta . . . . . . . . . . . . . . . . 1 . . . + . . . . . . . . . . . i i i potentilla pedata . . . . . . . . . . . . . . . . . . . . . . . . . . . + 1 + + + i b i o l o g i c a n y s s a n a 8 (1 )  s e p te m b e r 2 0 1 7 : 6 1 -7 2 ć u š te re v s k a , r .  a rm e rio ru m e lic a e -p o te n tillio n m ic e v s k i 1 9 7 8 … 6 4 potentilla recta subsp. recta . . . . . . . . . . . . . . . . . + . . . . . . . . . . . . . . i i scleranthus annuus subsp. annuus . . + + . . . . . . . . . . . . . . . . . . . + . . . . . . . . i sedum urvillei . . + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i i trifolium arvense . . + . . . . . . . . . . + + + . . . . 1 . . . . . . . . . . . i iii iii vicia lathyroides . . + + . + . . . . . . . . . . . + . . . . . . . . . . . . . . i rumex acetosella + . . . . . . + + . . . . + . . . . . . . . . . . . . . . . + . i iv ii v iii scabiosa triniifolia . . . . . . . . + . . . + . . . . . . . . . . . . . . . . . . . i i i iv viola tricolor subsp. macedonica . . . . . . . . . . . . . . . . . . . . . . . . . . + . . . . . i iv other species dianthus cruentus 1 + + 1 1 2 2 1 2 1 1 + 2 1 1 1 1 + 1 + 1 + + + + + 1 + + 1 + + v ii iv galium verum 1 2 1 1 + + 1 1 1 + + + + 1 + 1 + 1 1 1 1 1 + + + + 1 1 + + 1 1 v ii iv ii iii pilosella officinarum 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 + 1 + + 1 1 1 1 1 1 v v v thymus sibthorpii 2 . 1 + 1 1 1 1 2 1 1 + + + 2 2 2 1 1 1 1 1 1 1 1 + 1 1 1 1 1 1 v v campanula patula subsp. patula 1 1 + 1 . . 1 1 1 1 1 + 1 1 + + + + + + + + 1 1 1 1 1 1 + 1 + + v iii v rhinanthus minor 1 + + 1 + . + . . . 1 + . + + + + + . 1 1 + 1 + . + + 1 . 1 + + iv v potentilla detommasii 1 1 1 + 1 1 1 1 + . 1 + + + . 1 + 1 1 + 1 + + + + + + 1 . . . . v ii stellaria graminea + + + + + + + . . . + . . . . . . + . . . + + . . . 1 1 1 + + + iii iii v trifolium pratense . + . . + . + . + . + + . . . . . + + + + . . . . . + + + + + . iii ii i i v cerastium brachypetalum subsp. roeseri 1 + 1 + + 1 1 + + . . . . . . . . . . . . . . + + . 1 + + . . . iii silene sendtneri subsp. balcanica . . . . . . . . . + + + . . + + + + . + + + + + . 1 + . . + + . iii potentilla arenaria + + + + + . . . . . . . + + . . . . . + . + 1 + . + . . . . . . ii achillea chrysocoma . . + + + . + . + . + + 1 2 1 1 1 . . . . . . . . . . . . . . . ii koeleria splendens . . . . 1 1 + 1 1 . 1 + + + + + + . . . . . . . . . . . . . . . ii genista tinctoria . . . . . . . 1 + . 1 1 + . . . 1 + . . . . + . . . 1 1 . + . + ii i stipa balcanica . . . . . . . . . . . . . . . . . . + . 1 + + . + 1 4 4 3 4 4 4 ii dichoropetalum oligophyllum . . . . . . . . + . . + . . . . . 1 . . . . . + 1 + + 1 1 + . + ii ii i lotus tenuis . . . . . . . . . . . . . + 1 1 + + . + + . . + . . . 1 + + . . ii v ii asphodelus albus . . . . . . . . . . . . . . . . . + . . . + + + + + 1 + + + + . ii cirsium eriophorum + + + + . . + . . . . . . . . . . . + . . . . + + + . + . . . . ii primulla veris subsp. columnae + + + + . . . + . . . . . . . . . . . . . . . . . . . + . + + + ii i noccaea praecox . + + + . . . . . . + + + . . . + . . . . . . . . . 1 . + . . . ii i euphorbia verrucosa . . . . . . . . . . . . . . . . . . . + . . . . 1 1 2 1 1 1 1 + ii secale strictum + . + + . . . . . . . . . . . . + . . . . + + . + 1 . . . . . . ii scorzoneroides cichoriacea . + + + 1 + + . . . . . . . . . . + . . + . . . . . . . . . . . ii silene viscaria . . . . + . . . . 2 1 . . + + 1 1 . . . . . . . . . . . . . + . ii i achillea setacea . . . . . . . . . . . . . . . . . . . + . + . . . . + 1 + + 1 + ii v v carduus tmoleus . . . . . . . 1 + . . . . + . . . . . + + . . . . + . . . . . 1 ii ii galium verticillatum . . . . . . . . + . . + + + + + 1 . . . . . . . . . . . . . . . ii knautia arvensis . . . . . . . . . . . + . . . . . . . . . . . . 1 + 1 + . . + 1 ii ii iii verbascum longifolium subsp. pannosum + . . + . . . . . . . . . . . . . . . . . . . . . . . + + + . + i ii euphorbia myrsinites . . + . + + + . . . . . . . . . . . . . + . + . . . . . . . . . i i b i o l o g i c a n y s s a n a 8 (1 )  s e p te m b e r 2 0 1 7 : 6 1 -7 2 ć u š te re v s k a , r .  a rm e rio ru m e lic a e -p o te n tillio n m ic e v s k i 1 9 7 8 … 6 5 carex halleriana . . . . . . . . . . . + 1 + 1 + + . . . . . . . . . . . . . . . i seseli peucedanoides + + + 1 . . + . . . . . . . . . . . . . . . . . . . . . . . . . i ii trifolium phleoides . + + + 1 . 1 . . . . . . . . . . . . . . . . . . . . . . . . . i muscari racemosum . + + + . . . . + . . . . . . . . . . . . . . . . . + . . . . . i ranunculus psilostachys . . + + + + + . . . . . . . . . . . . . . . . . . . . . . . . . i pimpinella tragium subsp. lithophila . . . . . . . 1 1 . . + + . . . + . . . . . . . . . . . . . . . i trisetum flavescens . . . . . . . 1 1 . . . . . . . . . . . . . . 1 + . + . . . . . i i iv i thesium arvense . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 + 1 + i i alyssum strigosum . . + + 1 + . . . . . . . . . . . . . . . . . . . . . . . . . . i festuca rubra subsp. rubra + + . . . . . + 1 . . . . . . . . . . . . . . . . . . . . . . . i erysimum kummerlei + . + + . . . . . . . . . . . . . . . . + . . . . . . . . . . . i i cruciata laevipes + . . . 1 . . . . . . . . . . . . + . . . . . . . . + . . . . . i malva moschata + . . . . . . . . . . . . . . . . . . . . . + . . . + . . . + . i i poa angustifolia + . . . . . . . . . . . . . . . . . . . . . . + 1 1 . . . . . . i i sanguisorba minor . + . . + + + . . . . . . . . . . . . . . . . . . . . . . . . . i iv i ii ii iii galium divaricatum . . + + 1 . + . . . . . . . . . . . . . . . . . . . . . . . . . i iv ii iii bromus squarrosus . . + + + . + . . . . . . . . . . . . . . . . . . . . . . . . . i trifolium alpestre . . . . . 1 . . . . . . . . . . . . . . + . . . . . . . . + + . i ii iii ii ii trifolium velenovskyi . . . . . . . . . + . . . . . . . + . . + . 1 . . . . . . . . . i iii i v briza minor . . . . . . . . . . . . . . . . . . . . . . . . . . . + + + + . i juniperus communis . . . . . . . . . . . . . . . . . . . . . . . . . . . . + + + + i iv veronica chamaedrys + 1 . . + . . . . . . . . . . . . . . . . . . . . . . . . . . . i i i i clinopodium alpinum subsp. meridionale + . + . + . . . . . . . . . . . . . . . . . . . . . . . . . . . i i teucrium chamaedrys . . 1 + 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . i ii i i phleum montanum . . . + 1 + . . . . . . . . . . . . . . . . . . . . . . . . . . i eryngium amethistinum . . . . 1 1 + . . . . . . . . . . . . . . . . . . . . . . . . . i i ranunculus oreophilus . . . . . . . 1 1 + . . . . . . . . . . . . . . . . . . . . . . i asperula aristata subsp. scabra . . . . . . . . 1 . . . . . . . . + . . + . . . . . . . . . . . i iii ornithogalum orthophyllum subsp. kochii . . . . . . . . . . . + + . . . + . . . . . . . . . . . . . . . i i stachys germanica subsp. heldreichi . . . . . . . . . . . . . . . . . . . + . + . . . + . . . . . . i polygala nicaeensis . . . . . . . . . . . . . . . . . . . + . . . . . + + . . . . . i ii polygala vulgaris . . . . . . . . . . . . . . . . . . . + . . . . . . . + . . + . i i ii iv helleborus odorus + . . . + . . . . . . . . . . . . . . . . . . . . . . . . . . . i i schedonorus pratensis + . . . . . . . . . . . . . . . . . . . . . . . . + . . . . . . i medicago lupulina . . + + . . . . . . . . . . . . . . . . . . . . . . . . . . . . i geum montanum . . . . . . . . + . . . . . . . . . . . . . . . + . . . . . . . i carthamus lanatus . . . . . . . . . + . . . . . . . . . . . . . . . . . . . + . . i thymus longicaulis . . . . . . . . . . . . . . . . . . . + + . . . . . . . . . . . i v iv ii v elytrigia intermedia . . . . . . . . . . . . . . . . . . . + . . . . . + . . . . . . i digitalis ferruginea . . . . . . . . . . . . . . . . . . . . . . + . . . + . . . . . i dactylis glomerata + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i b i o l o g i c a n y s s a n a 8 (1 )  s e p te m b e r 2 0 1 7 : 6 1 -7 2 ć u š te re v s k a , r .  a rm e rio ru m e lic a e -p o te n tillio n m ic e v s k i 1 9 7 8 … 6 6 bromopsis cappadocica . . . . 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . i orlaya daucorlaya . . . . + . . . . . . . . . . . . . . . . . . . . . . . . . . . i xeranthemum annuum . . . . + . . . . . . . . . . . . . . . . . . . . . . . . . . . i scandix australis subsp. australis . . . . . + . . . . . . . . . . . . . . . . . . . . . . . . . . i alyssum repens subsp. trychostachyum . . . . . . + . . . . . . . . . . . . . . . . . . . . . . . . . i i orlaya daucoides . . . . . . . . + . . . . . . . . . . . . . . . . . . . . . . . i prunella laciniata . . . . . . . . + . . . . . . . . . . . . . . . . . . . . . . . i i ii odontites luteus . . . . . . . . . . . . . . 1 . . . . . . . . . . . . . . . . . i silene nemoralis . . . . . . . . . . . . . . . + . . . . . . . . . . . . . . . . i cytisus austriacus subsp. heuffelii . . . . . . . . . . . . . . . + . . . . . . . . . . . . . . . . i clinopodium alpinum subsp. hungaricum . . . . . . . . . . . . . . . . . . . + . . . . . . . . . . . . i trifolium tenuifolium . . . . . . . . . . . . . . . . . . . . + . . . . . . . . . . . i delphinium fissum . . . . . . . . . . . . . . . . . . . . . + . . . . . . . . . . i silene bupleuroides subsp. staticifolia . . . . . . . . . . . . . . . . . . . . . + . . . . . . . . . . i gentiana cruciata . . . . . . . . . . . . . . . . . . . . . . . + . . . . . . . . i allium rotundum . . . . . . . . . . . . . . . . . . . . . . . . + . . . . . . . i eryngium campestre . . . . . . . . . . . . . . . . . . . . . . . . + . . . . . . . i v i ii lathyrus nissolia . . . . . . . . . . . . . . . . . . . . . . . . + . . . . . . . i rumex alpestris . . . . . . . . . . . . . . . . . . . . . . . . . . + . . . . . i i brachypodium pinnatum . . . . . . . . . . . . . . . . . . . . . . . . . . . + . . . . i stachys officinalis . . . . . . . . . . . . . . . . . . . . . . . . . . . + . . . . i veronica orbelica . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + . i b i o l o g i c a n y s s a n a 8 (1 )  s e p te m b e r 2 0 1 7 : 6 1 -7 2 ć u š te re v s k a , r .  a rm e rio ru m e lic a e -p o te n tillio n m ic e v s k i 1 9 7 8 … biologica nyssana 8 (1)  september 2017: 61-72 ćušterevska, r.  armerio rumelicae-potentillion micevski 1978… 67 the plots were located within targeted remnant patches of dry grasslands in such a way that edge effects (close forest edge, roads and settlements) were excluded. the sampled 32 relevés and the rest 108 from literature sources were entered into turboveg (h e n n e k e n s & s c h a m i n é e , 2001). classification was made using the pc-ord 4 package (m c c u n e & m e f f o r d , 1999) integrated in juice software, using flexible beta (clustering) and relative euclidean distance (known also as chord distance) as the measure of resemblance among relevés. the table sorting based on this clustering was then performed in juice 7.0 (t i c h ý & h o l t , 2006). diagnostic species of each cluster (interpreted as associations) were defined in juice by calculating the fidelity of each species to each group using the phi coefficient as the fidelity measure (c h y t r ý et al., 2002). the threshold of the phi value was selected at level 0.55. diagnostic species were identified by juice using the phi coefficient greater than 0.6. in order to assist ecological interpretation of the ordinated patterns, average bioindicator values (p i g n a t t i , 2005) for the relevés were calculated based on presence-absence data and overlaid as supplementary environmental data. we used r a u n k i a e r ’ s (1934) system of life forms and we determined the chorological spectra using data of g a j i ć (1980) and p i g n a t t i (2005). the chorological spectrum is presented as percentages of each group of species within the entire species composition. in the analytic table (tab. 1) are presented the results of the cluster analysis and subsequent juice tabular sorting. the syntaxonomic affiliation of taxa other than diagnostic ones was decided upon using expert knowledge and the database of the european vegetation checklist (m u c i n a et al., 2016). the nomenclature follows in principle the flora europaea (t u t i n et al., 1964-1993) however it was adjusted using the med-check list (g r e u t e r et al., 19841989; g r e u t e r et al., 2008) and new taxonomic and nomenclatural findings as featured in the euro+med database (www.emplantbase.org). some taxonomic concepts follow regional floras (h a y e k , 1927, 1931, 1933; j o r d a n o v , 1963-1979; j o s i f o v i ć , 1970-1986; v e l c h e v , 1982-1989; m i c e v s k i , 1985-2005; k o z u h a r o v , 1995; m a t e v s k i , 2010). formation of the names of the new syntaxa follows the international code of phytosociological nomenclature (w e b e r et al., 2000). results and discussion the optimal level of the clustering was determined by the optimclass 1 method. the cluster analysis suggested six well separated clusters (fig. 2). since they show a high degree of floristic (and geographic) integrity, five already described associations were confirmed. while, after the made extensive comparative analysis, relevés of cluster 5, showed the greatest syntaxonomic affinity with ass. euphrasioplantaginetum holostei because of the presence of its characteristic species. fig. 2. graphical representation of the classification results of the armerio rumelicae-potentillion vegetation (flexible beta clustering method, relative euclidean distance). legende: cluster 1: genisto-agrostietum byzanthinae, cluster 2: koelerio-festucetum stojanovii, cluster 3: plantagini subulatae-agrostietum capillaris, cluster 4: diantho-armerietum rumeliacae, cluster 5: euphrasio-plantaginetum holostei subass. festucetosum hirtovaginatae subass. nova., cluster 6: euphrasio-plantaginetum holostei biologica nyssana 8 (1)  september 2017: 61-72 ćušterevska, r.  armerio rumelicae-potentillion micevski 1978… 68 the association euphrasio-plantaginetum holostei has been described and for the first time registered on bistra mountain, developing exclusively on silicate substrate with a thin layer of soil, so that in some places subsoil breaks out on the surface. this association on bistra mountain is differentiated into 3 subassociations subass. typicum, subass. festucetosum nigrescentis (over the village galičnik) and subass. agrostidetosum capilaris (from kičevo side of the mountain bistra, above knežino). in the floristic composition of the vegetation relevés (cluster 5, fig. 2) made in our research on the move asan gjura-dofa, all three characteristic species of the association were registered: euphrasia pectinata, arenaria serpyllifolia and plantago holosteum. none of diagnostic taxa of existing subassociations of the ass. euphrasio-plantaginetum holostei, was registered. but therefore, with analysis "analysis of constancy columns in synoptic table" of the program package juice, were separated taxa over which the community that is developing on the galičica mountain, differs from existing community euphrasioplantaginetum holostei. diagnostic species: achillea seidlii, bupleurum falcatum subsp. cernuum, centaurea deusta, festuca hirtovaginata, hypericum barbatum, rhinanthus minor, verbascum speciosum constant species: anthoxanthum odoratum, euphrasia pectinata, galium verum, pilosella officinarum, plantago lanceolata, thymus sibthorpii dominant species: agrostis castellana, festuca hirtovaginata, festuca valesiaca, plantago holosteum, stipa balcanica because of this, the need for extraction of new subassociation festucetosum hirtovaginatae subass. nova was imposed (holotypus hoc loco: tab. 1/23) (tab. 1). subassociation is named by the species festuca hirtovaginata, which has a dominant presence and define its physiognomy. subass. festucetosum hirtovaginatae, unlike the other subassociations of ass. euphrasio-plantaginetum holostei develope on a limestone substrate, on deep soil, at the move asan gjura-dofa (fig. 3). diagnostic species of subassociation are: achillea seidlii, centaurea deusta, hypericum barbatum, rhinanthus minor, verbascum speciosum, festuca hirtovaginata and bupleurum falcatum subsp. cernuum. as far as the ecological characteristics of this community, subass. festucetosum hirtovaginatae, develops at an altitude of 1455-1500 m, in habitats with very low inclination (most vegetation relevés are made on a completely flat surface, almost without inclination) (app. 1). from the spectrum of life forms (fig. 4), it may be noted that the percentage of hemicryptophytes is 66%, which is due to the dominant presence of representatives of fam. poaceae: agrostis castellana, festuca hirtovaginata, festuca valesiaca, stipa balcanica and others. from the spectrum of floral elements it can be seen that the most common is the eurasian floral element (46%) (fig. 5), while the balkan floral elements are second group by representation (19%), fig. 3. ass. euphrasio-plantaginetum holostei micevski 1994 subass. festucetosum hirtovaginatae subass. nova – asan gjura (galičica mt.) fig. 4. ass. euphrasio-plantaginetum holostei micevski 1994. subass. festucetosum hirtovaginatae subass. nova. – spectrum of life forms biologica nyssana 8 (1)  september 2017: 61-72 ćušterevska, r.  armerio rumelicae-potentillion micevski 1978… 69 with the most dominant balkan-apennine (35%) (fig. 6). using the data from "worldclim", we received the following bioclimatic variables for the researched community: bio1 = annual mean temperature (6,6), bio2 = mean diurnal range (mean of monthly (max temp – min temp)) (9,8), bio3 = isothermality (bio2/bio7) (0,34), bio4 = temperature seasonality (standard deviation *100) (62,9), bio5 = max temperature of warmest month (22,1), bio6 = min temperature of coldest month (-6), bio7 = temperature annual range (bio5–bio6) (28.1), bio8 = mean temperature of wettest quarter (2.6), bio9 = mean temperature of driest quarter (14.7), bio10 = mean temperature of warmest quarter (14.7), bio11 = mean temperature of coldest quarter (-1.4) bio12 = annual precipitation (930), bio13 = precipitation of wettest month (117), bio14 = precipitation the driest month (48), bio15 = precipitation seasonality (coefficient of variation) (27), bio16 = precipitation of wettest quarter (311), bio17 = precipitation of driest quarter (149), bio18 = precipitation of warmest quarter (149), bio19 = precipitation of coldest quarter (272); conclusion this paper represents an oveview of the distribution and syntaxonomic position of the supratemperate and submediterranean, silicicolous perennial grasslands south-central balkan, which are covered by the alliances armerio-potentillion micevski 1978. with numerical analyses it has also been shown that own unpublished data from mountain galičica sintaxonomically have proved that they are closest to the ass. euphrasio-plantaginetum holostei micevski 1994. the fact that species festuca hirtovaginata was significantly dominanted in the reaserched stands led us to distinguish a new subassociation festucetosum hirtovaginatae subass. nova which was confirmed with additional ecological analyses. this study complements the picture of south-centarl balkan supratemperate submediterranean silicicolous perennial grasslands with additional data from the republic of macedonia, but there are still areas that are not sufficietly researched. references braun-blanquet, j. 1936: über die trockenrasengesellschaften des festucion vallesiacae in den ostalpen. berichte der schweizerischen botanischen gesellschaft, 46: 169-189. braun-blanquet, j. 1964: pflanzensoziologie: grundzuge der vegetationskunde. 3. neu bearb. aufl. springer-verlag, 866 pp., wien. braun-blanquet, j., r. molinier, wagner h. 1940: prodrome des groupements végétaux. prodromus der pflanzengesellschaften. cisto-lavanduletea (landes siliceuses à cistes et lavandes). 53 pp. montpellier. braun-blanquet, j., tüxen, r. 1943: übersicht der höheren vegetationseinheiten mitteleuropas. fig. 5. ass. euphrasio-plantaginetum holostei micevski 1994. subass. festucetosum hirtovaginatae subass. nova. – spectrum of geo-elements (balk balkan and sub balkan; smed steno mediterranean; emed eurimediterranean; medmt – mediterranean mountainous; eas eurasian; ose orophil-south european; bor boreal; gwd geoelements with wider distribution) fig. 6. ass. euphrasio-plantaginetum holostei micevski 1994. subass. festucetosum hirtovaginatae subass. nova. – percentage representation of balkan geo-elements (phytogeographic spectrum) (balk balkan (sensu stricto); sbalk south balkan; scpind scardo-pindus; balk-apen balkanapennine; sbalk-am south balkan-asia minor) balk 19% smed 2% emed 11% medmt 7% eas 46% ose 6% bor 7% gwd 2% balk 35% sbalk 25% scpnd 6% balkapen 16% sbalk-am 18% biologica nyssana 8 (1)  september 2017: 61-72 ćušterevska, r.  armerio rumelicae-potentillion micevski 1978… 70 communications de la station internat, de géobotanique, 84: 1-11. chytrý, m., tichý l., holt, j., botta-dukát, j. 2002: determination of diagnostic species with statistical fidelity measures. journal of vegetation sciences, 13: 79–90. di pietro r., theurillat j.-p., capelo j., fernándezgonzález f., terzi m., čarni a., mucina l. 2015. nomenclature and syntaxonomic notes on some high-rank syntaxa of the european grassland vegetation. lazaroa, 36: 79-106 gajić, m. 1980: pregled vrsta flore s.r. srbije sa biljno-geografskim oznakama. glasnik šumarskog fakulteta, serie a ”šumarstvo”, 54: 111-141. beograd. greuter, w, burdet, h.m., long, g. (eds.). 2008: med-checklist 2. – optima secretariat, palermo; med-check trust of optima, genève; euro+med plantbase secretariat, berlin. greuter, w., burdet, h.m., long, g. (ed.). 1984– 1989: med-checklist. 1, 3, 4. conservatoire et jardin botanique de la ville de genève, genève. hayek, a. 1927: prodromus florae peninsulae balcanicae. i. repertorium specierum novarum regni vegetabilis 30 (1). verlag des repertoriums, dahlem bei berlin. hayek, a. 1931: prodromus florae peninsulae balcanicae. i. repertorium specierum novarum regni vegetabilis 30 (2). verlag des repertoriums, dahlem bei berlin. hayek, a. 1933: prodromus florae peninsulae balcanicae. i. repertorium specierum novarum regni vegetabilis 30 (3). verlag des repertoriums, dahlem bei berlin. hennekens, s.m., schaminée, j.h.j. 2001: turboveg, comprehensive data base management system for vegetation data. journal of vegetation science 12: 589-591. jordanov, d. (ed.). 1963–1979: flora reipublicae popularis bulgaricae. vols. 1–7. in: aedibus academiae scientarum bulgaricae, serdicae (sofia). josifović, m. (ed.). 1970–1986: flora sr srbije, vol. 1-10. sanu, beograd. klika, j., hadač, e. 1944: rostlinná společenstva střední evropy. (dokončeni.) příroda, 36: 281– 295. kojić, m., 1957: chrysopogono-danthonion calycinae nova sveza iz reda festucetalia valesiacae br. bl. et tx. zbornik radova poljoprivrednog fakulteta zemun, 5 (2): 51-55 kozhuharov, s. (ed.). 1995: flora reipublicae popularis bulgaricae. vol. 10. in aedibus academiae scientarum bulgaricae, serdicae (sofia). matevski, v. (ed.). 2010: flora na republika makedonija 2(1). macedonian academy of sciences and arts, skopje. mccune, b., mefford, m.j. 1999: pc-ord multivariate analysis of ecological data, version 5. mjm software design, glenede beach, oregon, usa. micevski, k. 1970: astragalo-potentilletalia, nov vegetaciski red na brdskite pasišta vo makedonija. prilozi, 2 (2): 15–23. micevski, k. 1978: tipološki istražuvanja na vegetacijata na livadite i pasištata vo maleš i pijanec. macedonian academy of sciences and arts, skopje. micevski, k. (ed.). 1985–2005: flora na republika makedonija. macedonian academy of sciences and arts, skopje. micevski, k. 1971: "stepska" vegetacija vo makedonija. godišen zbornik pmf-biologija, 23: 131–150. micevski, k. 1994: visokoplaninska vegetacija na planinata bistra. bistra 3, macedonian academy of sciences and arts, skopje. milosavljević, v.n., randelović, v.n., zlatković, b., randelović, n.v. 2008: phytocenologic diversity of krajište in southern serbia. natura montenegrina, 7: 193-204. mucina, l., bültmann, h., dierssen, k., theurillat, j.-p., raus, t., čarni, a., šumberová, k., willner, w., dengler, (...), tichý, l. 2016: vegetation of europe: hierarchical floristic classification system of vascular plant, bryophyte, lichen, and algal communities. applied vegetation science, 19: 3–264. pedashenko, h., apostolova, i., boch, s., ganeva, a.; janišová, m., sopotlieva, d., todorova, s., ünal, a., vassilev, k., velev, n., dengler, j. 2013: dry grasslands of nw bulgarian mountains: first insights into diversity, ecology and syntaxonomy. tuexenia, 33: 309 – 346. pignatti, s. 2005: valori di bioindicazione delle piante vascolaridella flora d‘italia. braun blanquetia, 39: 1-97. ranđelović, v.n., zlatković, b.k. 2010: flora and vegetation of vlasina plateau. prirodnomatematički fakultet, niš. ranđelović, v.n., zlatković, b.k., milosavljević, v.n., ranđelović, n.v. 2008: the endemic flora of bosilegrad surroundings (krajište region) in se serbia. phytologia balcanica, 14: 367-375. ranđelović, n. 1978: fitocenološko-ekološke karakteristike brdskih travnjaka jugoistočne srbije. doctorska disertacija, prirodoslovnomatematički fakultet, zagreb: 145 pp. biologica nyssana 8 (1)  september 2017: 61-72 ćušterevska, r.  armerio rumelicae-potentillion micevski 1978… 71 raunkiaer, c, 1934: the life forms of plants and statistical plant geography. oxford, charendon press. rivas goday, s., rivas-martínez, s. 1963: estudio y clasificación de los pastizales españoles. publ. ministerio de agricultura. madrid. tichy l., holt j. 2006: juice program for management analysis and classification of ecological data. program manual. brno 98 pp. http://www.sci.muni.cz/botany/juice/jc06_ man.htm (accessed 11.02.2009). tutin, t.g., burges, d.m., chater, a.o., edmonson, j.r., heywood, v.h., moore, d.m., valentine, d.h., walters, s.m., webb, d.a. (eds.) 1993: flora europaea volume 1: psilotaceae to platanaceae. 2nd ed. cambridge university press. tutin, t.g., heywood, v.h., burges, d.m., valentine, d.h., walters, s.m., webb, d.a. (eds.) 1972: flora europaea volume 3: diapensiaceae to myoporaceae. cambridge university press. tutin, t.g., heywood, v.h., burges, d.m., valentine, d.h., walters, s.m., webb, d.a. (eds.) 1976: flora europaea volume 4: plantaginaceae to compositae (and rubiaceae). – cambridge: cambridge university press. tutin, t.g., heywood, v.h., burges, d.m., valentine, d.h., walters, s.m., webb, d.a. (eds.) 1980: flora europaea volume 5: alismataceae to orchidaceae (monocotyledones). cambridge university press. tutin, t.g., heywood, v.h., burges, n.a., moore, d.m., valentine, d.h., walters, s.m., webb, d.a. (eds.) 1968: flora europaea volume 2: rosaceae to umbelliferae. cambridge university press. velchev, v. (ed.). 1982–1989: flora reipublicae popularis bulgaricae. vols. 8 & 9. in aedibus academiae scientarum bulgaricae, serdicae (sofia). venables, w.n., ripley, b.d. 2000: s programming. new york: springer-verlag. [v, 2, 12, 20, 30, 41, 56, 60, 67, 68] weber, h.e., moravec, j., theurillat, j.p. 2000: international code of phytosociological nomenclature. 3rd edition. journal of vegetation sciences, 11: 739-768. appendix review of metadata. running number, locality, latitude, longitude, sampling date, area, altitude (m), aspect, inclination (%) and cover for all reléves shown in the table 1. 1. from asan gjura to krstec, 41,028498, 20,860192, 25.06.2009, 100, 1498, s, 5, 100; 2. from asan gjura to krstec, 41,028141, 20,863512, 25.06.2009, 100, 1480, nw, 3, 100; 3. from asan gjura to krstec, 41,027695, 20,860687, 25.06.2009, 100, 1480, n, 8, 96; 4. from asan gjura to krstec, 41,027817, 20,861515, 25.06.2009, 100, 1492, nw, 3, 100; 5. from asan gjura to krstec, 41,028471, 20,860517, 25.06.2009, 100, 1499, s, 10, 98; 6. from asan gjura to krstec, 41,025218, 20,866777, 25.06.2009, 100, 1483, sw, 5, 100; 7. from asan gjura to krstec, 41,024437, 20,866423, 25.06.2009, 100, 1467, ne, 3, 100; 8. from sheepfold below krstec to asan gjura, 41,068493, 20,859072, 25.07.2009, 100, 1468, w, 5, 100; 9. from sheepfold below krstec to asan gjura, 41,068493, 20,859072, 25.07.2009, 100, 1462, w, 5, 100; 10. from sheepfold below krstec to asan gjura, 41,067209, 20,858302, 25.07.2009, 100, 1460, 0, 0, 100; 11. from sheepfold below krstec to asan gjura, 41,065362, 20,857134, 25.07.2009, 100, 1455, 0, 0, 100; 12. from sheepfold below krstec to asan gjura, 41,064284, 20,856815, 25.07.2009, 100, 1461, 0, 0, 100; 13. from sheepfold below krstec to asan gjura, 41,062178, 20,856931, 25.07.2009, 100, 1457, 0, 0, 100; 14. from sheepfold below krstec to asan gjura, 41,056869, 20,856586, 25.07.2009, 100, 1455, 0, 0, 100; 15. from sheepfold below krstec to asan gjura, 41,056341, 20,856325, 25.07.2009, 100, 1451, 0, 0, 100; 16. from sheepfold below krstec to asan gjura, 41,053895, 20,855478, 25.07.2009, 100, 1458, 0, 0, 100; 17. from sheepfold below krstec to asan gjura, 41,051082, 20,855352, 25.07.2009, 100, 1460, 0, 0, 100; 18. from asan gjura to dofa, 41,031984, 20,863158, 26.07.2009, 100, 1483, se, 5, 100; 19. from asan gjura to dofa, 41,032361, 20,854084, 26.07.2009, 100, 1480, 0, 0, 100; 20. from asan gjura to dofa, 41,032862, 20,859263, 26.07.2009, 100, 1481, se, 2, 100; 21. from asan gjura to dofa, 41,032597, 20,855601, 26.07.2009, 100, 1481, 0, 0, 100; 22. from asan gjura to dofa, 41,032404, 20,851892, 26.07.2009, 100, 1475, 0, 0, 100; 23. from asan gjura to dofa, 41,033543, 20,848666, 26.07.2009, 100, 1495, 0, 0, 100; 24. from asan gjura to dofa, 41,0366, 20,847866, 26.07.2009, 100, 1481, 0, 0, 100; 25. from asan gjura to dofa, 41,041671, 20,854218, 26.07.2009, 100, 1473, 0, 0, biologica nyssana 8 (1)  september 2017: 61-72 ćušterevska, r.  armerio rumelicae-potentillion micevski 1978… 72 100; 26. from asan gjura to dofa, 41,041567, 20,855939, 26.07.2009, 100, 1468, 0, 0, 100; 27. from asan gjura to dofa, 41,039669, 20,860384, 26.07.2009, 100, 1484, e, 2, 100; 28. from asan gjura to dofa, 41,035231, 20,856573, 29.07.2009, 100, 1480, ne, 3, 100; 29. from asan gjura to dofa, 41,035491, 20,856483, 29.07.2009, 100, 1469, ne, 3, 100; 30. from asan gjura to dofa, 41,036381, 20,856884, 29.07.2009, 100, 1472, e, 8, 100; 31. from asan gjura to dofa, 41,037793, 20,857846, 29.07.2009, 100, 1467, ne, 5, 100; 32. from asan gjura to dofa, 41,038896, 20,856981, 29.07.2009, 100, 1462, ne, 3, 100; jakšić, p.: a contribution to the knowledge of the lepidoptera fauna of eastern serbia. biologica nyssana, 8 (1), september 2017 biologica nyssana 8 (1)  september 2017: 113-122 jakšić, p.  a contribution to the knowledge of the lepidoptera… 113 original article received: 02 july 2017 revised: 18 august 2017 accepted: 30 august 2017 a contribution to the knowledge of the lepidoptera fauna of eastern serbia predrag jakšić university of niš, faculty of science and mathematics, department of biology and ecology, višegradska 33, niš, serbia * e-mail: jaksicpredrag@gmail.com abstract: jakšić, p.: a contribution to the knowledge of the lepidoptera fauna of eastern serbia. biologica nyssana, 8 (1), september 2017: 113-122. the faunistic review of lepidoptera, heterocera in the eastern serbia area, with ecological characteristics and zoogeographical distribution is presented. author gives details of lepidoptera species collected in the years 1998, 2014 and 2016 respectively. in total, 44 species are registered, of which there were 1tineidae, 1 limacodidae, 1 cossidae, 1 pterophoridae, 1 thyrididae, 2 lasiocampidae, 1 sphingidae, 1 drepanidae, 13 geometridae, 4 notodontidae, 8 erebidae, 8 noctuiodae and 1 nolidae species. some of them show narrow distribution, of which 15 are recorded for the second, or third, time in serbia. illustration of some habitats, adults and genitalia slides are given. key words: lepidoptera, heterocera, eastern serbia apstrakt: jakšić, p.: prilog poznavanju faune lepidoptera u istočnoj srbiji. biologica nyssana, 8 (1), septembar 2017: 113-122. prikazani su rezultati faunističkih istraživanja lepidoptera, heterocera u istočnoj srbiji, sa njihovim ekološkim i zoogeografskim osobenostima. detalji se odnose na vrste lepidoptera sakupljenih tokom 1998., 2014. i 2016. godine. ukupno je prikazano prisustvo 44 vrste, među kojima su brojčano po familijama predstavnici 1tineidae, 1 limacodidae, 1 cossidae, 1 pterophoridae, 1 thyrididae, 2 lasiocampidae, 1 sphingidae, 1 drepanidae, 13 geometridae, 4 notodontidae, 8 erebidae, 8 noctuiodae i 1 nolidae vrsta. neke od utvrđenih vrsta pokazuju usku distribuciju, a 15 njih pronađeno je po drugi ili treći put u srbiji. date su i ilustracije staništa, pojedinih utvrđenih vrsta in situ i genitalne armature mužjaka. ključne reči: lepidoptera, heterocera, istočna srbija introduction the basin of nišava river in eastern serbia, with the associated mountain ranges, is very interesting biogeographically. on the other site, basin is still insufficiently explored when it comes to its lepidoptera fauna. the first field research from this area were compiled by m. hilf in 1894-1896. it is the area between niš and bela palanka (then called akpalanka). hilf collected a total of 109 species of 8 (1) • september 2017: 113-122 doi: 10.5281/zenodo.964535 biologica nyssana 8 (1)  september 2017: 113-122 jakšić, p.  a contribution to the knowledge of the lepidoptera… 114 lepidoptera, of which 58 species are moths and 51 are butterflies. the material was processed and published by r e b e l (1903, 1904). the following contribution to the knowledge of the fauna was given than by the hungarian natural history museum's preparator józsef uhl who collected material in the vicinity of niš and pirot. the results were published in 1903. t o d o r o w a & p e t k o f f (1915) published faunal studies of dimitrovgrad (then named caribrod) and its environment. r a d o s a v l j e v i ć (1924) gives a contribution to the knowledge of harmful lepidoptera species in this area. after the world war ii v a s i ć (1948) published a contribution of gradation (outbreaks) of harmful moths. the works of those authors can be considered as the pioneer contributions. material and methods specimens were collected with butterfly net and light trap, using “philips” mercury vapor lamp, 125 w. the localities and coordinates where the lepidoptera were collected were obtained using garmin-trex vista gps device. the material was sampled on the following localities: pirot, vidlič mt., hotel “stara”, 1040 m, fn38, 43° 10' 46" (n), 22° 41' 30" (e); pirot, dojkinci, ponor, 1550 m, fn49, 43° 15' 24" (n), 22° 48' 34" (e) (fig. 1); pirot, vidlič mt., crni vrh, 1116 m, fn38, 43° 11' 08" (n), 22° 38' 58" (e); pirot, vidlič, kitka – čuka, 957 m, fn38, 43° 11' 16" (n), 22° 38' 35" (e) (fig. 2); suva planina, bojanine vode, 850 m, en88, 43° 13' 19" (n), 22° 06' 39" (e); suva planina, devojački grob, 1317 m, en88, 43° 11' 59" (n), 22° 08' 14" (e) and knjaževac, staro selo village, dvoja vrata cave, 451 m, fp13, 43° 40' 34" (n), 22° 22' 55" (e). fig. 1. stara planina mt. – ponor near dojkinci the photos in situ of specimens were taken using nikon camera with af-s micro nikkor lens. after setting, we determined the specimens by the wing-patterns. male genitalia were examined for species that cannot be reliably identified based exclusively on wing morphology. the preparations were carried out following the well-known standard procedure: maceration by boiling in potash, dissecting and cleaning, clearing in xylolum and mounting in canada balsam. the photographs of the genital structure were taken using a "leica dm 1000" microscope with a "camera leica dfc 290". all the material (specimens and genitalia slides) is deposited in the author's collection. fig. 2. vidlič mt. near pirot the taxonomic order is done according to n i e u k e r k e n et al. (2011) and f i b i g e r et al. (2012). nomenclature and id number before the species follows k a r s h o l t & r a z o w s k i (1996) ecological preferences and biotopes were done according to c a r l et al. (2005). estimation by analogy for missing species was done by author. fieldwork in protected areas was realized in agreement with permits provided by the ministry of environment, mining and spatial planning, republic of serbia, no. 353-01-1559-2011-03, dated from 8. 06. 2011; no. 353-01-1070/2012-03, dated from 12. 06. 2012.; no. 353-01-916/2014-08, dated from 29.05.2014.; no. 353-01-356/2015-17, dated from 27. 04. 2015 and no. 353-01-389/2016-17, dated from 08. 04. 2016. results ordo lepidoptera fam. tineidae 724. euplocamus anthracinalis (scopoli, 1763) material examined: pirot, vidlič mt., crni vrh, 1130 m, 30. v 2016., 1 male. (fig. 3). this species is well known in serbia (j a k š i ć , 2016).the larva is mycophagous, inhabit rotting wood, which they eat bracket fungi growing on it and dead wood. habitat: e. anthracinalis inhabit humid woodland, as well as biologica nyssana 8 (1)  september 2017: 113-122 jakšić, p.  a contribution to the knowledge of the lepidoptera… 115 thermophilous forests of fagus, quercus and crataegus. fig. 3. euplocamus anthracinalis (scopoli, 1763), pirot, vidlič mt., crni vrh, 1130 m, 30. v 2016. fam. limacodidae 3907. apoda limacodes (hufnagel, 1766) material examined: pirot, vidlič mt., hotel “stara”, 1040 m, 6.vii 2016., 1 male, 5 females. this species is well known in serbia (j a k š i ć , 2016). caterpillars is oligophagous, feed on quercus and fagus species. according to ecological amplitude this species is eurytope (wide ecological amplitude). habitat: a. limacodes inhabit deciduous forests. fam. cossidae 4151. cossus cossus (linnaeus, 1758) material examined: pirot, vidlič mt., hotel “stara”, 1040 m, 6.vii 2016., 1 male. this species is well known in serbia (j a k š i ć , 2016). the larva is oligophagous, feed in the branches of a wide variety of trees. ecological amplitude: eurytope. habitat: woodland biotope, deciduous forests. fam. pterophoridae 5378. gillmeria ochrodactyla ([denis & schiffermüller], 1775) (syn.: g. tatradactyla (linnaeus, 1758)) material examined: pirot, vidlič mt., crni vrh, 1130 m, 6. vi 2016., 1 male (fig. 4). so far, this species is known in serbia only from žljeb mt. (r e b e l , 1917b). vidlič mt. is second known locality of g. ochrodactyla in serbia. this is a monophagous species, larval food-plant is tanacetum vulgare. m a r k o v i ć (2014) reported both t. vulgare, t. corymbosum and t. macrophyllum on vidlič mt. they have restricted ecological amplitude. this species inhabit woodland biotopes along edges of thermophilous forest. fig. 4. gillmeria ochrodactyla denis & schiffermüller, 1775, pirot, vidlič mt., crni vrh, 1130 m, 6. vi 2016 fam. thyrididae 5562. thyris fenestrella (scopoli, 1763) material examined: suva planina mt., devojački grob, 1317 m, 4. vii 2016., 1 male. this species is well known in serbia (j a k š i ć , 2016). the larva is monophagous, feed on clematis vitalba. j o v a n o v i ć (1980) reported this plant species on suva planina mt. this is a species with restricted ecological amplitude. habitat: hedgerows and related biotopes. fam. lasiocampidae 6744. malacosoma castrensis (linnaeus, 1758) material examined: pirot, vidlič mt., hotel “stara”, 1040 m, 6.vii 2016., 1 male. so far, b e s h k o v (2015c) reported this species for western serbia, prijepolje region, zvijezda village, savina voda near jabuka pass, 1117 m. the caterpillars are oligophagous and feed on caryophyllaceae, chenopodiaceae, betulaceae, plantaginaceae et cetera. malacosoma castrensis is eurytope, it has wide ecological amplitude. it inhabits woodland biotopes, beech dominated forest. 6834. hyloicus pinastri (linnaeus, 1758) material examined: pirot, vidlič mt., hotel “stara”, 1040 m, 6.vii 2016., 1 male. this species is well known in serbia (z e č e v i ć , 1996). hyloicus pinastri is polyphagous species, larval food plants are different conifer species. they have wide ecological biologica nyssana 8 (1)  september 2017: 113-122 jakšić, p.  a contribution to the knowledge of the lepidoptera… 116 amplitude, inhabit pine forest, fir-tree dominated forest, spruce forests, et cetera. fam. sphingidae 6843. macroglossum stellatarum (linnaeus, 1758) material examined: suva planina mt., devojački grob, 1317 m, 4. vii 2016., 1 male. this species is well known in serbia (z e č e v i ć , 1996). for this polyphagous species larval food plants are galium, rubiatinctoria and stellaria. j o v a n o v i ć (1980) reported g. aparine, g. mollugo, g. sylvaticum, g. aristatum, s. holostea, and others on suva planina mt. ecological amplitude: eurytope, wide ecological amplitude. they inhabit woodlands biotopes. fam. drepanidae 7505. watsonalla cultraria (fabricius, 1775) material examined: pirot, vidlič mt., hotel “stara”, 1040 m, 6.vii 2016., 1 male (genitalia slide sr2855). so far, only g r a d o j e v i ć (1926) reported this species for serbia, south kučaj mt. the larvae is monophagous, feed on fagus species. ecological amplitude: eurytope, wide ecological amplitude. biotope: woodland biotopes, deciduous forests. fam. geometridae 7522. abraxas grossulariata (linnaeus, 1758) material examined: pirot, vidlič mt., hotel “stara”, 1040 m, 6.vii 2016., 1 male. this species is well known in serbia (z e č e v i ć , 1996). recorded foodplants for their caterpillars are ribes rubrum, r. nigrum, prunus spinosa, crataegus, corylus, salix and euonymus europaeus, this is a polyphagous species. ecological amplitude: eurytope, wide ecological amplitude. biotope: woodland biotopes, deciduous forests. 7537. heliomata glarearia ([denis & schiffermüller], 1775) material examined: pirot, vidlič mt., hotel “stara”, 1040 m, 6.vii 2016., 1 male. r e b e l (1904) reported this species on the basis of hilf's material. the caterpillars are olygophagous, feed on different fabaceae (trifolium, medicago, lathyrus, hippocrepis). heliomata glarearia is an stenotope species, with restricted ecological amplitude. they inhabit meadows and pastures biotopes. 7559. narraga tessularia (metzner, 1845) material examined: pirot, vidlič mt., crni vrh, 1100-1116 m, 30.v 2016., 1 female (genitalia slide sr-2850); 6.vi 2016., 3 males. so far, only beshkov (2017) reported this species for the same area, as a new for serbia. the species is locally distributed in the forest belt, up to 1000 m above sea level, occurring in forest margins. it is monophagous, larva feed on artemisia maritima and a. campestris. our photo shown adult specimens on a. alba turra (fig. 5a). variation in the signum shape in female genitalia (fig. 5b) is significant, our specimen is closely related to female from austria (s k o u & s i h v o n e n , 2015). they have restricted ecological amplitude. this species inhabit meadows biotopes along edges of thermophilous forest. fig. 5. narraga tessularia (metzner, 1845): a-adult specimens (pirot, vidlič mt., crni vrh, 1100-1116 m, 30.v 2016) (photo p. jakšić); b-the signum (genitalia slide sr-2850, photo ivan gnjatović) biologica nyssana 8 (1)  september 2017: 113-122 jakšić, p.  a contribution to the knowledge of the lepidoptera… 117 7790. cleorodes lichenaria (hufnagel, 1767) material examined: vidlič mt., hotel “stara”, 1040 m, 6.vii 2016., 1 male, 1 female. this species is well known in serbia (z e č e v i ć , 1996). cleorodes lichenaria is lichen-eating moth. ecological amplitude: stenotope, restricted ecological amplitude. this species inhabit woodland biotopes. 7961. aplasta ononaria (fuessly, 1783) material examined: pirot, vidlič mt., hotel “stara”, 1040 m, 6.vii 2016., 1 male (genitalia slide sr-2857, fig. 6). male genitalia are illustrated in c a n (2010), our specimen belong to the same type. this species is well known in serbia (z e č e v i ć , 1996). the larva is oligophagous, feed on different papilionoceae: sarothamnus scoparius and ononis repens. m a r k o v i ć (2014) reported ononis pusilla, o. spinosa and o. arvensis on vidlič mt. ecological amplitude is wide. they inhabit woodland biotopes of mesophilous forests. fig. 6. aplasta ononaria (fuessly, 1783), pirot, vidlič mt., hotel “stara”, 1040 m, 6.vii 2016., (genitalia slide sr-2857, photo ivan gnjatović) 8269. catarhoe putridaria (herrich-schäffer, 1852) material examined: pirot, vidlič mt., hotel “stara”, 1040 m, 6.vii 2016., 1 male (genitalia slide sr-2856, fig. 7). in serbia this species is known only from the same locality, described by b e s h k o v (2015) as a new for serbia. the larval food-plants are different rubiaceae, this is oligophagous species. according to m a r k o v i ć (2014) there are 21 rubiaceae species on vidlič mt. ecological amplitude: eurytope. biotope: wetland biotopes, woodland biotopes with deciduous forests and hedgerows and related biotopes. fig. 7. catarhoe putridaria (herrich-schäffer, 1852), pirot, vidlič mt., hotel “stara”, 1040 m, 6.vii 2016. (genitalia slide sr-2856, photo ivan gnjatović) 8272. epirrhoe tristata (linnaeus, 1758) material examined: pirot, vidlič mt., hotel “stara”, 1040 m, 6.vii 2016., 1 male. this species is well known in serbia (z e č e v i ć , 1996). the larval foodplants are galium species, like previous moth species. hibernates as pupa in a cocoon on the ground. ecological amplitude: eurytope. biotope: woodland biotopes with deciduous forests. 8319. cosmorhoe ocellata (linnaeus, 1758) material examined: pirot, vidlič mt., hotel “stara”, 1040 m, 6.vii 2016., 1 male, 1 female. this species is well known in serbia (z e č e v i ć , 1996). caterpillars are monophagous, feeding on a single genus – galium species, like previous moth species. ecological amplitude: eurytope species. biotope: wetland biotopes, woodland biotopes, meadows and pastures, as well as intensively used agricultural areas. 8350. cidaria fulvata (forster, 1771) material examined: vidlič mt., hotel “stara”, 1040 m, 6.vii 2016., 1 male, 1 female. this species is well known in serbia (z e č e v i ć , 1996). c. fulvata is monophagous species, the larval food-plants are different rosaceae species. ecological amplitude: eurytope. biotope: woodland biotopes and cultural biotopes (gardens, parks, groves). 8427. triphosa sabaudiata (duponchel, 1830) material examined: knjaževac, staro selo village, dvoja vrata cave (pećina dvoja vrata), 9.ix 1998., 2 males, 2 females. the adults hibernate and live from july to may. hibernation takes place in rather dry caves, like dvoja vrata cave. this species is well biologica nyssana 8 (1)  september 2017: 113-122 jakšić, p.  a contribution to the knowledge of the lepidoptera… 118 known in serbia (z e č e v i ć , 1996). the larval-food plants are rhamnus cathartica, r. saxatilis, r. orbiculata, r. alpina and frangula alnus, this is monophagous species. ecological amplitude: stenotope. biotope: areas with rock formations. 8457. perizoma hydrata (treitschke, 1829) material examined: pirot, vidlič mt., hotel “stara”, 1040 m, 6.vii 2016., 1 male. this species is well known in serbia (z e č e v i ć , 1996). oligophagous species, the larval food-plants are silene nutans, s. inflata, lychnis viscaria and melandrium species, well presented in the flora of vidlič mt. (m a r k o v i ć , 2014). ecological amplitude: stenotope. biotope: rock formations, scree formations. 8620. aplocera plagiata (linnaeus, 1758) material examined: pirot, vidlič mt., hotel “stara”, 1040 m, 6.vii 2016., 1 female. this species is well known in serbia (z e č e v i ć , 1996). the larva of this polyphagous species feed on solidago virgaureae, calluna vulgaris, silene vulgaris, echium vulgare centaurea species and cirsium species. ecological amplitude: eurytope. biotope: deciduous forests and coniferous forests. 8624. aplocera praeformata (hübner, 1826) material examined: pirot, vidlič mt., hotel “stara”, 1040 m, 6.vii 2016., 1 female. this species is well known in serbia (z e č e v i ć , 1996). aplocera praeformata is monophagous species. the larva feed on different hypericum species. according to m a r k o v i ć (2014) there are 8 hypericum species on vidlič mt. ecological amplitude: eurytope. biotope: hedgerows and related biotopes, as well as meadows and pastures. fam. notodontidae 8709. furcula bicuspis (borkhausen, 1790) material examined: pirot, vidlič mt., hotel “stara”, 1040 m, 6.vii 2016., 1 male. d o d o k (1997) reported that f. bicuspis has been found at užice and jelova gora mt. this is oligophagous species: species feeding on a single family. larval food-plants are betula pendula and alnus glutinosa. m a r k o v i ć et al. (2010) reported both species for area of eastern serbia. ecological amplitude: eurytope. biotope: beech dominated forests, mountainous green alder shrubs. 8738. ptilodon capucina (linnaeus, 1758) material examined: stara planina mt., dojkinci, ponor, 1550 m, 5. vii 2016., 1 female. this species is well known in serbia (z e č e v i ć , 1996). caterpillars of this olygophagous species feed on carpinus, corylus, quercus, fagus, acer, betula, alnus and other. ecological amplitude: eurytope. biotope: woodland biotopes and cultural biotopes (gardens, parks, groves). 8739. ptilodon cucullina ([denis & schiffermüller], 1775) material examined: pirot, vidlič mt., hotel “stara”, 1040 m, 6.vii 2016., 1 female. l a z a r e v i ć (1898) reported this species for the vicinity of beograd. p. cucullina is a polyphagous species. larval foodplants are acer pseudoplatanus and acer campestre, both present on explored area (m a r k o v i ć , 2014). ecological amplitude: eurytope. biotope: deciduous trees/shrubs. 8750. phalera bucephala (linnaeus, 1758) material examined: pirot, vidlič mt., hotel “stara”, 1040 m, 6.vii 2016., 1 female. this species is well known in serbia (z e č e v i ć , 1996). caterpillars live gregariously and feed on a number of different deciduous trees (tilia, quercus, corylus, salix spp.). ecological amplitude: eurytope. biotope: deciduous trees/shrubs. fam. erebidae 8874. catocala nupta (linnaeus, 1767) material examined: knjaževac, staro selo village, dvoja vrata cave (= pećina dvoja vrata), 9.ix 1998., 1 female. this species is well known in serbia (z e č e v i ć , 1996). the larval food-plants are salix alba, s. fragilis and populus nigra. it is polyphagous species – species feeding on a single family. ecological amplitude: eurytope. biotope: wetland biotopes, woodland biotopes. 8882. catocala promissa(schiffermüller, 1775) material examined: pirot, vidlič mt., hotel “stara”, 1040 m, 6.vii 2016., 1 female. this species is well known in serbia (z e č e v i ć , 1996). the caterpillars are monophagous, feed on quercus species. ecological amplitude: eurytope. biotope: woodland biotopes, deciduous forests: thermophilous forests. 8888. catocala nymphagoga (esper, 1787) material examined: pirot, vidlič mt., hotel “stara”, 1040 m, 6.vii 2016., 1 female. b e s h k o v (2015c) recently reported this species from suva planina mt., bojanine vode. the caterpillars are monophagous, feed on quercus species. ecological amplitude: eurytope. biotope: woodland biotopes, deciduous forests: thermophilous forests. biologica nyssana 8 (1)  september 2017: 113-122 jakšić, p.  a contribution to the knowledge of the lepidoptera… 119 8984. scoliopteryx libatrix (linnaeus, 1758) material examined: knjaževac, staro selo village, dvoja vrata cave (pećina dvoja vrata), 9.ix 1998., 2 females. the adults hibernate in caves. scoliopteryx libatrix is well known in serbia (z e č e v i ć , 1996). during the winter s. libatrix hibernates in dark, cool places. the larval food-plants are salix and populus species, this is oligophagous species. ecological amplitude: eurytope. biotope: woodland biotopes: floodplain forests, moorland forests, as well as cultural biotopes (gardens, parks, groves). 10375. lymantria monacha (linnaeus, 1758) material examined: pirot, vidlič mt., hotel “stara”, 1040 m, 6.vii 2016., 1 male. this species is well known from western serbia (z e č e v i ć , 1996). lymantria monacha is a major pest of broadleaved and coniferous trees in europe and asia (pinus, picea, larix and abies are preferred hosts), feed on needles or leaves and can defoliate host trees. this is polyphagous species. ecological amplitude: stenotope. biotopes: woodland biotopes. 10489. eilema lurideola (zincken, 1817) material examined: pirot, vidlič mt., hotel “stara”, 1040 m, 6.vii 2016., 2 males (genitalia slides sr2745 and sr-2746). this species is well known from western serbia (z e č e v i ć , 1996; j a k š i ć & d i m o v i ć , 2000). the caterpillars are mycophagous, feeds on various lichens, including parmelia. ecological amplitude: eurytope. biotopes: woodland biotopes. 10579. rhyparia purpurata (linnaeus, 1758) material examined: suva planina mt., devojački grob, 1317 m, 25. vii 2014., 1 female. b e s h k o v (2015c; b e s h k o v & n a h i r n i ć , 2016) reported this species from pirot, kamenolom kitka and pirot, krupac village, as well as from starac mt., preševo district. the caterpillars feed calluna and other herbaceous plants and deciduous trees, they are polyphagous. ecological amplitude: stenotope. biotopes: woodland biotopes, hedgerows and related biotopes. 10603. callimorpha dominula (linnaeus, 1758) material examined: pirot, vidlič mt., hotel “stara”, 1040 m, 6.vii 2016., 1 female. this species is well known from western serbia (z e č e v i ć , 1996). the caterpillars feed on herbaceous plants, especially symphytum spp. m a r k o v i ć (2014) reported s. tuberosum and s. officinale on vidlič mt. ecological amplitude: stenotope. biotopes: this species inhabits humid clearings in wet forests, bog margins, beech dominated forests et cetera. fam. noctuidae 9307. amphipyra pyramidea (linnaeus, 1758) material examined: pirot, vidlič mt., hotel “stara”, 1040 m, 4. vii 2016., 1 male and 6.vii 2016., 1 female. this species is well known in serbia (zečević, 1996). the larva is polyphagous, feeds on a variety of trees and schrubs: fraxinus, ligustrum, lonicera, malus, quercus, rhododendron, rosa, sorbus, syringa et cetera. ecological amplitude: eurytope. biotope: woodland biotopes: floodplain forests, moorland forests, deciduous forests. 9453. hoplodrina respersa ([denis & schiffermüller], 1775) material examined: pirot, vidlič mt., hotel “stara”, 1040 m, 6.vii 2016., 1 male. so far, only v a s i ć (1969) reported this species from deliblato sands. the caterpillars are active at night and can be found on hippocrepis comosa. a d a m o v i ć (1909) reported this species for eastern serbia. ecological amplitude: stenotope. biotope: thermophilous forests, pine forests, scree formations and xerophilous meadows and pastures. 9515. actinotia polyodon (clerck, 1759) material examined: suva planina mt., bojanine vode, 850 m, 4.vii 2016., 1 female. so far, this species is known only from deliblato sands (g r a d o j e v i ć , 1963). the caterpillar feed on hypericum perforatum and astragalus glycyphyllos, they are polyphagous. j o v a n o v i ć (1980) reported this plant species for suva planina mt.ecological amplitude: stenotope. biotope: hedgerows and related biotopes. 9550. cosmia trapezina (linnaeus, 1758) material examined: pirot, vidlič mt., hotel “stara”, 1040 m, 6.vii 2016., 1 male (genitalia slide sr2780). this species is well known in serbia (z e č e v i ć , 1996). the caterpillars are polyphagous, feed on carpinus, betula, quercus, fagus, ulmus, pyrus, corylus avellana, and others. ecological amplitude: eurytope. biotope: woodland biotopes: floodplain forsts, moorland forests, deciduous forests. 9642. brachylomia viminalis (fabricius, 1777) material examined: pirot, vidlič mt., hotel “stara”, 1040 m, 6.vii 2016., 1 male, 2 females. this species is well known from western serbia (z e č e v i ć , biologica nyssana 8 (1)  september 2017: 113-122 jakšić, p.  a contribution to the knowledge of the lepidoptera… 120 1996). the larvae are monophagous, feed on salix species. ecological amplitude: eurytope. biotope: woodland biotopes. 9781. oligia versicolor (borkhausen, 1792) material examined: pirot, vidlič mt., hotel“stara”, 1040 m, 6. vii 2016., 3 males, 1 female. b e s h k o v (2015c) reported this species from suva planina, bojanine vode; pirot, stone-pit kitka and pirot, krupac village. the larvae is polyphagous, feed on deciduous tree and schrubs, brachypodium sylvaticum, carex sp., poaceae, luzula luzuloides et cetera. ecological amplitude: stenotope. biotope: wetland biotopes, floodplain forests, moorland forest. 10100. noctua fimbriata (schreber, 1759) material examined: pirot, vidlič mt., hotel “stara”, 1040 m, 6.vii 2016., 1 female. this species is well known from western serbia (z e č e v i ć , 1996). the larvae are polyphagous and feed on ligustrum, primula, rubus, rumex, salix, trifolium and urtica species. ecological amplitude: eurytope. biotope: woodland biotopes, meadows and pastures. 10372. colocasia coryli (linnaeus, 1758) material examined: pirot, vidlič mt., hotel “stara”, 1040 m, 6.vii 2016., 2 males, 1 female. this species is well known from western serbia (z e č e v i ć , 1996). the larval food-plants are carpinus betulus, corylus avellana, fagus sylvatica, quercus robur, et cetera, this is polyphagous species. ecological amplitude: eurytope. biotopes: floodplain forests, moorland forests, deciduous forests, hedgerows and related biotopes. fam. nolidae 10427. nola cucullatella (linnaeus, 1758) material examined: pirot, vidlič mt., hotel “stara”, 1040 m, 6.vii 2016., 1 males (genitalia slide sr2860). male genitalia are illustrated in h a c k e r et al. (2012). this species is well known from western serbia (z e č e v i ć , 1996). the caterpillars feed on crataegus sp., malus sp. and prunus spinosa. ecological amplitude: eurytope. biotopes: woodland biotopes. discussion the results are related to geographical area of east serbia that gravitates to catchment of nišava river basin. the area is located in the contact zone of three mountain systems: rhodopy mountain system, carpathian mountain system and balkan mountain range. climate impact also varies from continental climate to the sub-mediterranean climate. nišava river basin is influenced by pontic and mediterranean region through struma river valley. its position suggests faunal richness but its lepidoptera fauna has not been sufficiently explored to the present day. results given by b e s h k o v (2017), as well as results shown here suggest existence of significant faunal richness. presence of 43 species of lepidoptera (heterocera) from 13 families is shown. among the established species 14 (35%) are rare and found only in 2-3 locations in serbia so far: gillmeria ochrodactyla, malacosoma castrensis, watsonalla cultraria, heliomata glarearia, narraga tessularia, catarhoe putridaria, epirrhoe tristata, furcula bicuspis, ptilodon cucullina, catocala nymphagoga, hoplodrina respersa, actinotia polyodon, oligia versicolor and rhyparia purpurata. rich fauna is significant part conditioned by the existence of differentiated biotope and ecological niches. the good examples are the species related to the calcareous rocky habitats. besides there is noticeable connection with neighboring mountain areas such as dinarides (which are also limestone surface). although geographically distant, the two systems have common elements of lepidoptera fauna. for example we can mention species of adscita albanica distributed on mountain paštrik mt. (dinarides) and suva planina mt. (rhodope) (n a h i r n i ć et al., 2016); gillmeria ochrodactyla distributed on žljeb mt. (dinarides) and vidlič mt. (rhodope); malacosoma castrensis distributed on prijepolje, zvijezda (dinarides) and vidlič mt. (rhodope). it is interesting to point out that listed species in geographical area close to kopaonik mt. and šar-planina mt. have not been found because it is not the limestone bedrock. biogeographical connection of the tested area exists with carpathian system based again on the limestone. examples of such species are watsonalla cultraria present in kučaj mt. and vidlič. thanks to erosion processes in the limestone cavernicolous facilities are developed. therefore the list of these species are also petrophilous species: triphosa sabaudiata, catocala nupta and scoliopteryx libatrix. all these species are troglophile. however the real number of species of environmental group is much higher. in neighboring bulgarian caves b e s h k o v and p e t r o v (1996) have determined the presence of 32 species of lepidoptera. special environmental group is comprised by species whose caterpillars are fed by lichen, lichen eating moths. typical representatives are: cleorodes lichenaria and eilema lurideola. environmentally biologica nyssana 8 (1)  september 2017: 113-122 jakšić, p.  a contribution to the knowledge of the lepidoptera… 121 specific species is also euplocamus anthracinalis, whose caterpillars are mycophagous. to summarize we can say that shown fauna species represent significant contribution to the knowledge of its distribution in serbia. also these findings indicate a high potential for faunal species of this area. this indicates that further faunistic research will result in new and significant findings. acknowledgements. i am thankful to dr bojan zlatković for the primary identification of artemisia alba turra. i also thank to ana nahirnić and ivan gnjatović for their valuable assistance and useful advices. this investigation was partly supported by scientific society biodat alpin from innsbruck, austria, as well as university of maribor, faculty of natural sciences and mathematics (project biodiversity of the green lacewing in serbia, grants nos. rp biodiv chryser 2015). references adamović, l. 1909: die vegetationsverhältnisse der balkanländer (mösische länder). in: engler, a. & drude, o. (eds.): die vegetation der erde. band xi. leipzig. beshkov, s. 2015a: some new for serbia and rare lepidoptera species collected at light in eastern serbia. the entomologist's record and journal of variation, 127: 127–134. beshkov, s. 2015b: a significant range extension of erannis declinans (staudinger, 1879) in europe (lep.: geometridae). the entomologist's record and journal of variation, 127: 271–277, map 1, figs 2. beshkov, s. 2015c: eight new and some rare for serbia nocturnal lepidoptera species collected at light. the entomologist's record and journal of variation, 127: 212–227, pl. 10. beshkov, s. 2017: contribution to knowledge of the lepidoptera fauna of the balkan peninsula. the entomologist's record and journal of variation, 129: 9-33. beshkov, s., petrov, b. 1996: a catalogue of the bulgarian lepidoptera species reported and collected from the caves and galleries in bulgaria. atalanta, 27 (1/2): 433-448. beshkov, s., nahirnić, a. 2016: new and rare nocturnal lepidoptera species for serbia from pčinja river valley hot spots for biodiversity (insecta: lepidoptera). atalanta, 47 (1/2): 139-149. can, f. 2010: evaluation of morphological characters and malegenitalia features of emerald moths (lepidoptera: geometridae, geometrinae) from turkey. african journal of agricultural research, 5 (9): 867-873, carl, m., huemer, p., zanetti, a., salvadori, c. 2005: ecological assessment in alpine forest ecosystems: bioindication with insects (auchenorrhyncha, coleoptera (staphylinidae), lepidoptera). studi trentini di scienze naturali. acta biologica, 81, suppl. 1: 167-217. dodok, i. 1997: new butterfly species in the fauna of serbia (lepidoptera: notodontidae, drepanidae and geometridae). acta entomologica serbica, 2 (1-2): 153–158. fibiger, m., yela, j.l., zilli, a., varga, z., ronkay, g., ronkay, l. 2012: check list of the quadrifid noctuoidea of europe. in: witt, t. and ronkay, l. (editors): noctuidae europaeae, vol. 13: lymantriinae and arctiinae. entomological press. gradojević, m., 1926: kratak izveštaj o dosadašnjem radu na prikupljanju i proučavanju leptirova srbije. spomenica i kongresa entomologa kraljevine s.h.s. glasnik entomološkog društva kraljevine srba, hrvata i slovenaca, i (1): 39–44. beograd. gradojević, z. 1963: naselja arthropoda travnih zajednica deliblatske peščare i njihova sukcesija. doktorska disertacija. beograd. hacker, h.h., schreier, h.-p., goater, b. 2012: revision of the tribe nolini of africa and the western palaearctic region (lepidoptera, noctuoidea, noctuidae, nolinae). esperiana, 17: 1-614. schwanfeld. jakšić, p. 2016: tentative check list of serbian microlepidoptera. ecologica montenegrina, 7: 33-258. jakšić, p., dimović, d. 2000: pregled utvrđenih vrsta rodova eilema hübner, [1804] i lithosia fabricius, 1798 bora i susednih područja (lepidoptera: arctiidae, lithosiinae). zaštita prirode, 52 (1): 47–63. jovanović, b. 1980: šumske fitocenoze i staništa suve planine (waldphytocenosen und standorte der suva planina). glasnik šumarskog fakulteta, posebno izdanje, 55: 1–216 + 1–14, tabs. i–xv, 1 map. beograd. karsholt, o., razowski, j. 1996: the lepidoptera of europe. a distributional checklist. apollo book. stenstrup. lazarević, r. 1898: prilozi za građu entomologije kralj. srbije. makrolepidoptere okoline beograda ii heterocera. glas srpske kraljevske akademije, lvi, prvi razred 20: 185–235. marković, m. 2014: uticaj požara na floru planine vidlič. univerzitet u nišu, prirodno-matematički fakultet, str. 1–314. niš. mаrković, m., mаtović, m., pаvlović, d., zlаtković, b., mаrković, a., jotić, b., stаnkov-jovаnović, v. 2010: resources of medicinal plants and herbs biologica nyssana 8 (1)  september 2017: 113-122 jakšić, p.  a contribution to the knowledge of the lepidoptera… 122 collector’s calendar of pirot county (serbia). biologica nyssana, 1 (1–2): 9–21. nahirnić, a., jakšić, p., marković, m., zlatković, b. 2016: new data on rare zygaenidae and their habitats from eastern serbia. xv international symposium on zygaenidae 11–18 september 2016 mals/malles, südtirol/alto adige, italy. book of abstracts, p. 29. nieukerken v. et al. 2011: order lepidoptera linnaeus, 1758. in: zhang, z. q. (ed.) animal biodiversity: an outline of higher-level classification and survey of taxonomic richness. zootaxa, 3148: 211-221. radosavljević, d. 1924: bolesti i štetočine kulturnih biljaka u 1922. g. na teritoriji kraljevine shs. glasnik ministarstva poljoprivrede i voda, ii (6): 93–117. rebel, h. 1903: studien über die lepidopterenfauna der balkanländer. i. teil. bulgarien und ostrumelien. annalen des k.k. naturhistorischen hofmuseums, xviii (2. und 3): 123-347 + taf. iii, wien. rebel, h. 1904: studien über die lepidopterenfauna der balkanländer. ii. teil. bosnien und herzegowina. annalen des naturhistorischen hofmuseums in wien xix: 97-377, taf. 4, 5. rebel, h., 1917a: neue lepidopterenfunde in nordalbanien, mazedonien und serbien. jahresbericht des naturwissenschaftlichen orientvereins, 21: 17-24. wien. rebel, h. 1917b: lepidopteren aus neumontenegro. sitzungsberichte der akademie der wissenschaften mat.-nat. klasse, 126: 765-813. skou, p., sihvonen, p. 2015: ennominae i. in: a. hausmann (ed.): the geometrid moths of europe 5: 1–657. brill, leiden. todorowa, w., petkoff, p. 1915: макролепидоптерната фауна на царибродско и трьнско. arbeiten der bulgarischen naturforschenden gesselschaft, viii: 128–147. uhl, j. 1903: adalék szerbia lepke-faunajahoz. rovartani lapok, budapest, x: 38-40. vasić, k. 1948: gradacija štetnih sovica u srezu nišavskom u godinama 1946 i 1947 [(la gradation des noctuelles polyphages (agrotis sp.) dans l'arrondissement de nishava (pirote)]. godišnjak poljoprivredno–šumarskog fakulteta, 1: 331–348. vasić, k. 1969: prilog poznavanju sovica (noctuidae, lepidoptera) deliblatskog peska. zbornik radova “deliblatski pesak” 1: 199–214. beograd. zečević, m. 1976: novi nalazi leptira u timočkoj krajini. zavod za poljoprivredu, pp. 209–225. zaječar. zečević, m. 1990: nove vrste leptira u fauni timočke krajine (istočna srbija) nađene u period 1985 – 1989. godine. razvitak, 2: 26–33. zaječar. zečević, m. 1993: nove vrste leptira u fauni timočke krajine (istočna srbija) nađene u periodu 1954 – 1992. godine. razvitak, xxxiii (1–2): 22–30. zaječar. zečević, m., 1996: pregled faune leptira srbije. nauka i institut za istraživanja u poljoprivredi srbija. pp. 1–157. beograd. zečević, m., radovanović, s. 1974: leptiri timočke krajine (makrolepidoptera). zavod za poljoprivredu zaječar i novinska ustanova “timok”. str. 1–183. zaječar. anticancer compounds from medicinal plants biologica nyssana 2 (2)  december 2011: 00-00 ljupković r.b. et al..  removal cu(ii) ions from water... 21 review article checklist of the genus bracon (hymenoptera: braconidae) in serbia vladimir žikić 1* , saša s. stanković 1 , marijana ilić 1 1 faculty of science and mathematics, department of biology and ecology, university of niš, višegradska 33, 18000 niš, serbia * e-mail: vzikic@yahoo.com abstract: žikić, v., stanković, s., ilić, m.: checklist of the genus bracon (hymenoptera: braconidae) in serbia, biologica nyssana, 3 (1), september 2012: 21-29. this is the first checklist of the genus bracon for the territory of serbia. here we present 80 species of this cosmopolitan genus of the subfamily braconinae which is one of the cyclostome braconid groups. we connected the parasitoids with their hosts where the data about them were available. there are 290 host species in total belonging to 39 families of four insect orders: diptera, coleoptera, hymenoptera and lepidoptera. key words: cyclostome braconidae, bracon, serbia, checklist introduction braconinae belongs to the cyclostome group of the family braconidae. this subfamily assembles ectoparasitoid wasps which lay their eggs on the host body. according to their biology, ectoparasitic braconid wasps belong to the group of idiobionts; which means that they do not permit the host to continue its development after oviposition (m a t h e w s , 1974). oviposition in this group is split in two acts: paralisation of the host, than laying eggs which is opposite in koinobions. as a cosmopolitan group of parasitoids they represent a large and powerful biological weapon (eg. b a i r d , 1958; l e w i s et al., 1990) against various pests from the following holometabolous insect orders: diptera, coleoptera, hymenoptera and lepidoptera. the species of the genus bracon show different degree of the host specificity, ranging from a strict specialization to only one host species, whereas other can be polyphagous, when parasitism occurs in more than a hundred hosts in different types of habitats and geographical areas (s h a w & h u d d l e s t o n , 1991). because of their important role in biological control, biology of some species is very well studied: for b. intercessor (van a c h t e r b e r g et al., 1990; g e o r g i e v et al., 2005), for b. brevicornis (t e m e r a k , 1982), or many publications for b. hebetor and its host plodia interpunctella (pyralidae) (r e i n e r t et al., 1971; t a y l o r , 1988a, b; y u et al., 1999; m i l o n a s , 2005; d w e c k et al. 2010). according to the fauna europaea (v a n a c h t e r b e r g , 2012), about 250 species of the genus bracon are recorded in europe. the present publication stemmed from the need to make the list of all registered bracon species on the territory of serbia after many years of investigation and also to connect the parasitoids with their hosts. investigation of the genus bracon in serbia in different aspects is presented in following publications by the authors b e l o k o b y s k i j & ž i k i ć (2009); b r a j k o v i ć (1989); b r a j k o v i ć et al. (1991, 1994); s t a n k o v i ć et al. (2010) and ž i k i ć et al. (2000, 2010, 2012). 3 (1) • september 2012: 21-29 biologica nyssana 3 (1)  september 2012: 21-29 žikić, v. et al.  checklist of the genus bracon (hymenoptera: braconidae)… 22 materials and methods the majority of analyzed specimens were taken from the collection of the faculty of biology, university of belgrade and the faculty of sciences and mathematics, university of niš, serbia. a part of specimens is deposited at the zoological institute ras (st. petersburg, russia) and the natural history museum of budapest, hungary. the data about braconid hosts are compiled using the available literature; the list of references is bellow. the basic key for the identification of the species we used is one by tobias et al. (1986), updated fauna europaea (http://faunaeur.org) and the database taxapad (y u et al. 2012). a lot of information about the hosts and the distribution of the investigated genus we collected from p a p p (1973, 1977, 2008) and b e y a r s l a n et al. (2005). results we present 80 species of the genus bracon registered on the territory of serbia. the data regarding parasitoids are connected with the adequate data about their hosts. the species of bracon are listed alphabetically according to their subgenera. for 25 braconid species there were no host records for serbia nor for other parts of europe. in addition, we give the geographical distribution of all registered parasitoids in continental part of europe and russia. bracon (bracon) alutaceus szepligeti 1901 – no host data. distribution: austria, great britain, bulgaria, cyprus, czech republic, denmark, germany, hungary, lithuania, poland, russia (northwestern), slovakia, spain, sweden, ukraine, serbia. bracon (bracon) crassiceps thomson 1892 – no host data. distribution: austria, finland, germany, hungary, russia (northwestern), sweden, switzerland, serbia. bracon (bracon) crassungula thomson 1892 – no host data. distribution: austria, hungary, italy, sweden, switzerland, serbia. bracon (bracon) filicornis thomson 1892 – no host data. distribution: austria, sweden, serbia. bracon (bracon) flagellaris thomson 1892 – no host data. distribution: croatia, germany, hungary, romania, russia (south), slovenia, sweden, switzerland, ukraine, serbia. bracon (bracon) fulvipes nees 1834 on apion minimum, cistapion cyanescens (apionidae); curculio villosus, mononychus punctumalbum (curculionidae); tetramesa brevicornis t. hyalipennis (eurytomidae); coleophora coronillae (coleophoridae); paranthrene tabaniformis (sesiidae). distribution: austria, belgium, great britain, bulgaria, croatia, turkey, finland, france, germany, greece, hungary, ireland, italy, latvia, lithuania, poland, russia (central and south), slovakia, slovenia, spain, sweden, switzerland, netherlands, serbia. bracon (bracon) grandiceps thomson 1892 on bembecia scopigera (sesiidae). distribution: austria, croatia, turkey, finland, france, germany, greece, hungary, italy, macedonia, poland, russia (south), slovenia, sweden, serbia. bracon (bracon) intercessor nees 1834 on apion opeticum (apionidae); agapanthia villosoviridescens (cerambycidae); anthonomus pedicularius, a. pomorum, a. sorbi, lixus brevirostris, l. incanescens, l. junci, l. scabricollis, microlarinus lareynii, m. lypriformis (curculionidae); rhynchites bacchus (rhynchitidae); tetramesa hyalipennis, t. rossica (eurytomidae); haplochrois theae (agonoxenidae); augasma atraphaxidellum (coleophoridae); synanthedon culiciformis, paranthrene tabaniformis (sesiidae); sparganothis pilleriana (tortricidae). distribution: albania, austria, great britain, bulgaria, croatia, cyprus, turkey, france, germany, greece, hungary, italy, latvia, lithuania, macedonia, norway, poland, romania, russia (central, east, northwestern and south), slovakia, slovenia, spain, sweden, switzerland, netherlands, ukraine, serbia. bracon (bracon) laetus wesmael 1838 on anthonomus pomorum, magdalis nitida (curculionidae); amblypalpis tamaricella (gelechiidae); argyresthia chrysidella, prays citri; prays oleae (yponomeutidae). distribution: belgium, great britain, bulgaria, croatia, cyprus, czech republic, france, germany, greece, hungary, spain, serbia. bracon (bracon) leptus marshall 1897 on metzneria lapella (gelechiidae). distribution: austria, great britain, bulgaria, croatia, cyprus, germany, greece, hungary, lithuania, macedonia, romania, russia (central and south), slovakia, slovenia, spain, switzerland, netherlands, serbia. bracon (bracon) longicollis wesmael 1838 on chlorops pumilionis (chloropidae). distribution: austria, belgium, great britain, bulgaria, croatia, cyprus, turkey, finland, france, germany, greece, hungary, italy, lithuania, macedonia, norway, poland, russia (central, northwestern and south), biologica nyssana 3 (1)  september 2012: 21-29 žikić, v. et al.  checklist of the genus bracon (hymenoptera: braconidae)… 23 slovenia, spain, sweden, switzerland, netherlands, serbia. bracon (bracon) longulus thomson 1892 on oscinella frit (chloropidae). distribution: austria, bulgaria, croatia, cyprus, czech republic, denmark, finland, france, germany, greece, hungary, italy, macedonia, norway, slovenia, spain, sweden, netherlands, serbia. bracon (bracon) luteator spinola 1808 on urophora solstitialis (tephritidae); metzneria aestivella, m. lappella (gelechiidae). distribution: austria, great britain, bulgaria, croatia, cyprus, turkey, france, greece, hungary, italy, latvia, macedonia, russia (central, northwestern and south), slovakia, spain, switzerland, ukraine, serbia. bracon (bracon) mariae dalla torre 1898 on chaetorellia jaceae (tephritidae). distribution: bulgaria, croatia, germany, hungary, italy, russia (south), slovakia, spain, sweden, switzerland, ukraine, serbia. bracon (bracon) mediostriatus papp 1968 – no host data. distribution: hungary, serbia. bracon (bracon) nigratus wesmael 1838 on chaetostomella cylindrica (tephritidae); coleophora millefolii (coleophoridae); grapholita compositella (tortricidae); zygaena loti (zygaenidae). distribution: austria, belgium, great britain, croatia, czech republic, france, germany, hungary, ireland, italy, lithuania, romania, russia (central and north), slovakia, slovenia, spain, sweden, switzerland, netherlands, serbia. bracon (bracon) pallicarpus thomson 1892 – no host data. distribution: great britain, bulgaria, finland, germany, hungary, italy, spain, sweden, serbia. bracon (bracon) pectoralis wesmael 1838 on chrysobothis affisinis, sphenoptera gossypii, s. lobicollis, s. montana (buprestidae); plagionotus arcuatus, plagionotus bobelayei (cerambycidae). distribution: austria, belgium, great britain, bulgaria, croatia, cyprus, turkey, france, germany, greece, hungary, italy, macedonia, romania, russia (central and south), slovakia, spain, switzerland, serbia. bracon (bracon) scabriusculus dalla torre 1898 – no host data. distribution: austria, germany, hungary, italy, romania, russia (central), spain, serbia. bracon (bracon) scutellaris wesmael 1838 on anobium abietis (anobiidae); euura mucronata, pontania pedunculi, p. viminallis (tenthredinidae). distribution: belgium, great britain, finland, france, hungary, ireland, italy, poland, sweden, netherlands, serbia. bracon (bracon) speerschneideri schmiedeknecht 1897 on argyresthia thuiella, ocnerostoma sp. (yponomeutidae). distribution: austria, great britain, france, germany, norway, sweden, switzerland, serbia. bracon (bracon) subglaber szepligeti 1901 on noeeta pupillata, tephritis leontodontis, terellia serratulae, urophora cardui, u. quadrifasciata, u solstitialis (tephritidae); metzneria lappella (gelechidae); epichnopterix sieboldi (psychidae); paranthrene tabaniformis, pennisetia hylaeiformis (sesiidae); aethes williana (tortricidae). distribution: croatia; germany; hungary; italy, kazakhstan, moldova, russia (russia-krasnodar, yaroslavl oblast), slovenia, switzerland, ukraine, serbia. bracon (bracon) trucidator marshall 1888 on metzneria lappella (gelechiidae). distribution: albania, austria, belgium, great britain, bulgaria, croatia, denmark, turkey, finland, france, germany, greece, hungary, italy, latvia, poland, romania, russia (central, northwestern and south), slovenia, spain, switzerland, netherlands, serbia. bracon (bracon) variegator spinola 1808 on ernobius abietis (anobiidae); coleophora iutipennella (coleophoridae); agonopterix propinquella, (depressariidae); anarsia lineatella, recurvaria nanella, teleiodes luculella (gelechiidae); phyllonorycter klemannella, ph. mespillela (gracillariidae); endrosis sarcitrella (oecophoridae); acrobasis obsutella (pyralidae); archips rosana, cydia strobilella, tortrix viridana (tortricidae); yponomeuta padella (yponomeutidae). distribution: albania, austria, croatia, cyprus, france, hungary, italy, macedonia, russia (south), sardinia, spain, switzerland, ukraine, serbia. bracon (cyanopterobracon) sabulosus szepligeti 1896 on tephritidae. distribution: bulgaria, croatia, cyprus, greece, hungary, macedonia, romania, russia (south), serbia. bracon (cyanopterobracon) urinator (fabricius 1798) on larinus saussureae, l. sturnus, rhinocyllus conicus (curculilonidae); tephritis pulchra (tephritidae). distribution: albania, austria, belgium, bosnia and herzegovina, great britain, bulgaria, croatia, cyprus, turkey, france, germany, greece, hungary, italy, macedonia, portugal, romania, russia (central, east, biologica nyssana 3 (1)  september 2012: 21-29 žikić, v. et al.  checklist of the genus bracon (hymenoptera: braconidae)… 24 northwestern and south), slovakia, spain, sweden, switzerland, netherlands, ukraine, serbia. bracon (cyanopterobracon) uromelas costa 1888 – no host data. distribution: albania, austria, croatia, cyprus, france, hungary, italy, macedonia, russia (south), sardinia, spain, switzerland, ukraina, serbia. bracon (glabrobracon) abbreviator nees 1834 on anthonomus pomorum (curculionidae); lipara lucens (chloropidae); cephus pygmaeus (cephidae); coleophora trogldytella (coleophoridae); mesapamea secalis, oria musculosa, sesamia nonagrioides (noctuidae); pennisetia hylaeiformis (sesiidae); cochylis posterna, pandemis heparana, rhyacionia buoliana (tortricidae). distribution: austria, great britain, croatia, cyprus, czech republic, finland, germany, hungary, poland, romania, slovakia, spain, sweden, switzerland, netherlands, serbia. bracon (glabrobracon) arcuatus thomson 1892 – no host data. distribution: austria, great britain, bulgaria, croatia, cyprus, czech republic, germany, greece, hungary, slovenia, sweden, netherlands, serbia. bracon (glabrobracon) atrator nees 1834 on omphalapion buddebergi (apionidae); gymnetron villosulum (curculionidae); amauromyza flavifrons (agromyzidae); tephritis conura, t. separata (tephritidae); coleophora coronillae (coleophoridae). distribution: austria, belgium, great britain, bulgaria, croatia, cyprus, turkey, finland, france, germany, greece, hungary, ireland, italy, latvia, lithuania, macedonia, poland, russia (central, northwestern and south), slovakia, spain, sweden, switzerland, netherlands, serbia. bracon (glabrobracon) caudatus ratzeburg 1848 on hylesinus fraxini (curculionidae). distribution: austria, great britain, bulgaria, czech republic, germany, greece, hungary, poland, serbia. bracon (glabrobracon) caudiger nees 1834 on coleophora obscenella (coleophoridae); aphelia amplana, cydia splendana, c. strobilella (tortricidae). distribution: austria, belgium, czech republic, turkey, france, germany, hungary, italy, poland, romania, russia (central, north, northwestern and south), sweden, the netherland, serbia. bracon (glabrobracon) colpophorus wesmael 1838 on coleophora sp. (coleophoridae). distribution: austria, belgium, great britain, czech republic, france, germany, hungary, italy, moldova, poland, romania, russia (northwestern), netherlands, ukraine, serbia. bracon (glabrobracon) coniferarum fahringer 1928 on millieria dolosalis (choreutidae); coleophora gallipennella, c. serratella (coleophoridae). distribution: great britain, bulgaria, czech republic, germany, italy, poland, switzerland, netherlands, serbia. bracon (glabrobracon) delibator haliday 1833 on gymnetron campanulae, hylobius piceus (curculionidae); olibrus aeneus (phalacridae); urophora cuspidata (tephritidae); cydia strobilella (tortricidae). distribution: austria, belgium, bosnia and herzegovina, great britain, bulgaria, croatia, cyprus, czech republic, turkey, finland, france, germany, greece, hungary, ireland, italy, macedonia, poland, russia (central and south), slovakia, slovenia, spain, sweden, switzerland, netherlands, ukraine, serbia. bracon (glabrobracon) delusor spinola 1808 – no host data. distribution: austria, croatia, france, germany, italy, latvia, poland, russia (northwestern), serbia. bracon (glabrobracon) dichromus wesmael 1838 on gymnetron asellus (curculionidae); platyedra subcinerea (gelechidae); grapholita funebrana (tortricidae). distribution: austria, belgium, bulgaria, croatia, cyprus, france, germany, hungary, italy, poland, romania, slovakia, spain, serbia. bracon (glabrobracon) discoideus wesmael 1838 on saperda populnea (cerambycidae); anthonomus pomorum, a. spilotus, ceutorhynchus assimilis, c. quadridens, curculio crux, c. nucum, c. pyrrhoceras, c. salicivorus, pissodes notatus (curculionidae); byctiscus betulae, b. populi, rhynchites pauxillus (rhynchitidae); chlorops pumilionis (chloropidae); biorhiza pallida (cynipidae); euura mucronata, pontania bridgmanii, p. collactanea, p. foetidae, p. pedunculi, p. proxima, p. vesicator, p. viminalis (tenthredinidae); anticlea badiata (geometridae); adoxophyes orana, rhyacionia buoliana (tortricidae). distribution: austria, belgium, great britain, bulgaria, czech republic, france, germany, hungary, ireland, italy, poland, romania, russia (northwestern), slovakia, slovenia, sweden, switzerland, netherlands, serbia. bracon (glabrobracon) fumatus szepligeti 1901 – no host data. distribution: austria, croatia, cyprus, turkey, france, greece, hungary, russia (south), spain, switzerland, serbia. bracon (glabrobracon) fuscipennis wesmael 1838 on gastrophysa polygoni, g. viridula, plagiodera versicolora (chrysomelidae). distribution: austria, http://www.faunaeur.org/full_results.php?id=11049 http://www.faunaeur.org/full_results.php?id=11175 biologica nyssana 3 (1)  september 2012: 21-29 žikić, v. et al.  checklist of the genus bracon (hymenoptera: braconidae)… 25 belgium, croatia, france, germany, hungary, italy, poland, slovenia, serbia. bracon (glabrobracon) hyalinipennis szepligeti 1901 – no host data. distribution: hungary, serbia. bracon (glabrobracon) immutator nees 1834 on anthonomus rubi, ceutorhynchus pleurostigma, cryptorhynchus lapathi, curculio villosus, pissodes notatus (curculionidae); urophora stylata (tephritidae); biorhiza pallida, (cynipidae). distribution: austria, belgium, bosnia and herzegovina, great britain, croatia, cyprus, czech republic, finland, france, germany, hungary, italy, macedonia, poland, romania, russia (central, north, and northwestern), slovenia, sweden, switzerland, netherlands, ukraine, serbia. bracon (glabrobracon) larvicida wesmael 1838 – no host data. distribution: austria, belgium, great britain, bulgaria, croatia, turkey, france, germany, greece, hungary, italy, poland, romania, russia (central, northwestern and south), slovakia, slovenia, serbia. bracon (glabrobracon) marshalli szepligeti 1901 on trachys sp. (buprestidae); myopites inulaedyssentericae (tephritidae); cydia nigricana (tortricidae). distribution: great britain, bulgaria, croatia, cyprus, denmark, finland, germany, greece, hungary, italy, norway, portugal, romania, slovakia, slovenia, spain, sweden, switzerland, serbia. bracon (glabrobracon) minutator (fabricius 1798) on anthonomus pomorum (curculionidae); lipara lucens (chloropidae); trypeta jaceae (tephritidae); cephus pygmaeus (cephidae); caloptilia syringella (gracillariidae); mesapamea secalis, oria musculosa, sesamia nonagrioides (noctuidae); pennisetia hylaeiformis (sesiidae); cochylis posterana, leguminivora glycinivorella, rhyacionia buoliana (tortricidae). distribution: albania, austria, belgium, great britain, bulgaria, cyprus, czech republic, denmark, turkey, finland, france, germany, greece, hungary, ireland, italy, latvia, lithuania, macedonia, norway, poland, russia (central and south), slovakia, slovenia, spain, sweden, switzerland, netherlands, serbia. bracon (glabrobracon) obscurator nees 1811 on trachys pumilus, t. troglodytes (buprestidae); hylesinus fraxini, ips typographus, orthotomicus suturalis, trypodendron domesticum, t. signatum (curculionidae); coleophora caespititiella (colephoridae); epermenia fulviguttella (epermeniidae); homoeosoma sinuella (pyralidae). distribution: austria, belgium, bosnia and herzegovina, great britain, bulgaria, croatia, cyprus, czech republic, denmark, turkey, finland, france, germany, greece, hungary, italy, macedonia, poland, romania, russia (central, northwestern and south), slovakia, slovenia, spain, sweden, switzerland, netherlands, serbia. bracon (glabrobracon) osculator nees 1811 on millieria dolosalis (choreutidae); coleophora albicostella, c. alticolella, c. caespititiella, c. colutella, c. discordella, c. flaviella, c. frischella, c. gallipennella, c. lutipennella, c. milvipennis, c. obscenella, c. ochrea, c. saponariella, c. serenella, c. serratella, c. solitariella, c. spinella, co. spiraeella, c. therinella, c. trifariella, c. vibicella, c. vicinella, c. vulpecula, coleophora sp.1, coleophora sp.2 (coleophoridae); cosmopterix eximia (cosmopterigidae); elachista contaminatella, elachista sp. (elachistidae); phyllonorycter sp. (gracillariidae); mompha conturbatella (momphidae); ectoedemia agrimoniae (nepticulidae); retinia perangustana (tortricidae). distribution: austria, belgium, bosnia and herzegovina, great britain, bulgaria, croatia, cyprus, czech republic, denmark, turkey, finland, france, germany, greece, hungary, ireland, italy, latvia, lithuania, macedonia, poland, romania, russia (central, east, northwestern and south), slovakia, slovenia, spain, sweden, switzerland, netherlands, serbia. bracon (glabrobracon) peroculatus wesmael 1838 on cnephasia chrysantheana (tortricidae). distribution: belgium, finland, france, germany, hungary, italy, russia (central), serbia. bracon (glabrobracon) picticornis wesmael 1838 on clytus sp., plagionotus arcuatus (cerambicidae); nematus bipartitus, n. salicis, pontania pedunculi, p. proxima, p. vesicator, p. viminallis (tenthredinidae); cochylis pallidana (tortricidae). distribution: austria, belgium, great britain, bulgaria, turkey, finland, france, germany, greece, hungary, ireland, italy, lithuania, poland, russia (central, northwestern and south), slovakia, spain, sweden, switzerland, netherlands, serbia. bracon (glabrobracon) pineti thomson 1892 on ernobius abietis, e. longicornis (anobiidae); cydia strobilella (tortricidae). distribution: austria, bulgaria, croatia, czech republic, finland, germany, hungary, lithuania, moldova, poland, russia (central, north, northwestern and south), slovakia, sweden, serbia. bracon (glabrobracon) ratzeburgii dalla torre 1898 on ceutorhynchus maculaalba, hylesinus fraxini, scolytus rugulosus (curculionidae). distribution: great britain, germany, italy, serbia. biologica nyssana 3 (1)  september 2012: 21-29 žikić, v. et al.  checklist of the genus bracon (hymenoptera: braconidae)… 26 bracon (glabrobracon) terebella wesmael 1838 on miarus campanulae (curculionidae); cephus pygmaeus, trachelus tabidus (cephidae). distribution: austria, belgium, great britain, denmark, finland, germany, hungary, italy, lithuania, poland, romania, russia (central,northwestern and south), slovakia, sweden, switzerland, serbia. bracon (glabrobracon) titubans wesmael 1838 – no host data. distribution: austria, belgium, bulgaria, finland, france, germany, hungary, italy, poland, russia (northwestern), slovakia, sweden, switzerland, netherlands, serbia. bracon (glabrobracon) tornator marshall 1885 – no host data. distribution: austria, great britain, bulgaria, croatia, finland, germany, greece, hungary, italy, sweden, switzerland, serbia. bracon (glabrobracon) variator nees 1811 on bruchidius lividimanus, bruchus atomarius, b. laticollis, b. lentis, b. viciae (bruchidae); anthonomus pomorum, baris chlorizans, b. cuprirostris, b. laticollis, ceutorhynchus punctiger, gymnetron asellus, g. tetrum, larinus jaceae, l. turbinatus, magdalis rufa, miarus campanulae, pissodes validirostris, sibinia viscariae, sitona longulus (curculionidae); chaetostomella cylindrica, noeeta pupillata, sphenella marginata, tephritis leontodontis (tephritidae); hoplocampa brevis, h. flava (tenthrenidae); colephora coronillae, c. medelichenis (coleophoridae); mesophleps corsicella, pexicopia malvella, platyedra subcinerea, (gelechiidae); phyllonorycter mespilella (gracillariidae); hadena bicruris (noctuidae); dioryctria abietella, etiella zinckenella, myelois circumvoluta (pyralidae); synathedon andrenaeformis (sesiidae); barbara herrichiana, cydia strobilella, eucosma cana, grapholita funebrana, g. lunulana, pandemis cerasana, retinia resinella (tortricidae). distribution: austria, belgium, bosnia and herzegovina, great britain, bulgaria, canary is., croatia, cyprus, czech republic, turkey, finland, france, germany, greece, hungary, ireland, italy, latvia, lithuania, macedonia, moldova, poland, romania, russia (central, east, north, northwestern and south), slovakia, slovenia, spain, sweden, switzerland, netherlands, ukraine, serbia. bracon (habrobracon) brevicornis wesmael 1838 on sesamia cretica (noctuidae). distribution: belgium, great britain, bulgaria, czech republic, france, germany, hungary, italy, poland, portugal, romania, russia (central and south), slovakia, spain, switzerland, netherlands, serbia. bracon (habrobracon) hebetor say 1836 on ostrinia nubilalis (crambidae); pexicopia malvella (gelechiidae); helicoverpa armigera, heliothis peltigera, spodoptera littoralis (noctuidae); ephestia elutella, e. kuehniella, etiella zinckenella, galleria mellonella, plodia interpunctella (pyralidae); adoxophyes orana, lobesia botrana (tortricidae). distribution: austria, belgium, great britain, bulgaria, croatia, cyprus, czech republic, turkey, finland, france, germany, greece, hungary, italy, lithuania, macedonia, poland, russia (central and south), slovakia, slovenia, spain, netherlands, serbia. bracon (lucobracon) apricus schmiedeknecht 1897 – no host data. distribution: germany, greece, serbia. bracon (lucobracon) exarator marshall 1885 – no host data. distribution: great britain, germany, serbia. bracon (lucobracon) femoralis brullé, – no host data. distribution: cyprus, greece, hungary, spain, switzerland, serbia. bracon (lucobracon) fortipes wesmael 1838 on plagionotus floralis (cerambycidae); lixus anguinus (curculionidae); gortyna xanthenes (noctuidae); bembecia scopigera (sesiidae). distribution: belgium, bulgaria, croatia, cyprus, france, germany, greece, hungary, italy, poland, slovenia, switzerland, serbia. bracon (lucobracon) fumarius szepligeti 1901 – no host data. distribution: austria, croatia, cyprus, hungary, romania, serbia. bracon (lucobracon) hungaricus (szepligeti 1896) on clytus arietis (cerambycidae). distribution: bulgaria, croatia, turkey, hungary, russia (central and south), slovakia, ukraine, serbia. bracon (lucobracon) hylobii ratzeburg 1848 on hylobius abietis, h. piceus, pissodes harcyniae, p. notatus, p. piceae, p. pini, polygraphus polygraphus (curculionidae). distribution: austria, great britain, bulgaria, czech republic, denmark, germany, hungary, italy, lithuania, poland, russia (central and northwestern), sweden, switzerland, netherlands, serbia. bracon (lucobracon) megapterus wesmael 1838 on glaphyra umbellatarum (cerambycidae); chaetorellia jaceae (tephritidae). distribution: belgium, bulgaria, czech republic, finland, france, germany, sweden, serbia. bracon (lucobracon) nigriventris wesmael 1838 on plagionotus floralis (cerambycidae); hylobius piceus, pissodes pini (curculionidae). distribution: biologica nyssana 3 (1)  september 2012: 21-29 žikić, v. et al.  checklist of the genus bracon (hymenoptera: braconidae)… 27 albania, austria, belgium, bulgaria, croatia, cyprus, turkey, germany, hungary, italy, poland, romania, russia (south), slovakia, switzerland, ukraine, serbia. bracon (lucobracon) ochraceus szepligeti 1896 – no host data. distribution: croatia, hungary, russia (south), spain, switzerland, serbia. bracon (lucobracon) planiceps telenga 1936 – no host data. distribution: russia (north), serbia. bracon (lucobracon) romani fahringer 1927 on chamaesphecia bibioniformis, paranthrene tabaniformis, pennisetia hylaeiformis, sesia apiformis, s. bembeciformis, synanthedon culiciformis, s. spheciformis (sesiidae). distribution: greece, hungary, slovakia, slovenia, sweden, netherlands, serbia. bracon (lucobracon) thuringiacus schmiedeknecht 1896 – no host data. distribution: great britain, germany, greece, italy, serbia. bracon (lucobracon) triangularis nees 1834 on synanthedon tipuliformis (sesiidae). distribution: great britain, bulgaria, czech republic, finland, germany, hungary, ireland, italy, poland, slovakia, switzerland, netherlands, serbia. bracon (orthobracon) epitriptus marshall 1885 on cryptorhynchus lapathi, hylobius transversovittatus, pissodes spp. (curculionidae); agromyza flaviceps (agromyzidae); iteomyia capreae (cecidomyiidae); fenusa pumila (tenthredinidae). distribution: austria, great britain, bulgaria, germany, greece, hungary, italy, lithuania, poland, russia (central, northwestern and south), slovakia, slovenia, switzerland, netherlands, serbia. bracon (orthobracon) erraticus wesmael 1838 on gastrophysa viridula, phaedon cochleariae, phyllotreta nemorum (chrysomelidae); chaetostomella cylindrca, urophora eriolepidis, terellia serratulae (tephritidae); coleophora laricella, c. lutipennella (coleophoridae); metzneria lappella (gelechiidae); pennisetia hylaeiformis (sesiidae); acleris rhombana (tortricidae). distribution: austria, belgium, bosnia and herzegovina, great britain, bulgaria, croatia, cyprus, turkey, finland, france, germany, greece, hungary, ireland, italy, lithuania, macedonia, poland, russia (central, east, north, northwestern, and south), slovakia, slovenia, spain, sweden, switzerland, netherlands, ukraine, serbia. bracon (orthobracon) exhilarator nees 1834 on omphalapion hookerorum (apionidae); nanna sp. (scatophagidae); acleris rhombana (tortricidae). distribution: belgium, great britain, bulgaria, cyprus, turkey, finland, france, germany, hungary, italy, latvia, lithuania, norway, poland, russia (central, north, northwestern and south), slovakia, sweden, switzerland, netherlands, serbia. bracon (orthobracon) laevigatissimus dalla torre 1898 on plagionotus arcuatus (cerambycidae); pontania pedunculi, p. proxima, p. vesicator, p. viminalis (tenthredinidae). distribution: austria, great britain, finland, france, germany, italy, poland, romania, switzerland, netherlands, serbia. bracon (orthobracon) ochropus nees 1834 – no host data. distribution: austria, france, germany, italy, serbia. bracon (orthobracon) roberti wesmael 1838 on synanthedon andrenaeformis (sesiidae); cydia strobilella (tortricidae). distribution: austria, belgium, great britain, turkey, france, germany, ireland, russia (northwestern), netherlands, serbia. bracon (orthobracon) subcylindricus wesmael 1838 – no host data. distribution: belgium, great britain, france, poland, russia (northwestern), serbia. bracon (orthobracon) tenuicornis wesmael 1838 on phloetribus scarabaeoides (curculionidae). distribution: belgium, great britain, france, hungary, russia (south), sweden, netherlands, serbia. bracon (striobracon) idrianus fahringer 1934 on hylesinus oleiperda (curculionidae). distribution: serbia. discussion the present account is the first summary of all available data about the genus bracon and its hosts until now on the territory of serbia. out of total number 250 bracon species in europe, 80 species are present in serbian fauna. we have registered 290 hosts for 55 parasitoid species connected in 409 trophic associations. the hosts come from four insect orders. the most frequent hosts belong to lepidoptera and coleoptera, whereas diptera and hymenoptera are less parasitized. numbers in brackets refer to the number of registered host species. 1. diptera (28) with five families: agromyzidae (2); chloropidae (3); cecidomyiidae (1) scatophagidae (1) and tephritidae (21). 2. coleoptera (109) with nine families: anobiidae (3); apionidae (5); bruchidae (5); buprestidae biologica nyssana 3 (1)  september 2012: 21-29 žikić, v. et al.  checklist of the genus bracon (hymenoptera: braconidae)… 28 (7); cerambycidae (12); chrysomelidae (6); curculionidae (66); phalacridae (1) and rhynchitidae (4). 3. hymenoptera (26) with four families: cephidae (2); cynipidae (1); eurytomidae (3) and tenthredinidae (20). 4. lepidoptera (128) with 21 families: agonoxenidae (1); choreutidae (1); coleophoridae (37); cosmopterigidae (1); crambidae (1); depressariidae (1); elachistidae (1); epermeniidae (1); gelechiidae (11); geometridae (1); gracillariidae (4); momphidae (1); nepticulidae (1); noctuidae (5); oecophoridae (1); psychidae (1); pyralidae (9); sesiidae (14); tortricidae (24); yponomeutidae (6) and zygaenidae (1). the most suitable host species in these trophic relations parasitoid-host, belongs to the coleopteran family curculionidae and lepidopteran family coleophoridae especially the genus coleophora which has 11 parasitoids registered in this work. the torticid moth cydia strobilella has remarkably wide range of parasitoids, six bracon species were registered here as parasitoids of this moth (b. (bracon) variegator, b. (glabrobracon) caudiger, b. (glabrobracon) delibator, b. (glabrobracon) pineti, b. (glabrobracon) variator and b. (orthobracon) roberti). we noted that bracon idrianus is present only on the territory of serbia. this basic research shows better insight into the trophic interactions about each parasitoid species which can help in further biological control programs. nonetheless, it can ease the identification of some parasitoid species according to their hosts. acknowledgements. the authors wish to express their deepest gratitude to dr. sergey a. belokobylskij from the zoological institute ras (st. petersburg, russia) and dr. jenő papp from the natural history museum of budapest, hungary for the identification of parasitoid specimens and to dr. aleksandar ćetković from the faculty of biology, university of belgrade for the loan of some specimens from his collection. for the present work the authors were supported by the ministry of science and environment protection of the republic of serbia (grant no. iii43001). references achterberg, c. van 2012: fauna europaea: braconidae. in: van achterberg (ed.): fauna europaea: hymenoptera: symphyta + ichneumonoidea. fauna europaea, version 2.5. http://www.faunaeur.org achterberg, c. van, hemminga, m.a., soelen, j. van 1990: new host record of bracon intercessor nees f. megasomides strand (hymenoptera: braconidae), a parasite of agapanthia villosoviridescens degeer (coleoptera: cerambycidae) in salt marshes. zoologische mededelingen, 64 (3): 25-29. achterberg, c. van. 1985: notes on braconidae iiv. zoologische mededelingen, 59 (15): 163167. baird, a.b. 1958: biological control of insects and plant pests in canada. in: baecker, e.c., (ed.), proceedings of 10 th international congress of entomologist, ottawa, 4: 483-485. belokobylskij, s.a., žikić, v. 2009: new data on cyclostome braconid subfamilies doryctinae, exothecinae, rogadinae and braconinae (braconidae: hymenoptera) of serbia and neighbouring territories. acta entomologica serbica, 14 (1): 65-71. beyarslan, a., çetin erdoğan, ö., aydoğdu, m. 2005: a survey of braconinae (hymenoptera, braconidae) of turkish western black sea region. linzer biologische beiträge, 37 (1): 195–213. brajković, m., 1989: knowledge of the braconidae (hymenoptera) fauna in yugoslavia. glasnik prirodnjačkog muzeja, b (43/44): 127-138. brajković, m., krunić, m., tomanović, ž., 1991: a contribution to the braconid fauna (braconidae: hymenoptera) of yugoslavia. in: hawksworth, d.l. (ed.): proceedings of the fourth european congress of entomology and the xiii internationale symposium für entomofaunistik mitteleuropas, hungarian natural history museum, budapest, (2): 424-428. brajković, m., krunić, m., tomanović, ž., 1994: research of braconids (braconidae: hymenoptera) of deliblatska peščara. in: marinković, p. (ed.): proceedings of deliblatski pesak, srbijašume, 6: 197-502 dweck, h.k., svensson, g.p., gündüz, e.a., anderbrant, o. 2010: kairomonal response of the parasitoid, bracon hebetor say, to the maleproduced sex pheromone of its host, the greater waxmoth, galleria mellonella (l.). journal of chemical ecology, 36: 171-178. fischer, m. 1959: neue und wenig bekannte braconiden aus jugoslawien (hymenoptera). acta musei macedonici scientiarum naturalium, 6 (1): 53: 1-25. georgiev, g. 2005: bioecological characteristics of bracon intercessor nees (hymenoptera: braconidae) as a parasitoid of the poplar clearwing moth, paranthrene tabaniformis http://www.faunaeur.org/full_results.php?id=11049 biologica nyssana 3 (1)  september 2012: 21-29 žikić, v. et al.  checklist of the genus bracon (hymenoptera: braconidae)… 29 (rott.) (lepidoptera: sesiidae) in bulgaria. journal of pest science, 78: 161-165. lewis, w.j., vet, l.e.m., tumlinson, j.h., van lenteren, j.c., papaj, d.r. 1990: variations in parasitoid foraging behavior: essential element of a sound biological control theory. environmental entomology, 19: 1183-1193. mathews, r.w. 1974: biology of braconidae. annual review of entomology, 19: 15-32. milonas, p.g. 2005: influence of initial egg density and host size on the development of the gregarious parasitoid bracon hebetor on three different host species. biocontrol, 50: 415-428. papp, j. 1973: contributions to the braconid fauna of yugoslavia (hymenoptera, braconidae) i. acta musei macedonici scientarium naturalium, skopje, 14 (1: 119): 1-23. papp, j. 1977: contribution to the braconid fauna of yugoslavia. (hymenoptera: braconidae) iii. folia entomologica hungarica (series nova), 30: 105-117. papp, j. 2008: a revision of the bracon (subgenera bracon s. str., cyanopterobracon, glabrobracon, lucobracon, osculobracon, subgen. n., pigeria) species described by szépligeti from the western palaearctic region. biologia, 65 (1): 110-112. reinert, j.a., king, e.w. 1971. action of bracon hehetor say as a parasite of plodia interpunctella at controlled densities. annals of the entomological society of america, 64: 1335 1340. shaw, m.r. and huddleston, t. 1991: classification and biology of braconid wasps (hymenoptera: braconidae). royal entomological society of london, london. 126p. stanković, s., žikić, v., ilić, m. 2010: betula species as host plants for various insects parasitized by braconids (hymenoptera: braconidae) in serbia. biologica nyssana, 1 (12): 117-122. taylor, a.d. 1988a: host effects on larval competition in the gregarious parasitoid bracon hehetor. journal of animal ecology, 57: 163172. taylor, a.d. 1988b: host effects on functional and ovipositional responses of bracon hebetor. journal of animal ecology 57: 173-184. temerak, a.s. 1982: laboruntersuchungen zur wirtshabitat-wahl von bracon brevicornis wesm. (hym., braconidae), einem parasitoiden der raupen von sesamia cretica led. (lep., noctuidae) in sorghum-stengeln. anzeiger für schädlingskunde, berlin und hamburg, 55: 152154. tobias, v.i., belokobylskij, s.a., kotenko, a.g. 1986: family braconidae. in: medvedev g.s. (ed.). a key to insects of the european part of the ussr. hymenoptera 3: 4. leningrad: nauka. 500 p. [in russian]. yu, d.s., achterberg, c. van, horstmann, k. 2012: world ichneumonoidea 2011. taxonomy, biology, morphology and distribution (braconidae). taxapad (scientific names for information management) interactive catalogue on dvd/cdrom. vancouver. yu, s.-h., ryoo, m.ii., na, j.h. 1999: life history of bracon hebetor (hymenoptera: braconidae) on plodia interpunctella (lepidoptera: pyralidae) on a dried vegetable commodity. journal of asia-pacific entomology, 2 (2): 149-152. žikić, v., achterberg, c. van, stanković, s. 2010: a contribution to braconidae, hybrizontidae (ichneumonoidea: hymenoptera) and stephanidae (stephanoidea: hymenoptera) from the southwest balkans. acta entomologica serbica, 15 (2): 227-235. žikić, v., brajković, m., tomanović, ž. 2000: preliminary results of braconid fauna research (braconidae: hymenoptera). acta entomologica serbica, 5 (1/2): 95-111. žikić, v., stanković, s.s., ilić, m., kavallieratos, n.g. 2012: braconid parasitoids (hymenoptera: braconidae) on poplars and aspen (populus spp.) in serbia and montenegro. north-western journal of zoology [in press]. biologica nyssana 3 (1)  september 2012: 21-29 žikić, v. et al.  checklist of the genus bracon (hymenoptera: braconidae)… 30 damjanović-vratnica, b. et al.  composition and antimicrobial studies… biologica nyssana 6 (2)  december 2015: 67-73 damjanović-vratnica, b. et al.  composition and antimicrobial studies… 67 original article received: 23 november 2015 revised: 17 december 2015 accepted: 20 december 2015 composition and antimicrobial studies of essential oil of thymus vulgaris from montenegro biljana damjanović-vratnica1*, danka caković2, svetlana perović2 1faculty of metallurgy and technology, university of montenegro, bul. džordža vašingtona bb., 81000 podgorica, montenegro 2faculty of natural sciences and mathematics, university of montenegro, bul. džordža vašingtona bb., 81000 podgorica, montenegro * e-mail: biljanad@ac.me abstract: damjanović-vratnica, b., caković, d., perović, s.: composition and antimicrobial studies of essential oil of thymus vulgaris from montenegro. biologica nyssana, 6 (2), december 2015: 67-73. chemical composition of the hydrodistilled essential oil of thymus vulgaris l. (thyme) from montenegro was analyzed by gas chromatography-mass spectrometry and its antimicrobial activity was evaluated against 10 microorganisms, including reference and clinically isolated strains. t. vulgaris essential oil yield was 0.42% (v/w, based on the dry leaves weight) whereas the analysis showed that major components, amongst 22 identified in the oil, were geraniol (25.66%), geranyl-acetate (20.34%), linalool (10.89%) and caryophyllene oxide (9.89%). the results of the antimicrobial activity tests revealed that the essential oil of t .vulgaris from montenegro has rather strong antimicrobial activity, especially against staphylococcus aureus, escherichia coli, candida albicans and klebsiella pneumoniae. these results confirm the potential use of t. vulgaris essential oil in food products the as well as for therapeutic applications. key words: thymus vulgaris l., essential oil, chemical composition, antimicrobial activity apstrakt: damjanović-vratnica, b., caković, d., perović, s.: sastav i antibakterijska studija etarskog ulja thymus vulgaris iz crne gore. biologica nyssana, 6 (2), december 2015: 67-73. hemijski sastav etarskog ulja thymus vulgaris l. (timijan) sa područja crne gore analiziran je gasno-masenom spektrometrijom i ispitano je antimikrobno dejstvo ulja na 10 mikroorganizama, uključujući referentne i klinički izolovane sojeve. prinos etarskog ulja t. vulgaris bio je 0.42% (v/w, računato na težinu suvog lišća), dok su, od ukupno 22 identifikovanih komponenti, najzastupljenije: geraniol (25.66%), geranil acetat (20.34%), linalol (10.89%) i kariofilen oksid (9.89%). dobijeni rezultati antimikrobnog ispitivanja pokazuju da etarsko ulje t. vulgaris sa područja crne gore ima prilično snažno antimikrobno dejstvo, posebno na staphylococcus aureus, escherichia coli, candida albicans i klebsiella pneumoniae. ovi rezultati potvrđuju mogućnost korišćenja etarskog ulja t. vulgaris u prehrambenoj industriji kao i u terapeutske svrhe. key words: thymus vulgaris l., etarsko ulje, hemijski sastav, antimikrobna aktivnost 6 (2) • december 2015: 67-73 biologica nyssana 6 (2)  december 2015: 67-73 damjanović-vratnica, b. et al.  composition and antimicrobial studies… 68 introduction the genus thymus, member of the lamiaceae family, contains about 400 species of perennial aromatic, evergreen or semi-evergreen herbaceous plants with many subspecies, varieties, subvarieties and forms (d e m a r t i n o et al., 2009). t. vulgaris, also known as common thyme, is indigenous in the mediterranean region and has long history as a source of the essential oil (thyme oil) and other constituents (e.g. thymol, flavonoids, caffeic acid and labiatic acid) derived from the different parts of the plant (hudaib et al., 2002). it has been grown commercially in a number of countries for the production of the dried leaves, thyme oil, thyme extracts, and oleoresins. thyme oil is among the world’s top 10 used essential oils, which are also utilized as a preservative for food. thymus species are commonly used as herbal tea, flavoring agents (condiments and spices) and for medicinal purposes (s t a h l b i s k u p & s a e z , 2002). studies have found that the main chemical compositions and the concentrations of essential oil of thyme are highly variable (j o r d á n et al., 2006). seven thyme chemotypes have been described whose principal volatile components are: 1,8-cineole, lanolool, α-terpineol, geraniol, trans-thujan-4-ol, terpinen-4-ol, thymol and carvacrol (d i a z m a r o t o et al., 2006). many researchers investigated chemical composition and biological properties of t. vulgaris essential oil and extracts from various origins (c o s e n t i n o et al., 1999; l e t c h a m o et al., 1999; d o r m a n & d e a n s , 2000; a z a z et al., 2004; d e b u r t , 2004; h u d a i b & a b u r j a i , 2007; b a k k a l i et al., 2008; s o k o v i ć et al., 2008; i m e l o u a n e et al., 2009; l i s i et al., 2011; r o b y et al., 2013; s t o j k o v i ć et al., 2013; b o r u g ă et al., 2014; m a r t i n s et al., 2015). it was found that, both the isolation yield and the chemical composition of the eos can be influenced by environmental and management factors such as temperature, water stress, soil fertility, light, pest pressure, cutting date and plant maturity (l e t c h a m o et al., 1999). in the mediterranean environment, there are several ecotypes of wild-growing thyme, which differ in morphological characteristics, distinguished by a strong and penetrating odor and sometimes a very evident balsamic and spicy flavor (d e l i s i at al., 2011). the genus thymus is represented by 15 wildgrowing species in the flora of montenegro (r o h l e n a , 1942; p u l e v i ć , 2005). essential oils composition of two species were determined (c o u l a d i s et al., 2004; s l a v o v s k a et al., 2006), but there are no studies on the chemical profile of the best known species of this genus. the aim of this study is to determine the chemical composition together with the antimicrobial properties of the essential oil from leaves of cultivated t. vulgaris l. from montenegro, as natural sources of antiseptics with potential applications in the pharmaceutical and food industry. material and methods preparation of herb material: fresh leaves of t. vulgaris were collected manually from the collection site in the podgorica region (komani n 42°27'43.40'', e 19°06'27.46''; central part of montenegro) in june 2014. the initial water inherent in the herb leaves found to be 8.9% (w/w) using a dean and stark apparatus with n-heptane as the reflux solvent. herb material was milled in a domestic coffee mill and, after sieving in erweka set of sieves, sample with a mean particle diameter size of 0.9 mm was obtained. a prepared batch was kept in an airtight resalable polypropylene bag and stored at 6 ºc for maximum 3 days before use, in order to avoid losses of volatile compounds. essential oil preparation: herb material (70 g) was submitted to hydrodistillation in a clevenger-type apparatus for 2 hours according to yugoslav pharmacopoeia iv. the obtained oil was dried over anhydrous sodium sulphate, measured, poured in hermetically sealed dark-glass containers and stored in refrigerator at 4 c until analyzed by gc-ms. gas chromatography mass spectrometry (gcms): the gc-ms analyses were carried out using a shimadzu 2010+ gas chromatograph-mass spectrometer equipped with a zb-5 ms (30 m x 0,25 mm x 0,25 μm) capillary column. the column temperature was programmed from 35 c (5 min) to 300 c at 5 c/min. the injection port temperature was 260 c, while the interface temperature was 305 c. the samples of oil were injected by splitting and the split ratio was adjusted to 1:100. helium was used as the carrier gas at a flow rate of 1.2 ml/min and 61.8 kpa inlet pressure. the ms conditions were: the ionisation voltage 70 ev, scanning interval 1.5 s, detector voltage 1.0 kv and m/z range 40 500. the components were identified by comparing their mass spectral data with those in the wiley229 and the nist107 mass spectra libraries, as well as by comparison of the fragmentation patterns of the mass spectra with those reported in the literature and whenever possible, by co-injection with authentic standards (fluka, great britain). biologica nyssana 6 (2)  december 2015: 67-73 damjanović-vratnica, b. et al.  composition and antimicrobial studies… 69 microbial strains: in order to evaluate the activity of the essential oil of t. vulgaris, the following microorganisms were used: reference strains staphylococcus aureus atcc 25923, escherichia coli atcc 25922, pseudomonas aeruginosa atcc 27853 and candida albicans atcc 10231 (torlak, belgrade); and clinical isolates of staphylococcus aureus, escherichia coli, pseudomonas aeruginosa, klebsiella pneumoniae and candida albicans. the microorganisms were isolated from clinically treated or hospitalized patients of medical health centre (podgorica). antimicrobial screening: the agar disc diffusion method was employed to determine the antimicrobial activity of the essential oil (e r e l et al., 2012). briefly, fresh overnight cultures of the tested microorganism were adjusted with sterile saline to a concentration of approximately 1106 cfu/ml (spectrophotometry was used to determinate optical density of cultures) and spread on the solid media plates. the above-mentioned bacteria were cultured on nutrient broth (torlak, belgrade) at 370.1 c while the fungi was grown on sabouraud dextrose broth (merck, germany) at 200.1 c. filter paper discs (6 mm in diameter) were individually impregnated with undiluted t. vulgaris essential oil (4.5, 9 and 18 g) and placed on the incubated plates. the plates were kept at room temperature for 30 min and then incubated at 37 c for 20 h (for bacterial strains) and 30 c for 72 h (for fungi). reading of the results was carried out by measuring diameters of zones of inhibition, in mm. in addition, reference antibiotic discs (provided by the institute for serums, vaccines and diagnostic preparations torlak, belgrade): ampicillin (10 g), ceftriaxone (30 g), erythromycin (15 g), amykacine (30 g), tetracycline (30 g), for bacteria and nystatin for fungi (100 g), were used for comparison, at the same condition as in the essential oil experiment. all tests of inhibitory activity were carried out in duplicate and the developing inhibition zones were compared with those of reference disks. the essential oil was also subjected to the test of sterility and was found to be free of microorganisms. results and discussion hydrodistillation of the leaves of t. vulgaris yielded 0.42% of essential oil (v/w, based on the dry weight of the adult leaves) with a spicy aromatic odor. the results obtained are higher than essential oil yield reported in the literature 0.09% in wild growing thyme in southern italy (m a n c i n i et al., 2015), and rather similar to those reported in the literature where yield was 0.15-1.2% in cultivated thyme from italy (h u d a i b et al., 2002) and 0.75% in cultivated thyme in iran (p i r b a l o u t i et al., 2013). obtained essential oil yield was smaller compared to the results reported in the literature where yield was 0.9% in wild-growing thyme in romania (g r i g o r e et al., 2010), 1.0% in wild-growing thyme in morocco (i m e l o u a n e et al., 2009), and significantly smaller compared to yield of 2.0% in wild-growing thyme in spain (a r r a i z a et al., 2009) and 3.7-5.6% and 1.1-2. 0% in wild-growing and cultivated thyme, respectively, in jordan (h u d a i b & a b u r j a i , 2007). table1. chemical composition of t. vulgaris essential oil (mi method of identification) no compound % mi* 1 α-pinene 0.15 2,3 2 camphene 0.23 2,3 3 sabinene 0.9 2,3 4 -pinene tr 2,3 5 α-terpenine 0.43 2 6 p-cymene 1.67 2,3 7 cis-ocimene 0.22 2 8 γ-terpenene 0.69 2,3 9 linalool 10.89 1,2,3 10 camphor 0.28 2,3 11 borneol 1.21 2,3 12 terpene-4-ol 5.21 1,2,3 13 α-terpineol 0.34 2,3 14 nerol 2.75 2,3 15 neral 0.48 2 16 geraniol 25.66 1,2,3 17 geranial 2.82 2,3 18 carvacrol 0.78 2 19 thymol 0.08 2,3 19 neryl-acetate 1.12 2 20 geranyl-acetate 20.34 1,2,3 21 -caryophyllene 1.05 2,3 22 bicyclogermacrene tr 2 23 elemol 5.36 2,3 24 caryophyllene oxide 9.89 1,2,3 25 viridiflorol 0.52 2,3 26 -eudesmol 2.87 2,3 total 95.94 monoterpene hydrocarbons 4.29 oxygenated monoterpenes 71.96 sesquiterpene hydrocarbons 1.05 oxygenated sesquiterpenes 18.64 * 1 – co-injection with authentic compounds; 2 – ms; 3 – literature comparison; tr < 0.1 it is acknowledged that yield and chemical composition of herbal extracts are determined by a series of factors including herb genetic, climate, elevation, and topography as well as by interaction of various factors (p i r b a o i l i t i et al., 2013). thus, biologica nyssana 6 (2)  december 2015: 67-73 damjanović-vratnica, b. et al.  composition and antimicrobial studies… 70 yield and composition of aromatic plants essential oil are influenced by harvesting time, ecological and climatic conditions. it was previously found that many species of the thymus genus present different intraspecific chemotypes and that chemical composition of the essential oils is variable in relation to the harvesting time, to the stage of development of the plant, and to the field environmental conditions (d e l i s i et al., 2011). the chemical composition of the hydrodistilled thyme essential oil is shown in tab. 1. gc-ms analyses revealed the presence of 26 compounds representing 95.94% of the total oil. the major components were geraniol (25.66%), geranyl-acetate (20.34%), linalool (10.89%) and caryophyllene oxide (9.89%). other major compounds (1.0% of the identified portion) in the gained oil were geranial (2.82%), elemol (5.36%), terpinen-4-ol (5.21%), -eudesmol (2.87%), nerol (2.75%), p-cymene (1.67%), caryophyllene (1.05%), borneol (1.21%) and nerylacetate (1.12%). it was previously reported than t. vulgaris species has seven genetically distinct chemotypes that can be distinguished on the basis of the dominant monoterpene produced in glandular trichomes on the surface of the leaves (d i a z m a r o t o et al., 2006). in southern france, six chemotypes were found and named after its dominant monoterpene: geraniol (g), α-terpineol (a), thuyanol-4 (u), linalool (l), carvacrol (c), and thymol (t) (t h o m p s o n et al., 2003). the six monoterpenes are all produced from geranyl pyrophosphate through a series of changes in configuration and have quite similar molecular structures. a major distinction is the phenolic nature of carvacrol and thymol, and the nonphenolic nature of the four other monoterpenes. it was found that the mean values of the proportion of dominant monoterpenes in the α-terpineol, geraniol, and linalool chemotypes exceeded those of the thuyanol, carvacrol, and thymol chemotypes (t h o m p s o n et al., 2003). m a n c i n i et al. (2015) found that the most abundant compounds in t. vulgaris from southern italy were thymol (46.2%–67.5%), carvacrol (5.7%–7.3%) and caryophyllene oxide (1.7%–7.3%), however de lisi et al. (2011) reported that the predominant compounds of essential oil were geraniol, thymol and linalool (d e l i s i et al., 2011). o z c a n & c h a l c h a t (2004) found that essential oil from wild-growing t. vulgaris in turkey has high content of thymol (46.2%). it was previously reported (i m e l o u a n e et al., 2009) that the oil of t. vulgaris from morocco contained camphor (38.54%), camphene (17.19%), α-pinene (9.35%) while thyme oil from jordan was characterized by high content of phenolic monoterpenoids (mainly thymol and carvacrol) in the range 70.8-89.0% (h u d a i b & a b u r j a i , 2007). g r o s s o et al. (2010) found that in thyme essential oil from spain the most abundant compounds were thymol (35.441.6%) and p-cymene (28.9-34.8%). chemical composition of obtained t. vulgaris essential oil from montenegro showed that the most abundant compounds were geraniol and geranylacetate, thus examined t. vulgaris belongs to chemotype geraniol. the presence of phenolic compounds thymol and carvacrol in the oil was insignificant, while content of p-cymene was only 1.58%, which significantly deferred from most results from literature survey. the thyme oil from montenegro consisted mostly of oxygenated monoterpenes (71.96%) and oxygenated sesquiterpenes (18.64%). table 2. antimicrobial activity of the t. globulus essential oil and some standard antibiotics inhibition zone (mm)* t. vulgaris oil standard antibiotics (g)** (g) amp ctr er amyx te ny microorganism 4.5 9 18 10 30 15 30 30 100 staphylococcus aureus 28 37 50 22 14 31 24 13 s. aureus atcc 25923 30 43 56 26 32 31 28 40 escherichia coli 29 37 43 36 26 e. coli atcc 25922 34 43 56 27 34 22 30 29 pseudomonas aeruginosa 14 17 16 21 16 p. aeruginosa atcc 27853 18 22 26 19 31 candida albicans 31 35 41 18 c. albicans atcc 10231 33 37 43 20 klebsiella pneumoniae 38 41 52 15 35 15 26 25 * includes diameter of disc; (-) not active; **amp: ampicillin; ctr: ceftriaxone; er: erythromycin; amyk: amykacine; te: tetracycline; ny: nystatin. biologica nyssana 6 (2)  december 2015: 67-73 damjanović-vratnica, b. et al.  composition and antimicrobial studies… 71 the antimicrobial activity of t. vulgaris essential oil is due to the presence of a mixture of monoterpenes and oxygenated monoterpenes and sesquiterpenes (most of the antimicrobial activity in the oils has been attributed to the oxygenated monoterpenes). identification of such compounds with wide biological activity is critical for mankind. it helps in the search for chemical structures that should assist in designing new drugs as therapeautics against human pathogens (d a m j a n o v i ć v r a t n i c a et al., 2011).the antimicrobial plate diffusion assay for thymus vulgaris essential oil, as summarized in the tab. 2, showed that different microorganisms tested had different susceptibility to the same essential oil. the essential oil activities against tested microorganisms were increased in both cases (for atcc strains and clinically isolated strains) with increased amount of investigated essential oil. thyme oil was very potent against all chosen microorganisms, except pseudomonas aeruginosa (at the lowest oil concentrations), whether as clinically isolated strain or as atcc strain. since pseudomonas species are known to have ability to metabolize a wide range of organic compounds and for this fact is used extensively in bioremediation, this may explain their high level of resistance. they may simply metabolize the compounds in the oils that are inhibitory to many of the other bacteria (chao et al., 2000). because of that fact, the obtained activity of 18 g of thyme oil against p. aeruginosa for clinically isolated and atcc strains is noteworthy (17 and 22 mm, respectively). for tested atcc strains, t. vulgaris essential oil showed the strongest antimicrobial activity against e. coli atcc 25922, followed by s. aureus atcc 25923 and fungi c. albicans atcc 10231. regarding clinically isolated bacterial strains, the highest inhibition zone values were observed against medically important pathogens staphylococcus aureus and escherichia coli, ranged from 28 to 50 mm and from 29 to 43 mm, respectively. for comparison, used standard antibiotics diameters of growth inhibition zones ranged from 13 to 31 mm (for s. aureus) and 26 to 36 mm (for e. coli). very high antibacterial activity thyme essential oil showed against klebsiella pneumoniae, which were significantly susceptible to the essential oil at concentration of 9 and 18 g, with significant diameters of growth inhibition zones (29 and 38 mm), respectively. oil exhibited very strong activity against fungi candida albicans at all concentrations, which could be significant since c. albicans invades different areas of the human body causing cutaneous, mucocutaneous and opportunistic infections. in our experiment, diameters of growth inhibition zones ranged from 29 to 40 mm, giving two times higher effect in comparison to nystatin. it is probably due to the high amount of monoterpene alcohols and aldehydes, already known as components that inhibit the growth of fungi, especially c. albicans (d a l l e a u at al., 2008; l e i t e et al., 2015). most of the antimicrobial activity of the essential oils has been attributed to the oxygenated monoterpenes (b a k k a l i et al., 2008). in the literature, it was reported that various chemical compounds have direct activity against many species of bacteria, such as terpenes and a variety of aliphatic hydrocarbons (alcohols, aldehydes and ketones). the lipophilic character of their hydrocarbon skeleton and the hydrophilic character of their functional groups are of the main importance in the antimicrobial action of essential oils components (m a n c i n i et al., 2015). in addition, the components present in lower amount in thyme essential oil, such as p-cymene, carvacrol, camphor and terpinen-4-ol, could also contribute to the antimicrobial activity of the oil. it was previously found that terpinen-4-ol exhibits very high antimicrobial effect (b a r e l et al., 1991, c a r s o n & r i l e y , 1995; c a r s o n et al., 2006; p a z y a r et al., 2013). the antimicrobial activity of the thyme essential oil could also be associated with presence of borneol and linalool, well-known chemicals with their pronounced antimicrobial properties (v i l j o e n et al., 2003). in fact, it is also possible that the components which are present in lower amount might be involved in some type of synergism with the other active compounds. antibacterial activity of essential oils from many herb species has been extensively surveyed in last decades (r i o s & r e c i o , 2005), but their antimicrobial mechanism has not been reported in excessive details. it was found that most active antimicrobial compounds of essential oils are terpenes and phenolics and, thus, their mode of action might be similar to that of other phenolic compounds (s h u n y i n g et al., 2005). individual essential oil contains complex mixtures of such compounds however, a little is known about the effect of interaction between individual constituents on antimicrobial activity. interactions between constituents may lead to additive, synergistic or antagonistic effects (d e l a q u i s et al., 2002). conclusion the obtained results revealed that examined thymus vulgaris from montenegro belongs to chemotype geraniol. hydrodistillation of the leaves of t. vulgaris yielded 0.42% of essential oil (v/w, biologica nyssana 6 (2)  december 2015: 67-73 damjanović-vratnica, b. et al.  composition and antimicrobial studies… 72 based on the dry weight of the adult leaves) with a spicy aromatic odor. the major components, identified by gc-ms, were geraniol (25.66%), geranyl-acetate (20.34%), linalool (10.89%) and caryophyllene oxide (9.89%). this study has shown that t. vulgaris essential oil possesses significant activity against different microorganisms, including human pathogens, food poisoning and spoilage bacteria and blastomycete opportunistic fungi c. albicans. these results confirm the potential use of t. vulgaris essential oil in food products as well as for therapeutic applications. references arraiza, m.p., andrés, m.p., arrabal, c., lópez, j.v. 2009: seasonal variation of essential oil yield and composition of thyme (thymus vulgaris l.) grown in castilla-la mancha (central spain). journal of essential oil research, 21 (4): 360-362. azaz, a.d., irtem, h.a., kurkcuoğlu, m., baser, k.h. 2004: composition and the in vitro antimicrobial activities of the essential oils of some thymus species. zeitschrift für naturforschung c, 59 (1-2): 75-80. bakkali, f., averbeck, s., averbeck, d., idaomar, m. 2008: biological effects of essential oil: a review. food and chemical toxicology, 46 (2): 446-475. barel, s., segal, r., yashphe, j. 1991: the antimicrobial activity of the essential oil from achillea fragrantissima. journal of ethnopharmacology, 33 (1-2): 187-191. borugă, o., jianu, c., mişcă, c., goleţ, i., gruia, a.t., horhat, f.g. 2014: thymus vulgaris essential oil: chemical composition and antimicrobial activity. journal of medicine and life, 19 (7): 56–60. burt, s. 2004: essential oils: their antibacterial properties and potential applications in foods a review. international journal of food microbiology, 94 (3): 223-253. carson, c.f., hammer, k.a. 1995: antimicrobial activity of the major components of the essential oil of melaleuca alternifolia. journal of applied bacteriology, 78(3): 264-9. carson, c.f., hammer, k.a., riley, t.v. 2006: melaleuca alternifolia (tea tree) oil: a review of antimicrobial and other medicinal properties. clinical microbiology reviews, 19(1): 50–62. chao, s.c., young, d.g., oberg, c.j. 2000: screening for inhibitory activity of essential oils on selected bacteria, fungi and viruses. journal of essential oil research, 12 (5): 639-649. cosentino, s., tuberoso, c.i.g., pisano, b., satta, m., mascia, v., arzedi, e., palmas, f. 1999: in vitro antimicrobial activity and chemical composition of sardinian thymus essential oils. letters in applied microbiology, 29 (2): 130-135. couladis, m., tzakou, o., kujundzić, s., soković, m., mimica-dukić, n. 2004: chemical analysis and antifungal activity of thymus striatus. phytotherapy research, 18 (1): 40-42. dalleau, s., cateau, e., bergès, t., berjeaud, j.m., imbert, c. 2008: in vitro activity of terpenes against candida biofilms. international journal of antimicrobial agents, 31(6): 572–576. damjanović-vratnica, b., đakov, t., šuković, d., damjanović j. 2011: antimicrobial effect of essential oil isolated from eucalyptus globules labill. from montenegro. czech journal of food science, 29 (3): 277–284. delaquis, p.j., stanich, k., girard, b., mazza g. 2002: antimicrobial activity of individual and mixed fractions of dill, cilantro, coriander and eucalyptus essential oils. international journal of food microbiology, 74 (1-2): 101-109. de lisi, a., tedone, l., montesano, v., sarli, g., negro, d. 2011: chemical characterisation of thymus populations belonging from southern italy. food chemistry, 125 (4): 1284–1286. de martino, l., bruno, m., formisano, c., de feo, v., napolitano, f., rosselli, s., senatore, f. 2009: chemical composition and antimicrobial activity of the essential oils from two species of thymus growing wild in southern italy. molecules, 14 (11): 4614-4624. diaz-maroto, m.s., perez-coello, s., esteban, j., sanz, j. 2006: comparison of the volatile composition of wild fennel samples (foeniculum vulgare mill.) from central spain. journal of agriculture and food chemistry, 54 (18): 68146818. dorman, h.j.d., deans s.g. 2000: antimicrobial agents from plants: antibacterial activity of plant volatile oils. journal of applied microbiology, 88 (2): 308-316. erel, s.b., gottfried, r., şenol, s.g., yavaşoğul, n.k., konyalioğlu, s., zeybek a.u. 2012: antimicrobial and antioxidant properties of artemisia l. species from western anatolia. turkish journal of biology, 36(1): 75-84. grigore, a., paraschiv, i., colceru-mihul, s., bubueanu, c., draghici, e., ichim, m. 2010: chemical composition and antioxidant activity of thymus vulgaris l. volatile oil obtained by two different methods. romanian biotechnological letters, 15 (4): 5436-5443. grosso, c., figueiredo, a.c., burillo, j., mainar, a.m., urieta, j.s., barroso, j.g., coelho, j.a., palavra, a.m.f. 2010: composition and antioxidant activity of thymus vulgaris volatiles: comparison between supercritical fluid extraction biologica nyssana 6 (2)  december 2015: 67-73 damjanović-vratnica, b. et al.  composition and antimicrobial studies… 73 and hydrodistillation. journal of separation science, 33 (14): 2211-2218. hudaib, m., speroni, e., di pietra a. m, cavrini, v. 2002: gc/ms evaluation of thyme (thymus vulgaris l.) oil composition and variations during the vegetative cycle. journal of pharmaceutical and biomedical analysis, 29 (4): 691–700. hudaib, m., aburjai, t. 2007: volatile components of thymus vulgaris l. from wild-growing and cultivated plants in jordan. flavour and fragrance journal, 22 (4): 322–327. imelouane, b., amhamdi, h., wathelet, j.p., ankit, m., khedid k., el bachiri, a. 2009: chemical composition of the essential oil of thyme (thymus vulgaris) from eastern morocco. international journal of agriculture &. biology, 11 (2): 205– 208. jordán, m.j., martinez, r.m., goodner, k.l., baldwin, e.a., sotomayor, j.a. 2006: seasonal variation of thymus hyemalis lange and spanish thymus vulgaris l. essential oils composition. industrial crops and products, 24 (3): 253–263. letchamo, w., gosselin, a., hoelzl, j., marquard, r. 1999: the selection of thymus vulgaris cultivars to grow in canada. journal of essential oil research, 11 (3): 337–342. leite, m.c., de brito bezerra, a.p., de sousa, j.p., de oliveira lima, e. 2015: investigating the antifungal activity and mechanism(s) of geraniol against candida albicans strains. medical mycology, 53: 275-284. mancini, e., senatore, f., del monte, d., de martino, l., grulova d., scognamiglio m., snoussi, m., de feo, v. 2015: antimicrobial and antioxidant activities of five thymus vulgaris l. essential oils. molecules, 20: 12016-12028. martins, n., barros l., santos-buelga, c. silva, s., henriques, m., ferreira, i. 2015: decoction, infusion and hydroalcoholic extract of cultivated thyme: antioxidant and antibacterial activities, and phenolic characterisation. food chemistry, 167 (3): 131-137. pazyar, n., yaghoobi, r., bagherani, n. and kazerouni, a. 2013: a review of applications of tea tree oil in dermatology. international journal of dermatology, 52(7): 784–790. pirbalouti, a.g., hashemi, m., ghahfarokhi, f.t. 2013: essential oil and chemical compositions of wild and cultivated thymus daenensis celak and thymus vulgaris l. industrial crops and products, 48: 43– 48. pulević, v. 2005: građa za vaskularnu floru crne gore. dopuna “conspectus florae montenegrinae”. posebno izdanje republičkog zavoda za zaštitu prirode crne gore, podgorica. 218 p. rios, j.l., recio, m.c. 2005: medicinal plants and antimicrobial activity. journal of ethnopharmacology, 100 (1-2): 80-84. roby, m.h.h., sarhan, m. a., abdel-hamed selim, k., khalel, k. i. 2013: evaluation of antioxidant activity, total phenols and phenolic compounds in thyme (thymus vulgaris l.), sage (salvia officinalis l.), and marjoram (origanum majorana l.) extracts. industrial crops and products, 43: 827-831. rohlena, j. 1942: conspectus florae montenegrinae, preslia, xx-xxi, 506 p. shunying, z., yang, y., huaidong, y., yue, y., guolin, z. 2005: chemical composition and antimicrobial activity of the essential oils of chrysanthemum indicum. journal of ethnopharmacology, 96 (1-2): 151-158. slavovska, v., lakušić, b., jančić, r., mimicadukić, n., vujičić, đ. 2006: chemical composition of the essential oil of the thymus bracteosus vis. ex bentham (lamiaceae). journal of essential oil research, 18 (3): 310311. soković, m., glamočlija, ćirić, a., kataranovskia, d., marin, p.d., vukojević, j., brkić, d. 2008: antifungal activity of the essential oil of thymus vulgaris l. and thymol on experimentally induced dermatomycoses. drug development and industrial pharmacy, 34 (12): 1388-1393. stahl-biskup, e., saez, f., 2002: thyme, the genus thymus, taylor and francis, london, 331 p. stojković, d., glamočlija, j., ćirić, a., nikolić, m. 2013: investigation on antibacterial synergism of origanum vulgare and thymus vulgaris essential oils. archive of biological science belgrade, 65(2): 639-643. thompson, j.d., chalchat, j.c., michet, a., linhart, y.b., ehlers, b. 2003: qualitative and quantitative variation in monoterpene cooccurrence and composition in the essential oil of thymus vulgaris chemotypes. journal of chemical ecology, 29 (4): 859–880. viljoen, a., vuuren, s.v., ernst, e., klepser, m., demirci, b., baser, h., wyk, b.e.v. 2003: osmitopsis asteriscoides (asteraceae) the antimicrobial and essential oil composition of a cape-dutch remedy. journal of ethnopharmacology, 88 (2-3): 137-143. yugoslavian pharmacopoeia iv. (1984). national institute for health protection, belgrade, yugoslavia, vol. i: 126-128. biologica nyssana 6 (2)  december 2015: 67-73 damjanović-vratnica, b. et al.  composition and antimicrobial studies… 74 raca, i., ljubisavljević, i., jušković, m., ranđelović, n., ranđelović, v.: comparative anatomical study of the taxa from series verni mathew (crocus l.) in serbia. biologica nyssana, 8 (1), september 2017 biologica nyssana 8 (1)  september 2017: 15-22 raca, i. et al.  comparative anatomical study of the taxa from… 15 original article received: 27 june 2017 revised: 25 july 2017 accepted: 12 august 2017 comparative anatomical study of the taxa from series verni mathew (crocus l.) in serbia irena raca*, irena ljubisavljević, marina jušković, novica ranđelović, vladimir ranđelović university of niš, faculty of science and mathematics, department of biology and ecology, višegradska 33, niš, serbia * e-mail: raca.irena@gmail.com abstract: raca, i., ljubisavljević, i., jušković, m., ranđelović, n., ranđelović, v.: comparative anatomical study of the taxa from series verni mathew (crocus l.) in serbia. biologica nyssana, 8 (1), september 2017: 15-22. the comparative leaf anatomy of three crocus l. taxa from series verni mathew (crocus heuffelianus herb., crocus tommasinianus herbert and crocus kosaninii pulević) in serbia is introduced. the general outlook of the cross sections of the leaves was defined with microphotographs. more precisely, leaf shape, leaf surface, mesophyll parenchyma and vascular bundle features were examined. the biggest differences were found at the level of vascular bundles (xylem area, phloem area, sclerenchyma area) and leaf blade features (section height, section length, arm length, white stripe width, lacuna area). in general, listed parameters had the lowest values in c. tommasinianus population, while the highest values could be found in population of c. heuffelianus. further investigations should be focused on discovering more localities followed by surveying the ecological factors of the habitats. key words: crocus, series verni, cross section, leaf apstrakt: raca, i., ljubisavljević, i., jušković, m., ranđelović, n., ranđelović, v.: uporedna anatomska studija taksona serije verni mathew (crocus l.) u srbiji. biologica nyssana, 8 (1), septembar 2017: 15-22. komparativna anatomska analiza listova tri taksona roda crocus l. a serije verni mathew (crocus heuffelianus herb., crocus tommasinianus herbert i crocus kosaninii pulević) iz srbije prikazana je na mikrofotografijama. konkretnije, razmatrani su oblik poprečnog preseka lista, površinske strukture listova, lisni mezofil i karakteristike provodnih snopića. najveće razlike zabeležene su na nivou provodnih snopića (površina ksilema, floema i sklerenhima) i parametara koji se odnose na oblik preseka (visina i dužina preseka, dužina ručice, širina bele pruge, površina centralnog parenhima). generalno, navedeni parametri imaju najniže izmerene vrednosti u populaciji vrste c. tommasinianus, dok su najviše izmerene vrednosti zabeležene u populaciji vrste c. heuffelianus. komparativna anatomska analiza obezbeđuje dodatne informacije o karakterima značajnim za diferencijaciju vrsta. kako god, buduća istraživanja bi trebalo da budu fokusirana na pronalaženje novih lokaliteta populacija od interesa, kao i na praćenje ekoloških uslova njihovih habitata. ključne reči: crocus, serija verni, poprečni presek, list 8 (1) • september 2017: 15-22 doi: 10.5281/zenodo.963053 biologica nyssana 8 (1)  september 2017: 15-22 raca, i. et al.  comparative anatomical study of the taxa from… 16 introduction saffrons are known as “golden spice” and ornamentals (c a n d a n , 2007). moreover crocus species have antitumor, antimutagenic, cytotoxic activities and inhibitory effect on nucleic acid synthesis in human malignant cells (f a t e h i et al., 2003). as a matter of fact, the genus crocus contains more than 200 taxa (r u k š ā n s , 2017) from the west mediterranean to central asia with poland as northern limit of distribution. in the latest detailed revision of the genus (m a t h e w et al., 2009), the genus was divided into: section crocus mathew and section nudiscapus mathew. the balkan peninsula is known as one center of diversity (m a t h e w , 1982). in serbia, there are 17 noticed species (r a n đ e l o v i ć et al., 1990). they belong to section crocus mathew (series verni mathew, scardici mathew, crocus mathew) and section nudiscapus mathew (series reticulati mathew, biflori mathew, flavi mathew) (r a n đ e l o v i ć et al., 1990). however, there are many contentious questions related to systematic positions of crocus taxa. one of them certainly refers to taxa from series verni. series verni is represented by three species in serbia: crocus heuffelianus herb.(fig. 1a), crocus kosaninii pulević (fig.1b) and crocus tommasinianus herb. (fig. 1c) (r a n đ e l o v i ć et al., 1990). since good differential markers can be found on the level of micromorphological aspects of the leaves, there have been several investigations about leaf anatomy of crocus taxa from different parts of its distribution range (r u d a l l & m a t h e w , 1990; a k a n , 2007; e r o l & k u ç u k e r , 2007; s a t i l , 2007; c a n d a n , 2007, 2015; k a n d e m i r , 2009, 2010, 2012; c o ş k u n , 2010; ö z d e m i r , 2010; y e t i ş e n , 2013). as can be seen in the literature, no detailed comparative study of series verni has been available, yet. therefore, the initial objective of this paper is to introduce the comparison of leaf anatomy in cross sections of series verni taxa in serbia and to identify their possible differences. material and methods plant samples were collected from natural populations in the flowering time. the sampling localities (fig. 2) with the associated information are given below (tab. 1). taxonomic description of the species was made according to r a n đ e l o v i ć et al. fig. 1. verni taxa in serbia: 1a. crocus heuffelianus, 1b. c. kosaninii, 1c. c. tommasinianus table 1. localities with associated information species crocus heuffelianus crocus kosaninii crocus tommasinianus locality velika ivanča strojkovce mađere n lat/e long 44º 42' 33" 20º 59' 20" 42º 54' 00" 21º 55' 00" 43º 41' 29" 21º 30' 34" altitude 173 m 305 m 207 m https://www.google.rs/url?sa=t&rct=j&q=&esrc=s&source=web&cd=7&cad=rja&uact=8&ved=0ahukewjtw_rjv_pmahviexokhauua0cqfghimay&url=https%3a%2f%2fwww.facebook.com%2foznur.kucuker&usg=afqjcngod6h_3lba3ebsio7vj928hxsvog&sig2=hl-gjbxdry4czpuo-bjrwg biologica nyssana 8 (1)  september 2017: 15-22 raca, i. et al.  comparative anatomical study of the taxa from… 17 (1990). specimens were deposited in herbarium moesiacum niš. five individuals per population were fixed in 50% alcohol for anatomical study, which was done in the laboratory for plant systematics and ecology, faculty of science and mathematics, university of niš. manual microtome (g l i g o r i j e v i ć & p e j č i n o v i ć , 1983) was utilized in order to make cross sections of the leaves. 30 transverse sections per population were stained with safranin alcian blue and examined by leica dm 1000 microscope afterwards. following parameters were considered in leaf cross sections: section height, section length, arm length, white stripe width, lacuna area, adaxial epidermis cells height and width, palisade cells height and width, palisade tissue height, spongy cells height and width, spongy tissue height, abaxial epidermis cells height and width, number of big vascular bundles, xylem area, phloem area and sclerenchyma area. listed anatomical features were measured with imagej software. anatomy characteristics were also described by microphotograps. results and discussion typical microphotographs of leaf cross sections for all of three verni species from serbia are presented in fig. 3. the first part of collage refers to the general outline of leaf cross sections. lateral arm details are given in the middle. the last part of collage represents the structure of big bundles positioned at the end of the arms. leaf blade: crocus leaves cross sections have a unique shape with a square and almost rectangular central keel and two lateral arms recurved towards the keel (r u d a l l & m a t h e w , 1990). however, there are some exceptions where the keel and arms are of the same length (c. scharojanii, c. carpetanus, c. scardicus). the leaf type was declared as morphologicaly and physiologicaly bifacial (e r o l , 2007). the keel is squared with wide base and acute corners in all verni cross sections. the length of arms and their curving degree differ amongst the species (fig. 3a) (tab. 2). the papillae are detected on the adaxial side at the end of the arms. also, the abaxial side of the corner of keel might have hair. the mentioned hair abundance pattern is characteristic of each of three species. leaf surface: adaxial epidermis cells are rectangular, while abaxial cells are elliptical in shape. the cuticle is thicker on the adaxial side of the leaf. stomata occurs on the abaxial surface in the region of arms and lateral sides of the keel. the stomata type is anomocytic (e r o l , 2007). mesophyll: large, almost round parenchyma cells are located in the central part of the keel. because of their thin walls, they might break down, forming an air space called lacuna. disintegration of parenchyma cell walls varies between species (fig. 3a, tab. 2). since parenchyma cells of the keel lack chloroplasts, lacuna area can be noticed as typical white stripe all along the central part of the crocus leaves (e r o l , 2007; y e t i ş e n , 2013). there are two types of parenchyma cells in the mesophyll of arms – palisade and spongy cells. palisade is oriented to the adaxial surface and it is made of two layers of polygonal cells. spongy parenchyma is three – four layered and consists elliptical irregular shaped cells sometimes with intercellular space within (fig. 4). two layers of palisade cells are noticed at the base of the keel as well. vascular bundles: collateral vascular bundles are located between the palisade and spongy cells in one row. there are four big vascular bundles (two of them are placed at the ends of the lateral arms, while two others are positioned in the base of the central keel) (fig. 3a). the number of small and the smallest vascular bundles depends on the leaf dimensions. additionally, they are located all along fig. 2. the geographical position of the investigated populations in serbia (1-crocus heuffelianus; 2-c. tommasinianus; 3-c. kosaninii) biologica nyssana 8 (1)  september 2017: 15-22 raca, i. et al.  comparative anatomical study of the taxa from… 18 the arms and lateral and basal side of the keel. the xylem is oriented towards the adaxial side with the phloem beneath. sclerenchyma “caps” below the phloem are well developed (fig. 5). the anatomy features measurements expressed through the average values and standard deviation are given in the table below (tab. 3). apparently, the highest values of leaf blade features (section height and length, arm length and lacuna area) are noticed in c. heuffelianus population. in the other two species values of those parameters are much lower. the white stripe is narrow in c. kosaninii cross sections (fig. 6). both adaxial and abaxial epidermis cells were found to be fig. 3. anatomic characters of leaves of crocus ser. verni species from serbia: a-leaf cross sections (5x), b-arm details (20x) and c-big bundles (40x) (a-c. heuffelianus; b-c. kosaninii; c-c. tommasinianus) table 2. qualitative anatomic characters of leaves of crocus ser. verni species from serbia crocus heuffelianus crocus kosaninii crocus tommasinianus leaf shape arms the longest with the highest curving degree shorter and slightly recurved keel rectangulared, with ± acute corners squared, with ± acute corners squared, with conspicuously acute corners epidermis cell shape adaxial rectangular, abaxial eliptical papillae finger-like, at the end of the arms and in the corner of the keel mesophyll parenchyma lacuna area large round cells disintegration of parenchyma cell walls is more frequent palisade 2 layers of polygonal cells spongy 3 4 layers of elliptical – irregular shaped cells big vascular bundles 2 of them at the ends of the lateral arms, 2 others at the base of the central keel sometimes one more pair in the middle of the arms biologica nyssana 8 (1)  september 2017: 15-22 raca, i. et al.  comparative anatomical study of the taxa from… 19 the largest in c. kosaninii population. palisade tissue characters are of a similar value. the thinnest spongy layer was observed in the investigated c. tommasinianus population. the most developed vascular tissue features (xylem, phloem and sclerenchyma cap area) were found in c. heuffelianus cross sections. crocus tommasinianus vascular bundles are the smallest, while c. kosaninii big bundles are middle-sized (fig. 6). the objective of this study was to introduce and compare leaf anatomy of the three crocus species from series verni present in serbia. leaf blade, epidermis, mesophyll and vascular bundle features were analyzed. as might be expected (r u d a l l & m a t h e w , 1990), those species tend to correspond closely in their leaf anatomy. however, major differencies can be found on the level of big vascular bundles and their examined parameters (xylem area, phloem area and sclerenchyma area). moreover, leaf blade features (section height and length, arm length, white stripe width and lacuna area) are good distinguishing characters as well. the table 3. quantitative anatomic characters of leaves of crocus ser. verni species from serbia species crocus heuffelianus crocus kosaninii crocus tommasinianus character mean ± stdev min max mean ± stdev min max mean ± stdev min max section height (µm) 881 ± 173 570 1210 654 ± 124 494 1017 630 ± 125 382 916 section length (µm) 7003 ± 1095 3611 9035 4527 ± 946 2810 6298 4152 ± 959 2391 5598 arm length (µm) 3139 ± 492 1684 4263 1908 ± 429 1109 2725 1701 ± 462 892 2467 white stripe width (µm) 752 ± 122 500 996 590 ± 93 434 792 740 ± 111 584 955 lacuna area (µm2) 371375 ± 105510 193667 648276 209992 ± 69767 111999 335380 248603 ± 74234 141373 395804 adaxial epidermis cell height (µm) 20 ± 3 16 28 22 ± 2 19 27 16 ± 2 11 20 adaxial epidermis cell width (µm) 18 ± 3 15 30 19 ± 2 16 25 17 ± 2 13 20 palisade cell height (µm) 37 ± 6 27 55 34 ± 4 27 42 34 ± 4 28 43 palisade cell width (µm) 15 ± 2 11 21 14 ± 1 12 17 16 ± 2 13 20 palisade tissue height (µm) 59 ± 13 35 88 59 ± 9 45 82 54 ± 7 40 66 spongy cell height (µm) 22 ± 3 16 29 22 ± 2 17 27 16 ± 2 12 20 spongy cell width (µm) 19 ± 3 15 26 20 ± 2 18 23 24 ± 3 17 30 spongy tissue height (µm) 68 ± 11 38 91 69 ± 16 38 107 47 ± 9 27 66 abaxial epidermis cell height (µm) 19 ± 2 14 24 22 ± 2 15 25 14 ± 2 11 18 abaxial epidermis cell width (µm) 18 ± 2 14 21 22 ± 2 17 27 16 ± 3 12 23 number of big bundles 4 ± 0 4 4 4 ± 0 4 4 4 ± 0 4 4 sclerenchyma cap area (µm2) 2614 ± 636 1323 3710 2219 ± 375 1596 2929 1459 ± 409 895 2303 phloem area (µm2) 1230 ± 245 669 1645 720 ± 337 338 1965 609 ± 87 442 768 xylem area (µm2) 2477 ± 586 1657 3946 1234 ± 491 621 2323 1059 ± 249 496 1509 biologica nyssana 8 (1)  september 2017: 15-22 raca, i. et al.  comparative anatomical study of the taxa from… 20 length of arms and their curving degree differ amongst the species (e r o l , 2007). crocus heuffelianus cross sections have the longest arms with conspicuously high curving degree. this population is also characterized by the most prominent vascular bundles. the features referred to leaf blade are of a higher value as well (section height and length, arm length, white stripe width, lacuna area). while c. tommasinianus can be distinguished from the other two species by the smallest vascular bundles, thinnest spongy layer and wide white stripe, c. kosaninii has middle sized vascular bundles and the biggest epidermal cells (both adaxial and abaxial). also, disintegration of parenchyma cell walls in lacuna area is more frequent in c. tommasinianus and c. kosaninii populations in comparision to c. heuffelianus. further researches should be carried out including more populations, other closely related species and surveying also the ecological conditions in each locality. this would allow us to evaluate which impact habitat factors have on the anatomy of leaves as well as the potential of leaf anatomy features as diagnostic characters. conclusion as could be seen from the literature, no detailed comparative study of series verni had been done before. therefore, the major aim of this paper was to introduce a leaf anatomy comparison of series verni taxa in serbia and to highlight the differences between them. this study is a basis for further researches within series verni. acknowledgements. the work was funded by the ministry of education, science and technological development of republic of serbia (project no. 173030). fig. 4. arm details on leaves cross sections of crocus ser. verni species from serbia (20x): a-c. heuffelianus; b-c. kosaninii; c-c. tommasinianus (ade-adaxial epidermis, p-palisade parenchyma, s-spongy parenchyma, abe-abaxial epidermis, st-stomata) fig. 5. big bundles on leaves cross sections of crocus ser. verni species from serbia (40x): a-c. heuffelianus; b-c. kosaninii; c-c. tommasinianus; (sc-sclerenchyma cap; ph-phloem; xy-xylem) biologica nyssana 8 (1)  september 2017: 15-22 raca, i. et al.  comparative anatomical study of the taxa from… 21 fig 6. box plots of quantitative anatomic characters of leaves of crocus ser. verni species from serbia: a-c. heuffelianus; b-c. kosaninii; c-c. tommasinianus (ad ec-adaxial epidermal cell; ab ec-abaxial epidermal cell) 200 700 1200 c b a section height (µm) 2000 4000 6000 8000 10000 c b a section length (µm) 500 1500 2500 3500 4500 c b a arm length (µm) 400 600 800 1000 c b a white stripe width (µm) 0,1 0,2 0,3 0,4 0,5 0,6 0,7 c b a lacuna area (mm2) 10 15 20 25 c b a ad ec height (µm) 12 17 22 27 32 c b a ad ec width (µm) 10 15 20 25 30 c b a ab ec height (µm) 10 15 20 25 30 c b a ab ec width (µm) 25 35 45 55 c b a palisade cell height (µm) 10 15 20 c b a palisade cell width (µm) 30 55 80 c b a palisade tissue height (µm) 10 20 30 c b a spongy cells height (µm) 10 20 30 c b a spongy cells width (µm) 20 45 70 95 c b a spongy tissue height (µm) 400 1400 2400 3400 c b a xylem area (µm2) 200 700 1200 1700 c b a phloem area (µm2) 500 2500 4500 c b a sclerenchyma cap area (µm2) biologica nyssana 8 (1)  september 2017: 15-22 raca, i. et al.  comparative anatomical study of the taxa from… 22 references akan, h., eker, i., satil, f. 2007: the morphological and anatomical properties of endemic crocus leichtlinii d. dewar bowles (iridaceae) in turkey. pakistan journal of botany, 39 (3): 711-718. akyol, y., durmuskahya, c., kocabaş, o., pekönür, s., özdemir, c. 2014: the morphological and anatomical investigation of two endemic crocus l. (iridaceae) species of turkey. pakistan journal of botany, 46 (3): 833-839. akyol, y., yetişen, k., özdemir, c., bozdağ, b., kocabaş, o. 2012: the morphological and anatomical studies on crocus biflorus miller subsp. tauri (maw) mathew (iridaceae) in turkey. journal of the institute of science and technology, 2 (ek: a): 15-20. candan, f. 2015: comparative morphological and leaf anatomical investigations of crocus flavus weston from turkey. international journal of agriculture, forestry and fisheries, 3 (3): 99104. candan, f. 2015: morphological and leaf anatomical investigations on 2 yellow flowered endemic taxa of crocus l. (crocus ancyrensis, crocus siehenaus) from turkey. international journal of agriculture, forestry and fisheries, 3 (3): 93-98. erol, o., küçüker, o. 2007: leaf anatomy of some endemic crocus l. (iridaceae) taxa from western anatolia. international journal of botany, 3 (3): 290-295. gligorijević, s., pejčinović, d. 1983: contribution to the methodology of anatomical sections preparation. acta biologiae et medicinae experimentalis, priština, 8: 43-45. kandemir, n. 2009: morphology, anatomy and ecology of critically endangered endemic crocus pestalozzae boiss. (iridaceae) in north-west turkey. bangladesh journal of botany, 38 (2): 127-132. kandemir, n. 2010: a morphological and anatomical investigation about two rare and endemic crocus taxa (iridaceae) from southern anatolia. eurasian journal of biosciences, 4: 54-62. kandemir, n. 2011: comparative leaf anatomy of some endemic crocus l. taxa from turkey. bangladesh journal of botany, 40 (2): 155-162. kandemir, n., çelik, a., yayla, f. 2012: comparative anatomic and ecologic investigation on some endemic crocus taxa (iridaceae) in turkey. pakistan journal of botany, 44 (3): 10651074. mathew, b. 1982: the crocus. a revision of the genus crocus (iridaceae). batsford ltd, london. mathew, b., petersen, g., seberg, o. 2009: a reassessment of crocus based on molecular analysis. the plantsman, 8 (1): 50-57. özdemir, c., akyol, y., yetişen, k., bozdağ, b., kocabaş, o. 2013: morphological and anatomical study on crocus chrysanthus (herbert) herbert (iridaceae). journal of the institute of science and technology, 3 (1): 25-30. özdemir, c., akyol, y., alçitepe, e. 2014: morphological and anatomical studies on two endemic crocus species of turkey area. pakistan journal of botany, 36 (1): 103-113. ranđelović, n., hill, d. a., ranđelović, v. 1990: the genus crocus l. in serbia. the serbian academy of sciences and arts, belgrade. rudall, p. 1990: leaf anatomy in crocus (iridaceae). kew bulletin, 45 (3): 535-544. rukšāns, j. 2017: the world of crocuses. latvian academy of sciences, riga. satil, f. & selami, s. 2007: an anatomical and ecological study of some crocus l. taxa (iridaceae) from the west part of turkey. acta botanica croatica, 66 (1): 25-33. stjepanović-veseličić, l. 1976. rod crocus l. in: josifović, m. (ed.), flora sr srbije viii: 2-12, srpska akademija nauka i umetnosti, beograd. yetişen, k., şen, u., yıldırım, t., özdemir, c. 2013: morphological and anatomical study on endemic crocus olivieri gay subsp. istanbulensis mathew subspecies (iridaceae). anadolu university journal of science and technology-c life sciences and biotechnology, 3 (1): 31-37. stanković, n., joković, n., mihajilov krstev, t., pejčić, m., dimitrijević, m.: frequency and antibiotic resistance of bacteria in urinary tract infections in south serbia. biologica nyssana, 8 (2) biologica nyssana 8 (2)  december 2017: 137-144 stanković, n. et al.  frequency and antibiotic resistance of bacteria… 137 original article received: 20 november 2017 revised: 22 december 2017 accepted: 23 december 2017 frequency and antibiotic resistance of bacteria in urinary tract infections in south serbia nikola stanković*, nataša joković, tatjana mihajilov krstev, milica pejčić, marina dimitrijević university of niš, faculty of science and mathematics, department of biology and ecology, višegradska 33, niš, serbia * e-mail: nikola.stankovic@pmf.edu.rs abstract: stanković, n., joković, n., mihajilov krstev, t., pejčić, m., dimitrijević, m.: frequency and antibiotic resistance of bacteria in urinary tract infections in south serbia. biologica nyssana, 8 (2), december 2017: 137-144. urinary tract infection (uti) refers to the presence of microbial pathogens within the urinary tract. the aim of this study was to determine frequency and antibiotic resistance of bacteria that cause urinary tract infections in south serbia during the year 2015. from 4784 analyzed urine samples 2367 isolates of pathogenic bacteria were obtained. the most frequent cause of uti was escherichia coli (43.0%) followed by enterococcus spp. (31.0%). also, there was a significant number of proteus mirabilis (11.0%) and klebsiella spp. (7.0%) isolates. in addition, remaining 8.0% of isolated bacteria belonged to genera staphylococcus spp., enterobacter spp., citrobacter spp., providencia spp., acinetobacter spp. and to species pseudomonas aeruginosa and proteus vulgaris. gram-negative bacterial isolates were the most resistant to penicillin antibiotics, and the most sensitive to cephalosporins. enterococcus spp., which are gram-positive bacteria showed sensitivity to ampicillin in significant percentage and also to vancomycin (glycopeptide antibiotic), while fosfomycin (quinolone antibiotic) showed the lowest potency against these isolates. key words: antibiotic resistance, urinary tract infections, escherichia coli, enterococcus spp. apstrakt: stanković, n., joković, n., mihajilov krstev, t., pejčić, m., dimitrijević, m.: učestalost i antibiotska rezistencija bakterija izazivača urinarnih infekcija u južnoj srbiji. biologica nyssana, 8 (2), decembar 2017: 137-144. infekcije urinarnog trakta (uti) predstavljaju prisustvo patogenih mikroorganizama u urinarnom traktu. cilj ovog rada je bio da se utvrdi učestalost i rezistencija na antibiotike uzročnika urinarnih infekcija na teritoriji južne srbije u toku 2015. godine. ispitivanje je vršeno u mikrobiološkoj laboratoriji poliklinike “human” u nišu. iz ukupno analiziranih 4784 uzoraka urina bilo je 2367 bakterijskih izolata. najčešći uzročnik urinarnih infekcija je bila bakterija escherichia coli (43.0%), a zatim enterococcus spp. (31.0%). u značajnijem broju je bilo i izolata proteus mirabilis (11.0%) i klebsiella spp. (7.0%). pored navedenih bakterija izolovane su i staphylococcus spp., pseudomonas aeruginosa, proteus vulgaris, enterobacter spp., citrobacter spp., providencia spp., acinetobacter spp., koje zajedno čine 8.0% izolata iz analiziranih uzoraka. gram-negativni 8 (2) • december 2017: 137-144 doi: 10.5281/zenodo.1135959 biologica nyssana 8 (2)  december 2017: 137-144 stanković, n. et al.  frequency and antibiotic resistance of bacteria… 138 bakterijski izolati su bili najrezistentniji na penicilinske antibiotike, dok su najveću osetljivost pokazali na cefalosporine. enterococcus spp., koje su inače gram-pozitivne bakterije, su u velikom procentu pokazale osetljivost na ampicilin, kao i vankomicin (glikopeptidni antibiotik), dok je primećena najveća rezistencija na fosfomicin (hinolonski antibiotik). ključne reči: antibiotska rezistencija, infekcije urinarnog trakta, escherichia coli, enterococcus spp. introduction urinary tract infections (uti) are among the most common infectious diseases occurring in either the community or healthcare settings. uti are caused by bacteria which, in case that they are present in significant number in urinary system, result by occurrence of different symptoms in patients (h o o t o n , 2000; n i c o l l e , 2005). according to f o x m a n (2000), utis are up to 10 times more frequent in women than in men. among the bacterial species escherichia coli account to 80% to 85% of the infection (v a s u d e v a n , 2014). except of e. coli, other bacteria can be the cause of uti including klebsiella spp., proteus spp., enterococcus spp., staphylococcus spp., pseudomonas aeruginosa (l i n h a r e s et al., 2013). resistance to antibiotics is a rising problem worldwide and many studies reported results on the main causative agents and their antibiotic resistance patterns in different regions (c h o w , 2000; a i b i n u et al., 2004; r u d y et al., 2004; v e l i č k o v i ć r a d o v a n o v i ć et al., 2009; h e i n t z et al., 2010; s w a m i n a t h a n & a l a n g a d e n , 2010; e l b o u a m r i et al., 2014). local antimicrobial susceptibility patterns of urinary isolates should be known in order to achieve a satisfactory therapeutic effect (h u a n g et al., 2014). usually, the first step in treating uti is an empiric antibiotic selection, partially determined by local resistance patterns. because of increasing resistance of bacteria to most common agents used in empiric therapy (trimethoprim/sulfamethoxazole), it is necessary to isolate, identify and make a susceptibility test on bacterial causative agent of infection for an adequate treatment of uti (n i c o l l e , 2005; h a n d e et al., 2005). the aim of this research was to to isolate and identify bacterial species that are the most common causes of uti in south serbia, as well as to determine whether the incidence of infection is related to gender. resistance to antibiotics of obtained isolates was also done in order to determine the local resistance patterns. material and methods samples urine samples from patients of polyclinic “human” in niš, were investigated in this study. a total of 4784 urine samples collected in the period january – december 2015. were analyzed. mediums and antibiotics cultivation and isolation of pathogenic bacteria from urine samples were performed on following microbiological culture media: uti agar, sheep blood agar, macconkey agar, mannitol salt agar base, esculin bile agar, kligler iron agar, peptone water, simmons citrate agar, christensen urea agar, parisian mannitol, mueller-hinton agar (titan media, india). kovacs reagent and gram staining reagents were used for identification purposes. testing the resistance of isolates was done using antibiotic discs (himedia, india), by list and concentration recommended by clsi standards (clsi, 2010). gram-negative isolates were tested to following antibiotics: ampicillin (10 µg), amoxicillin/clavulanic acid (30 µg), cefuroxime (30 µg), cefotaxime (30 µg), cefixime (30 µg), gentamicin (10 µg), nitrofurantoin (300 µg), ciprofloxacin (5 µg), and trimethoprim/ sulphamethoxazole (25 µg). isolates of enterococcus spp. were tested to: ampicillin (10 µg), levofloxacin (15 µg), doxycycline (30 µg), fosfomycin (50 µg), gentamicin (120 µg), ciprofloxacin (5 µg), nitrofurantoin (300 µg), and vancomycin (30 µg). isolation and identification of bacteria isolation of bacteria from urine samples was performed by plating samples on the uti agar followed by 24 h incubation on 35-37 °c. bacterial colonies with different macromorphological characteristics were further transferred on different selective and differential media in order to identify isolates on the basis of their biochemical characteristics. small blue-green colonies on uti agar were plated on blood and esculin agar. bacteria which had hemolytic activity on blood agar and decomposed bile salts on esculin agar were identified as biologica nyssana 8 (2)  december 2017: 137-144 stanković, n. et al.  frequency and antibiotic resistance of bacteria… 139 enterococcus spp. large white colonies on uti agar were further transferred on mannitol salt agar. isolates that formed yellow colored colonies and were positive on coagulase test (rabbit plasma coagulase test) were identified as s. aureus (k a r a k a š e v i ć , 1989). small white-light blue colonies on uti agar were transferred on blood agar in order to examine hemolytic activity. confirmation of streptococcus spp. was done by using agglutination strepto-kit (microgen, bioproducts). on uti agar, gram-negative bacteria form a large purple, light to dark blue, or white to cream colored, mostly slimy colonies. identification of these bacteria was performed using standard biochemical tests. susceptibility testing of bacteria agar plates were inoculated with a standardized inoculum (mcfarland standard 0.5) of the bacteria and antimicrobial disks were placed on the inoculated agar plate according to guidelines of the clinical and laboratory standards institute (clsi, 2010). the disks used for a disk diffusion assay contains a standardized known amount of an antimicrobial agent (table 1 and 2), which diffuses into the agar when is in contact with the agar surface. the plates were incubated inverted at 36±1°c for 18 to 24 h. following incubation, the diameter of this zone was measured, and the results were interpreted as resistant (r), intermediate (i), or susceptible (s) using standard guidelines. the isolates that were intermediary sensitive to certain antibiotics were classified into sensitive, because intermediate represents the sensitivity to a particular antibiotic in a smaller extent. results frequency of bacteria in uti in order to examine the dominant groups of bacteria in patients with uti, 2367 bacterial strains from the 4784 urine samples were isolated and identified. the most common pathogen in urine samples was e. coli (43.0%), while significant percentage of enterococcus spp. (31.0%), proteus mirabilis (11.0%) and klebsiella spp. (7.0%) was observed. also, staphylococcus spp., pseudomonas aeruginosa, proteus vulgaris, enterobacter spp., citrobacter spp., providencia spp., acinetobacter spp. were isolated but in smaller percentage (8.0% in total). considering that gender of the patients is the most common factor that affects the urinary infections, an analysis of the frequency of isolation of the pathogen from urine in relation to this factor was performed. analyses related to the gender of the patients were done for four most frequent bacteria causing uti and the results are presented in tab. 1. from a total of 2367 isolates from urine samples,1737 (73.4%) isolates were isolated from female patients, 630 (26.6%) isolates from male patients. e. coli was significantly represented in females than males (82.4% and 17.6% respectively). other pathogens were also more common in females than in males (tab. 1). in male patients, enterococcus spp. were most frequent (n=225, 35.7%), followed by e. coli (n=181, 28.7%), p. mirabilis (n=128, 20.3%) and klebsiella spp. (n=37, 5.9%). when it comes to female patients, e. coli was most frequent (n=846, 48.7%), followed by enterococcus spp. (n=503, 29.1% ), p. mirabilis (n=142, 8.2%), and klebsiella spp. (n= 127, 7.3%). antibiotic resistance of isolates antibiotic susceptibility testing was performed for e. coli, p. mirabilis, klebsiella spp., and enterococcus spp. (tab. 2). the following groups of antibiotics were tested: penicillins (apicillin and amoksicillin/clavulanic acid), sulfonamides (trimethoprim/sulphamethoxazole), quinolones (ciprofloxacin and levofloxacin), cephalosporins (cefixime, cefotaxime and cefuroxime), aminoglycosides (gentamicin), nitrofuran derivates (nitrofurantoin), glycopeptide (vancomicin), tetracyclines (doxycycline), and fosfomicin. table 1. number and frequency of isolates considering gender of the patients male female bacteria total number frequency number % of isolates by gender number % of isolates by gender escherichia coli 1027 43.0% 181 17.6 846 82.4 enterococcus spp. 728 31.0% 225 30.9 503 69.1 proteus mirabilis 270 11.0% 128 47.4 142 52.6 klebsiella spp. 164 7.0% 37 22.6 127 77.4 other bacteria 178 8.0% 59 33.1 119 66.8 biologica nyssana 8 (2)  december 2017: 137-144 stanković, n. et al.  frequency and antibiotic resistance of bacteria… 140 among the penicillins all isolates were more resistant to ampicillin in comparison to amoxicillin/clavulanic acid. klebsiella species showed the highest resistance to ampicillin (98.1%), followed by e. coli (72.7%) and p. mirabilis (58.2%). the same order of frequencies for amoxicillin clavulanic acid was observed, but with a much smaller percentage of resistant isolates (53.4%, 36.5%, and 24.9% respectively). it was noticed that resistance to trimethoprim/sulfamethoxazole was significant. resistance to trimethoprim/sulfamethoxazole for p. mirabilis was 49.6%, for klebsiella spp. 48.4%, and for e. coli 41.8%. resistance to ciprofloxacin for klebsiella spp. was 40.7%, for e. coli 25.0%, and for p. mirabilis 16.5%. in enterococcus spp. resistance to ciprofloxacin was 35.7%. table 3. antibiotic resistance of enterococcus spp. antimicrobials % resistant enterococcus spp. ampicillin 2.5 ciprofloxacin 35.7 levofloxacin 29 doxycycline 32 vancomycin 1.1 nitrofurantoin 7.7 fosfomycin 79.5 gentamicin 38.4 in relation to cephalosporins resistance was: to cefixime in klebsiella spp. 41.4%, in e. coli 18.5%, and p. mirabilis 13.4%; to cefuroxime in klebsiella spp. 44.4%, in p. mirabilis 16.2%, and in e. coli 15.6%; to cefotaxime in klebsiella spp. 45.3%, in p. mirabilis 16.0%, and in e. coli 14.7%. resistance to gentamicin in klebsiella spp. was 41.4%, in p. mirabilis 25.0%, and in e. coli 22.9%. resistance to nitrofurantoin in klebsiella spp. was 53.7%, in p. mirabilis 47.6%, and in e. coli 7.7%. resistance to pipemidic acid was identified in 47.4% of klebsiella spp., in p. mirabilis 41.0%, and in e. coli 36.3%. enterococcus spp. isolates were also tested to fosfomicyn, doxycycline, levofloxacin, and vancomycin, and resistance to named antibiotics were 79.5%, 32%, 29%, and 1.1% respectively (tab. 3). discussion infections of the urinary tract are one of the most common bacterial infections and one of the most common reasons for prescribing antimicrobial drugs. antibiotics are a group of effective and commonly used drugs. unfortunately, bacteria have developed extremely genetic mechanisms of antibiotic resistance. the most important factor that leads to the development of bacterial resistance to antibiotics is their overuse, especially in cases where their use is not necessary (f o x m a n , 2010). in our study, of total 2378 isolated bacterial strains from urine samples obtained of patients from south serbia, the most common cause of uti was e. coli, as expected. our study showed that e. coli was less frequent than in most european regions (s c h i t o et al., 2009; m a l m a r t e l & g h a s a r o s s i a n , 2015) but more frequent than in ivory coast (m o r o h et al., 2013). the frequency of p. mirabilis was 11%, which is similar to bosnia (m a h m u t o v i ć v r a n i ć & u z u n o v i ć , 2016). unlike the other members of enterobacteriaceae, p. mirabilis is not a common pathogen that causes table 2. antibioticresistance of gram-negative pathogens, (%) resistant antimicrobials e. coli p. mirabilis klebsiella spp. ampicillin 72.7 58.2 98.1 amoxicillin clavulanic acid 36.5 24.9 53.4 trimethoprim/sulphamethoxazole 41.8 49.6 48.4 ciprofloxacin 25 16.5 40.7 cefixime 18.5 13.4 41.4 cefuroxime 15.6 16.2 44.4 cefotaxime 14.7 16 45.3 gentamicin 22.9 25 41.4 nitrofurantoin 7.7 47.6 53.7 biologica nyssana 8 (2)  december 2017: 137-144 stanković, n. et al.  frequency and antibiotic resistance of bacteria… 141 urinary tract infections in normal hosts (c h e n at al., 2012). the prevalence of klebsiella spp. infection was similar to studies carried out in portugal (6.013.45%), reported by l i n h a r e s et al. (2013). other pathogens (staphylococcus spp., p. aeruginosa, p. vulgaris, enterobacter spp., citrobacter spp., providencia spp., acinetobacter spp.) that we isolated were not investigated because they all together accounted 8 percent of the total number of isolates. our research has shown that urinary infections are three times more common in women. that was expected because studies worldwide reported that uti is more common in females (f o x m a n et al., 2000; a y e g o r o et al., 2007; o m o r e g i e et al., 2008). these data on the frequency of the gender coincide with the data obtained in the other regions worldwide (c u n h a et al., 2016). the lowest in vitro efficiency on pathogens has been shown by ampicillin, where only 1 of 164 klebsiella spp. isolates were susceptible to this antibiotic. escherichia coli, as the most prevalent pathogen had also high rate of resistance to ampicillin, followed by p. mirabilis. resistance of gram negative pathogens to ampicillin observed in this study was like those observed in south africa, israel, hong-kong, philippines, iran and bosnia, where range of resistance were 62.0 – 84.0% (d e s e n c l o s et al., 1988, k a z e m n i a et al., 2014; m a h m u t o v i ć -v r a n i ć & u z u n o v i ć , 2016). minor resistance of enterococcus spp. to ampicillin was observed. that was expected, because enterococci are typically susceptible to ampicillin, because of the lack of of beta-lactamase (h o l l e n b e c k & r i c e , 2012). resistance of e. coli and other gram-negative pathogens to trimethoprim/sulphametho-xazole was similar, in the range of 41.8 –49.6%. high percentage of resistance to this antibiotic was reported in european countries and brazil, in the range of 38.9% 50.6% (n i c k e l , 2007; c u n h a et al., 2016). the reason for this high resistance may be due to the wide use of these antimicrobials in the treatment of community-acquired infections (c u n h a et al., 2016). guidelines of the american infectious diseases society and the european society for microbiology and infectious diseases suggest that antimicrobials with a resistance rate above 20% should not be prescribed empirically to patients with uncomplicated cystitis, unless susceptibility is determined by priorisolation in culture (g u p t a et al., 2011). fluoroquinolones are widely used for empirical treatment of uti (r o c h a et al., 2012). it can be noticed that resistance to ciprofloxacin in our study is dramatically high comparing with studies from region and another european countries. in bosnia, the most common pathogen e. coli was resistant to ciprofloxacin in 4.3% of cases, comparing with our study where 25% of e. coli isolates were resistant. in 9 european countries its reported that e. coli was resistant in 8.8% of cases (n i c k e l , 2007). the highest percentage of resistance to ciprofloxacin was reported in nigeria, with 65.7% (o l u r u n m o l a et al., 2013). it is considered that ciprofloxacin has only modest activity against enterococci (p e r r y et al., 1994). results for the resistance of enterococcus spp. isolates are in accordance with with results from other studies (a b d u l l a & a b d u l l a , 2006; g i l h o , 2013). overuse of one of the fluoroquinolone leads to the development of resistance to the whole group of quinolone antibiotics (m a h m u t o v i ć -v r a n i ć & u z u n o v i ć , 2016). resistance of gram-negative pathogens to cephalosporins of second (cefuroxime) and third generation (cefixime and cefotaxime) was relatively low comparing with other antibiotics. there was no significant diference in a resistance patern within bacterial species based on cephalosporin clasification. the most resistant were klebsiella spp. isolates, and the least resistant were p. mirabilis. the most common pathogen e. coli was resistant to cephalosporins in range of 14.7-18.5%. m e y e r et al. (2010), reported dramatic increase of third generation cephalosporin-resistant e. coli in german intensive care units in a period of 8 years, where this pathogen developed resistance from 1.2% to 19.7%. gentamicin was only aminoglycoside antibiotic used in this study. klebsiella spp., p. mirabilis and e. coli have shown significant resistance. rate of resistance presents double value comparing with brazil (c u n h a et al., 2016). compared with region, resistance rate of e. coli in bosnia was 2.15%, which presents 10 times lower value (m a h m u t o v i ć -v r a n i ć & u z u n o v i ć , 2016). higher rate of resistance in e. coli to gentamicin was observed in egypt and iran, 40% and 36% respectively (g a d et al., 2011; k a z e m n i a et al., 2014). in our study, nitrofurantoin was the most efficient antibiotic against two most common pathogens, e. coli and enterococcus spp.. similar data were obtained in bosnia, brazil, nigeria, and portugal (o l u r u n m o l a et al., 2013; l i n h a r e s et al., 2013; m a h m u t o v i ć -v r a n i ć & u z u n o v i ć , 2016; c u n h a et al., 2016). the rising prevalence of vancomycin-resistant enterococci (vre) is of particular concern within many institutions because of its association with increased mortality and health care costs, as well as limited treatment options (h e i n t z et al., 2010). biologica nyssana 8 (2)  december 2017: 137-144 stanković, n. et al.  frequency and antibiotic resistance of bacteria… 142 present study detected vre but in a very small percentage. fosfomycin (monural) is very frequently prescribed at first symptoms of utis, but high resistance to this agent was observed, showing unjustified use of this tretment. resistance to gentamicin was also significant, and explanation of rising level of resistance to amynoglicosides at general is that enterococci have acquired aminoglycoside resistance genes that mediate production of aminoglycoside-modifying enzymes (c h o w , 2000). conclusion the results obtained in this study suggest that e. coli is the most common cause of urinary tract infections, followed by enterococcus spp. urinary tract infections occur more often in women than in men. nitrofurantoin is the most efficient agent against bacterial uropathogens and represent effective option for empirical therapy. ampicillin presents the least effective antibiotic against gram-negative uropathogens, but still effective against enterococcus spp. maximum resistance to all applied antibiotics showed klebsiella spp. isolates, wherein the resistance to ampicillin was almost absolute. it can be concluded that the cephalosporins in vitro are more efficient than other groups of antibiotics to gram-negative uti pathogens, and it can be assumed that cephalosporins are the most effective in the treatment of urinary tract infections. also, conclusion is that ampicillin (penicillins) is still highly efficient to enterococcus spp., while fosfomycin and aminoglycosides cannot be used as the first-choice treatment due to low efficiency against these isolates. we reported unique local pattern of frequency and resistance to antibiotics of bacterial uropathogens in south serbia, which can help in definition of empirical treatment for uti. besides, it is more efficient to perform culture and susceptibility tests on isolated pathogen prior to treat, in order to avoid failure of therapy. references abdulla, f.e., abdulla, e.m. 2006: antibiotic options for enterococcus faecalis infections. pakistan journal of medical sciences, 22 (3): 286-290. aibinu, i., aednipekun, e., odugbemi, t. 2004: emergence of quinolone resistance amongst escherichia coli strains isolated from clinical infections in some lagos state hospitals in nigeria. nigerian journal of health and biomedical sciences, 3 (2): 73-78. ayegoro, o.a, igbinosa, o.o., ogunmwonyi, i.n., odjadjare, e.e., igbinosa, o.e., okoh a.i. 2007: incidence of urinary tract infections (uti) among children and adolescents in ile-ife, nigeria. african journal of microbiology research, 1: 1319. chen, c.y., chen, y.h., lu, p.l., lin, w.r., chen, t.c., lin, c.y. 2012: proteus mirabilis urinary tract infection and bacteremia: risk factors, clinical presentation, and outcomes. journal of microbiology, immunology and infection, 45(3): 228-36. chow, j.w. 2000: aminoglycoside resistance in enterococci. clinical infectious diseases, 31 (2): 586–589. clinical and laboratory standards institute, 2010: performance standards for antimicrobial susceptibility testing. clsi m100-s20. clinical and laboratory standards institute, wayne, pa. cunha, m.a., assuncao, g.l.m., medeiros i.m., freitas, m.r. 2016: antibiotic resistance patterns of urinary tract infections in a northeastern brazilian capital. journal of the são paulo institute of tropical medicine; 58(2). desenclos, j.c., zergabachew, a., desmoulins, b., chouteau, l., desve, g., admassu, m. 1988: clinical, microbiological and antibiotic susceptibility patterns of diarrhoea in korem, ethiopia. journal of tropical medicine and hygiene, 91 (6): 296-301. el bouamri, m.c., arsalane, l., el kamouni, y., zouhair, s. 2015: antimicrobial susceptibility of urinary klebsiella pneumoniae and the emergence of carbapenem-resistant strains: a retrospective study from a university hospital in morocco, north africa. african journal of urology, 21 (1): 36-40. foxman, b., barlow, r. d. arcy, h., gillespie, b., sobel, j. d. 2000: urinary tract infection; selfreported incidence and associated costs. annals of epidemiology, 10: 509 – 513. foxman b. 2010: the epidemiology of urinary tract infection. nature reviews urology, 7(2): 653660. gad, g.f., mohamed, h.a., ashour, h.m. 2011: aminoglycoside resistance rates, phenotypes, and mechanisms of gram-negative bacteria from infected patients in upper egypt. plos one, 6 (2): e17224. gilho, l. 2013: ciprofloxacin resistance in enterococcus faecalis strains isolated from male patients with complicated urinary tract infection. korean journal of urology, 54 (6): 388–393. gupta, k., hooton, t.m., naber, k.g., wullt, b., colgan, r., miller, l. g. 2011: international http://cid.oxfordjournals.org/search?author1=joseph+w.+chow&sortspec=date&submit=submit biologica nyssana 8 (2)  december 2017: 137-144 stanković, n. et al.  frequency and antibiotic resistance of bacteria… 143 clinical practice guidelines for the treatment of acute uncomplicated cystitis and pyelonephritis in women: a 2010 update by the infectious diseases society of america and the european society for microbiology and infectious diseases. clinical infectious diseases, 52 (5): 103-120. hande, a., ӧzlem, a., ӧnder, e., funda, t. 2005: risk factors for ciprofloxacin resistance among escherichia coli strains isolated from communityacquired urinary tract infections in turkey. journal of antimicrobial chemotherapy, 56 (5): 914–918. heintz, b.h., halilović, j., christensen, c.l. 2010: vancomycin-resistant enterococcal urinary tract infections. pharmacotherapy: the journal of human pharmacology and drug therapy, 30 (11): 1136-1149. hooton, t.m. 2000: pathogenesis of urinary tract infection: an update. journal of antimicrobial chemotherapy, 46 (s1): 1–7. hollenbeck, b.l. & louis, b.r. 2012: intrinsic and acquired resistance mechanisms in enterococcus. virulence, 3 (5): 421–433. huang, l.f., lo, y.c., su, l.h., chang, c.l. 2014: antimicrobial susceptibility patterns among escherichia coli urinary isolates from community-onset health care-associated urinary tract infection. journal of the formosan medical association, 113 (12): 970-973. karakašević, b. 1989. mikrobiologija i parazitologija. medicinska knjiga, beogradzagreb. kazemnia, a., ahmadi m., dilmaghani, m. 2014: antibiotic resistance pattern of different escherichia coli phylogenetic groups isolated from human urinary tract infection and avian colibacillosis. iranian biomedical journal, 18 (4): 219-224. linhares, i., raposo, t., rodrigues, a., almeida a. 2013: frequency and antimicrobial resistance patterns of bacteria implicated in community urinary tract infections: a ten-year surveillance study (2000–2009). bmc infectious diseases, 13: 19. mahmutovic-vranic s., uzunovic, a. 2016: antimicrobial resistance of escherichia coli strains isolated from urine at outpatient population: a single laboratory experience. materia socio-medica, 28 (2): 121-124. malmartel, a., ghasarossian, c. 2015: bacterial resistance in urinary tract infections in patients with diabetes matched with patients without diabetes. journal of diabetes and its complications, 30 (4): 705–709. meyer, e., schwab, f., schroeren-boersch, b., gastmeier, p. 2010: dramatic increase of thirdgeneration cephalosporin-resistant e. coli in german intensive care units: secular trends in antibiotic drug use and bacterial resistance, 2001 to 2008. critical care, 14 (3): r113. moroh, j-l.a., fleury, y., tiac, h., bahi, c., lietard, c., coroller, l., edoh, v., coulibaly, a., labia, r., leguerinel i. 2014: diversity and antibiotic resistance of uropathogenic bacteria from abidjan. african journal of urology, 20 (1): 18– 24. nickel, c.j. 2007: urinary tract infections and resistant bacteria. reviews in urology, 9 (2): 7880. nicolle, l. 2005: complicated urinary tract infection in adults. canadian journal of infectious diseases and medical microbiology, 16 (6): 349360. olorunmola, f.o., kolawole, d.o., lamikanra, a. 2013: antibiotic resistance and virulence properties in escherichia coli strains from cases of urinary tract infections. african journal of infectious diseases, 7 (1): 1-7. omoregie, r., erebor, j.o., ahonkhai, i., isibor, j. o., ogefere, h.o. 2008: observed changes in the prevalence of uropathogens in benin city, nigeria. new zealand journal of medical laboratory science, 62: 29-31. perry, d.j., ford, m., gould, f.k. 1994: susceptibility of enterococci to ciprofloxacin. journal of antimicrobial chemotherapy, 34 (2): 297–298. rocha, j.l., tuon, f.f., johnson, j.r. 2012: sex, drugs, bugs, and age: rational selection of empirical therapy for outpatient urinary tract infection in an era of extensive antimicrobial resistance. brazilian journal of infectious diseases, 16 (2): 115-121. rudy, m., zientara, m., bek, t., martirosian, g. 2004: occurrence of antibiotic resistant enterococci in clinical specimens from a pediatric hospital. polish journal of microbiology, 54 (1): 77-80. schito, g.c., naber, k.g., botto, h., palou, j., mazzei, t., gualco, l., marchese, a. 2009: the aresc study: an international survey on the antimicrobial resistance of pathogens involved in uncomplicated urinary tract infections. international journal of antimicrobial agents, 34 (5): 407-413. swaminathan, s., alangaden, g. j. 2010: treatmant of resistant enterococcal urinary tract infections. current infectious disease reports, 12 (6): 455464. vasudevan, r. 2014: urinary tract infection: an overview of the infection and the associated risk biologica nyssana 8 (2)  december 2017: 137-144 stanković, n. et al.  frequency and antibiotic resistance of bacteria… 144 factors. journal of microbiology & experimentation, 1 (2): 00008. veličković-radovanović, r., petrović, j., kocić, b., antić, s., ranđelović, g. 2009: correlation between antibiotic consumption and bacterial resistance as quality indicator of proper use of these drugs in inpatients. vojnosanitetski pregled, 66 (4): 307–312. gospodinov, natcheva, 2020, biologica nyssana 11(1) 11 (1) september 2020: 31-33 doi: 10.5281/zenodo.4060289 chromosome studies of some thalloid liverworts in bulgaria original article galin gospodinov department of plant and fungal diversity and resources, institute of biodiversity and ecosystem research, bulgarian academy of sciences, sofia, bulgaria gospodinov.bryol@gmail.com (corresponding author) rayna natcheva department of plant and fungal diversity and resources, institute of biodiversity and ecosystem research, bulgarian academy of sciences, sofia, bulgaria renimoss@bio.bas.bg received: october 01, 2019 revised: january 22, 2020 accepted: january 26, 2020 abstract: here, we report for the first time chromosome counts for bryophytes from bulgaria. the mitotic chromosomes from gametophytes of aneura pinguis, pellia epiphylla, pellia neesiana and riccia fluitans were studied. all counts correspond to previous reports from other parts of the species’ range. key words: chromosome counts, liverworts, bulgaria apstract: studija hromozoma nekih taloidnih jetrenjača u bugarskoj ovde prvi put izveštavamo o broju hromozoma za briofite iz bugarske. ispitivani su mitotički hromozomi iz gametofita aneura pinguis, pellia epiphilla, pellia neesiana i riccia fluitans. svi brojevi odgovaraju prethodnim izveštajima iz drugih delova areala vrste. ključne reči: brojanje hromozoma, jetrenjače, bugarska introduction chromosome studies are a useful tool for taxonomists (fritsch, 1978). unfortunately, chromosome studies concerning bryophytes in europe are sporadic and represent chromosome numbers from a limited number of local populations. although chromosome numbers in bryophytes are fairly uniform and karyotype analyses are not often informative (newton, 1979), chromosome counts are a useful tool to distinguish morphologically similar haplodiploid species pairs. the knowledge on bryophyte chromosome counts was summarized by fritsch (1991). notably, information from south-eastern europe is missing. few other publications deal with chromosomes of certain genera of hepatics (berrie, 1960; boisselier-dubaylee & bischler, 1998; zheng & zhu, 2009; ochyra et al., 2016). the aim of this study was to report chromosome numbers of some thalloid liverworts from bulgaria. material and methods the liverworts were collected during different excursions and kept alive in a greenhouse, for the purpose of the research. after a period of adaptation a modified gomori’s haematoxylin staining method was used (melander & wingstrand, 1953). fresh actively growing thallus tips were placed in 0.01% solution of colchicine for 90 min. after washing in distilled h2o they were fixed in clarke’s solution (3:1 ethyl alcohol/glacial acetic acid) for 2 h at room temperature or in a fridge for 24 h. after washing in distilled h2o the thalli were placed in 1m hydrochloric acid for 40min at 60 °c followed by washing in distilled h2o. the samples were incubated in hydrochloric acid /ether in ratio 1:1 for 15 min at 60 °c, washed in distilled h2o the stained in gomori’s hematoxylin for 1.45/2 h at 60 °c. samples were squashed in 45% acetic acid and observed under a light microscope. © 2020 gospodinov, natcheva. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work noncommercially under the same license as the original. 31 results and discussion our results were in accordance with already published chromosome numbers for the respective species. for bulgaria this is the first chromosome report for bryophyte species. aneuraceae aneura pinguis (l.) dumort., n = 10 (fig. 1). only one exception in the a. pinguis chromosome number is known up to now (inoe, 1975) where the species shows chromosome number of n = 20. pelliaceae pellia epiphylla (l.) corda, n=9 (fig. 2). in europe, diploid number n=18 is present in pellia borealis lorb, an allopolyploid species with two haploid cryptic species within p. epiphylla as parental taxa (orzechowska et al., 2010). the presence of p. borealis in bulgaria was expected but could not be proven by the present study. pellia neesiana nees, n=9 (fig. 3) most previous studies for this species showed haploid chromosome count of n=9 in mitotic counts (fritsh, 1991) with a single old report of diploid number of n=18 (showalter, 1927). ricciaceae riccia fluitans l., n=8 (fig. 4) the species shows uniformity of chromosome numbers within its range as far as it is studied (fritsch 1991). the only polyploid reports most likely belong to its close relative riccia rhenana (heitz, 1927, lorbeer, 1934). acknowledgements. this research is part of project кп-06-н21/15/19.12.2018 „cryptic species in bulgarian flora – molecular species delimitation in the aneura pinguis complex” financed by the national science fund of bulgaria. references berrie, g. 1960: the chromosome numbers of liverworts (hepadcae and anthocerotae). transactions of the british bryological society, 3(5): 688-705. fritsch r. 1991: index to bryophyte chromosome counts. bryophyta bibliothek, 40: 1–352. fritsch r. 1978: chromosome numbers of some hungarian liverworts. abstracta botanica, vol v, suppl. 3. heitz e. 1927: über multiple und aberrante chromosomenzahlen. abhandlungen des naturwissenschaftlicher verein hamburg, 21(3-4): 47-57. lorbeer g. 1934: zytologie der lebermoose mit besonderer be-rücksichtigung allgemeiner chromosomenfragen. i. teil. jahrbücher für wissenschaftliche botanik, 80: 567-818. marie‐catherine, b., bischler, h. 1998: 32 biologica nyssana ● 11 (1) september 2020: 31-33 gospodinov, natcheva ● chromosome studies of some thalloid liverworts in bulgaria fig. 1. mitotic chromosomes of: a aneura pinguis, b pellia epiphylla, cpellia neesiana, d riccia fluitans 33 biologica nyssana ● 11 (1) september 2020: 31-33 gospodinov, natcheva ● chromosome studies of some thalloid liverworts in bulgaria allopolyploidy in the thalloid liverwort corsinia (marchantiales). botanica acta, 111: 490-496. melander y., wingstrand k.g. 1953: gomori’s haematoxylin as a chromosome stain. stain technology, 28: 217-223. newton m.e. 1979: chromosome morphology and bryophyte systematics. in: clarke g.c.s., duckett j. g. (eds.) bryophyte systematics. acad. press, london, new yourk, pp. 207 229. ochyra, r., przywara, l., kuta, e. 1982: karyological studies on some antarctic liverworts. journal of bryology, 12(2): 259-263. orzechowska, m., siwinska, d., maluszynska, j. 2010: molecular cytogenetic analyses of haploid and allopolyploid pellia species. journal of bryology 32, 113–121. showalter, a.m. 1927: hermaphroditism in a dioicous hepatic. pnas, 13(6): 369-372. zheng, m., zhu, r.-l. 2009: karyological studies on some species of radula (radulaceae, jungermanniopsida, marchantiophyta). nova hedwigia, 88: 229-244. stanković, s. et al.  potential candidates for biological control of the black... biologica nyssana 6 (1)  september 2015: 49-54 stanković, s. et al.  potential candidates for biological control of the black... 49 original article received: 08 july 2015 revised: 13 july 2015 accepted: 01 august 2015 potential candidates for biological control of the black bean aphid aphis fabae in serbia saša s. stanković*, marijana ilić milošević, vladimir žikić university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia * e-mail: sasasta@gmail.com abstract: stanković, s.s., ilić milošević, m., žikić, v.: potential candidates for biological control of the black bean aphid aphis fabae in serbia. biologica nyssana, 6 (1), september 2015: 49-54. the black bean aphid is widely spread aphid species in the palaearctic, known to attack over 1150 plant species. because some of the host plants are of great agricultural interest, aphis fabae represent a very important pest. we assembled all data concerning the presence of this pest and connected it in tritrophic associations. in the period of 24 years investigation on the territory of serbia it has been recorded in 107 trophic associations. in total there are 145 findings of a. fabae parasitized by 19 taxa of aphidiinae (brackonidae) from seven genera. the most suitable biocontrol agents for the black bean aphid are lysiphlebus fabarum, binodoxys angelicae, lipolesis gracilis and the introduced species lysiphlebus testaceipes. key words: aphis fabae, parasitoids, aphidiinae, tritrophic associations apstrakt: stanković, s.s., ilić milošević, m., žikić, v.: potencijalni kandidati za biološku kontrolu crne repine vaši aphis fabae u srbiji. biologica nyssana, 6 (1), septembar 2015: 49-54. crna repina vaš je rasprostranjena širom palearktika. ova biljna vaš je zabeležena na preko 1150 vrsta biljaka. s obzirom na to da su mnoge biljke domaćini veoma bitne sa agrikulturnog stanovišta, aphis fabae predstavlja veoma vašnu štetočinu u zasadima gajenih biljaka. prikupljeni su podaci o prisustvu crne repine vaši u periodu od 24 godine na teritoriji srbije, a rezultati su predstavljeni u vidu tritrofičkih asocijacija. tokom istaraživanog perioda zabeleženo je 107 asocijacija. ukupno je registrovano 145 nalaza a. fabae koju je parazitiralo ukupno 19 taksona potfamilije aphidiinae (braconidae) iz sedam rodova. najpogodnije vrste za biološku kontrolu crne repine vaši su lysiphlebus fabarum, binodoxys angelicae, lipolesis gracilis, a takođe i introdukovana vrsta lysiphlebus testaceipes. key words: aphis fabae, parasitoidi, aphidiinae, tritrofičke asocijacije introduction the black bean aphid, aphis fabae scopoli 1763 (aphididae) is one of the most common aphid in europe. it is notorious for attacking many crops, especially broad bean vicia faba (fabaceae) and beet beta vulgaris (amaranthaceae), but also many other host plants which are of agricultural importance. according to h o l m a n (2008) a. fabae has been reported on over 1150 host plants. this species has almost global distribution; it is especially common in temperate regions (b l a c k m a n & e a s t o p , 2008). regarding its wide plant host range it is very difficult to comprehend biology and ecology of this particular group. therefore a. fabae (sensu lato) has long been 6 (1) • september 2015: 49-54 biologica nyssana 6 (1)  september 2015: 49-54 stanković, s. et al.  potential candidates for biological control of the black... 50 considered as a species complex and there were many attempts to resolve this problem (m ü l l e r & s t e i n e r , 1986; t h i e m e & d i x o n , 1996; r a y m o n d et al., 2001). today there are five recognized taxa of the a. fabae complex, three of them considered on a subspecies level: a. fabae fabae scopoli 1763, a. fabae cirsiiacanthoidis scopoli 1763, a. fabae mordvilkoi börner & janisch 1922; while a. evonymi fabricius 1775 and a. solanella theobald 1914 have the species status (v a n e m d e n & h a r r i n g t o n , 2007). beside direct damages inflicted by probing and sucking plant juices, a. fabae is an important vector of many plant viruses among other aphid species. one of the most important viruses is byv (beet yellows virus) which causes a yellowing disease in beta vulgaris and some other plants, but also bmv (brome mosaic virus) which primarily infects plants from poaceae family and cause damages to wheat plants. traditionally, aphids are treated chemically to prevent damages on agricultural plants; however, nowadays the biological control concept is of the highest importance. biologically, aphid number is controlled by predators, such are coccinellids, some hoverfly larvae; parasites, such are entomopathogenic nematodes, fungi and bacteria; and also by parasitoids, where the most important are wasps from the family braconidae, precisely the subfamily aphidiinae which are exclusive parasitoids of aphids. many aphidiinae species are economically very important as biological control agents in agroecosystems, therefore it is of great importance to know their taxonomy, biology and ecology for their successful usage against aphids (h å g v a r & h o f s v a n g , 1991; v ö l k l & m a c k a u e r , 2000). one of the most common parasitoid of a. fabae is an aphidiine wasp lysiphlebus fabarum (marshall 1896) which has been frequently reared from a. fabae feeding on crops and other plants (v ö l k l & s t e c h m a n n , 1998). beside l. fabarum, the black bean aphid is attacked by many other aphidiines such are ephedrus plagiator (nees 1811), binodoxys angelicae (haliday 1833), praon abjectum (haliday 1833) and others (s t a r ý , 1970). along native and common parasitoid wasps, a. fabae is very frequently parasitized by lysiphlebus testaceipes (cresson 1880) which is now rather invasive species previously introduced into the mediterranean area for biological control of citrus aphids (s t a r ý et al., 1988; ž i k i ć et al., 2015). research on a. fabae on the territory of serbia has been conducted since 1952 (m i l e t i ć , 1952a, 1952b), also aphid parasitoids were studied intensively in serbia (v u k a s o v i ć , 1928; t o m a n o v i ć & b r a j k o v i ć , 2001; t o m a n o v i ć et al., 1998; k a v a l l i e r a t o s et al., 2004, 2010; p e t r o v i ć et al., 2009). one of the recent surveys of aphidiine parasitoids in serbia was done three years ago where several parasitoids were recorded to attack a. fabae (ž i k i ć et al., 2012). having in mind the economic importance of a. fabae in crop productions, as well as its natural enemies such is wasps from the family aphidiinae, the authors strived to present tritrophic associations of the black bean aphid and its aphidiine parasitoids, but as well the host plant species on which aphids were found. also, we think this paper will serve as a good starting point toward practical usage of afidiinae parasitoids for biological control of a. fabae in serbia and beyond. material and methods the material was collected in a period of 24 years (1990-2014) research and collecting throughout the territory of serbia. most of the material was collected during 2000s, although some of the samples date from 1990s (table 1). infested plants were cut and placed into plastic cups along with aphids, a muslin cloth were put on the top of the cups to prevent the exit of insects but allowing ventilation. such prepared plastic cups were kept in laboratory condition with the constant temperature of 23 °c, relative humidity of 65%, and the photoperiod (l:d) 16:8h. each plant was prepared and photographed for later identification following j o s i f o v i ć (1972) and s a r i ć & d i k l i ć (1986). live aphids were preserved in 90% ethyl-alcohol and identified, following nomenclature r e m a u d i è r e & r e m a u d i è r e (1997). parasitoid wasps were preserved as dry material but most of them were put in plastic tubes with 90% ethyl-alcohol. morphological terminology of parasitoids follows s h a r k e y & w h a r t o n (1997). some of the wasp specimens were dissected and mounted onto microscopic slides in order to facilitate identification on the species level. results here we present 145 findings of a. fabae on 50 plant taxa. eight plant specimens were identified to the generic level, while for one plant specimen we have data only to the family level (tab. 1). in total, a. fabae was parasitized by 19 aphidiinae taxa from seven genera; aphidius, binodoxys, ephedrus, lipolexis, lysiphlebus, praon and trioxys. twelve taxa were identified to the species level, and the remaining seven taxa were identified to the generic biologica nyssana 6 (1)  september 2015: 49-54 stanković, s. et al.  potential candidates for biological control of the black... 51 table 1. the list of aphis fabae findings with its host plants and parasitoids. legend: ♂– male; ♀ – female; (+) – over the presented number; xx – no data about number of specimens. legator abbreviations: ap – anđeljko petrović, mđ – maja đorđević, mm – majda mehanović, ss – saša stanković, vž – vladimir žikić, zk – zorana kojičić, žt – željko tomanović, x – no data about legator. plant parasitoid no of spec. locality, date, legator initials aegopodium podagraria praon sp. 2♂ 3♀ niš, trošarina; 11.06.2014; vž lysiphlebus testaceipes 4♂ niš, trošarina; 11.06.2014; vž amaranthus retroflexus lipolexis gracilis 1♀ lebane, konjino; 06.07.2010; ss lysiphlebus fabarum 17♀ 20♂ lebane, konjino; 06.07.2010; ss anthriscus sylvestris lysiphlebus fabarum 50+♀ tara, perućac; 25.06.2014; ss apiaceae lipolexis gracilis 3♀ 2♂ brzeće; 29.07.2010; vž arctium lappa binodoxys angelicae 50+♀ sićevačka klisura, ostrovica; 28.05.2013; mđ lipolexis gracilis 2♀ sićevačka klisura, ostrovica; 28.05.2013; mđ lysiphlebus cardui 100+♀♂ sićevačka klisura, ostrovica; 28.05.2013; mđ lysiphlebus cardui 2♀ 6♂ sićevačka klisura; 23.06.2012; ss lysiphlebus fabarum 50+♀ 1♂ sićevačka klisura; 23.06.2012; ss lysiphlebus cardui 1♂ tara, mitrovac; 25.06.2013; vž beta vulgaris lysiphlebus fabarum 13♀ 7♂ zemun; 27.09.1990; x bifora radians lysiphlebus testaceipes 6♀ 5♂ niš, trošarina; 22.05.2013; mđ calendula officinalis binodoxys angelicae 1♀ 2♂ kruševac; 18.06.2013; zk capsella bursa pastoris lysiphlebus fabarum 3♂ vlasina, čemernik; 04.08.2011; vž carduus acanthoides lysiphlebus fabarum 13♀ slankamen; 24.06.2011; x carduus sp. lysiphlebus fabarum 1♀ petnica; 12.06.2011; ap lysiphlebus fabarum 22♀ kragujevac, ilićevo; 06.06.2011; x centaurea cyanus lysiphlebus fabarum 13♀ 7♂ lebane, konjino; 31.05.2014; ss chenopodium album aphidius matricariae 1♀ niš, popovac; 22.05.2010; vž binodoxys acalephae 23♀ 14♂ niš, popovac; 22.05.2010; vž ephedrus plagiator 1♂ niš, popovac; 22.05.2010; vž lipolexis gracilis 11♀ niš, popovac; 22.05.2010; vž lysiphlebus fabarum 1♀ niš, popovac; 22.05.2010; vž lysiphlebus fabarum 18♀ 4♂ sićevačka klisura; 04.06.2011; vž binodoxys angelicae 1♀ sićevačka klisura; 04.06.2011; vž lysiphlebus cardui 18♀ 3♂ sićevačka klisura, sićevo; 17.07.2013; ss binodoxys angelicae 1♂ sićevačka klisura, sićevo; 17.07.2013; ss binodoxys angelicae 11♀ niš, popovac; 25.06.2010; vž lipolexis gracilis 9♀ 3♂ niš, popovac; 24.07.2010; vž lysiphlebus cardui 23♀ 11♂ sićevačka klisura, sićevo; 23.06.2012; vž lipolexis gracilis 2♀ 5♂ sićevačka klisura, sićevo; 23.06.2012; vž lysiphlebus fabarum 2♀ 3♂ sićevačka klisura; 06.06.2013; vž binodoxys angelicae 2♀ 8♂ sićevačka klisura; 06.06.2013; vž lysiphlebus orientalis 4♀ slankamen; 24.06.2011; x praon volucre 1♀ 1♂ slankamen; 24.06.2011; x lysiphlebus fabarum 3♀ lebane; 01.06.2013; ss lysiphlebus fabarum 21♀ 1♂ sićevačka klisura, sićevo; 17.07.2013; ss binodoxys sp. 1♂ sićevačka klisura, sićevo; 17.07.2013; ss lysiphlebus fabarum 2♀ surčin; 15.06.2010; ap lysiphlebus fabarum 20♀ zemun; 17.10.1991; x lysiphlebus fabarum 14♀ zemun; 15.06.2011; ap lysiphlebus orientalis 28♀ zemun, galenika; 07.06.2011; ap binodoxys angelicae 1♀ zemun, galenika; 07.06.2011; ap lysiphlebus testaceipes 21♀ 26♂ niš; 03.06.2014; ss lysiphlebus fabarum 1♀ prokuplje, bresničić; 15.06.2014; ss cichorium sp. lipolexis gracilis 12♀ 2♂ sićevačka klisura; 04.06.2011; ss binodoxys angelicae 2♀ sićevačka klisura; 04.06.2011; ss cirsium arvense lysiphlebus fabarum 116♀ 37♂ vlasina; 03.08.2011; ss lysiphlebus fabarum 83♀ 85♂ vlasina; 03.08.2011; ss lysiphlebus fabarum 15♀ 4♂ vlasina, čemernik; 07.08.2010; ss lysiphlebus fabarum 1♀ 14♂ brzeće; 29.07.2010; ss lipolexis gracilis 6♀ 4♂ brzeće; 29.07.2010; ss lysiphlebus fabarum 1♀ 2♂ merošina, oblačinsko jezero; 15.06.2014; ss lysiphlebus fabarum 22♀ 1♂ ostružnica; 06.07.2011; x lysiphlebus testaceipes 8♂ 9♀ niš; 25.05.2014; ss lysiphlebus confusus 7♂ 2♀ niš; 25.05.2014; ss biologica nyssana 6 (1)  september 2015: 49-54 stanković, s. et al.  potential candidates for biological control of the black... 52 cirsium arvense lysiphlebus fabarum 8♀ 4♂ vlasina; 21.07.2013; ss lysiphlebus fabarum 15♀ 3♂ vlasina, čemernik; 07.08.2010; ss lysiphlebus fabarum 2♀ vlasina; 15.06.2013; ss lysiphlebus cardui 66♀ 16♂ suva planina, bojanine vode; 11.07.2010; vž lysiphlebus fabarum 7♀ 1♂ vlasina; 11.08.2006; vž lysiphlebus fabarum 62♂ vlasina; 28.06.2012; vž lysiphlebus fabarum 27♀ 15♂ vlasina; 06.08.2010; vž binodoxys acalephae 3♀ zlatibor; 26.06.2013; ss cirsium eriophorum lipolexis gracilis 4♀ 2♂ dukat; 07.08.2011; ss cirsium palustre lysiphlebus fabarum 9♀ 24♂ goč; 13.07.2011; x cirsium sp. lysiphlebus fabarum 12♀ 21♂ kopaonik; 17.07.2013; ss coreopsis verticillata lysiphlebus fabarum xx zemun; 17.10.1991; žt coronilla varia lysiphlebus testaceipes 2♀ niš, pantelej; 10.07.2014; vž digitalis ambigua lysiphlebus fabarum 12♀ tara, derventa; 03.07.2012; vž lysiphlebus cardui 8♀ tara, derventa; 03.07.2012; vž eryngium campestre lipolexis gracilis 7♀ 5♂ jerma canyon; 19.07.2013; vž euonymus japonicus lysiphlebus fabarum 11♀ 12♂ lebane, konjino; 31.05.2014; ss euonymus europaeus trioxys sp. xx beograd, radmilovac; 21.04.1995; x binodoxys angelicae 5♂ obedska bara; 01.04.2001; x galium aparine lysiphlebus testaceipes 1♂ 2♀ niš, gornji matejevac; 25.05.2014; vž lysiphlebus fabarum 37♀ 9♂ niš, tvrdjava; 05.06.2013; mđ ephedrus sp. 30♀ sićevačka klisura, ravni do; 29.05.2014; vž lipolexis sp. 7♀ 4♂ sićevačka klisura, ravni do; 29.05.2014; vž lysiphlebus cardui 10♂ vlasina; 05.08.2011; vž binodoxys sp. 2♀ 1♂ niš, bubanj; 28.05.2014; ss lactuca serriola lysiphlebus cardui 89♂ vlasina; 05.08.2011; ss levisticum officinale lysiphlebus fabarum 7♀ užice; 14.08.1999; žt matricaria chamomilla lipolexis sp. 2♀ 4♂ grdelička klisura; 05.06.2014; ss lysiphlebus fabarum 4♀ 1♂ grdelička klisura; 05.06.2014; ss lysiphlebus cardui 3♀ sićevačka klisura, ostrovica; 28.05.2013; mđ binodoxys angelicae 3♀ 1♂ lebane; 01.06.2013; ss praon volucre 3♀ 1♂ lebane; 01.06.2013; ss lysiphlebus fabarum 6♀ 2♂ lebane; 01.06.2013; ss lysiphlebus testaceipes 4♀ 10♂ lebane; 01.06.2013; ss lysiphlebus testacepies 50+♀ lebane, konjino; 31.05.2014; ss lysiphlebus fabarum 50+♀ lebane, konjino; 31.05.2014; ss lysiphlebus confusus 50+♀ lebane, konjino; 31.05.2014; ss matricaria perforata lysiphlebus fabarum 88♀ 60♂ vlasina; 05.08.2011; ss musa sapientum lysiphlebus fabarum 21♀ 4♂ kruševac; 21.06.2012; zk oenanthe stenoloba lysiphlebus fabarum 75♀ 31♂ vlasina; 05.08.2011; ss onopordum acanthium lysiphlebus fabarum 2♀ niš, lalinac; 08.06.2013; ss orlaya grandiflora lysiphlebus testaceipes 4♀ niš, gornji matejevac; 25.05.2014; vž papaver dubium praon sp. 1♀ niš, gornji matejevac; 25.05.2014; vž papaver rhoeas binodoxys angelicae 1♀ 1♂ niš, tvrdjava; 05.06.2013; mđ lysiphlebus fabarum 3♂ niš, tvrdjava; 05.06.2013; mđ lysiphlebus orientalis 1♀ 2♂ niš, tvrdjava; 05.06.2013; mđ praon volucre 1♀ niš, tvrdjava; 05.06.2013; mđ lysiphlebus testaceipes 2♀ 3♂ niš, tvrdjava; 05.06.2013; mđ lysiphlebus testaceipes 3♀ 1♂ niš, gornji matejevac; 25.05.2014; vž papaver sp. binodoxys angelicae 3♀ beograd, botanička bašta; 06.12.2013; x lysiphlebus orientalis 4♀ beograd, botanička bašta; 06.12.2013; x praon abjectum 3♀ 2♂ beograd, botanička bašta; 06.12.2013; x pastinaca sativa lysiphlebus fabarum 3♀ 1♂ beograd, radmilovac; 22.08.1996; x binodoxys angelicae 1♀ vlasina; 03.08.2011; ss pastinaca sp. binodoxys angelicae 1♀ nova varoš, suljova česma; 07.07.2013; x praon sp. 3♀ nova varoš, suljova česma; 07.07.2013; x philadelphus coronarius aphidius matricariae 1♂ niš; 05.05.2009; vž aphidius sp. 2♂ niš; 07.05.2014; ss praon sp. 2♂ 2♀ niš; 07.05.2014; ss binodoxys sp. 3♀ 5♂ niš; 07.05.2014; ss lysiphlebus testaceipes 1♀ 1♂ niš; 07.05.2014; ss lysiphlebus sp. 1♂ niš; 25.05.2014; ss aphidius sp. 1♂ niš; 25.05.2014; ss praon sp. 2♂ 2♀ niš; 25.05.2014; ss lysiphlebus testaceipes 1♂ 1♀ niš; 20.05.2014; ss biologica nyssana 6 (1)  september 2015: 49-54 stanković, s. et al.  potential candidates for biological control of the black... 53 philadelphus coronarius binodoxys sp. 1♀ srb, niš; 20.05.2014; ss plantago lanceolata lysiphlebus fabarum 9♀ 19♂ vlasina; 05.08.2011; ss polygonum aviculare lysiphlebus fabarum 42♀ 23♂ zemun; 06.10.2012; ss ranunculus sp. binodoxys angelicae 10♀ 5♂ sićevačka klisura, ostrovica; 28.05.2013; mđ lysiphlebus cardui 1♀ sićevačka klisura, ostrovica; 28.05.2013; mđ rumex sp. lysiphlebus fabarum 40♀ padinska skela; 06.04.2009; x lysiphlebus testaceipes 1♀ lebane, konjino; 31.05.2014; ss lysiphlebus confusus 2♂ lebane, konjino; 31.05.2014; ss lysiphlebus fabarum 4♂ niš, trošarina; 22.05.2013; mđ lysiphlebus fabarum 10♀ tara, derventa; 03.07.2012; ss lysiphlebus cardui 6♀ tara, derventa; 03.07.2012; ss solanum nigrum binodoxys angelicae 29♀ 16♂ niš; 08.10.2010; vž lysiphlebus fabarum 1♀ niš; 08.10.2010; vž lysiphlebus fabarum 2♀ obedska bara; 15.06.1996; žt tamarix sp. lysiphlebus testaceipes 30+♀ niš, niška banja; 29.05.2014; vž torilis microcarpa ephedrus plagiator 1♀ dukat; 07.08.2011; ss urtica dioica lysiphlebus fabarum 7♀ 2♂ smederevo; 27.05.2011; x valeriana officinalis lysiphlebus fabarum 2♀ 5♂ vlasina; 21.07.2013; ss lysiphlebus testaceipes 1♀ 5♂ vlasina; 21.07.2013; ss yucca filamentosa lysiphlebus testaceipes 7♀ 2♂ niš; 02.07.2014; mm zea mays lysiphlebus fabarum 2♀ 1♂ beljanica, resavska pećina; 30.07.1996; x lysiphlebus cardui 10♀ 10♂ lebane, konjino; 24.07.2012; ss level (tab. 1). in this survey we found 107 tritrophic associations; plant/aphid/parasitoid. the results of this research are given in a table form where all listed plants were infested by a. fabae, and the very same species was parasitized by the listed aphidiinae taxa (tab. 1). although the fauna of aphidiinae parasitoids has been relatively well investigated in serbia, we want to highlight the three new associations of a. fabae and its parasitoids since the last checklist of aphidiinae in serbia (ž i k i ć et al., 2012). aphidius matricariae haliday 1834 is now newly recorded to parasitize a. fabae in serbia, two specimens, one male and one female were found on chenopodium album (chenopodiaceae) and philadelphus coronarius (hydrangiaceae) (tab. 1). also, two invasive species from the genus lysiphlebus are reported to attack black bean aphid. lysiphlebus orientalis stary & rakhshani 2010, which was originally described from the northeast china as a specialized parasitoid of the soybean aphid (aphis glycines matsumura 1917) was recently recorded on the territory of serbia where as a host, among other aphid species, was a. fabae (p e t r o v i ć et al., 2013). also, lysiphlebus testaceipes, known to attack many aphids primarily in the mediterranean area, in some recent researches was recognized as a very competitive introduced species in the european mainland as well as in serbia (ž i k i ć et al., 2015). sublimating the results of this investigation we found that the most suitable biological control agent of a. fabae is lysiphlebus fabarum, but also binodoxys angelicae, lipolesis gracilis foerster 1862 and the previously mentioned introduced species l. testaceipes. acknowledgements. the authors wish to express great thankfulness to dr željko tomanović and dr anđeljko petrović from the faculty of biology, university of belgrade, also to maja đorđević, zorana kojičić and majda mehanović for sample collection. this investigation was supported by grant iii 43001 (the ministry of science, education and technological development of the republic of serbia). references blackman, r.l., eastop, v.f. 2008: aphids on the world's herbaceous plants and shrubs. john wiley & sons. 1460p. hågvar, e.b., hofsvang, t. 1991: aphid parasitoids (hymenoptera, aphidiidae): biology, host selection and use in biological control. biocontrol news and information, 12 (1): 13-42. holman, j. 2008: host plant catalog of aphids. springer, netherlands. 1216p. josifović, m. 1972: flora sr srbije. srpska akademija nauka i umetnosti. beograd. kavallieratos, n.g., tomanović, ž., starý, p., athanassiou, c.g., sarlis, g.p., petrović, o., niketić, m., veroniki, m.a. 2004: a survey of aphid parasitoids (hymenoptera: braconidae: aphidiinae) of southeastern europe and their aphid-plant associations. applied entomology and zoology, 39: 527563. kavallieratos, n.g., tomanović, ž., starý, p., žikić, v., petrović-obradović, o. 2010: parasitoids (hymenoptera: braconidae: biologica nyssana 6 (1)  september 2015: 49-54 stanković, s. et al.  potential candidates for biological control of the black... 54 aphidiinae) attacking aphids feeding on solanaceae and cucurbitaceae crops in southeastern europe: aphidiine-aphid-plant associations and key. annals of the entomological society of america, 103: 153164. milietić, r. 1952a: prilog poznavanju biologije crne repine lisne vaši. zaštita bilja, 12: 74-78. milietić, r. 1952b: zimski domaćini crne repine lisne vaši. zaštita bilja, 10: 66-72. müller, f.p., steiner, h. 1986: beitrag zur vergleichenden morphologie und bionomie von aphis euonymi f. deutsche entomologische zeitschrift, 33: 257-262. petrović, a., mitrović, m., starý, p., petrovićobradović, o., žikić, v., tomanović, ž., vorburger, c. 2013: lysiphlebus orientalis (hymenoptera, braconidae), a new invasive aphid parasitoid in europe–evidence from molecular markers. bulletin of entomological research, 103 (4): 451-457. petrović, a., tomanović, ž., žikić, v., kavallieratos, n.g., starý, p. 2009: new records of aphidiinae (hymenoptera: braconidae) from serbia and montenegro. acta entomologica serbica, 14: 219-224. raymond, b., searle, j.b., douglas, a.e. 2001: on the processes shaping reproductive isolation in aphids of the aphis fabae (scop.) complex (aphididae: homoptera). biological journal of the linnean society, 74: 205-215. remaudière, g., remaudière, m. 1997: catalogue des aphididae du monde. editions quae. 478p. sarić, m., diklić, n. 1986: flora sr srbije. srpska akademija nauka i umetnosti, beograd. starý, p. 1970: biology of aphid parasites (hymenoptera: aphidiidae) with respect to integrated control. series entomologica., 6. dr. w. junk, publishers, the hague, the netherlands. 291p. starý, p., lyon, j.p., leclant, f. 1988: biocontrol of aphids by the introduced lysiphlebus testaceipes (cress.) (hym., aphidiidae) in mediterranean france. journal of applied entomology, 105 (1‐5): 74-87. thieme, t., dixon, a.f.g. 1996: mate recognition in the aphis fabae complex: daily rhythm of release and specificity of sex pheromones. entomologia experimentalis et applicata, 79: 85-89. tomanović, ž., brajković, m., krunić, m. 1998: a check list of aphid parasitoids (hymenoptera: aphidiidae) in yugoslavia. acta entomologica serbica, 3 (1/2): 95-106. tomanović, ž., brajković, m. 2001: aphid parasitoids (hymenoptera, aphidiidae) of agroecosystems of the south part of the pannonian area. archives of biological sciences, 53: 57-64. van emden, h.f., harrington, r. 2007: aphids as crop pests. cabi. 717p. völkl, w., mackauer, m. 2000: oviposition behaviour of aphidiine wasps (hymenoptera: braconidae, aphidiinae): morphological adaptations and evolutionary trends. the canadian entomologist, 132: 197-212. völkl, w., stechmann, d.h. 1998: parasitism of the black bean aphid (aphis fabae) by lysiphlebus fabarum (hym., aphidiidae): the influence of host plant and habitat. journal of applied entomology, 122 (1‐5): 201-206. vukasović, p. 1928: prilog poznavanju entomofagnih insekata parazita. glas srpske kraljevske akademije, 131: 45-72. wharton, r.a., marsh, p.m., sharkey, m.j., marsh, p.m. 1997: manual of the new world genera of the family braconidae (hymenoptera). the international society of hymenopterists, washington, dc. 439p. žikić, v., ilić milošević, m., stanković, s., petrović, a., petrović-obradović, o., kavallieratos, n.g., tomanović, ž. 2012: aphidiinae (hymenoptera: braconidae) of serbia and montenegro—tritrophic interactions. acta entomologica serbica, 17: 83-105. žikić, v., stanković, s. s., ilić milošević, m., petrović-obradović, o., petrović, a., starý, p., tomanović, ž. 2015: first detection of lysiphlebus testaceipes (cresson) hymenoptera: aphidiinae) in serbia: an introduced species invading europe. northwestern journal of zoology, 11 (1): 97-101. mihajilov-krstev, t. et al.  antimicrobial and antioxydant potential… biologica nyssana 6 (2)  december 2015: 55-58 mihajilov-krstev, t. et al.  antimicrobial and antioxydant potential… 55 original article received: 15 july 2015 revised: 12 august 2015 accepted: 01 october 2015 antimicrobial and antioxidant potential of wild growing silene baccifera (l.) roth. (caryophyllaceae) fruits juice tatjana mihajilov-krstev1*, bojan zlatković1, marija ilić2, vesna stankov-jovanović2, violeta mitić2 1university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia 2university of niš, faculty of sciences and mathematics, department of chemistry, višegradska 33, 18000 niš, serbia * e-mail: tatjanamk@pmf.ni.ac.rs abstract: mihajilov-krstev, t., zlatković, b., ilić, m., stankov-jovanović, v., mitić, v.: antimicrobial and antioxidant potential of wild growing silene baccifera (l.) roth (caryophyllaceae) fruits juice. biologica nyssana, 6 (2), december 2015: 55-58. silene baccifera is widespread plant species in europe, asia and north africa, registered in the list of medicinal plants of india. insufficiently known, biological activities of juice obtained from fresh fruits of this plant were investigated in this study. antimicrobial activity of juice was tested against pathogenic gastrointestinal microbial strains, using microwell-dilution method, while antioxidant properties were evaluated employing dpph and total phenolic and flavonoid content assays. to our knowledge, this is the first study of the juice from fruits of this plant species. key words: silene baccifera, fruit juice, antimicrobial, antioxidant activity apstrakt: mihajilov-krstev, t., zlatković, b., ilić, m., stankov-jovanović, v., mitić, v.: antimikrobni i antioksidativni potencijal soka plodova divljerastuće vrste silene baccifera (l.) roth (caryophyllaceae). biologica nyssana, 6 (2), december 2015: 55-58. silene baccifera predstavlja rasprostranjenu biljnu vrstu na području evrope, azije i severne afrike. registrovana je na listi lekovitih biljnih vrsta indije. u ovom radu je ispitivana biološka aktivnost soka dobijenog ceđenjem svežih plodova pomenute vrste. antimikrobna aktivnost je testirana mikrodilucionom metodom protiv patogena gastrointestinalnog trakta. antioksidantna aktivnost je procenjena dpph metodom i određivanjem ukupnog sadržaja fenola i flavonoida. prema našem saznanju, ovo je prva takva studija soka plodova ove biljne vrste. key words: silene baccifera, sok od plodova, antimikrobna, antioksidativna aktivnost introduction silene baccifera (l.) roth. (syn. cucubalus baccifer l.) is widely spread plant species in the area of europe, asia and north africa, especially in the regions with a temperate climate (g a j i ć , 1970). it is perennial plant with long, prostrate or ascending (50-150 cm), well-branched stems and white or 6 (2) • december 2015: 55-58 biologica nyssana 6 (2)  december 2015: 55-58 mihajilov-krstev, t. et al.  antimicrobial and antioxydant potential… 56 greenish nodding flowers sorted in lateral brunches of the inflorescence. comparatively large (up to 15 mm in diameter), subglobose, fleshy, blackish-red berries are distinctly exerted from the calyx during the ripening period. blossoms appear in june to august, fruits ripen from july to october (k o n r a d v o n w e i h e , 1972). silene baccifera is common plant species that usually grows along roadsides and weedy places in the human settlements. this plant species is registered in the list of indian medicinal plants (www.docslide.us) and is used in traditional medicine for treatment of nephritis, hydropsy, bone-fractures, pulmonary tuberculosis and scrofula (c h e n g et al., 2001). to the best of the author’s knowledge, there is no literature or field data on its use in official either traditional medicine in serbia or balkan countries. the chemical composition of the underground part of the s. baccifera has been poorly investigated. previously isolated constituents from the whole plant were oligosaccharides (c o u r t o i s & a r i y o s h i , 1960; 1962), tocopherol and tocotrienol (i v a n o v & a i t z e t m u e l l e r , 1998) and phytoecdysterones and cucubalugenin a (c h e n g et al., 2001a; 2001b). to the best of our knowledge, antioxidant and antimicrobial activity of this plant species were not investigated. in this paper, the first results of antioxidant and antimicrobial activity of the juice obtained from fresh fruits of s. baccifera are presented. material and methods plant material ripe fruits of wild growing silene baccifera were collected on the territory of serbia (kamenica village, stara planina, e serbia) and identified by dr bojan zlatković. a voucher specimens, under the acquisition number 6859, were deposited at the herbarium collection of department of biology and ecology, faculty of science and mathematics in niš (hmn). preparation of juice cleaned, ripe fruits were tap washed, followed by washing with distilled water and subsequent drying. the fruits were separated from the seeds, cut in small pieces, arranged in clean plastic containers and frozen. melted at room temperature, the softened material was grounded in mechanical blender to a fine mash prior to filtration and the juice extraction. antimicrobial assay microbial strains. in vitro antimicrobial activity of the tested samples have been investigated against the commonest human gastrointestinal pathogenic microbial strains: salmonella enteritidis (atcc 13076), shigella sonnei (atcc 25931), escherichia coli (atcc 25922), pseudomonas aeruginosa (atcc 9027), listeria monocytogenes (atcc 7644), bacillus cereus (atcc 10786), staphylococcus aureus (atcc 6538) and candida albicans (atcc 10031) using microwell-dilution method. microwell dilution method. determination of the minimum inhibitory concentration (mic) and minimum microbicidal concentration (mmc) was carried out according to the method described by clsi (2005) with some modifications. an overnight culture of tested strains were used for the preparation of 0.5 mcfarland standard turbidity suspension (corresponding 108 cfu/ml). a serial doubling dilutions of tested juice were prepared in the range 500.00-0.25 µl/ml, in a 96/well microtiter plate with inoculated mueller-hinton broth (mhb) for bacteria and sabouraud dextrose broth (sdb) for yeast. the final volume was 100 µl and the final concentration of bacterial cells was 106 cfu/ml in each well. the plates were incubated for 24 h at 37 °c. chloramphenicol at concentrations ranging from 10.00-0.002 µg/ml was used as positive control. all determinations were performed in triplicate. microbial growth was determined by adding 20 μl of 0.5% triphenyl tetrazolium chloride (ttc) aqueous solution. mic was defined as the lowest concentration of juice at which microorganisms showed no visible growth. in order to determine mmc, broth was taken from each well and inoculated on mueller-hinton agar (mha) for bacteria and sabouraud dextrose agar (sda) for yeast. the plates were incubated for 24 h at 37°c. the mmc is defined as the lowest concentration of the juice at which 99.9% of inoculated microorganisms were killed. antioxidant assay the antioxidant properties of the tested juice were evaluated employing dpph and total phenolic and flavonoid content assays. all the assays were carried out in triplicate and average value was considered. 1,1-diphenyl-2-picrylhydrasyl (dpph) radical scavenging assay. relatively stable organic radical dpph has been widely used in the determination of antioxidant activity of single compounds as well as the different plant extracts (k u l i š i ć , 2004). the dpph˙-assay was performed as described (s t o j a n o v i ć , 2010). samples of juice methanol solution (10 μl, 232.4, 232.5 and 232.6 mg/ml respectively) were mixed with 90 μmol/l dpph˙in methanol (1.0 ml), and these biologica nyssana 6 (2)  december 2015: 55-58 mihajilov-krstev, t. et al.  antimicrobial and antioxydant potential… 57 solutions were diluted up to 4.0 ml. after shaking mixtures vigorously, they were stored in darkness for 60 min at room temperature and the absorbances were measured at 515 nm (perkin-elmer lambda 15 uv-vis spectrophotometer). radical scavenging activity of the samples was calculated applying following equation: dpph rsc (%) = 100 (a0−a1/a0) where: a0-absorbance of the blank; a1absorbance of the sample. total phenolic content determination. the total phenolic concentration was determined spectrophotometrically according to modified folinciocalteu method (d i m a j o , 2008). to the samples of juice (100 µl) portions of 1 ml of folinciocalteu reagent (purchased from “mol” belgrade, serbia) and 4 ml of sodium carbonate (20% v/v) were added and diluted with distilled water up to 20 ml. the mixture was allowed to stand at room temperature in dark place for 30 min and the absorbance of the solution at 750 nm was measured with a perkin elmer lambda 15 uv/vis spectrophotometer. the total phenolic concentrations were calculated from a calibration curve using gallic acid as a standard. gallic acid was provided by sigma aldrich (darmstadt, germany). data were expressed as gallic acid equivalents per 1 mg of juice. the levels of total infusions in juice determined according to the folin–ciocalteu method are not absolute measurements of the amounts of phenolic materials, but are in fact based on their chemical reducing capacity relative to an equivalent reducing capacity of gallic acid. determination of total flavonoid content. the amount of total flavonoids was determined with aluminium chloride (alcl3) colorimetric assay according to a known method (r i c e e v a n s et al., 1996). briefly, 0.5 ml of juice was made up to a final volume of 1 ml with reaction medium (meo/h2o/ch3cooh=14:5:1). prepared solution was mixed with alcl3 reagent (4 ml, 133 mg of alcl3x6h2o and 400 mg of ch3coona dissolved in 100 ml h2o). after 5 min, the absorbance level was measured versus prepared reagent blank (containing the same chemicals, except for the sample) at 430 nm (perkin-elmer lambda 15 uv-vis spectrophotometer). total flavonoid content was calculated on the basis of the calibration curve of rutin and expressed by mg rutin/g dry extract. the total flavonoid assay was measured in triplicate. results and discussion freshly drained juice from the fruits have shown antimicrobial activity on all tested strains of patogenic microbes (tab. 1). the obtained minimal inhibitory concentrations were in the range 31.2-500.0 µl/ml, and minimal microbicidal concentrations ranged from 125.0-500.0 µl/ml. the tested juice exhibited the highest activity against p. aeruginosa and s. enteritidis (mic/mmc=31.2/125 and 62.5/125 µl/ml, respectively). also, good effect was observed against e. coli (mic=mmc=125 µl/ml). the juice was the least active against s. aureus and c. albicans (mic=mmc=500 µl/ml). table 1. antimicrobial activity of juice from s. baccifera fruits and reference antibiotics (tested strains: sb s. baccifera fruits juice, mic/mmc in µl/ml; a antibiotic, mic/mmc in µg/ml; bacterial strains: se – salmonella enteritidis; ss shigella sonnei, ec escherichia coli, pa pseudomonas aeruginosa, lm listeria monocytogenes, bc bacillus cereus, sa staphylococcus aureus; ca – candida albicans) tested strains se ss ec pa lm bc sa ca sb 62.5/125 250/250 125/125 31.2/125 250/500 125/500 500/500 500/500 a 0.035* 0.035* 0.035* 0.078* 0.035* 0.035* 0.035* 0.035** *chloramphenicol, **nystatin table 2. the antioxidant activity of juice from s. baccifera fruits tested samples rsa (ec 50 u µg/ml)** ppc (gallic acid equivalents, µg/mg of dry matter) pc (rutin equivalents, µg/mg of dry matter) silene baccifera* 0.65 6.00 15.18 *juice was prepared by dissolving fruit in water; ** ec 50 for bht as a standard is 0.63 µg/ml biologica nyssana 6 (1)  september 2015: 55-58 mihajilov-krstev, t. et al.  antimicrobial and antioxydant potential… 58 the obtained results of antioxidant activity of juices are summarized and presented in tab. 2. comparative study of antimicrobial and antioxidant activities of the fresh juices obtained from the fruits of plant species: viburnum lantana, viburnum opulus, sambucus nigra, cornus sanguinea and paliurus spina-christi, wild growing on the territory of serbia, showed that all tested juices had inhibitory concentrations in the range from 15.6500.0 μl/ml and microbicidal concentrations from mbc/mfc=62.5-500.0 μl/ml (m i h a j i l o v k r s t e v et al., 2011). generally, better activity than s. baccifera juice had only the juices from v. opulus and c. sanguinea. also, the juice of this species demonstrated the best antioxidant activity in comparison to the mentioned juices. conclusion the obtained results showed that s. baccifera fruit juice presents a natural source of antimicrobial and antioxidant components that can be applied in prevention and treatment of gastrointestinal diseases and oxidative stress in humans. in future studies, the chemical composition and biological activity of different extracts of certain parts of the plant should be investigated, as well as their cytotoxicity and acute toxicity. acknowledgements. the financial support of this work was provided by ministry of education and science of the republic of serbia (project 172047). references cheng, y.x., zhou, j., tan, n.h., ding, d.t. 2001a: indian medical plants in traditionl medicine. acta botanica sinica, 43:31. cheng, y.x., zhou j., dai, h.f., ding, d.t. 2001b: cucubalugenin a, a new triterpenoid from cucubalus baccifer. fitoterapia, 72: 848-849. clsi, 2005: clinical and laboratory standards institute, wayne, pa. m100-s22 courtois, j.e., ariyoshi, u. 1960: saccharose galactosides from the roots of cucubalus baccifer (caryophyllaceae). study of their structure. bulletin de la société de chimie biologique, 42:737-51. courtois, j.e., ariyoshi, u. 1962: the galactosides of saccharose in the roots of the carnation. bulletin de la société de chimie biologique, 44:23-30. di majo, d., guardia, m.l., giammanco, s., neve, l.l., giammanco, m. 2008: the antioxidant capacity of red wine in relationship with its polyphenolic constituents. food chemistry, 111:45–49. gajić, m. 1970: cucubalus l. [in. gajić, m.: caryophyllaceae juss.]. in: josifović, m. (ed.): flora srbije 2:240-241. srpska akademija nauka i umetnosti, beograd. ivanov, s.a., aitzetmuller, k. 1998: undersuchungen über die tocopherol-undtocotrienol zusammensetzung der samenlipide einiger arten der bulagarischen flora. fett/lipid, 100: 348-352. konrad von weihe, h. 1972: illustrierte flora. deutschland und angrenzende gebiete. gefäßkryptogamen und blütenpflanzen. begründet von august garcke. 23. auflage. paul parey, berlin/hamburg. kulišić, t., radonić, a., katalinić, v., miloš, m. 2004: use of different methods for testing antioxidative activity of oregano essential oil. food chemistry, 85:633–640. mihajilov-krstev, t., zlatković, b., ilić, m., stankov-jovanović, v., mitić, v. 2011: comparative study of antibacterial and activities activities of wild growing fruits juices. book of abstracts of international conference ''medicinal and aromatic plants in generating of new values in 21st century''. academy of sciences and arts of bosnia and herzegovina, special edition vol. cxl, department of natural sciences and mathematics, sarajevo, 9-12 november, 223. rice-evans, c.a., miller, n.j., paganga, g. 1996: structure antioxidant activity relationshipe of flavonoids and fenolic acids. free radical in biology and medicine, 20: 933-956. stojanović, g., stojanović, i., stankov-jovanović, v., mitić, v., kostić, d. 2010: reducing power and radical scavenging activity of four parmeliaceae species. open life sciences, 5(6):808–813. www.docslide.us anticancer compounds from medicinal plants biologica nyssana 2 (2)  december 2011: 00-00 ljupković r.b. et al..  removal cu(ii) ions from water... 37 original article contribution to the knowledge of jumping spiders (araneae: salticidae) from vicinity of jagodina, central serbia boban stanković 1* 1 city goverment of jagodina, department of environmental protection, kralja petra i, no 6, 35000 jagodina, serbia * e-mail: boban.stankovic035@gmail.com abstract: stanković, b.: contribution to the knowledge of jumping spiders (araneae: salticidae) from vicinity of jagodina, central serbia, biologica nyssana, 3 (1), september 2012: 37-42. during last 10 years, based on personal collectings, 21 species from 14 genera of salticidae (araneae) are recorded from vicinity of jagodina: ballus chalybeius, carrhotus xanthogramma, evarcha arcuata, evarcha falcata, heliophanus auratus, heliophanus cupreus, heliophanus flavipes, heliophanus kochii, icius hamatus, icius subinermis, leptorchestes berolinensis, macaroeris nidicolens, marpissa muscosa, marpissa nivoyi, mendoza canestrinii, pellenes tripunctatus, phintella castriesiana, phlegra fasciata, pseudeuophrys erratica, pseudeuophrys lanigera, salticus scenicus. all those species are provided with habitat notes and global distribution. new records for the spider fauna of serbia are heliophanus kochii (simon 1868), icius subinermis (simon, 1937), marpissa nivoyi (lucas, 1846) and mendoza canestrinii (ninni, 1868). key words: salticidae, jumping spiders, jagodina, serbia. introduction the salticidae blackwall, 1841 or jumping spiders, are the most diverse globally distributed spider family mostly tropical, with 5337 species placed in 573 genera (p l a t n i c k , 2011). in the spider fauna of europe, this family is represented with 396 species (h e l s d i n g e n , 2010). the salticid fauna of serbia is not yet fully known. according to d e l t s h e v et al. (2003), only 49 species of jumping spiders have been recorded in serbia. s t a n k o v i ć (2010) reported two species of salticidae as new for the fauna of serbia: icius hamatus (c. l. koch, 1846) and pseudeuophrys lanigera (simon, 1871). in this paper, original faunistic records and habitat notes of 21 species from 14 genera of jumping spiders from vicinity of jagodina with information of globally distribution and zoogeographic analysis are presented. new species records for the spider fauna of serbia are: heliophanus kochii (simon, 1868), icius subinermis (simon, 1937), marpissa nivoyi (lucas, 1846) and mendoza canestrinii (ninni, 1868). materials and methods study area. all specimens of the jumping spiders species were collected in wide vicinity of jagodina (the central part of serbia) (utm ep16,17,26,27; 21°11‘-21°21‘, 43°56‘-44°03‘; altitude range 109-300m). the study area included parts of middle morava valley and southeastern šumadija. it belongs to the peripannonic region of serbia. there are three landscapes types (biomes) in the study area: biome of submediterranean oak woodlands, biome of south european deciduous montane woodlands and biome of south european 3 (1) • september 2012: 37-42 http://www.faunaeur.org/full_results.php?id=182682 http://www.faunaeur.org/full_results.php?id=182682 biologica nyssana 3 (1)  september 2012: 37-42 stanković, b.  contribution to the knowledge… 38 deciduous woods in lowland and inundated areas (m a t v e j e v & p u n c e r , 1989). the climate is moderately continental. according to the relevant meteorological station in ćuprija, the average annual air temperatures are between 11.2 and 11.7°c approximately, and the average annual rainfall is 619 mm. during the eight month period of march-november, the average monthly air temperatures were higher to 10°c. collection, preservation and identification of specimens. the material has been collected mainly during last 10 years by hand, sweep net and pitfall trapping by the author. material was preserved in 70% ethyl alcohol with added glycerin (9:1 by volume) and kept in the author’s collection. determinations, photographs and drawings were made using stereomicroscope (btc stm-1 x20) and light microscope (meopta) from preserved specimens. for new records, photos or original drawings of the male palp or female epigyne are presented. determinations were made mainly according to p r ó s z y ń s k i (1976, 1979, 1997, 2003, 2005), h e i m e r & n e n t w i g (1991), r a k o v & l o g u n o v (1996a), r a k o v (1997), l o g u n o v (1996, 1998b, 1999b), ż a b k a (1997) and m e t z n e r (1999, 2011). the nomenclature follows p l a t n i c k (2011). results and discussion ballus chalybeius (walckenaer, 1802) material examined: 1♂, đurđevo brdo near jagodina, 17.06.2007; 1♂, đurđevo brdo – ćelijan, 2.07.2007; 1♂, đurđevo brdo, 23.04.2010; 1♂, jagodina, 13.05.2011. distribution: europe, north africa to central asia (p l a t n i c k , 2011). habitat: high density shrubs of ligustrum vulgare, prunus spinosa, rosa sp., crataegus monogyna, also urban habitats, gardens (present data). carrhotus xanthogramma (latreille, 1819) material examined: 1♂, panjevački rit, on young leaves of verbascum nigrum, 11.05.2005; 1♀, đurđevo brdo, 4.06.2006; 1♂, road for vill. gornje štiplje, in oak wood, 9.07.2008; 1♂, rit vill.bukovče, 28.05.2009. distribution: amphi-eurasian subborealsubtropical species (l o g u n o v & g u s e i n o v , 2001). habitat: low vegetation, meadows, on oak trunk, shrubs and bushes (present data). evarcha arcuata (clerck, 1757) material examined: 1♂, crni vrh, near vill.gornje štiplje, 14.07.2005; 2♀♀, đurđevo brdo, 5.20.04.2007; 2♀♀, đurđevo brdo, 10.-20.07.2007; 1♂, panjevački rit, 14.05.2008; 1♂, jagodina, graveyard, 5.06.2010. distribution: trans-euroasian temperate species (l o g u n o v & g u s e i n o v , 2001). habitat: low vegetation, bushes, grassy habitat, meadows (present data). evarcha falcata (clerck, 1757) material examined: 1♂, rit-vill.ribnik, 28.05.2009; 1♀, lipar, 4.07.2010. distribution: euro-siberian temperate species (l o g u n o v & g u s e i n o v , 2001). habitat: meadows, grass and bushes in oak woods (quercetum confertae-cerris rudski) (present data). heliophanus auratus (c.l.koch, 1835) material examined: 1♀, jagodina, near r.belica, 1.06.2007; 1♂, rit-vill.ribnik, 28.05.2009; 1♂, glogovačke bare, 17.07.2010. distribution: euro-siberio-central asian temperate species (l o g u n o v & g u s e i n o v , 2001). habitat: wet meadows-grass vegetation, agricultural fields (present data). heliophanus cupreus (walckenaer, 1802) material examined: 1♂, vicinity of jagodina, 3.08.2005; 1♂, đurđevo brdo, 4.06.2006; 1 juv., jagodina, 19.06.2006; 2♂♂, đurđevo brdo, 17.06.2007; 2♂♂, vill.ribare, bank of velika morava, 10.08.2008; 1♀, rit vill. ribnik, r.belica valley, 28.05.2009; 1♂, lipar, 25.04.2010. distribution: euro-caucasian (r a k o v & l o g u n o v , 1996a). habitat: meadows, shrubs, gardens, vineyards, agricultural fields (present data). heliophanus flavipes (hahn, 1832) material examined: 1♀, jagodina – vill. rakitovo, along bank of river lugomir, 19.04.2006; 1♂, vill.bukovče, 28.05.2009; 2♀♀, panjevački rit, 31.05.2009; 1♂ juvenile, vill.končarevo, near r.velika morava, 23.07.2010. distribution: trans palearctic temperate species (l o g u n o v & g u s e i n o v , 2001). habitat: high grass meadows, wet grassy habitat, in the litter of populus woods (present data). biologica nyssana 3 (1)  september 2012: 37-42 stanković, b.  contribution to the knowledge… 39 a b c figure 1. heliophanus kochii, male, dorsal view (a), male palp, ventral (b), lateral view (c). heliophanus kochii (simon 1868), fig. 1. material examined: 1♂, vill.ribare, 30.06.2003; 1♂, jagodina – vill.rakitovo, along bank of river lugomir, 1.05.2005; 1♀, jagodina, near river belica, 2.08.2007; 1♂, vill.končarevo, lake predor, 29.05.2009. distribution: palearctic (p l a t n i c k , 2011). new for serbia. habitat: grassy meadows (present data). icius hamatus (c. l. koch, 1846) material examined: only previous records (stanković, 2010): 1♂, jagodina, 21.07.1998; 1♀, đurđevo brdo near jagodina in dry grassy habitat, 12.06.2005. distribution: widely distributed in mediterranean regions (southern europe, mediterranean islands and north africa) (a l i c a t a & c a n t a r e l l a 1993). habitat: urban habitat, in grass (present data). icius subinermis (simon, 1937), fig. 2. material examined: 1♀, jagodina, 19.06.2004. distribution: presumably western mediterranean range (a l i c a t a & c a n t a r e l l a , 1993), although the species has been found in germany (p l a t n i c k , 2011), slovenia (k o s t a n j š e k & f i š e r , 2005) and switzerland (b l i c k e t a l ., 2004). new for serbia. habitat: i.subinermis favors moist habitats, for example near streams or on moist meadows. it builds a silken retreat in infructescences of rushes or under rocks near rivers or creeks. it will retreat there when the weather is unfavorable (b e l l m a n n , 1997); urban habitat (present data); remarks: the records of mediterranean species i.subinermis, indicates the possibility of the introduction with imported fruits and spread into synanthropic habitats, like second mediterranean species from this genus icius hamatus, recorded in central and west europe (t o m a s i e w i c z & w e s o ł o w s k a , 2006). however, another possibility, may be indicates spread of distribution from macedonia. figure 2. icius subinermis, epigyne. a b figure 3. leptorchestes berolinensis, male palp, ventral (a), lateral view (b). leptorchestes berolinensis (c. l. koch, 1846), fig. 3 material examined: the suburb of jagodina, road for vill.štiplje: (2♂♂, 1♀), 16.05.2002; (1♀, 1 juvenile), 22.06.2002; (1♂, 1♀), 2.07.2003; 1♂, 9.06.2004. biologica nyssana 3 (1)  september 2012: 37-42 stanković, b.  contribution to the knowledge… 40 distribution: europe to turkmenistan (p l a t n i c k , 2011). this is a second records of l.berolinensis in serbia. habitat: sunny walls, tree barks, wood, in grass and fences (present data). macaroeris nidicolens (walckenaer, 1802) material examined: 1♂ juvenile, đurđevo brdo, 23.06.2004; 1♂, bank of velika morava, vill.ribare, 14.06.2005; 1♂ juvenile, jagodina, 13.07.2005; 1♂ juvenile, jagodina – rusko groblje, 5.07.2008; 1♀ juvenile, đurđevo brdo, 28.06.2009. distribution: west palearctic subboreal species (l o g u n o v & r a k o v , 1998), occuring from the canary islands and madeira (w u n d e r h i c h , 1991) to turkmenistan (f e t , 1983). habitat: grassy vegetation, woods (present data). marpissa muscosa (clerck, 1757) material examined: jagodina: 1♀, 12.05.2002; 1♀, 25.04.2004; 2♀♀, 17.08.2006; 1♀, ?.07.2008. distribution: european temperate species (l o g u n o v & g u s e i n o v , 2001). habitat: this species was collected only in urban habitat at shadow and dry places around the houses, external walls of the houses (present data). a b figure 4. marpissa nivoyi, female dorsal view adult (a) and juvenile (b). marpissa nivoyi (lucas, 1846), fig.4. material examined: jagodina: 1♀ juvenile, 11.05.2011; 1♀, 22.05.2011. distribution: south european – central asian subboreal species (l o g u n o v & g u s e i n o v , 2001). new for serbia. habitat: urban habitat: gardens, tree-line, on leave of tilia sp. (present data). mendoza canestrinii (ninni, 1868), fig. 5. material examined: 1♂, rit-vill. ribnik (river belica valley), 3.05.2009. distribution: trans-eurasian subboreal-subtropical species (l o g u n o v & g u s e i n o v , 2001), with the easternmost localities in china (s c h e n k e l , 1963; w e s o ł o w s k a , 1981). new for serbia. habitat: swamp, on leave of iris sp. (present data). figure 5. mendoza canestrinii, male lateral view pellenes tripunctatus (walckenaer, 1802) material examined: 1♀, lipar, 28.05.2003. distribution: euro-siberian temperate species (l o g u n o v & g u s e i n o v , 2001). habitat: in grass of edge of oak woods (present data). phintella castriesiana (grube, 1861) material examined: 1♀, vicinity of jagodina, weeds near road, 11.06.2006; 1♀, jagodina, near r.belica, 26.05.2008; 1♂, vicinity of jagodina, on leaves of dock, 19.04.2011. distribution: amphi-eurasian subboreal species (l o g u n o v & g u s e i n o v , 2001). habitat: in broad-leaved vegetation, weeds (present data). phlegra fasciata (hahn, 1926) material examined: 1♀, đurđevo brdo – ćelijan, in grass, 2.07.2007. distribution: trans-eurasian temperate-subtropical species (l o g u n o v & g u s e i n o v , 2001). habitat: at sunny and dry places, in grass, on stones and on banks of waters (n e n t w i g e t a l ., 2010). pseudeuophrys erratica (walckenaer, 1826) material examined: 1♀, jagodina, 29.04.2002; 1♀, jagodina, near r.belica, 3.06.2004; 1♂, panjevački rit, 17.06.2008; 1♀, đurđevo brdo, 1.07.2008; 1 biologica nyssana 3 (1)  september 2012: 37-42 stanković, b.  contribution to the knowledge… 41 juvenile, jagodina, 23.04.2009; 1♂, rit vill.bukovče, 15.05.2011; 1♂, jagodina, 24.08.2011. distribution: trans-eurasian temperate species (l o g u n o v & g u s e i n o v , 2001). in nearctic found in new jersey (l o g u n o v , 1998b). habitat: lowland grassy vegetation, meadows in the river valley, also urban habitat (present data). pseudeuophrys lanigera (simon, 1871) material examined: jagodina, indoors in my apartment on the 8 th floor: (4 ♀♀ juvenile, 1♀) unknown data; 1♀, ?.09.2002; (1♂, 1♀ juvenile), 04.2003; 1♂, ?.08.2004; (1♀, 1♂), 11.2004; 1♀, 3.10.2009; 1♀ juvenile, 11.03.2010; 1♀, 5.10.2010; (1♂, 1♀), 12.04.2011; 1♂, 7.08.2011. distribution: western, central and southern europe, eastward to the caucasus mts (l o g u n o v 1998b, l o g u n o v & g u s e i n o v , 2001). habitat: synanthropic species, walls of buildings (present data). salticus scenicus (clerck, 1757) material examined: jagodina: 1♀, 12.05.2002; 1♂, 4.08.2003; 1♀, 24.07.2006; 1♂, 7.07.2009; 1♂, vill.bukovče, on wall of school, 21.07.2005; 1♀, đurđevo brdo, 13.05.2007. distribution: holarctic temperate species (l o g u n o v & g u s e i n o v , 2001). habitat: on sunny walls and barks (present data). conclusion total of 21 species from 14 genera of family salticidae are presented. new records for the spider fauna of serbia are: heliophanus kochii (simon 1868), icius subinermis (simon, 1937), marpissa nivoyi (lucas, 1846) and mendoza canestrinii (ninni, 1868). from the results presented in this study, a total of 55 species of salticidae registered in serbia. in the area and vicinity of jagodina registered 38.2% of total. zoogeographical analysis of species found in the region jagodina (fig. 6), it can be concluded that the widespread species (holarctic, palearctic) most represented with 18 species or 86%. the largest number of species with transpalearctic distribution, 8 species or 38,1%, followed by 7 westpalearctic species (33.3%), 3 mediterranean species (14,3%), 2 amphipalearctic species (9.5%), and the 1 holarctic species (4.8%). jumping spiders are grouped into zoogeographical categories according to p r ó s z y ń s k i (1976), l o g u n o v (1992), s z u t s e t a l . (2003). 38,1 33,3 14,3 9,5 4,8 0 5 10 15 20 25 30 35 40 tp al w pa l m ed ap al ho l figure 6. – zoogeografical composition of jumping spider fauna (araneae: salticidae) from vicinity of jagodina. (tpal – transpalearctic, wpal – westpalearctic, med – mediterranean, apal – amphipalearctic, hol – holarctic). acknowledgements. i wish to express my warmest thanks to dr. dmitri v. logunov (curator of arthropods, the manchester museum) for reviewing the manuscript and confirmed determinations of the new species records. references alicata p., cantarella t. 1993: the euromediterranean species of icius (araneae, salticidae): a critical revision and description of two new species. animalia, 20(1/3): 111-131. bellmann, h. 1997: spinnentiere europas. kosmosatlas, stuttgart, franckh-kosmos verlags-gmbh & co. blick, t., bosmans, r., buchar, j., gajdos, p., hänggi, a., helsdingen, p., van, ruzicka,v., starega, w., thaler, k. 2004: checkliste der spinnen mitteleuropas (arachnida: araneae). version 1. dezember 2004. http://www.arages.de/checklist.html#2004_aran eae deltshev, c., ćurčić, b. p. m., blagoev, g. a. 2003: spiders of serbia. institute of zoology, belgrade, monographs 7., 832pp. fet, v., ya 1983: the fauna of aranei of the southwestern kapetdagh//ent. obozr. 62(4): 835-845. heimer, s., nentwig, w. 1991: spinnen mitteleuropas. verlag paul parey, berlin & hamburg. helsdingen, p.j. van, 2010: araneae, in: fauna europaea database european spiders and their distribution distribution version 2010.1, online at www.european-arachnology.org. kostanjšek, r., fišer, c. 2005: new records of jumping spiders (araneae: salticidae) for slovenia. natura sloveniae, 7(1): 5-11. http://www.faunaeur.org/full_results.php?id=182682 http://www.faunaeur.org/full_results.php?id=182682 biologica nyssana 3 (1)  september 2012: 37-42 stanković, b.  contribution to the knowledge… 42 logunov, d. v. 1992: preliminary report on the euro-siberian faunal connections of jumping spiders (araneae, salticidae). annales zoologici fennici, 201: 71-76. logunov, d. v. 1996: notes on a jumping spider collection from israel (aranei salticidae). arthropoda selecta, 5(1/2): 55-61. logunov, d. v. 1998b: pseudeuophrys is a valid genus of the jumping spiders (araneae, salticidae). revue arachnologique, 12(11): 109128. logunov d.v. 1999b: redefinition of the genera marpissa c. l. koch, 1946 and mendoza peckham & peckham, 1894 in the scope of the hoalarctic fauna (araneae, salticidae). revue arachnologique, 13(3). 25-60. logonuv, d. v., guseinov, e. f. 2001: faunistic review of the jumping spiders of azerbaijan (aranei: salticidae), with additional faunistic records from neighboring caucasian countries. arthropoda selecta, 10: 243-260. logunov, d. v., rakov, s. y. 1998: miscellaneous notes on middle asian jumping spiders (aranei: salticidae). arthropoda selecta, 7(2): 117-144. matvejev, s., puncer, i. 1989: karta bioma – predeli jugoslavije. prirodnjački muzej, beograd. metzner, h. 1999: die springspinnen (araneae, salticidae) griechenlands. andrias, 14: 1-279. metzner, h. 2011: worldwide database of jumping spiders (arachnida, araneae, salticidae). http://www.jumping-spiders. com/site/ impressum. html nentwig, w., blick, t., gloor, d., hänggi, a., kropf, c. 2010: spiders of europe. www.araneae.unibe.ch. version of access date.araneae spinnen europas identification key to spiders of europe http://www.araneae.unibe.ch/ platnick, n.i. 2011: the world spider catalog, version 11.5. american museum of natural history, online at http://research.amnh.org/ entomology/spiders/ catalog. doi: 10.5531/db.iz. 0001. proszynski, j. 1976: studium systematyczno zoogeograficzne nad rdzina salticidae regionow palearktycznego i nearktycznego. wysza skola pedagogiczna w siedlcach, 6: 260 pp. proszynski, j. (1979): systematic studies on east palearctic salticidae iii. remarks on salticidae of the ussr. annales zoologici warszava, 34(11): 299-369. proszynski, j. 1997: salticidae (araneae) of the world, part i: diagnostic drawings libraryby. http://www.salticidae.org. proszynski, j. 2003: salticidae (araneae) of the levant. annales zoologici warszava, 53(1): 1180. prószyński, j. 2005: catalogue of salticidae (araneae). [http://salticidae.org/salticid/catalog/ thorell.htm]. rakov s.y, 1997: a review of the spider genus evarcha simon, 1902 in middle asia (aranei, salticidae). arthropoda selecta, 6(1/2): 105-112. rakov, s., yu., logunov, d.v. 1996a: a critical review of the genus heliophanus c.l.koch, 1833 from middle asian and the caucasus (aranei, salticidae). arthropoda selecta, 5(3-4): 67-104. schenkel, e. 1963: ostasiatische spinnen aus dem museum d'histoire naturelle de paris. memoires memoirs du museum d'histoire naturelle, 25(1): 1-481. stanković, b. 2010: two new species of salticidae (aranea) for the fauna of serbia. bulletin of the natural history museum, 3: 133-136. szuts, t., szinetar, c., samu, f., szita, e. 2003: check list of the hungarian salticidae with biogeographical notes. arachnologische mitteilungen basel, 25:45-61. tomasiewicz, b., wesolowska, w. 2006: icius hamatus (salticidae, araneae) in poland? polish journal of entomology, 75: 339-342. wesołowska, w. 1981: salticidae (aranei) from north korea, china and mongolia. annales zoologici warszava, 36: 45-83. wunderlich, j. 1991: höhlentiere der kanarischen inseln. mitt. dt. höhl.-karstf, 37(2): 33. żabka m. 1997: salticidae pajaki skaczace (arachnida: araneae). fauna polski 19. muzeum i instytut zoologii pan, warszawa. genomic dna from rat blood biologica nyssana 7 (1)  september 2016: 47-52 takić miladinov, d.  genomic dna from rat blood… 47 original article received: 22 january 2016 revised: 23 february 2016 accepted: 01 mart 2016 genomic dna from rat blood: a comparison of two extraction methods dijana takić miladinov1,2* 1department of molecular biology, institute for biological research, university of belgrade, bulevar despota stefana 142, 11060 belgrade, serbia 2department of biology and ecology, faculty of science and mathematics, university of niš, višegradska 33, niš, serbia * e-mail: takicdijana@gmail.com abstract: takić miladinov, d.: genomic dna from rat blood: a comparison of two extraction methods. biologica nyssana, 7 (1), september 2016: 47-52. in this study, two methods for dna extraction from fresh rat blood were compared. one is based on the use of cetyltrimethylammonium bromide (ctab method), while the other one is well-known salting out method. spectrophotometric analysis was employed to assess yield and purity of isolated dna, while agarose gel electrophoresis was carried out to evaluate dna integrity. the results have clearly demonstrated that the extraction method has significantly influenced the quantity and purity of isolated dna. by using the ctab method, a larger quantity of high-molecular weight dna with good purity is obtained which, along with time and cost-efficiency of the procedure, makes this method more suitable for the extraction of dna from rat whole blood. key words: dna extraction; ctab; salting out; rat whole blood apstrakt: takić miladinov, d.: genomska dnk iz krvi pacova: poređenje dveju metoda ekstrakcije. biologica nyssana, 7 (1), septembar 2016: 47-52. u ovoj studiji su upoređivane dve metode za ekstrakciju dnk iz sveže krvi pacova. jedna je zasnovana na upotrebi cetil trimetil amonijum bromida (ctab metoda), dok je druga dobro poznata metoda isoljavanja. za procenu prinosa i čistoće izolovane dnk korišćena je spektrofotometrijska analiza dok je agarozna gel elektroforeza korišćena za procenu integriteta dnk. rezultati su jasno pokazali da je način ekstrakcije značajno uticao na količinu i čistoću izolovane dnk. korišćenjem ctab metode dobijena je veća količina dnk velike molekulske težine i dobre čistoće što, zajedno sa efikasnošću u pogledu trajanja i ekonomičnosti procedure, čini ovu metodu pogodnijom za ekstrakciju dnk iz pune krvi pacova. key words: dnk ekstrakcija; ctab; isoljavanje; puna krv pacova 7 (1) • september 2016: 47-52 doi: 10.5281/zenodo.159103 biologica nyssana 7 (1)  september 2016: 47-52 takić miladinov, d.  genomic dna from rat blood… 48 introduction deoxyribonucleic acid (dna) isolation is a process of extraction of dna from wide variety of sources. the first dna was isolated from white blood cells in 1869 by swiss physician and biologist friedrich miescher (d a h m , 2008). currently, it is a routine procedure in molecular biology, forensic science and medical diagnostics. the preparation of high quality dna samples from various sources is the first and vital step for subsequent molecular biological techniques such as polymerase chain reaction (pcr), restriction fragment length polymorphism (rflp), mutation detection, cloning as well as genotyping (p h i l l i p s et al., 2000; w a n g et al., 2 0 0 3 ; t a n r i o v e r et al., 2010). although in some cases pcr can be performed without previously dna isolation step (k o v a c e v i c g r u j i c i c et al., 2012), it is usual to employ pure genomic dna in determination of genetic disorders, epigenetic studies and diagnostic tests (a n g e l i n i et al., 2000; p h i l l i p s et al., 2000; w a n g et al., 2003; l e w i s et al., 2005). various methods for dna isolation from blood samples have already been established (m i l l i g a n , 1998; a n g e l i n i et al., 2000) and many dna isolation kits are commercially available today. the choice of a method for preparing dna depends on the source as well as quantity, quality and purity of the dna desired for downstream applications. an ideal extraction technique should optimize yield, purity and integrity of isolated dna, and also be efficient in terms of cost, time and safety. the cetyltrimethyl ammonium bromide (ctab) method is successfully used for extraction of high quality genomic dna from wide variety of sources (g u s t i n c i c h et al., 1991; t h o m a s et al., 1997; d e s l o i r e et al., 2006; j a s b e e r et al., 2009; c h e n et al., 2010; f i l h o & a l m e i d a , 2013). also, a rapid salting out dna extraction method is well documented in the literature (m i l l e r et al., 1988; o l e r u p & z e t t e r q u i s t , 1992; a l j a n a b i & m a r t i n e z , 1997). the aim of this study was to determine whether these two methods are suitable for isolation of high-molecular weight dna from fresh rat blood. the two methods were compared in terms of yield, purity and quality of isolated dna as well as in terms of cost/time efficiency and environmental safety. material and methods dna extraction published protocols with slight modifications were performed for the ctab (g u s t i n c i c h et al., 1991) and salting out (o l e r u p & z e t t e r q u i s t , 1992) extraction methods. samples of fresh whole blood were obtained from rat tail vein and aliquoted in 1.5 or 2 ml tubes containing edta. for both methods, dna was extracted from four samples of fresh blood. in the final step of isolation, dna was resuspended in 50 µl of te buffer (10 mm tris-hcl, 1mm edta, ph 7.6) and stored at -20 °c until analysis by agarose gel electrophoresis. ctab method solution i: 8% ctab, 1.5 m nacl, 100 mm tris ph 8.5, 50 mm edta ph 8. solution ii: 5% ctab, 0.1 m nacl 500µl of solution i was pre-warmed at 68 °c, mixed with 250 µl of fresh blood and further incubated at 68°c for 30 min. afterwards, 750 µl of chloroform was added, inverted several times to mix and samples were centrifuged at 13 000 rpm for 5 min at room temperature (rt). dna containing upper phases were transferred into a new tubes, with no disturbing of proteins containing medium phase, and theirs volumes (v) were determined. aqueous phases were mixed with solution ii (v/50) and ddh2o (v) and centrifuged at 13 000 rpm for 5 min at rt. supernatants were discarded and pellets were resuspended in 250 µl 1.2 m nacl. to precipitate dna, 750 µl of ice cold absolute ethanol was added and samples were centrifuged at 13 000 rpm for 5 min at +4 °c. the pellets were washed twice with 750 µl of 70% ethanol, and centrifuged at the above mentioned conditions to remove residual salt. the pellets were air-dried at rt. salting out extraction method (se method) red blood cell lysis buffer (rbcl): 0.5 gr of khco3 and 4.14 gr of nh4cl were diluted in 500 ml of dh2o. digestion buffer: 100 mm nacl, 10 mm tris-hcl ph 8, 25 mm edta ph 8, 0.3 mg/ml proteinase k, 0.05% triton x-100. adjust final volume to 5 ml with dh2o. fresh blood (500 µl) was aliquoted in four sterile 1.5 ml tubes and centrifuged at 3 000 rpm for 10 min at rt. supernatants were discarded and pellets of the cells were resuspended in 1.5 ml of rbcl buffer. following incubation for 10 min at rt with occasional inverting of the tubes, the mixtures were centrifuged at the same conditions mentioned above. the lysis step was repeated for 3-4 times until the supernatants were nearly clear and the pellets were mostly white. after lysis, the pellets were resuspended in 100 µl of digestion buffer. two tubes with samples were digested at 37 °c overnight, while two others were digested at 65 °c for one hour. after digestion was complete, 240 µl of saturated 5m nacl was added to all samples and, after centrifugation at biologica nyssana 7 (1)  september 2016: 47-52 takić miladinov, d.  genomic dna from rat blood… 49 3 000 rpm for 10 min, precipitated protein pellets were left at the bottom while supernatants containing dna were transferred to the new tubes. ice-cold absolute ethanol was added in a double volume and, after centrifugation at 13 000 rpm for 20 min at +4 °c, supernatants were discarded and pellets were washed with 1 ml of 70% ethanol. after final centrifugation under the same conditions, the pellets were air-dried at rt. spectrophotometric analysis – concentration and purity determination concentration and purity of extracted dna samples were determined by biospectrometer (eppendorf, germany). two microliters of each dna sample was mixed with 48 µl of 1 × te buffer and put in a quartz cuvette. absorbance was measured at a wavelengths of 260, 280 and 230 nm (a260, a280 and a230) which are max absorption points for bases in nucleic acids, proteins and polyphenols/polysaccharides, respectively. dna was quantified by measuring uv absorbance at 260 nm, while the purity of genomic dna was evaluated on the basis of uv absorption ratios at 260 and 280 nm (a260/a280) and 260 and 230 nm (a260/a230). agarose gel electrophoresis – integrity determination the quality and integrity of all dna samples were assessed by agarose gel electrophoresis. about 650 ng of the isolated dna was analyzed on 0.8% agarose gel (applied biosystems, usa) in 1 × trisborate-edta buffer containing 0.5 µg/ml ethidium bromide, under conditions: 80v and 120 ma for one hour. the bands were visualized using chemidoc xrs uv transilluminator (biorad, usa). results the two different dna extraction methods were analyzed according to the following criteria: yield, purity, quality, environmental safety, time and cost efficiency. dna yield rates and purity table 1 summarizes the yield and purity ranges of dna samples extracted by two different methods. ctab method resulted in high mean yield of dna (1130.25 ng/µl; 22.6 µg of dna per 100 µl of blood). the dna yields for the se method were dependent on duration of digestion step. samples 1 and 2 were incubated in digestion buffer just 1 hour at 65 °c while samples 3 and 4 were digested overnight at 37 °c. prolonged digestion yielded 236.65 ng/µl dna (2.37 µg of dna per 100 µl of blood), while approach with short digestion resulted in approximately six times lower mean dna yield (39 ng/µl; 0.39 µg of dna per 100 µl of blood). according to data from the literature, it is common to table 1. dna yields, absorbance ratios, environmental safety, and estimated duration of the two different methods for dna extraction from fresh rat blood. concentration (ng/µl) a260/280 a260/230 yield (µg) per 100 µl of blood e n z y m e s c h lo r o fo r m d u r a ti o n ( h ) ctab method no yes ~ 1 sample 1 456 1.77 2.07 sample 2 1577 1.8 2.31 sample 3 1093 1.81 1.93 sample 4 1395 1.82 1.9 mean ± s.e.m. 1130.25 ± 245 1.8 ± 0.01 2.05 ± 0.09 22.6 se method (digestion for 1 h at 65 °c) yes no ~ 3.5 sample 1 40 1.76 1.57 sample 2 38 1.66 2.1 mean ± s.e.m. 39 ± 1 1.71 ± 0.05 1.83 ± 0.26 0.39 (digestion overnight at 37 °c) ~ 16 sample 3 220.9 1.51 1.12 sample 4 252.4 1.41 1.2 mean ± s.e.m. 236.65 ± 15.75 1.46 ± 0.05 1.16 ± 0.04 2.37 biologica nyssana 7 (1)  september 2016: 47-52 takić miladinov, d.  genomic dna from rat blood… 50 extract approximately 2 µg of genomic dna per 100 µl of rat/mouse whole blood by employing various extraction methods (h o f s t e t t e r et al., 1997; x i n g et al., 2007; c h a c o n -c o r t e s , 2012) and about 6 µg of dna by using commercial kits (n o e t h & d a s o v i c h -m o o d y , 1997; c h a c o n -c o r t e s et al., 2012). a260/a280 and a260/a230 absorbance ratios were employed to evaluate the purity of extracted dna samples. according to s a m b r o o k et al. (1989) pure dna extracts have 1.8 < a260/a280 < 2 and a260/a230 > 2. the data in table shows that there are some differences in dna extracts’ purity dependent on the method of extraction. the ctab method produced a260/a280 ratios ranged from 1.77 to 1.82, with a mean value of 1.8. such results indicate the pure dna samples with no protein or rna contaminations. furthermore, a260/a230 ranged from 1.9 to 2.31, with a mean value of 2.05, indicating the high purity of samples 1 and 2. samples 3 and 4 had slight lower a260/a230 ratios which means certain contamination with carbohydrates, salts or organic solvents. on the other side, se method produced dna samples with poor a260/a280 and a260/a230 ratios. all dna samples have been contaminated with proteins, salts or organic solvents. sample 3 was the only one which met criteria a260/a230 > 2, but had a poor a260/a280 ratio of 1.66 indicating protein contamination. the two techniques were found to be very different in terms of yield and purity of dna recovered from blood. ctab method resulted in much higher yield of pure dna compared to se method which produced dna samples of poor yield and purity. since source effect was reduced by using the same samples, variations in yield and purity can be attributed to the effects of the extraction methods. high dna yield and good purity obtained by ctab method were previously described in the literature for a variety of extraction sources (j a s b e e r et al., 2009; c h e n et al., 2010; f i l h o & a l m e i d a , 2013). on the contrary, salting out method often results in an insufficient purity of dna extracts (f i l h o & a l m e i d a , 2013). to improve purity, s a m b r o o k et al. (1989) suggested taking a special care when dispart supernatant, containing dna, from the sediment, containing so many contaminants. also, in a case of contamination of dna extract with salts, it is suggested to re-precipitate dna with ethanol. gel electrophoresis analysis of isolated dna the quality and integrity of genomic dna samples isolated by two different methods were examined by 0.8% agarose gel electrophoresis. this technique has been widely used for determining the size of dna samples (z i m m e r m a n n et al., 1998). as can be seen in figure 1, the main bands of dna were slowmigrating and located high above 3 kb. all samples contained high-molecular weight dna with no lowmolecular weight smear tails. although all lines should contain the same quantity of dna (650 ng), the lane 4 in se method had much lower dna content on the gel (fig. 1). one possible reason could be over-estimation of dna concentration in the spectrophotometric analysis. proteins, rna, salts, lipids and other contaminants can increase the spectrophotometric estimation of dna concentration (h a q u e et al., 2003). except rna molecules, the other contaminants can not be visualized on gel by staining with ethidium bromide, but one can notice the lower amount of dna on a gel in regard to the expected amount. the data from dna agarose gel electrophoresis supplement the data from absorbance readings indicating compatibility of the two techniques. fig. 1. agarose gel electrophoresis of total genomic dna samples isolated from rat fresh blood by two different extraction methods. l – 100 bp dna ladder (fermentas). environmental safety, time and cost efficiency the ctab method required approximately 1 h compared to 3.5 h or overnight incubation required for the se method. moreover, the use of proteinase k makes se method less cost-effective than ctab method. despite time and cost efficiency, ctab method employs chloroform, well-known hazardous organic solvent. conclusion yield, purity and quality of dna extracted from rat whole blood depend greatly on the extraction biologica nyssana 7 (1)  september 2016: 47-52 takić miladinov, d.  genomic dna from rat blood… 51 method. ctab method provided a high yield of pure dna and, according to time and cost efficiency, this method is suitable for simultaneous processing of a large number of samples. salting out extraction method yielded several times lower amount of dna of unsatisfactory purity and along with special care needed during execution procedure to avoid contamination, this method is much less efficient compared to ctab method. acknowledgements. this work was supported by the ministry of education, science and technological development of the republic of serbia, grant oi 173020. references aljanabi, s.m., martinez, i. 1997: universal and rapid salt-extraction of high quality genomic dna for pcr-based techniques. nucleic acids research, 25 (22): 4692-4693. angelini, a., di febbo, c., rullo, a., di ilio, c., cuccurullo, f., porreca, e. 2002: new method for the extraction of dna from white blood cells for the detection of common genetic variants associated with thrombophilia. pathophysiology of haemostasis and thrombosis, 32 (4): 180-183. chacon-cortes, d., haupt, l., lea, r., griffiths, l. 2012: comparison of genomic dna extraction techniques from whole blood samples: a time, cost and quality evaluation study. molecular biology reports, 39: 5961-5966. chen, h., rangasamy, m., tan, s.y., wang, h., siegfried, b.d. 2010: evaluation of five methods for total dna extraction from western corn rootworm beetles. plos one, 5 (8): 1-6. dahm, r. 2008: discovering dna: friedrich miescher and the early years of nucleic acid research. human genetics, 122 (6): 565–81. desloire, s., valiente moro, c., chauve, c., zenner, l. 2006: comparison of four methods of extracting dna from d. gallinae (acari: dermanyssidae). veterinary research, 37 (5): 725-732. filho, e.s., almeida, t. 2013: optimized protocols for extraction of dna in plant and blood tissues. international journal of scientific research, 2 (11): 57-58. gustincich, s., manfioletti, g., del sal, g., schneider, c., carninci, p. 1991: a fast method for high-quality genomic dna extraction from whole human blood. biotechniques, 11 (3): 298301. haque, k.a., pfeiffer, r.m., beerman, m.b., struewing, j.p., chanock, s. j., bergen, a.w. 2003: performance of high-throughput dna quantification methods. bmc biotechnology, 3: 20. hofstetter, j.r., zhang, a., mayeda, a.r., guscar, t., nurnberger, j.i., lahiri, d.k. 1997: genomic dna from mice: a comparison of recovery methods and tissue sources. biochemical and molecular medicine, 62: 197-202. jasbeer, k., son, r., ghazali, m.f., cheah, y.k. 2009: real-time pcr evaluation of seven dna extraction methods for the purpose of gmo analysis. international food research journal, 16: 329-341. kovačević grujičić, n., davidović, s., takić, d., mojsin, m., stevanović, m. 2012: direct pcr amplification of the hvsi region in mitochondrial dna from buccal cell swabs. archives of biological sciences, 64 (3): 851-858. lewis, c.m., cler, l.r., bu, d.w., zöchbauermüller, s., milchgrub, s., naftalis, e.z., leitch, a.m., minna, j.d., euhus, d.m. 2005: promoter hypermethylation in benign breast epithelium in relation to predicted breast cancer risk. clinical cancer research, 11 (1): 166-172. miller, s.a., dykes, d.d., polesky, h.f. 1988: a simple salting out procedure for extracting dna from human nucleated cells. nucleic acids research, 16 (3): 1215. milligan, b.g. 1998. total dna isolation. in: hoelzel, a.r. (ed.), molecular genetic analysis of population: a practical approach 2: 29–64, oxford, new york, tokyo: oxford university press. noeth, l., dasovich-moody, m. 1997: use of the dna isolation kit for mammalian blood for the purification of genomic dna. biochemica, 1: 25-27. olerup, o., zetterquist, h. 1992: hla-dr typing by pcr amplification with sequence-specific primers (pcr-sscp) in 2 hours: an alternative to serological dr typing in clinical practice including donor-recipient matching in cadaveric transplantation. tissue antigens, 39 (5): 225-235. phillips, h.a., howard, g.c.w., miller, w.r. 2000: p53 mutations as a marker of malignancy in bladder washing samples from patients with bladder cancer. british journal of cancer, 82 (1): 136-141. sambrook, j., fritsch e.f., maniatis t. 1989: in molecular cloning: a laboratory manual. cold spring harbor, ny: cold spring harbor laboratory press. tanriover, m.d., tatar, g.b., uluturk, t.d., erden, d.d., tanriover, a., kilicarslan, a., oz, s.g., yurter, h.e., sozen, t., guven, g.s. 2010: evaluation of the effects of vitamin d receptor and estrogen receptor 1 gene polymorphisms on bone mineral density in postmenopausal women. clinical rheumatology, 29: 1285-1293. biologica nyssana 7 (1)  september 2016: 47-52 takić miladinov, d.  genomic dna from rat blood… 52 thomas, j.c., khoury, r., neeley, c.k., akroush, a.m., davies, e.c. 1997: a fast ctab method of human dna isolation for polymerase chain reaction applications. biochemical education, 25 (4): 233-235. wang, s.s., thornton, k., kuhn, a.m., nadeau, j.g., hellyer, t.j. 2003: homogeneous real-time detection of single-nucleotide polymorphisms by strand displacement amplification on the bd probetec et system. clinical chemistry, 49 (10): 1599-1607. xing, c., dafu, c., changchun, l., hui, l., weixing, z. 2007: sample pretreatment microfluidic chip for dna extraction from rat peripheral blood. frontiers of chemistry in china, 2 (1): 74-78. zimmermann, a., lüthy, j., pauli, u. 1998: quantitative and qualitative evaluation of nine different extraction methods for nucleic acids on soy bean food samples. zeitschrift fur lebensmitteluntersuchung und forschung a, 207: 81-90. frequency of isolation and antibiotic resistance patterns of bacterial isolates from wound infections biologica nyssana 7 (2)  december 2016: 151-158 stojanović-radić, z. et al.  frequency of isolation and antibiotic… 151 original article received: 03 october 2016 revised: 20 november 2016 accepted: 29 november 2016 frequency of isolation and antibiotic resistance patterns of bacterial isolates from wound infections zorica stojanović-radić*, marina dimitrijević, nikola stanković, ana aleksić, milica pejčić department of biology and ecology, faculty of science and mathematics, university of niš * e-mail: zstojanovic@pmf.ni.ac.rs abstract: stojanović-radić, z., dimitrijević, m., stanković, n., aleksić, a., pejčić, m.: frequency of isolation and antibiotic resistance patterns of bacterial isolates from wound infections. biologica nyssana, 7 (2), december 2016: 151-158. six hundred and thirteen bacterial strains were isolated from wound swabs and the isolates were identified on the basis of growth on differential and selective media. in order to test the sensitivity of isolated strains to different antibiotics, the disc diffusion method, according to eucast protocol v 5.0 was used. the most common species isolated from wound swabs was staphylococcus epidermidis (18.4%), followed by staphylococcus aureus, pseudomonas aeruginosa and enterococcus faecalis (16.8%, 12.7% and 10.4%, respectively). the maximum resistance of gram-positive cocci was observed to penicillin and the lowest to linezolid. gram-negative bacteria showed the highest resistance to tetracyclines, while the same strains demonstrated the highest sensitivity to polypeptide antibiotics. comparison of the resistance patterns of gramnegative and gram-positive bacterial strains showed significant difference in the tetracycline efficiency. key words: skin infections, wound swab, antibiotic resistance, s. epidermidis, s. aureus, p. aeruginosa apstrakt: stojanović-radić, z., dimitrijević, m., stanković, n., aleksić, a., pejčić, m.: učestalost i rezistencija bakterijskih izolata iz infekcija rana. biologica nyssana, 7 (2), decembar 2016: 151-158. mikrobiološkom analizom uzoraka, izolovano je 613 bakterijskih sojeva iz briseva rana. izolati su identifikovani na osnovu rasta na diferencijalnim i selektivnim podlogama. ispitivanje osetljivosti izolovanih sojeva na različite antibiotike rađeno je metodom disk difuzije po eucast v 5.0 protokolu. najčešće izolovana bakterija iz briseva rana bila je staphylococcus epidermidis (18.4%), nakon koje su najučestalije izolovane bile staphylococcus aureus, pseudomonas aeruginosa i enterococcus faecalis. najveća rezistencija gram-pozitivnih koka primećena je na peniciline a najmanja na linezolid. gram-negativne bakterije su pokazale najveću rezistenciju na tetracikline, dok su isti sojevi ispoljili najveću osetljivost na antibiotike iz klase polipeptidnih antibiotika. poređenjem obrazaca rezistencije gram-negativnih i gram-pozitivnih bakterijskih sojeva utvrđena je značajna razlika u efikasnosti primene tetraciklina. key words: infekcije kože, bris rane, antibiotska rezistencija, s. epidermidis, s. aureus, p. aeruginosa 7 (2) • december 2016: 151-158 12th sfses • 16-19 june 2016, kopaonik mt doi: 10.5281/zenodo.200414 biologica nyssana 7 (2)  december 2016: 151-158 stojanović-radić, z. et al.  frequency of isolation and antibiotic… 152 introduction loss of skin integrity (trauma) caused by mechanical, biological or chemical agent provides a suitable environment for infectious agent entrance, colonization and consequenting acute/chronic infection (g i a c o me t t i et al., 2000; m a c e d o & s a n t o s , 2005). wound infections are very frequent in outpatients and clinical patients, where they constitute a major healing barrier, which affects patient's quality of life. such infected wounds are the cause of large patient's physical and mental discomfort owing to associated pain, hypersensitivity and accompanying unpleasant odour (k o t z et al., 2009). the most prevalent organisms that have been associated with acute and chronic wound infections are staphylococcus aureus (including mrsa – methycillin resistant staphylococcus aureus), pseudomonas aeruginosa and members of family enterobacteriaceae, such as escherichia coli, proteus vulgaris, klebsiella sp., yeast candida albicans and others (a ki n k u n mi et al., 2014; b e s s a et al., 2015). emerging occurence and spread of (multi)drug resistant microbial pathogens present a significant challenge in providing an effective health care and, therefore, a worldwide medical issue. extensive use of antimicrobial agents in the treatment of commonly isolated pathogens results with emergence of their increased resistance to these drugs. also, prescribing of broad spectrum antibiotics for uncomplicated infections presents one of the most responsible approaches for emerging of resistance (m c q u i s t o n h a s l u n d et al., 2013). among resistant microbes, those showing multiresistance (mdr – multiple drug resistance) are the main concern in antimicrobial treatment today, owing to accompanying morbidity, mortality and costs (v e l i č ko vi ć r a d o va n o vi ć et al., 2009). among isolates from wound infections, many of them represent multiresistant microorganisms. literature on this subject showed alarming data, with reported multiresistance of many bacterial species, such as p. aeruginosa, s. aureus (mrsa), as well as species from genera enterococcus and acinetobacter (c e t i n ka ya et al., 2000; r i c e , 2001; s a d e r et al., 2001; g u z ma n b l a n c o et al., 2009; m o n i r i et al., 2009). due to the fact that there are differences in recommendations considering the empiric first-choice antibiotic treatment among european countries (m c q u i s t o n h a s l u n d et al., 2013), diverse reports on isolation frequency and resistance patterns for the same species, but isolated in different countries, are not a surprising fact (s a d e r et al., 2001; s h i t t u et al., 2002; h i r a n s u t h i ku l et al., 2004; o gu n t i b e j u & n w o b u , 2004; m a c e d o & s a n t o s , 2005; a n gu z u & o l i l a , 2007; p o n d e i et al., 2013). in serbia, study in aleksinac (s t a n ko vi ć n e d e l j ko vi ć et al., 2012) investigated frequency of p. aeruginosa isolates from wound infection swabs and reported its high incidence rate of 36.6%. j o va n o vi ć et al. (2014) investigated isolation frequencies of surgical and traumatic wounds causative agents and confirmed that there are differences in incidences among different clinics (abdominal surgery clinic and orthopedy and traumatology clinic in zemun and belgrade). among isolated bacteria, the most prevalent ones were e. coli (48%), pseudomonas spp. (12%), klebsiella spp. (11%), acinetobacter spp. (7%) in abdominal wounds, while s. aureus (36.4%), pseudomonas spp. (20.1%) and acinetobacter spp. (7.1%) were the most common causative agents of traumatic wound infections. according to the presented facts, periodical surveillance of predominant pathogens and also reporting of their antimicrobial resistance patterns are neccessary actions in each country and its distant regions, whose results will be applied in adjusting therapies to the application of proper and effective antibiotics. therefore, the goal of this study was to investigate local (territory of niš) predominant pathogens, recovered from wound infections during a one year period (january to december, 2015). also, the study included determination of their resistance patterns in order to, based on these results, recommend the best antimicrobial therapy approach for the treatment of wound infections in niš region. material and methods specimen collection the present study has been conducted by analyzing 526 wound swab samples, collected from wound infections of polyclinic human (niš) outpatients. the wound samples were collected using a sterile cotton swab, which were used for gentle swabbing of the inner surface of the infected area, afterwards the swabs were aseptically transported to the laboratory. bacterial samples were collected during a one year period between january and december 2015. a total of 613 bacterial isolates were covered by analysis in order to determine the frequency and resistance patterns of bacterial strains. identification of the pathogenic strains isolation of bacteria from wound swabs was done by inoculating the samples on blood agar. the inoculated media were incubated at 35-37 ºc for 16 to 24 hours. bacterial colonies on the agar plates were transported to a panel of selective and differential biologica nyssana 7 (2)  december 2016: 151-158 stojanović-radić, z. et al.  frequency of isolation and antibiotic… 153 media (himedia, india) in order to complete their identification to the genus or species level, afterwards bacterial isolates were further identified using a battery of biochemical tests. endo and macconkey agar were used for identification of the strains belonging to the family enterobacteriaceae. morphological and cultural characteristics were observed on kligler's double sugar medium and peptone water to demonstrate the indole production. other biochemical tests included citrate and urea utilization, and also fermentation of mannitol according to bergey’s manual of determinative microbiology (2000). susceptibility of isolates to antibiotics susceptibility testing was performed according to the standardized eucast protocol v 5.0. briefly, overnight cultures of isolates on mueller hinton agar incubated at 37 °c for 24 h were used for making bacterial suspensions in 0.85% nacl (w/w) and adjusted to mcfarland 0.5 turbidity standards. prepared suspensions were used for inoculation of the mueller hinton agar plate’s surface, afterwards the antibiotic discs were placed on the inoculated agar surface. after the incubation period of 24 h at 37 °c, the inhibition zones around the discs were measured and interpretation of the inhibition zone values (s-sensitive / r resistant) was based on the eucast v 5.0 criteria. results and discussion the six hundred and thirteen strains were obtained from 526 samples of wound swabs collected during 2015. the most frequently isolated species was s. epidermidis (18.4%), which belongs to the grampositive cocci. two species of gram positive cocci, s. aureus and e. faecalis were also isolated with high frequency, 16.8% and 10.4% respectively. the most common gram-negative bacteria in samples were p. aeruginosa (12.7%) and e. coli (9.0%). the other bacteria were isolated in a relatively low percentage, and citrobacter sp. was the least detected isolate in the swabs (fig. 1). the results of our research indicate no correlation with previous studies on this subject, conducted in other countries, where s. epidermidis, reported here as the most dominant isolate showed relatively low isolation frequency (s a d e r et al., 2001; s h i t t u et al., 2002; h i r a n s u t h i ku l et al., 2004; o gu n t i b e j u & n w o b u , 2004; m a c e d o & s a n t o s , 2005; a n gu z u & o l i l a , 2007; p o n d e i et al., 2013). two different studies on isolation frequency of wound isolates in brasil (s a d e r et al., 2001; m a c e d o & s a n t o s , 2005) showed s. aureus and p. aeruginosa to be the dominant ones. investigation in thailand after tzunami (h i r a n s u t h i ka l et al., 2004) reported aeromonas sp. to be the most common wound pathogen, followed by e. coli and s. aureus. similar research in uganda (a n g u z u & o l i l a , 2007) showed that p. mirabilis ranks as the second one by its isolation frequency, right after s. aureus, which was the most frequent wound pathogen. in nigeria, investigation of s h i t t u et al. (2002) reported s. aureus (25.3%), e. coli (12.3%) and p. aeruginosa (9.3%) to be the most prevalent isolates from wounds. two more recent studies (o gu n t i b e j u & n w o b u , 2004; fig. 1. the frequency of bacteria isolated from wound swab 18.4% 16.8% 12.7% 10.4% 9.0% 6.2% 4.6% 4.2% 4.1% 3.6% 2.8% 2.8% 1.6% 1.0% 0.8% 0.3% 0.3% 0.2% 0.2% s. epidermidis s. aureus p. aeruginosa e. faecalis e. coli staphylococcus sp. p. mirabilis klebsiella sp. enterobacter sp. pseudomonas spp. acinetobacter spp. s. alfa haemolyticus p. vulgaris m. morganii serratia sp. s. beta haemolyticus p. rettgeri citrobacter sp. gbacilli biologica nyssana 7 (2)  december 2016: 151-158 stojanović-radić, z. et al.  frequency of isolation and antibiotic… 154 p o n d e i et al., 2013) in the same country, showed significant changes in these frequencies, where p. aeruginosa strains were the most dominant (33.3% and 32.6%, respectively). considering studies performed in serbia (s t a n ko vi ć n e d e l j ko vi ć et al., 2012; j o va n o vi ć et al., 2014), which reported gram negative bacteria p. aeruginosa and e. coli to be the most prevalent ones, there is also high disagreement with the obtained results in this study, where two gram positive strains presented those with the highest isolation frequency. the isolates of s. epidermidis were absolutely resistant (100.0%) to the penicillin antibiotics (penicillin g, ampicillin and amoxicillin), while a higher percentage of isolates showed sensitivity to amoxicillin/clavulanic acid and piperacillin. the carbapenems were significantly effective against the tested strains of s. epidermidis. the absolute fig. 2. frequency of antibiotic resistance of s. epidermidis pen g-penicillin, amp-ampicillin, amo-amoxicillin, amc-amoxicillin/clavulanic acid, taz-tazobactam, pippiperacillin/tazobactam, mer-meropenem, imi-imipenem, cef-cefalexin, cfc-cefaclor, cfx-ceftriaxone, cfrcefuroxime, cfm-cefotaxime, cld-clindamicin, g-gentamicin, cip-ciprofloxacin, t/s -trimethoprim/sulfametoxazol, fuc-fusidic acid, rif-rifampicin, tei-teicoplanin, chl-chloramphenicol, lin-linezolid, erm-erythromicin fig. 3. frequency of antibiotic resistance of s. aureus pen g-penicillin, amp-ampicillin, amo-amoxicillin, amc-amoxicillin/clavulanic acid, taz-tazobactam, pippiperacillin/tazobactam, mer-meropenem, imi-imipenem, lin-linezolid, cef-cefalexin, cfc-cefaclor, cfrcefuroxime, dox-doxycicline, chl-chlomphenicol, erm-erythromicin, cld-clindamicin, g-gentamicin, cipciprofloxacin, t/s-trimethoprim/sulfametoxazol, fuc-fusidic acid, rif-rifampicin, tei-teicoplanin 0,0% 10,0% 20,0% 30,0% 40,0% 50,0% 60,0% 70,0% 80,0% 90,0% 100,0% 0,00% 10,00% 20,00% 30,00% 40,00% 50,00% 60,00% 70,00% 80,00% 90,00% 100,00% biologica nyssana 7 (2)  december 2016: 151-158 stojanović-radić, z. et al.  frequency of isolation and antibiotic… 155 sensitivity was found in the cases of glycopeptide antibiotics (teicoplanin) and linezolid (fig. 2). the results of research conducted in mexico do not indicate similarity with the results of our study (c a s t r o -a l a r c o n et al., 2011). in 2014, study conducted on s. epidermidis isolates obtained from intensive care unit patients showed the highest resistance to erythromycin (59.4%), tetracycline (57.8%) and trimethoprim-sulfamethoxazole (53.1%) (n a j a r -p e e r a ye h et al., 2014). in the present study, percentage of the strains resistant to erythromycin and trimethoprim-sulfamethoxazole were also very high (74.1% and 63.2%, respectively) and had the similar relation (trimethoprimsulfamethoxazole demonstrated higher efficacy against isolates). considering s. aureus isolates, resistance to ampicillin, penicillin g and amoxicillin, which all belong to penicillins, amounted 98.7% for all three antibiotics. study of isolates obtained from the patients of the instutute for public health in ćuprija (serbia) reported similar data, showing high resistance of these strains to penicillin group of antibiotics (p e t r o vi ć -j e r e mi ć et al., 2008). all tested strains of s. aureus were sensitive to carbapenems (imipenem and meropenem), linezolid, doxycycline, fusidic acid and teicoplanin (fig. 3). investigation in nigeria (fa d e yi et al., 2008) found the highest resistance of s. aureus isolates to fluoroquinolones, which was not the case in our study. contrary to this, s a d e r et al. (2001) reported almost identical results with ours for the glycopeptide antibiotics (vancomycin and teicoplanin), which were the most active against all s. aureus isolates (100.0% susceptibility) in both studies. pseudomonas aeruginosa showed high rates of resistance to several classes of antibiotics (fig. 4). in this study, we established the maximum resistance (100.0%) to ampicillin, amoxicillin/clavulanic acid, cefuroxime, and trimethoprim/sulfometaxazol, while slightly lower number of isolates (92.9%) showed resistance against ceftriaxone. out of 78 p. fig. 4. frequency of antibiotic resistance of p. aeruginosa amp-ampicillin, amc-amoxicillin/clavulanic acid, taz-tazobactam, pip-piperacillin/tazobactam, imi-imipenem, mer-meropenem, cfx-ceftriaxone, cfd-ceftazidime, cfp-cefepime, cfr-cefuroxime, g-gentamicin, amkamikacin, tbr-tobramycin, nor-norfloxacin, cip-ciprofloxacin, lev-levofloxacin, t/strimethoprim/sulfametoxazol, col-colistin 0,00% 10,00% 20,00% 30,00% 40,00% 50,00% 60,00% 70,00% 80,00% 90,00% 100,00% fig. 5. frequency of antibiotic resistance of e. faecalis amp-ampicillin, lin-linezolid, dox-doxycicline, chlchloramphenicol, erm-erythromicin, g-gentamicin, g fgentamicin forte, cip-ciprofloxacin, lev-levofloxacin, rif-rifampicin, van-vancomycin, tei-teicoplanin 0,00% 10,00% 20,00% 30,00% 40,00% 50,00% 60,00% 70,00% 80,00% biologica nyssana 7 (2)  december 2016: 151-158 stojanović-radić, z. et al.  frequency of isolation and antibiotic… 156 aeruginosa isolates, 7.9% were resistant to imipenem and even lower percentage to carbapenems (3.5%). c a r me l i et al. (1999) got similar results about the susceptibility of p. aeruginosa to different classes of antibiotics. absolute sensitivity of the p. aeruginosa isolates has been observed toward tazobactam (penicillin group of antibiotics) and to polypeptide antibiotic colistin. similar data were obtained by the research of nosocomial infection in boston (t r o i l l e t et al., 1997). the strains of e. faecalis were in the highest percentage resistant to erythromycin (73.3%), which belongs to the macrolide group of antibiotics (fig. 5). the greatest sensitivity of the pathogen was determined to the glycopeptide antibiotics, vancomycin and teicoplanin, while all tested isolates of e. faecalis showed sensitivity to linezolid. all tested isolates were sensitive to vancomycin which is highly important, bearing in mind the fact that occurrence of multi-resistant enterococci, in particular vancomycin resistant enterococci (vre) is continuously reported (r i c e , 2001). in comparison to the survey conducted in kuwait (u d o et al., 2002), there was a great similarity in the results. examination of individual resistance of all other isolated bacteria wasn’t the part of the reported results of this research, because of their low isolation frequency from wound swabs and, therefore, low number for analysis performance. a small number of isolates wouldn’t give a true picture of the resistance/susceptibility of bacteria to different drugs. these isolates were taken into account in determining the total resistance of gram-positive and gram-negative bacteria to various classes of antibiotics (fig. 6). significant difference in the tetracycline efficiency was found when the resistance patterns of gram-negative and gram-positive bacterial strains were compared. gram-negative bacilli were resistant to tetracycline in 60.8% of samples, while grampositive cocci showed lower resistance (15.1%). the carbapenems were the most active against most of the gram-positive and gram-negative bacteria (>80.0% susceptible). these results are similar with research in uganda (a n gu z u & o l i l a , 2007). conclusion a total of 613 bacterial isolates were isolated and identified from 526 samples of wound swabs. the most common causative agents of wound infection were the members of the genus staphylococcus. staphylococcus epidermidis was the predominant species (18.4%), followed by s. aureus (16.8%), p. aeruginosa (12.7%) and e. faecalis (10.4%). the gram-positive cocci showed the highest resistance rates to penicillin, while gram-negative bacteria were mostly resistant to tetracycline. significant difference in the tetracycline efficiency was found when comparing the resistance patterns of gramnegative and gram-positive bacterial strains. according to the obtained results, antibiotic therapy of wound infections in niš and surrounding area should always include previous isolation and antibiogram of the pathogen, but when it is not possible, the treatment of choice should include carbapenems or their combination with tetracyclines in order to cover both possible groups of causative organisms. at the same time, this approach leaves a battery of the last-resort antibiotics (e.g. linezolid, fig. 6. comparison of the resistance patterns of gram-negative and gram-positive bacterial wound isolates 0,0% 10,0% 20,0% 30,0% 40,0% 50,0% 60,0% 70,0% 80,0% g+ gbiologica nyssana 7 (2)  december 2016: 151-158 stojanović-radić, z. et al.  frequency of isolation and antibiotic… 157 teicoplanin) for each group of bacteria, which are found to be very efficient against obtained isolates. acknowledgements. this work was funded by the ministry of education, science and technological development of the republic of serbia (project 172061). references akinkumi, e.o., adesunkanmi, a.r., & lamikanra, a. (2014). pattern of pathogens from surgical wound infections in a nigerian hospital and their antimicrobial susceptibility profiles. african health sciences, 14(4), 802–809. anguzu, j.r., olila, d. 2007: drug sensitivity patterns of bacterial isolates from septic postoperative wounds in a regional referral hospital in uganda. african health sciences, 7(1): 148-154. bergey, d.h., & holt, j.g. (2000). bergey’s manual of determinative microbiology. lippincott williams & wilkins, philadelphia. bessa, l.j., fazii, p., di giulio, m. and cellini, l. 2015. bacterial isolates from infected wounds and their antibiotic susceptibility pattern: some remarks about wound infection. international wound journal, 12: 47–52. carmelli, y., troillet, n., eliopoulos, m.g., samore, h.m. 1999: emergence of antibiotic-resistant pseudomonas aeruginosa: comparison of risks associated with different antipseudomonal agents. antimicrobial agents and chemotherapy, 43(6): 1379-1382. castro-alarcon, n., ribas-aparicio, r.m., silvasanchez, j., calderon-navarro, a., sanchezperez, a., parra-rojas, i., aparicio-ozores, g. 2011: molecular typing and characterization of macrolide, lincosamide and streptogramin resistance in staphylococcus epidermidis strains isolated in a mexican hospital. journal of medical microbiology, 60: 730-736. cetinkaya, y., falk, p., mayhall, c.g. 2000: vancomycin resistant enterococci. clinical microbiology reviews, 13(4): 686-707. fadeyi, a., adigun, a.i., rahman, a.g. 2008: bacteriological pattern of wound swab isolates in patients with chronic leg ulcer. international journal of health research, 1(4): 183-188. giacometti, a., cirioni, o., schimizzi, a.m., del prete, m.s., barchiesi, f., d'errico, m.m., scalise, g. 2000: epidemiology and microbiology of surgical wound infections. journal of clinical microbiology, 38(2): 918-922. guzman-blanco, m., mejia, c., isturiz, r., alvarez, c., bavestrello, l., gotuzzo, e., labarca, j., luna, m.c., rodriguez-noriega, e., salles, j.c.m., zurita, j., salles, c. 2009: epidemiology of meticillin resistant staphylococcus aureus (mrsa) in latin america. international journal of antimicrobial agents, 34: 304-308. hiransuthikul, n., tantisiriwat, w., lertutsahakul, k., vibhagool, a., boonma, p. 2005: skin and soft-tissue infections among tsunami survivors in southern thailand. clinical infectious diseases, 41(10): 93-96. stanković nedeljković, n.s, tiodorović, b., kocić, b., cirić, v., milojković, m., waisi, h. 2015. pseudomonas aeruginosa serotypes and resistance to antibiotics from wound swabs. vojnosanitetski pregled, 72(11): 996-1003. kotz, p., fisher, j., mccluskey, p., hartwell, s.d., dharma, h. 2009. use of a new silver barrier dressing, allevyn◊ ag in exuding chronic wounds. international wound journal, 6(3):186194. macedo, j.l.s.d., santos, j.b. 2005: bacterial and fungal colonization of burn wounds. memorias do instituto oswaldo cruz, 100(5): 535-539. mcquiston haslund, j., rosborg dinesen, m., sternhagen nielsen, a. b., llor, c., & bjerrum, l. 2013. different recommendations for empiric first-choice antibiotic treatment of uncomplicated urinary tract infections in europe. scandinavian journal of primary health care, 31(4), 235–240. moniri, r., kheltabadi farahani, r., shajari, g., nazem shirazi, m.h., ghasemi, a. 2009: molecular epidemiology of aminoglycosides resistance in acinetobacter spp. with emergence of multidrug-resistant strains. iranian journal of public health, 39(2): 63-68. oguntibeju, o., nwobu, r. 2004: occurrence of pseudomonas aeruginosa in post-operative wound infection. pakistan journal of medical sciences, 20(3): 187-191. petrović-jeremić, lj., kuljić kapulica, n., mirović, v., kocić, b. 2008. staphylococcus aureus methycillin-resistance mechanisms. vojnosanitetski pregled, 65(5): 377-382. pondei, k., beleudanyo, g.f., oluwatoyosi, o. 2013: current microbial isolates from wound swabs, their culture and sensitivity pattern at the niger delta university teaching hospital, okolobiri, nigeria. tropical medicine and health, 41(2): 4953. rice, b.l. 2001: emergence of vancomycinresistant enterococci. emerging infectious diseases, 7(2): 183-187. sader, s.h., gales, c.a., pffaler, a.m., mendes, e.r., zoccoli, c., barth, a., jones, n.r. 2001: pathogen frequency and resistance patterns in brazilian hospitals: summary of results from three years of the sentry antimicrobial https://www.ncbi.nlm.nih.gov/pubmed/?term=fisher%20j%5bauthor%5d&cauthor=true&cauthor_uid=19538192 https://www.ncbi.nlm.nih.gov/pubmed/?term=mccluskey%20p%5bauthor%5d&cauthor=true&cauthor_uid=19538192 https://www.ncbi.nlm.nih.gov/pubmed/?term=hartwell%20sd%5bauthor%5d&cauthor=true&cauthor_uid=19538192 https://www.ncbi.nlm.nih.gov/pubmed/?term=dharma%20h%5bauthor%5d&cauthor=true&cauthor_uid=19538192 biologica nyssana 7 (2)  december 2016: 151-158 stojanović-radić, z. et al.  frequency of isolation and antibiotic… 158 surveillance program. the brazilian journal of infectious diseases, 5(4): 200-214. shahin najar-peerayeh, s., moghadas, a. j., behmanesh, m. 2014. antibiotic susceptibility and meca frequency in staphylococcus epidermidis, isolated from intensive care unit patients. jundishapur journal of microbiology, 7(8): e11188. shittu, a.o., kolawole, d.o., oyedepo, e.a.r. 2002: a study of wound infections in two health institutions in ile-ife, nigeria. african journal of biomedical research, 5: 97-102. jovanović, s., radić-sekereš, m., jovanović, n., radenković, d. 2014. bacteriological analysis of surgical and traumatic wounds of patients treated at the clinical center of serbia. ii congress for chronic wounds treatment “old dilema-new solutions”, belgrade, serbia. the european committee on antimicrobial susceptibility testing. routine and extended internal quality control for mic determination and disk diffusion as recommended by eucast. version 5.0, 2015. http://www.eucast.org. troillet, n., samore, m.h., carmeli, y. 1997: imipenem-resistant pseudomonas aeruginosa: risk factors and antibiotic susceptibility patterns. clinical infectious diseases, 25(5): 1094-1098. udo, e.e., al-sweih, n., phillips, a.o., chugh, d.t. 2002: species prevalence and antibacterial resistance of enterococci isolated in kuwait hospitals. journal of medical microbiology, 52: 163-168. veličković-radovanović, r., petrović, j., kocić, b., antić, s., ranđelović, g. 2009: correlation between antibiotic consumption and bacterial resistance as quality indicator of proper use of these drugs i inpatients. vojnosanitetski pregled, 66(4): 307-312. hacıoğlu doğru, acar, 2019, biologica nyssana 10(2) 10 (2) december 2019: 181-187 doi: 10.5281/zenodo.3600203 effects of salt stress factors on antimicrobial activity of two triticum aestivum l. varieties original article nurcihan hacıoğlu doğru department of biology, faculty of arts and sciences, çanakkale onsekiz mart university, çanakkale, turkey nurcihan.n@gmail.com (corresponding author) okan acar department of biology, faculty of arts and sciences, çanakkale onsekiz mart university, çanakkale, turkey oacar@comu.edu.tr received: july 10, 2019 revised: september 10, 2019 accepted: september 13, 2019 abstract: salinity is one of the most common environmental stress factors that adversely affect plant growth and crop production in cultivated areas worldwide. herbal or ‘alternative’ medicine is gaining popularity and scientific research about wheat grass as a “functional food” is becoming more available and popular. wheat grass, triticum aestivum l. has a long history and is widely used as a health food supplement. it is found to be used as a treatment for minor ailments, and also as a preventive dietary supplement and therapeutic drug. current study was aimed at evaluate antimicrobial properties of the two varieties of t. aestivum l. [cv. tosunbey (drought tolerant) and cv. sultan 95 (drought sensitive)], grown in three different conditions [(1) control; not treated with salt or acetyl salicylic acid; (2) treatment with sea water; (3) sea water and pre-treatment of seeds with acetyl salicylic acid]. the antimicrobial activity of the ethanol extracts of the two varieties of t. aestivum were assayed against escherichia coli nrrl b-3704, pseudomonas aeruginosa atcc 27853, proteus vulgaris atcc 13315, acinetobacter baumanii atcc 19606, bacillus subtilis atcc 6633, staphylococcus aureus atcc 25923, s. haemolyticus atcc 43252 and candida albicans atcc 10231 test microorganisms by agar disc diffusion method and broth microdilution method. the results showed that the ethanol extracts from the different treatments showed antimicrobial activities, with the diameters of the inhibition zone ranging from 8 to 13 mm and 2.5 to 20 µg/ ml, respectively. the highest antimicrobial activity was demonstrated against p. aeruginosa atcc 27853 by the extract of t. aestivum cv. sultan 95, which grown in sea water and whose seeds were pre-treated with acetyl salicylic acid. key words: antibacterial activity, t. aestivum cv. tosunbey, t. aestivum cv. sultan 95 apstract: efekat sonog stresa na antibakterijsku aktivnost dva varijeteta triticum aestivum l. salinitet je jedan od najčešćih sredinskih stresnih faktora koji nepovoljno utiču na rast biljaka i produkciju useva u kultivisanim područjima širom sveta. biljna ili “alternativna” medicina dobija na popularnosti i naučna istraživanja na temu pšenice kao “finkcionalne hrane” postaju sve dostupnija i popularnija. pšenica, triticum aestivum l., ima dugu istoriju i veoma je zastupljena kao dodatak ishrani. utvrđeno je da se koristi kao tretman za blaža oboljenja i kao preventivni dodatak ishrani i terapeutski agens. ovo istraživanje imalo je za cilj da utvrdi antimikrobni potencijal dva varijeteta t. aestivum l. [cv. tosunbey (tolerantna na sušu) i cv. sultan 95 (osetljiva na sušu)], uzgajanih u tri različita uslova [(1) kontrola; netretirana semena; (2) tretman morskom vodom; (3) tretman morskom vodom i pretretman semena acetil salicilnom kiselinom]. antimikrobna aktivnost etanolnih ekstrakata dva varijeteta t. aestivum testirana je u odnosu na escherichia coli nrrl b-3704, pseudomonas aeruginosa atcc 27853, proteus vulgaris atcc 13315, acinetobacter baumanii atcc 19606, bacillus subtilis atcc 6633, staphylococcus aureus atcc 25923, s. haemolyticus atcc 43252 i candida albicans atcc 10231, korišćenjem metoda disk difuzije na agaru i mikrodilucije. rezultati su pokazali da etanolni ekstrakti iz različitih uslova gajenja poseduju antimikrobnu aktivnost, sa dijametrima inhibicionih zona od 8 to 13 mm i minimalnim inhibitornim aktivnostima od 2.5 do 20 µg/ml. najveća antimikrobna aktivnost, utvrđena u odnosu na p. aeruginosa atcc 27853, pokazana je od strane ekstrakta t. aestivum cv. sultan 95, dobijenog od biljaka gajenih u morskoj vodi i čija su semena pretretirana acetil salicilnom kiselinom. ključne reči: antibakterijska aktivnost, t. aestivum cv. tosunbey, t. aestivum cv. sultan 95 © 2019 hacıoğlu doğru, acar. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work noncommercially under the same license as the original. 181 13th symposium on the flora of southeastern serbia and neighboring regions introduction antibiotic resistance has become a global concern (westh et al., 2004) and clinical efficiency of many existing antibiotics is being threatened by the emergence of multi-drug resistant pathogens as reported by bandow et al. (2003). so, there is an urgent need to develop new antimicrobial compounds which are more active against newer and re-emerging infectious diseases (rojas et al., 1992). medicinal plants have bioactive compounds which are used for curing of various human diseases and play an important role in healing. biosynthesis and accumulation of substances such as flavonoids, phenolic acid and anthocyanin enhances the biological activity (antimicrobial, antioxidant, etc.) of plant material (karakaş and türker, 2013). wheat grass, triticum aestivum l. has a long history and is widely used as a health food supplement. it is found to be utilized as a treatment for minor ailments and serious life-threatening diseases, and as a preventive dietary supplement and therapeutic drug. salinity is one of the most common environmental stress factors that affect plant growth and crop production in cultivated areas worldwide. primary saline conditions appear naturally in environment, while anthropogenic activities are responsible for secondary salinity. increased urbanization and deforestation are two important human-derived activities for salinity. a number of reports on soil salinization and their influences on crop productivity, land degradation and ecological disturbances are reported worldwide. excess amount of salts affected physical, chemical, as well as the biological properties of soils. plant health in saline soils considerably declines owing to poor nutrition, osmotic stress and reduced microbial diversity (paul and lade, 2014; vimal et al., 2018). the response of plants to physiological stress conditions (e.g. salinity) activates genes encoding pathogen-related proteins (prs) (sehgal and mohamad, 2018). therefore, prs are commonly synthesized in plants not only under biotic stress conditions, but also under abiotic stress conditions (such as osmotic shock, chemical injury, and aging). in fact, there are inducible defense proteins classified into 17 prs families in plants (van loon et al., 2006; sehgal and mohamad, 2018). the relationship between antifungal (schmidt at al., 2019) and antimicrobial resistance (sharma et al., 2018) has been shown in proteins induced by oxidative stress caused by environmental stresses. in addition, phytoalexins synthesized in response to stresses such as antimicrobial attack and physical injury in plants (ejike et al., 2013) have been shown to increase antimicrobial activity in the rice plant in relation to the physiology of chloroplast and mitochondria (li et al., 2011). accordingly, it is believed that increases in antimicrobial activity are a consequence of oxidative stress caused by environmental stresses. in this study, the effects of seawater induced salt stress (24-26% nacl, 24 h) and salicylic acid (sa) priming combinations were investigated on antimicrobial activity in 21-day-old seedlings of the two wheat varieties [cv. tosunbey (drought tolerant) and cv. sultan 95 (drought sensitive)]. material and methods the plants material: the seeds were sterilized by washing with 5% sodium hypochlorite solution for 5 min (1 time) and with sterile distilled water for 2.5 min (3 times). seawater is taken from the coast of kepez (çanakkale), 30 m from the coast, and filtered in the laboratory to remove the particles. salt concentration of clear seawater is 24-26% (türkoğlu, 2006) and it was mixed with distilled water to obtain 50% concentration (sw 50%), while full concentration (sw 100%) was also used in experiments. seedlings were planted in perlite:peat mixture (1:3) containing pots. plants were watered with hoagland nutrient solution (100%; steward, 1983) for 21 days. seedlings of t. aestivum l. cv. tosunbey (drought tolerant) and cv. sultan 95 (drought sensitive) were divided into three groups: (1) control; not treated with salt or acetyl salicylic acid; (2) treatment with 100 ml sea water (50% and 100 %); (3) sea water and pre-treatment of seeds with 1000 mg acetyl salicylic acid (during 2 hours in 2 x 500 mg aspirin tablets solution). leaf sampling was collected after 24 hours of salt treatment of 21d old plants. test microorganisms gram-negative bacteria (escherichia coli nrrl b-3704, pseudomonas aeruginosa atcc 27853, proteus vulgaris atcc 13315, acinetobacter baumanii atcc 19606), gram-positive bacteria (bacillus subtilis atcc 6633, staphylococcus aureus atcc 6538, staphylococcus haemolyticus atcc 43252) and yeast (candida albicans atcc 10231) were used for determining the antimicrobial activities of the two t. aestivum varieties. preparation of extracts for antimicrobial activity air-dried samples of two wheat varieties were grounded into a fine powder in a grinding mill. pulverized plant samples (1 g) were extracted with 10 ml of 80% ethanol, (1:10 w/v) using an orbital shaker for 8 h at room temperature. the extract was separated from the solids by filtration with whatman no. 1 filter paper. the remaining solids 182 biologica nyssana ● 10 (2) december 2019: 181-187 hacıoğlu doğru, acar ● effects of salt stress factors on antimicrobial activity of two triticum aestivum l. varieties 183 biologica nyssana ● 10 (2) december 2019: 181-187 ta bl e 1. a nt im ic ro bi al a ct iv ity re su lts o f t he tw o va rie tie s of t . a es tiv um e xt ra ct s te st m ic ro or ga ni sm s p la nt e xt ra ct s *d is c d iff us io na (m m ) m ic (µ g/ m l) cv . t os un be y cv . s ul ta n 95 cv . t os un be y cv . s ul ta n 95 t1 t2 t3 t4 s 1 s 2 s 3 s 4 c on tr ol p 10 / n y 10 0 t1 t2 t3 t4 s 1 s 2 s 3 s 4 c on tr ol s t/ n y 10 0 e . c ol i n r r l b -3 70 4 8. 0 ± 0. 21 11 .0 ± 0. 2 8. 0 ± 0. 03 8. 0 ± 0. 03 12 .0 ± 0. 0 1 11 .0 ± 0. 03 7. 0 ± 0. 0 1 7. 0 ± 0. 0 1 16 .0 20 .0 2. 5 20 .0 20 .0 2. 5 2. 5 20 .0 20 .0 4. 0 p. a er ug in os a at c c 2 78 53 10 .0 ± 0. 11 9. 0 ± 0. 02 7. 0 ± 0. 2 10 .0 ± 0. 01 11 .0 ± 0. 12 7. 0 ± 0. 01 10 .0 ± 0. 02 13 .0 ± 0. 13 8. 0 5. 0 5. 0 20 .0 5. 0 2. 5 20 .0 10 .0 2. 5 1. 0 p. v ul ga ris at c c 1 33 15 13 .0 ± 0. 01 10 .0 ± 0. 0 1 8. 0 ± 0. 1 11 .0 ± 0. 14 12 .0 ± 0. 12 9. 0 ± 0. 03 9. 0 ± 0. 02 8. 0 ± 0. 01 13 .0 2. 5 5. 0 20 .0 5. 0 2. 5 10 .0 10 .0 20 .0 4. 0 a . b au m an ii at c c 1 96 06 10 .0 ± 0. 0 1 9. 0 ± 0. 12 8. 0 ± 0. 01 10 .0 ± 0. 12 10 .0 ± 0. 01 7. 0 ± 0. 03 10 .0 ± 0. 04 10 .0 ± 0. 12 12 .0 10 .0 20 .0 20 .0 10 .0 10 .0 20 .0 20 .0 20 .0 2. 0 b . s ub til is at c c 6 63 3 7. 0 ± 0. 14 8. 0 ± 0. 2 7. 0 ± 0. 12 9. 0 ± 0. 03 11 .0 ± 0. 12 9. 0 ± 0. 03 8. 0 ± 0. 12 10 .0 ± 0. 12 14 .0 20 .0 20 .0 20 .0 5. 0 2. 5 10 .0 20 .0 5. 0 4. 0 s . a ur eu s at c c 6 53 8p 11 .0 ± 0. 02 9. 0 ± 0. 12 8. 0 ± 0. 04 10 .0 ± 0. 1 10 .0 ± 0. 01 8. 0 ± 0. 01 12 .0 ± 0. 01 7. 0 ± 0. 0 1 15 .0 2. 5 5. 0 20 .0 5. 0 5. 0 20 .0 2. 5 20 .0 4. 0 s . h ae m ol yt ic us at c c 4 32 52 9. 0 ± 0. 03 10 .0 ± 0. 1 8. 0 ± 0. 12 10 .0 ± 0. 1 11 .0 ± 0. 1 10 .0 ± 0. 2 10 .0 ± 0. 0 1 8. 0 ± 0. 0 1 14 .0 5. 0 5. 0 20 .0 5. 0 5. 0 5. 0 5. 0 20 .0 5. 0 c an di da a lb ic an s at c c 1 02 31 7. 0 ± 0. 1 8 .0 ± 0. 1 10 ± 0. 1 11 .0 ± 0. 0 1 8. 0 ± 0. 1 7. 0 ± 0. 12 7. 0 ± 0. 03 7. 0 ± 0. 12 16 .0 20 .0 20 .0 10 .0 5. 0 20 .0 20 .0 20 .0 20 .0 2. 5 t: t . a es tiv um c v. t os un be y s : t . a es tiv um c v. s ul ta n 95 t1 : c on tro l; t2 : t re at m en t w ith a ce ty l s al ic yl ic a ci d; t 3: tr ea tm en t w ith s al in ity s tre ss ; t 4: s al in ity s tre ss a nd p re -tr ea tm en t o f s ee ds w ith a ce ty l s al ic yl ic a ci d s 1: c on tro l; s 2: tr ea tm en t w ith a ce ty l s al ic yl ic a ci d; s 3: tr ea tm en t w ith s al in ity s tre ss ; s 4: s al in ity s tre ss a nd p re -tr ea tm en t o f s ee ds w ith a ce ty l s al ic yl ic a ci d in hi bi tio n zo ne (m m ); a in cl ud es d ia m et er o f d is k (6 m m ); p 10 = p en ic ill in (1 0 ug /d is c) ; s t: s tre pt om yc in ; n y 10 0 n ys ta tin 1 00 u g/ di sc hacıoğlu doğru, acar ● effects of salt stress factors on antimicrobial activity of two triticum aestivum l. varieties were extracted twice with the same solvent and the extracts were combined. extracts were stored in a refrigerator (4 °c) until analyzed (sultana et al., 2007). screening of antimicrobial activity disc diffusion assay empty sterilized antibiotic discs having a diameter of 6 mm (schleicher and schull no. 2668, dassel, germany) were each impregnated with 50 μl of the extract (10 mg/disc). all the bacteria mentioned above were incubated at 35 ± 0.1 °c for 24 h by inoculation into nutrient broth (difco laboratories, mi, usa) and the yeast culture studied was incubated in malt extract broth (difco laboratories, mi, usa) at 25 ± 0.1 °c for 48 h. an inoculum containing 106 bacterial cells or 108 yeast cells/ml was spread on mueller hinton agar (mha) (oxoid ltd., hampshire, uk) plates (1 ml inoculum/plate). the discs injected with extracts were placed on the inoculated agar by pressing slightly. petri dishes were placed at 4 °c for 2 h, afterwards the plate inoculated with the yeast culture was incubated at 25 ± 0.1 °c and bacteria were incubated at 35 ± 0.1 °c for 24 h (collins et al., 1989). at the end of the period, inhibition zones formed on the medium were measured in millimeters. studies were performed in triplicate. treatments with penicillin (p10), and nystatin (nys30) served as positive controls and treatments with ethanol, without bacterial or fungal materials served as negative controls. minimum inhibitory concentration assay minimum inhibitory concentration (mic) was investigated as recommended by instructions of the clinical and laboratory standards institute (clsi, 2006). the lowest concentration of extracts inhibiting visible growth of each test microorganisms was taken as the mic. the medium, 0.1% (w/v) streptomycin (st), nystatin (nys100) and 10% dmso were used as the non-treated, positive and negative controls, respectively (teanpaisan et al., 2017). statistical analysis the results of antimicrobial assay were mean ± sd of the three parallel assays. results and discussion a total of eight plant extracts were tested against eight test microorganisms. ethanol was selected as extraction solvent, because it is one of the best solvents used for the extraction of antimicrobial substances (jonathan and fasidi, 2003). the results showed that the ethanol extracts from the different treatments studied have antimicrobial activities, with the diameters of the inhibition zone 184 ranging from 8 to 13 mm and 2.5 to 20 µg/ml, respectively (tab. 1). the highest antimicrobial activity was demonstrated against p. aeruginosa atcc 27853 by the extract of t. aestivum cv. sultan 95 which grown in sea water and whose seeds were pre-treated with acetyl salicylic acid (s4). however, the disc diffusion method is a qualitative technique and is mainly used for selecting extracts with antimicrobial activity, mostly when diameter zones of inhibition are ≥ 10 mm (sánchez et al., 2016). so, s. haemolyticus atcc 43252 was the second most susceptible strain against the tested plants extracts. the same was found when observing the results of micro-dilution method. although the disc diffusion method is one of the inexpensive and less laborious antimicrobial testing methods currently available, this procedure suffers with the demerit of not always producing reproducible results for plant extracts (eloff, 1998; vambe et al., 2018). this is primarily because some constituents do not diffuse readily in polarized agarose gels (vambe et al., 2018). when the same extracts were screened using microdilution method, five plant extracts, namely t1, t2, s1, s2, s3 demonstrated higher antibacterial activity than the control antibiotic. this discordance observed in the results obtained from these two bioassays could be attributed to several factors (vambe et al., 2018). it is possible that antibacterial activity was masked in the disk diffusion assay by low diffusion potential. wheat is a grain containing protein, starch (vida et al., 2014), fiber, phytochemicals and antioxidant substances which have a very important place in human nutrition (andersson et al., 2013). while meeting the daily nutrition requirements with protein and starch, it facilitates digestion with fibers and thus reduces the risk of developing colon cancer (huang et al., 2015). this plant has been also shown to have anti-inflammatory, antioxidant, anti-carcinogenic, immuno-modulatory, laxative, astringent, diuretic, antibacterial and anti-aging properties and is a part of formulations as a highly effective antimicrobial agent (saha et al., 2018). in the literature there are many investigations about wheat grass antimicrobial activity. pallavi et al. (2011), tested wheat grass extracts against the gram-positive bacteria s. aureus, b. subtilis and gram-negative e. coli, using amoxicillin as standard. certain extracts exhibited considerable activity against b. subtilis and moderate activity against s. aureus and e. coli. ashok (2011) also reported antibacterial activity against e. coli, p. aeruginosa and s. aureus. antifungal activity was also reported against c. albicans. sundaresan et al. (2015), tested antimicrobial properties of 7th, 14th, biologica nyssana ● 10 (2) december 2019: 181-187 hacıoğlu doğru, acar ● effects of salt stress factors on antimicrobial activity of two triticum aestivum l. varieties and 21st day wheat grass extracts obtained with five different solvents (water, ethanol, methanol, ethyl acetate and hexane). all these extracts showed antibacterial activity against seven food borne pathogens. amongst them, hexane extracts from the seven-day-old wheat grass showed maximum antibacterial activity, especially against yersinia enterocolitica and listeria monocytogenes. das et al. (2012) found that acetone (80%) extract of wheatgrass was effective against five foodborne microorganisms, including the fungus aspergillus niger, a common contaminant of food. there appears to be a lack of published scientific data on the salt stress effect to antimicrobial properties of wheat grass, thereby highlighting the need for continued research. plants are frequently subjected to a variety of harsh environmental stresses such as scarcity of water, extreme temperatures, high soil salinity, herbivore attack, and pathogen infection diminishing their productivity (sewelam et al., 2016). different salt concentrations induce physiological and metabolic changes in plants affecting their seed germination, growth, development, yield and also decreases the rate of respiration and photosynthesis in plants. in addition to the growth and yield, the composition of bioactive compounds present in the aromatic and medicinal plants is affected by salinity (gil et al., 2002). the increased levels of plant secondary metabolites such as phenols, flavonoids, tannins, alkaloids and others under the influence of increased salt concentrations as a part of defense mechanism have been reported (kate, 2008). reactive oxygen species (ros) are continuously produced under normal environmental conditions in plant cells. bioactive compounds also trigger metabolic pathways in plant cells and result in a higher ros accumulation. the common response observed in salt-stressed plants is the generation of ros, highly reactive and responsible in damaging cell structures, nucleic acids, lipids and proteins. plants possess medicinal value with anti-inflammatory and antimicrobial activities; acquired resistance to stress induced by ros is due to the presence of several bio-active compounds (foyer et al., 1994). in the scientific literature, there are no data on the effect of salt stress on wheat grass antimicrobial activity. kotagiri et al. (2017) investigated the secondary metabolites and antimicrobial potential of leaf, stem and root ethanol and chloroform extracts of five different coleus species; c. aromaticus, c. amboinicus, c. barbatus, c. forskohlii and c. zeylanicus subjected to salinity stress. the up-regulation in the content of plant bioactive compounds, along with the antimicrobial activities of ethanol and chloroform extracts under the influence of salinity stress have been observed during the study in coleus. the leaf, stem and root extracts of all the five coleus species showed good antimicrobial activity against the tested pathogenic strains. the leaf extracts of coleus showed higher inhibitory activity compared to the stem and root extracts. ethanol extracts showed higher antimicrobial activity ranging from 1.5-100.0 mg/ml when compared with the chloroform extracts ranging from 0.97-250 mg/ ml respectively. the leaf, stem and root extracts of coleus showed effective antimicrobial activity against the pathogenic strains even under saline conditions is due to the up regulation of secondary metabolites which provides a scope of developing novel drugs to treat infectious diseases. our results have a correlation with the results of kotagiri et al (2017) about increasing antimicrobial activity under saline stress and pre-treatment of seeds with acetyl salicylic acid conditions, which is probably related to the up-regulation of secondary metabolites which provides a scope for developing novel drugs to treat p. aeruginosa atcc 27853. conclusion the screening of the plant extracts for antimicrobial activity has shown that higher plants represent a potential source of new antiinfective agents. herbal medicines are a valuable and rapidly available resource for primary health care and complementary health care systems. the results of the present study provide evidence that t. aestivum plant varieties grown in different condition represent an important asset to the health care in communities. some infectious diseases could be potentially managed using t. aestivum plant varieties, based on monoor combination therapies. more pharmacological investigations including phytochemical analysis are necessary to find out which compounds are responsible for observed effects. acknowledgments. this paper was presented at the symposium on the flora of southeastern serbia and neighboring regions, june, 20-23 2019, serbia. references andersson, a.a.m., andersson, r., piironen, v., lampi, a.m., nystrom, l., boros, d., fras, a., gebruers, k., courtin, c.m., delcour, j.a., rakszegi, m., bedo, z., ward, j.l., shewry, p.r., aman, p. 2013: contents of dietary fibre components and their relation to associated bioactive components in whole grain wheat samples from the healthgrain diversity screen. food chemistry, 136: 1243-1248. ashok, s.a. 2011: phytochemical and pharmaco185 biologica nyssana ● 10 (2) december 2019: 181-187 hacıoğlu doğru, acar ● effects of salt stress factors on antimicrobial activity of two triticum aestivum l. varieties logical screening of wheat grass juice (triticum aestivum l.). international journal of pharmaceutical sciences review and research, 9: 159-164. bandow, j.e., brötz, h., leichert, l.i., labischinski, h., hecker, m. 2003: proteomic approach to understanding antibiotic action. antimicrobial agents and chemotherapy, 47: 94855. clsi 2006: clinical and laboratory standarts institute. methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard-seventh edition. m07a7. das, a,, raychaudhuri, u,, chakraborty, r. 2012: antimicrobial effect of edible plant extract on the growth of some foodborne bacteria including pathogens. nutrafoods, 12: 83-88. eloff, j.n. 1998: which extractant should be used for the screening and isolation of antimicrobial components from plants? j. ethnopharmacol, 60: 1-8. foyer, c.h, lelendais, m., kunert, k.j. 1994: photooxidative stress in plants. physiologia plantarum, 92: 696-717. gil, a., de la fuente, e.b., lenardis, a.e., loopez pereira, m., suaorez, s.a., bandoni, a., van baren, c., di leo lira, p., ghersa, c.m. 2002: coriander essential oil composition from two genotypes grown in different environmental conditions. journal of agricultural food chemistry, 50: 2870-2877. huang, t., xu, m., lee, a., cho, s., qi, l. 2015: consumption of whole grains and cereal fiber and total and cause-specific mortality: prospective analysis of 367,442 individuals. bmc medicine, 13:59. jonathan, s.g., fasidi, i.o. 2003: antimicrobial activities of two nigerian edible macrofungi, lycoperdon pusilum and l. giganteum. african journal of biomedical research, 6: 85-90. karakaş, f.p., turker, a.u. 2013: an efficient in vitro regeneration system for bellis perennis l. and comparison of phenolic contents of field-grown and in vitro-grown leaves by lcms/ms. industrial crops and products. 48: 162-170 kate, v.v. 2008: physiological and biochemical studies in some medicinal plants: tribulus terrestris l. and pedalium murex l. ph. d. thesis submitted to shivaji university, kolhapur, maharashtra, india. kotagiri, d., shaik, k.b., kolluru, v.c. 2017: secondary metabolites and the antimicrobial potential of five different coleus species in response to salinity stress. biorxiv plant biology https://doi. org/10.1101/220368 pallavi, k., kumarswammy, g., shruthi. b. 2011: pharmacognostic investigation and antibacterial activity of triticum aestivum. journal of pharmacy research, 4: 3355-3359. paul, d., lade, h. 2014: plant-growth-promoting rhizobacteria to improve crop growth in saline soils: a review. agronomy for sustainable development, 34: 737-752. rojas, a., hernandez, l., pereda-miranda, r., mata, r. 1992: screening for antimicrobial activity of crude drug extracts and pure natural products from mexican medicinal plants. journal of ethnopharmacology, 35: 275-83 saha, s., islam, z., islam, s., hossain, s., islam, s.m.s. 2018: evaluation of antimicrobial activity of wheat (triticum aestivum l.) against four bacterial strains. skuast journal of research, 20(1): 58-62. sultana, b., anwar, f., przybylski, r. 2007: antioxidant activity of phenolic components present in barks of azadirachta indica, terminalia arjuna, acacia nilotica, and eugenia jambolana lam. trees food chemistry, 104: 1106–1114. sundaresan, a., selvi, a., manonmani, h.k. 2015: the anti-microbial properties of triticum aestivum (wheat grass). international journal of biotechnology for wellness industries, 4(3): 84-91. teanpaisan, r., kawsud, p., pahumunto, n., puripattanavog, j. 2017: screening for antibacterial and antibiofilm activity in thai medicinal plant extracts against oral microorganisms. journal of traditional and complementary medicine, 7(2): 172–177. türkoğlu, m., baba, a., özcan, h. 2006: determination and evaluation of some physicochemical parameters in the dardanelles (canakkale strait, turkey) using multiple probe system and geographic information system. hydrology research, 37(3): 293-301. s ánchez, e. morales, c.r., castillo, s., leosrivas, c., garcía-becerra, l., ortiz martínez, d.m. 2016: antibacterial and antibiofilm activity of methanolic plant extracts against nosocomial microorganisms. evidence-based complementary and alternative medicine, 1-8. sewelam n, kemel k, peer ms. 2016: global plant stress signalling: reactive oxygen species at the cross-road. frontiers of plant science, 7: 187. steward f.c. 1983: plant physiology, academic 186 biologica nyssana ● 10 (2) december 2019: 181-187 hacıoğlu doğru, acar ● effects of salt stress factors on antimicrobial activity of two triticum aestivum l. varieties press, new york, 797p. vambe, m., aremu, a.o., chukwujekwu, j.c., finnie, j.f., van staden, j. 2018: antibacterial screening, synergy studies and phenolic content of seven south african medicinal plants against drugsensitive and -resistant microbial strains. south african journal of botany, 114: 250-259 vida, g., szunics, l., veisz, o., bedo, z., lang, l., arendas, t., bonis, p., rakszegi, m. 2014: effect of genotypic, meteorological and agronomic factors on the gluten index of winter durum wheat. 187 euphytica, 197:61-71 vimal, s.r., gupta, j., singh, j.s. 2018: effect of salt tolerant bacillus sp. and pseudomonas sp. onwheat (triticum aestivum l.) growth under soil: a comparative study. microbiology research, 9(7462): 26–32. westh, h., zinn, c.s., rosdahl, v.t. 2004: an international multicenter study of antimicrobial consumption and resistance in staphylococcus aureus isolates from 15 hospitals in 14 countries. microbial drug resistance, 10: 169-76. biologica nyssana ● 10 (2) december 2019: 181-187 hacıoğlu doğru, acar ● effects of salt stress factors on antimicrobial activity of two triticum aestivum l. varieties anticancer compounds from medicinal plants biologica nyssana 2 (2)  december 2011: 00-00 ljupković r.b. et al..  removal cu(ii) ions from water... 1 original article floristic diversity of plants spontaneously spreading in the botanical garden of the university of łódź (poland) anna bomanowska 1 , maria kurzac 1 , agnieszka stefaniak* 1 1 department of geobotany and plant ecology, university of łódź, banacha 12/16, pl-90237 łódź; * e-mail: stefa@biol.uni.lodz.pl abstract: bomanowska, a., kurzac, m., stefaniak, a.: floristic diversity of plants spontaneously spreading in the botanical garden of the university of łódź (poland), biologica nyssana, 3 (1), september 2012: 01-10. the aim of the paper is to characterize plants spontaneously spreading outside the flower beds within the territory of the botanical garden of the university of łódź. floristic studies rivealed that many cultivated species also appear outside their original planting sites. total of 124 taxa of vascular plants were found to be subject of spontaneous spreading. various directions of spontaneous migration of species from sites of their planting were observed within the garden: the “oak-hornbeam lawn”, shaded lawns, sunny lawns, shaded paths, sunny paths, limestone rock garden on hillside. most species occurred exclusively in one of the abovelisted sites. the analysed group of vascular plants includes a predominant representation of native species – 103 species (83.1 %) and predominance of herbal perennial plants – hemicryptophytes (80 species; 67.7 %). key words: botanical garden, migration patterns, plant collection, spontaneous spread introduction there are many reasons for growing plants in modern botanic gardens, including species conservation, scientific research, cultivar development, education, public amenity and pleasure, and cultural heritage (w y s e j a c k s o n & s u t h e r l a n d , 2000, m a u n d e r et al., 2001). historically, their main activities were horticultural and taxonomic research, but the urgent need for biodiversity conservation, biotechnological outreach and education have become an increasingly important focus (k o j s & k o j s , 2004, r y b c z y ń s k i et al., 2004, h a v e n s et al., 2006, c h a n g et al., 2008). for this reason, botanical gardens are areas of intensive cultivation of numerous plants and a key pathway for the introduction of new species. most plant species introduced to a new country or region has originated from ornamental horticulture (d e h n e n s c h m u t z & t o u z a , 2008, v i r t u e et al., 2008, g a l e r a & s u d n i k w ó j c i k o w s k a , 2010). botanical gardens, as well as private flower gardens are often in service to alien taxa as bridgeheads in the process of successful colonisation of new territories (r e i c h a r d & w h i t e , 2001, s m i t h et al., 2006, d e h n e n -s c h m u t z et al., 2007, d a w s o n et al., 2008, v i r t u e et al., 2008, g a l e r a & s u d n i k -w ó j c i k o w s k a , 2010). due to this phenomenon, botanical gardens might influence the plant species inventory that contributes to enrichment of country or regional flora with new, mainly exotic taxa. floristic studies carried out in the teaching and experimental botanic garden of the university of łódź, led to the observation that many cultivated species also appear outside of their original planting 3 (1) • september 2012: 01-10 biologica nyssana 3 (1)  september 2012: 01-10 bomanowska, a. et al.  floristic diversity of plants spontaneously… 2 sites. these observations supplemented by the analysis of data contained in the documentation of garden collections, formed the basis for the preparation of the present work, aiming to characterize those plants that spontaneously spread outside the flower beds within the territory of the university botanical garden. materials and methods the botanical garden of the university of lodz is situated in the centre of lodz (19° 29’ 05” e and 51° 46’ 40” n) and occupies total area of 1.02 ha. the garden does not constitute a separate legal entity, being internally managed by the faculty of biology and environmental protection. the object was established in 1985, and has status of a teaching and experimental garden, i.e. the collections are mainly used for teaching purposes and for scientific research. management duties include the selection of plant material, creation of necessary collections to be used for the education of biology and environmental protection students, and care of aesthetic qualities and the external image of the garden. the garden features herbaceous and ligneous plants, represented by species of both native and foreign origin (s t e f a n i a k & b o m a n o w s k a , 2011). collection of the garden includes 793 taxa: 295 species and varieties of trees and shrubs as well as 498 taxa of herbaceous plants (s t e f a n i a k & b o m a n o w s k a , 2011). the herbaceous collection is spread over 189 cultivation plots, each having an area of 3.75 m 2 . garden collections are a reflection of habitat and taxonomic diversity of the vascular flora of poland. numerous plants of alien origin (mainly mediterranean, american) are also cultivated in the garden. detailed floristic studies were carried out in the garden in the period of 2007-2008. observations were performed both on the cultivation plots and on the dividing paths and in the internal buffer zone of the garden. the present study includes selected data from these floristic observations, with some additional data from the years 2006 and 2009. only those cultivated species, for which individuals where observed to have spontaneously spread outside their original planting sites, were included in the analysis. the relevant individuals occurred outside the actual area of cultivation of the given plant species, on ground with a different land management profile, e.g. on a path, a lawn, in the garden buffer zone etc. in the case of vegetatively reproducing plants, only independent separated rametes were considered as spontaneously occurring individuals (f a l i ń s k a , 2002). biological classification of species was based on the system of raunkiaer’s life forms, with the life form for each species taken from z a r z y c k i et al. (2002) and from the „biolflor” database (http://www.ufz.de/biolflor). the remaining data on the biology of a species such as manner of reproduction, dispersal of diasporas and life strategies were compiled from available sources (v a n d e r p j l , 1972, h a n d e l & b e a t i e , 1990, l i n d a c h e r , 1995, t o k a r s k a -g u z i k , 2005) as well as from the on-line databases (biolflor, floraweb, l i u et al., 2008) and personal observations. the geographical and historical classification of the flora as proposed by k o r n a ś (1981) and modified by j a c k o w i a k (1990). status of species within this classification was determined using the studies of z a j ą c (1979), m i r e k et al. (2002), t o k a r s k a -g u z i k (2005), but it was related to local conditions. due to the history of ground usage and the group of analysed plants (exclusively species escaping from cultivation), all analysed species were treated as synanthropic ones. native species were ascribed the status of oekiophytic apophytes (oekiophytes), i.e. native species escaping from cultivation (s u d n i k -w ó j c i k o w s k a & k o ź n i e w s k a , 1988). species of alien origin, on the other hand, were classified as metaphytes (archaeophytes and kenophytes) if they are permanently naturalised in the polish flora and occur spontaneously in plant communities, or as diaphytes (ergasiophygophytes) if they are exclusively cultivated plants which appear in the spontaneous flora of poland transiently as a result of temporary “escape” from cultivation. the nomenclature of herbaceous vascular plants follows m i r e k et al. (2002) and g a w r y ś (2009), whereas names of woody species are from s e n e t a & d o l a t o w s k i (2000). results in the course of field research in the garden, 124 taxa of vascular plants were found to be subjects of spontaneous spreading, belonging to 47 families and 107 genera (table 1). in distinguished group the most numerously represented families are: asteraceae (12 species) and lamiaceae (11), as well as poaceae (8) and ranunculaceae (8) and rosaceae and apiaceae represented by seven species each (table 1). the majority of families (35) have only one or two species. the analysed group of vascular plants includes a predominant representation of native species, i.e. oekiophytes – 103 species (83.1%). among alien species, ergasiophygophytes (14 species; 11.3%) biologica nyssana 3 (1)  september 2012: 01-10 bomanowska, a. et al.  floristic diversity of plants spontaneously… 3 predominate over metaphytes (7; 5.6%). the comparision of the life form spectrum showed a predominance of herbal perennial plants – hemicryptophytes (80 species; 64.5%) over other life forms. 11 species (8.9%) are chamaephytes. the proportion of woody species (megafanerophytes, nanofanerophytes) and species which are both geophytes and hemicryptophytes is 7.3% for each of these groups. table 1. cultivated taxa spontaneously spreading in the experimental and teaching botanical garden of the university of łódź. abbreviations and symbols: status (affiliation to geographical-historical groups): oe – oekiophyte, me – metaphyte, ar – archaeophyte, ke – kenophyte, er – ergasiophygophyte; life forms: ch – chamaephyte, g – geophyte, h – hemicrytophyte, m – megaphanerophyte, n – nanophanerophyte, li – liana; life strategy: c – competitor, s – stres tolerator , r – ruderal; types of reproduction: s – seed, sv – seed and vegetatively, ssv – mosty by seed, rarely vegetatively, vvs – mosty vegetatively, rarely by seed, v – vegetatively; dispersal mode: ane – anemochory, aut – autochory, anthr – anthropochory, bar – barochory, dys – dyszoochory, end – endozoochory, epi – epizoochory, hyd – hydrochory, myr – myrmecochory; directions of spontaneous migration: ohl – oak-hornbeam lawn, shl – shaded lawns, sul – sunny lawns, shp – shaded paths, sup – sunny paths, lim – limestone rock, hill – hillside. no. species family status l ife fo rm l ife stra te g y r e p ro d u c tio n d isp e rsa l m o d e directions of spontaneous migration o h l s h l s u l s h p s u p l im h il l 1. abies alba l. pinaceae oe m c s ane + + + + + + + 2. acer platanoides l. aceraceae oe m c s ane + + + + + + + 3. acer pseudoplatanus l. aceraceae oe m c s ane + + + + + + + 4. aegopodium podagraria l. apiaceae oe h c vvs aut + 5. agrimonia eupatoria l. rosaceae oe h c s epi + 6. agrimonia procera wallr. rosaceae oe h cs sv epi + 7. ajuga reptans l. lamiaceae oe h csr sv myr, aut + 8. allium ursinum l. liliaceae oe g csr ssv end + 9. angelica archangelica l. subsp. archangelica apiaceae oe h cs s ane, hyd + 10. anthemis tinctoria l. asteraceae me [ar] h cs s aut, epi + 11. anthriscus sylvestris (l.) hoffm. apiaceae oe h c ssv epi, dys + 12. aquilegia x hybrida hort. ranunculaceae er h ? s ? + 13. arrhenatherum elatius (l.) p. beauv. ex j. presl et c. presl poaceae oe h c ssv ane, anthr + 14. asclepias syriaca l. asclepiadaceae me [ke] g, h c sv ane, anthr + 15. astragalus glycyphyllos l. fabaceae oe h c s epi, dys + 16. betonica officinalis l. lamiaceae oe h c sv ? + 17. betula pendula roth betulaceae oe m c s ane, epi + + + + + + + 18. borago officinalis l. boraginaceae er h cr s epi + 19. brachypodium sylvaticum (huds.) p. beauv. poaceae oe h cs sv epi + 20. campanula rapunculoides l. campanulaceae oe h csr sv aut + + + + + + + 21. cardamine pratensis l. brassicaceae oe h csr vvs aut + 22. carex brizoides l. cyperaceae oe g,h csr sv ane, aut + 23. carum carvi l. apiaceae oe h c s dys + 24. centaurea jacea l. asteraceae oe h c s dys, ane, aut + 25. centaurea stoebe l. asteraceae oe h csr s ane, epi, anthr + 26. cerastium tomentosum l. caryophyllaceae er ch c sv ane + 27. cerinthe minor l. boraginaceae me [ar] h csr s ? + 28. chaerophyllum hirsutum l. apiaceae oe h c sv epi, ane + 29. clematis vitalba l. ranunculaceae me [ke] n c s ane, epi, aut + + + + + + + 30. clinopodium vulgare l. lamiaceae oe h cs sv ane, epi, aut + biologica nyssana 3 (1)  september 2012: 01-10 bomanowska, a. et al.  floristic diversity of plants spontaneously… 4 31. convallaria majalis l. liliaceae oe g, h cs sv end, aut, anthr + 32. coronilla varia l. fabaceae oe h c ? epi, ane, aut + 33. corydalis cava (l.) schweigg. et körte fumariaceae oe g csr s aut, myr + 34. corynephorus canescens (l.) p. beauv. poaceae oe h cs s ane + 35. cynoglossum officinale l. boraginaceae oe h cs s epi + + + + + + + 36. deschampsia cespitosa (l.) p.beauv. poaceae oe h c s ane, anthr + + + + + + + 37. dianthus barbatus l. caryophyllaceae er ch ? s ane, anthr + 38. digitalis grandiflora mill. scrophulariaceae oe g, h c sv aut, ane, epi + 39. epiliobium hirsutum l. oenotheraceae oe h c sv ane + 40. epilobium roseum schreb. oenotheraceae oe h cs sv ane + 41. eranthis hyemalis (l.) salisb. ranunculaceae er g csr s ? + + + + + + + 42. euonymus europea l. celastraceae oe n c s end + 43. euphorbia cyparissias l. euphorbiaceae oe g,h csr sv aut, myr + 44. festuca ovina l. poaceae oe h csr s epi, dys, aut, anthr + + 45. ficaria verna huds. ranunculaceae oe g, h csr vvs aut, myr + 46. fragaria vesca l. rosaceae oe h csr sv end, epi, aut + 47. galium mollugo l. rubiaceae oe h c s dys + 48. galium verum l. rubiaceae oe h cs sv epi + + 49. genista tinctoria l. fabaceae oe ch cs s aut + + 50. geranium pratense l. geraniaceae oe h c sv aut, epi + 51. geum urbanum l. rosaceae oe h csr ssv epi + 52. hedera helix l. araliaceae oe n,ch sc sv end + 53. helianthemum nummularium (l.) mill. cistaceae oe ch cs ssv ? + 54. hepatica nobilis schreb. ranunculaceae oe h csr sv aut, myr + 55. heracleum sphondylium l. apiaceae oe h c ssv dys, epi, ane + 56. hieracium pilosella l. asteraceae oe h csr sv ane, aut, epi + 57. hieracium sabaudum l. asteraceae oe h c sv ane + 58. hippophaë rhamnoides l. eleagnaceae oe n c sv end, anthr + + + + + + + 59. holcus lanatus l. poaceae oe h c sv ane, end, anthr + + + + + + + 60. holcus mollis l. poaceae oe g, h csr vvs ane, anthr + + + + + + + 61. humulus lupulus l. cannabaceae oe h, li c sv ane + 62. inula helenium l. asteraceae me [ke] h c sv ane, epi, myr + + 63. junglans regia l. junglandaceae er m c s bar, ane, end + + + + + + + 64. knautia arvensis (l.) j.m.coult. dipsacaceae oe h c s end, epi + 65. lathyrus pratensis l. fabaceae oe h c ssv aut, ane, dys + 66. lathyrus vernus (l.) bernh. fabaceae oe g csr sv aut + 67. leontodon hispidus l. asteraceae oe h csr sv ane + + + + + + + 68. leucanthemum vulgare lam. asteraceae oe h c sv ane, epi, dys + 69. ligustrum vulgare l. oleaceae oe n c s end, anthr, aut + + + + + + + 70. linum perenne l. linaceae me [ke] h cs sv aut, epi, anthr + + 71. lychnis flos cuculi l. caryophyllaceae oe h csr sv ane, epi + 72. lysimachia vulgaris l. primulaceae oe h cs sv ane, epi, hyd, aut + 73. lythrum salicaria l. lythraceae oe h cs ssv ane, epi + + + + + + + 74. mahonia aquifolium (pursh) nutt. berberidaceae er n c sv anthr, end + + + + + + + 75. melica nutans l. poaceae oe g,h cs sv aut, myr + 76. melissa officinalis l. lamiaceae er h c sv ? + 77. mercurialis perennis l. euphorbiaceae oe g,h cs sv aut, myr + 78. origanum vulgare l. lamiaceae oe c,h csr sv ane, aut + + biologica nyssana 3 (1)  september 2012: 01-10 bomanowska, a. et al.  floristic diversity of plants spontaneously… 5 79. ornithogalum umbellatum l. liliaceae oe g csr vvs bar, myr, aut + + + + + + + 80. picea abies (l.) h.karst. pinaceae oe m c s ane, end + + + + + + + 81. pimpinella saxifraga l. apiaceae oe h cs s anthr, epi + 82. plantago media l. plantaginaceae oe h csr sv aut + 83. potentilla micrantha ramond ex dc. rosaceae oe h csr sv ane, aut + 84. primula veris huds. primulaceae oe h csr sv ane + 85. prunella vulgaris l. lamiaceae oe h csr sv hyd, epi, dys, aut + 86. pulmonaria obscura dumort. boraginaceae oe h csr sv myr + 87. pulmonaria officinalis l. boraginaceae oe h csr sv myr + 88. pyracantha coccinea m. roem. rosaceae er n ? ? end + + + + + + + 89. quercus robur l. fagaceae oe m c s ane, end, bar + + + + + + + 90. ranunculus cassubicus l. ranunculaceae oe h ? sv epi + + 91. ranunculus lanunginosus l. ranunculaceae oe h cs sv epi + 92. rudbeckia laciniata l. 'gold kugel' asteraceae me [ke] h c sv ane, aut, epi, myr + 93. rumex acetosa l. polygonaceae oe h c sv ane, epi, dys, hyd + + + + + + + 94. salvia sclarea l. lamiaceae er h ? ? aut + + + + + + + 95. salvia verticillata l. lamiaceae oe h csr ssv aut + + 96. sanguisorba minor scop. rosaceae oe h csr ssv ane, hyd, aut + 97. scabiosa ochroleuca l. dipsacaceae oe h csr s ane + + 98. scilla sibirica haw. liliaceae er g csr sv aut, myr, anthr + 99. scorzonera hispanica l. asteraceae er h csr s ? + 100. scropularia nodosa l. scrophulariaceae oe h cs sv ane, epi, aut + + 101. sedum acre l. crassulaceae oe ch s sv hyd, myr, aut + 102. sedum reflexum l. crassulaceae oe ch s sv hyd, myr, aut + 103. sedum sexangulare l. crassulaceae oe ch s sv hyd, myr, aut + 104. solanum dulcamara l. solanaceae oe n, li c sv end, epi, aut + 105. stachys recta l. lamiaceae oe h csr s ? + 106. stachys sylvatica l. lamiaceae oe h cs sv epi, ane, hyd, aut + 107. tanacetum balsamita l. asteraceae er h c sv ane, epi, anthr + 108. taxus x media rehder taxaceae er n ? ? end + + + + + + + 109. thalictrum minus l. ranunculaceae oe h cs sv ane, epi, hyd + + + + + + + 110. thymus kosteleckyanus opiz lamiaceae oe ch csr s aut + + 111. thymus pulegioides l. lamiaceae oe ch csr ssv aut, myr + + 112. tilia cordata mill. tiliaceae oe m c ssv ane + + + + + + + 113. tragopogon orientalis l. asteraceae oe h csr s ane, epi + 114. trifolium fragiferum l. fabaceae oe h csr ssv ane, epi + 115. ulmus levis pall. ulmaceae oe m c ssv ane + + + + + + + 116. valeriana officinalis l. valerianaeae oe h c sv ane, epi, hyd, aut + + + + + + + 117. verbascum densiflorum bertol. scrophulariaceae oe h c s ane, epi + 118. verbascum nigrum l. scrophulariaceae oe h c s ane, epi + 119. verbascum phlomoides l. scrophulariaceae oe h c s ane + 120. veronica teucrium l. scrophulariaceae oe t c s ane, aut, hyd, anthr + + 121. vinca minor l. apocynaceae oe ch cs sv aut, myr, anthr + 122. vincetoxicum hirundinaria medik. asclepiadaceae oe h cs sv ane, aut + 123. viola reihenbachiana jord. ex boreau violaceae oe h csr sv aut, myr + 124. viola riviniana rchb. violaceae oe h csr sv myr + biologica nyssana 3 (1)  september 2012: 01-10 bomanowska, a. et al.  floristic diversity of plants spontaneously… 6 as many as 49 escaped species are plants with competitive strategy. quite a large group (25 species) are species whose competitive abilities are limited by the stress (cs strategy). as many as 40 are species with the mixed, csr strategy. only one species (borago officinalis) represents the cr strategy. the distinguished group of escaped plants the most numerous are species that reproduce by seeds (40 species; 32.3%) or both by seeds and during vegetatively (75; 60.5%). only 5 species prefer vegetative reproduction: aegopodium podagraria, cardamine pratensis, ficaria verna, holcus mollis, ornithogallum umbellatum (table 1). the group of spontaneously spreading species in the garden is very diverse with respect to different ways of spreading. anemochorous plants prevail over others – 54 species (43.5%). the share of myrmeko-, baroand autochorous species is also significant. most species use several ways of dispersal. the migration of species appears to be multidirectional (fig.1, table 1). various directions of spontaneous migration of species from sites of their planting were observed within the garden: the “oakhornbeam lawn”, shaded lawns, sunny lawns, shaded paths, sunny paths, limestone rock garden on the hillside. most species (93; 75%) occurred exclusively in one of the above-listed sites. among abovementioned species, most have chosen shaded places, such as lawns, paths and “oak-hornbeam lawn” (43 species; 34.7%). escaped species appeared also quite numerously on the sunny paths and lawns (37 species; 29.8%; fig.1). only 13 species (10.5%) occurred spontaneously in limestone rock garden and hillside. there was also a relatively numerous group of plants (28 species; 22.6%) which spread all over the territory of the garden discussion the phenomenon of spontaneous spread of cultivated species, observed in the botanical garden of the university of łódź, confirms the tendencies observed in other botanical gardens (r e i c h a r d & w h i t e , 2001, g a l e r a , 2003, g a l e r a & s u d n i k -w ó j c i k o w s k a , 2004, 2010, d a w s o n et al., 2008, v i r t u e et al,. 2008). even though the number of plants escaping from cultivation is much smaller there (124) than in other gardens in poland (g a l e r a , 2003), this stems probably from the smaller size and less rich collection of the garden in łódź. still, on the local scale the “escaped species” flora is relatively rich, since it constitutes 15.5% of the whole plant collection in the garden. characteristic features of the described group are determined by the specific range of plants cultivated in the particular garden as well as biological propensities of some species for spreading. the influence of these local factors is figure 1. directions of spontaneous migrations of cultivated plants in the university botanical garden. abbreviations: see table 1. biologica nyssana 3 (1)  september 2012: 01-10 bomanowska, a. et al.  floristic diversity of plants spontaneously… 7 reflected both in the taxonomic structure and in the ecological characteristics of the investigated group of species. the high frequency of escaping plants from poaceae and asteraceae families is related to the fact that the majority of the herbaceous collection in the garden belongs to these families, like the majority of species in the polish flora (s t e f a n i a k & b o m a n o w s k a , 2011). moreover, biological characteristics of many species from these families (i.a. the ability to use various dispersal routes) predisposes them to spread and to occupy new diverse habitats (m i z i a n t y , 1995; p y š e k , 1997). the biological spectrum with its preponderance of hemicryptophytes is also a reflection of the species composition of polish flora and selection of species cultivated in the garden (z a r z y c k i et al., 2002, s t e f a n i a k & b o m a n o w s k a , 2011). the collection of herbaceous plants is composed mainly of perennials and a small number of biannual species. the lack of therophytes among spontaneously spreading plants may be explained by the fact that few plants from this group are cultivated in the university garden, with most of them being cultivars which produce no seeds. in other investigated polish botanical gardens this is numerous group of runaway species (g a l e r a & s u d n i k -w ó j c i k o w s k a , 2004). the most numerous group of the described flora is oekiophytes (native escaped species). the cultivated flora of university garden is also represented mainly by this group, while in the remaining botanical gardens in poland there is similar share of two groups: native species and ergasiophygophytes (g a l e r a & s u d n i k w ó j c i k o w s k a , 2004). it is probably because the garden collection is composed mainly of native species (s t e f a n i a k & b o m a n o w s k a , 2011). distribution of grime’s life strategies showing a similar share of species representing ctype and csr-type strategies in the described flora reflects the habitat conditions in the garden which is an anthropogenically transformed area, but with stable ecological conditions (with homogeneous manners of exploitation). this conclusion may be supported by the preference for generative reproduction shown by spontaneously spreading species, even though it requires significantly more ecological effort than vegetative reproduction (f a l i ń s k a , 2002). most plants that spread spontaneously in the university botanical garden uses several different ways of dispersion of propagules. the diversity of strategies helps species to spreadp0’’’, migrate and settle on new habitats (v a n d e r p i j l , 1972, k o l l m a n n , 2000, s o o n s & o z i n g a , 2000, v i t t o z & e n g l e r , 2007). composition of the described group is clearly dominated by anemochorous plants, including species from asteraceae and poaceae families, as well as some trees (e.g. maples). these results are in agreement with those obtained for warsaw botanical gardens (s u d n i k -w ó j c i k o w s k a & g a l e r a , 2005). species disseminated by the wind spread in different directions: the lawns and paths, both sunny and shady, and on limestone hills. in case of many plant species the main vector which spreads propagules are animals, including birds (a d a m o w s k i & k n o p i k , 1996, g o s p e r et al., 2005 and literature cited therein). garden of the university is a food source and resting site for many bird species, which eat the fleshy fruits of many species (e.g. taxus x media, euonymus europaea, mahonia aquifolia, pyracantha coccinea), thereby contributing to their spread. plants with seeds that have prehensile trichomes or hooks (e.g. agrimonia officinalis, borago officinalis) are spread around within the garden by mammals, especially by rodents, but also by house cats (own observations). seeds of species such as corydalis cava, scilla sibirica, viola reichenbachiana, v. riviniana, as well as some plants from asteraceae and lamiaceae families, which have elaiosome bodies, are spread by ants (v a n d e r p i j l , 1972, h a n d e l , 1990, g o r b et al., 2000). anthropochory is also of significant importance in spread of plants (f a l i ń s k i , 1972, v i t t o z & e n g l e r , 2007). the unintentional transport of diaspores of in the garden takes place both during gardening procedures and while teaching field classes with groups of students. in this manner, the following species may have spread: arrhenatherum elatius, centaurea stoebe, cynoglossum officinale. species spreading in zoochoric or anthropochoric way, most often spread on paths – places where most visitors walk, as well as living and penetrating it occasionally animals. among native species which spontaneously spread in the university garden particular speciallz significant is very rare species, e.g. potentilla micrantha, a vulnerable species (vu) included in the polish red data book of plants (k a ź m i e r c z a k o w a & z a r z y c k i , 2002). it is a characteristic species of broadleaf forests, and for this reason it has been planted in the most shaded site, the so-called “oak-hornbeam lawn”. secondary localities of this plant appeared spontaneously on lawns in other parts of the garden. this is another biologica nyssana 3 (1)  september 2012: 01-10 bomanowska, a. et al.  floristic diversity of plants spontaneously… 8 case of the positive function of botanical gardens in aiding the spread of plants. this escape of species from flower beds in the garden can be dangerous to the native flora if it contributes to expansion of invasive species (r e i c h a r d & w h i t e , 2001, d e h n e n s c h m u t z et al., 2007, d a w s o n et al., 2008, k ř i v á n e k & p y š e k , 2008, g a l e r a & s u d n i k -w ó j c i k o w s k a , 2010). it seems that this is not a real and present danger in the described case, since among the plants which appear spontaneously in the garden, few show any tendencies towards expansion or invasiveness. potentially, this description may fit only to asclepias syriaca and carex brizoides, two species which have a high reproductive potential, which grow quickly and reproduce both via seeds and vegetatively (l i u et al., 2008; biolflor database). the common milkweed is a new arrival in our flora (1872 – first record in polish territory; t o k a r s k a g u z i k , 2005), introduced as an ornamental plant, which has in recent times spread rather quickly in anthropogenic habitats (t o k a r s k a -g u z i k , 2005). in the university garden, individuals of the milkweed have also been observed to quickly occupy new habitats. the quaking grass sedge, although native to our flora, is considered an invasive species and an indicator of degeneration of oak-hornbeam forest phytocoenoses (c h m u r a & s i e r k a , 2007). the potential threat to the local flora may be derived from spontaneously spreading cultivars that are able to hybridise with native species (j a c k o w i a k , 1999). invasive species can show a number of patterns of hybridization with native species, from hybridization without introgression to complete admixture (r h y m e r & s i m b e r l o f f , 1996, a l l e n d o r f et al., 2001). among the species described here, this type of danger may be expected from the hybrid taxon aquilegia x hybrida. this plant undergoes intense generative reproduction and has a tendency to spread quickly, so that cultivating it in the vicinity of natural localities of the original native species aquilegia vulgaris carries the risk of introgressive hybridisation (g a l e r a , 2003). even though a realistic evaluation of the potential of these two species for further expansion is very difficult since the colonisation success of an alien species depends on numerous factors that are often difficult to predict (h e g e r & t r e p l , 2003, h i e r r o et al., 2005, s h a r m a et al., 2005), their spontaneous spread in the garden should not be treated too lightly. it is difficult to predict whether the spontaneous escape of cultivated plants from sites of cultivation within the łódź university botanical garden may be the beginning of their further spread outside its boundaries. the time course of observations was relatively short and it is unknown how long the escaped plants may persevere in the new sites where they grow, especially since most taxa were characterised by low frequency of occurrence. conclusion taking into account the composition of group of escaped plants (predominance of native species, high number of ergasiophygophytes among the alien species) and location of the garden in the centre of a major city, further spontaneous spread of described species seems rather doubtful. it can be supposed that the spontaneous appearance of most escaped plants was rather accidental – they appeared transiently and without a tendency for further spread. at the same time, information on first records of spontaneous occurrence of species escaping from cultivation may be valuable due to the future possibility of reconstruction of initial stages of migration (invasion) once it will have occurred. references adamowski, w. & knopik, a. 1996: ornithochorous species penetration onto abandoned farmland during secondary succession. phytocoenosis (n.s.) 4, seminarium geobotanicum, 8: 97-110. allendorf, f.w., leary, r.f.; spruell, p. & wenburg, j.k. 2001: the problems with hybrids: setting conservation guidelines. trends in ecology & evolution, 16: 613–622. biolflor. search and information system on vascular plants in germany database. ver. 1.1 dept. community ecology, ufz centre for environmental research leipzig-halle. http://www.ufz.de/biolflor/overview/gattung.jsp (accessed 22.02.2011). chmura, d. & sierka, e. 2007: the invasibility of deciduous forest communities after disturbance: a case study of carex brizoides and impatiens parviflora invasion. forest ecology and management, 242: 487–495. dawson, w., mndolwa, a.s.; burslem, d.f.r.p. & hulme, p.e. 2008: assessing the risks of plant invasions arising from collections in tropical botanical gardens. biodiversity & conservation, 17:1979–1995. biologica nyssana 3 (1)  september 2012: 01-10 bomanowska, a. et al.  floristic diversity of plants spontaneously… 9 dehnen-schmutz, k., touza, j., perrings, c. & williamson, m. 2007: the horticultural trade and ornamental plant invasions in britain. conservation biology, 21 (1): 224–231. dehnen-schmutz, k. & touza, j. 2008: plant invasions and ornamental horticulture: pathway, propagule pressure and the legal framework. 1521. in: teixeira da silva j.a. (ed), floriculture, ornamental and plant biotechnology: advances and topical issues. vol. v. global science books, isleworth, uk. falińska, k. 2002: przewodnik do badań biologii populacji roślin. wydawnictwo naukowe pwn, warszawa. 587 p. faliński, j.b. 1972: anthropochory in xerothermic grasslands in the light of experimental data. acta societatis botanicorum poloniae, 41(3): 357368. floraweb daten und informationen zu wildpflanzen und zur vegetation deutschlands. http://www.floraweb.de/index.html (accessed 19.01.2011). galera, h. 2003: rośliny występujące spontanicznie w polskich ogrodach botanicznych. biuletyn ogrodów botanicznych,12: 31-82. galera, h. & sudnik-wójcikowska, b. 2004: the structure and differentiation of the synanthropic flora of the botanical gardens in poland. acta societatis botanicorum poloniae, 73(2): 121128. galera, h. & sudnik-wójcikowska, b. 2010: central european botanic garden as centers of dispersal of alien plants. acta societatis botanicorum poloniae, 79(2): 147-156. gawryś, w. 2009: słownik roślin zielnych. officina botanica, kraków.200 p. gorb, s.n.; gorb, e.v. & punttila, p. 2000: effects of redispersal of seeds by ants on the vegetation pattern in a deciduous forest: a case study. acta oecologica, 21 (4-5): 293-301. gosper, c.r.; stansbury, c.d. & vivian-smith, g. 2005: seed dispersal of fleshy-fruited invasive plants by birds: contributing factors and management options. diversity and distributions, 11: 549–558. handel, s.n. & beattie, a.j. 1990: seed dispersal by ants. scientific american, 263: 54-61. heger, t. & trepl, l. 2003: predicting biological invasions. biological invasions, 5: 313-321. hierro, j.l.; maron, j.l. & callaway, r.m. 2005: a biogeographical approach to plant invasions: the importance of studying exotics in their introduced and native range. journal of ecology, 93: 5-15. jackowiak, b. 1990: antropogeniczne przemiany flory roślin naczyniowych poznania. wyd. naukowe uniwersytetu a. mickiewicza w poznaniu. ser. biol. 42: 1-208. jackowiak, b. 1999: modele ekspansji roślin synantropijnych i transgenicznych. phytocoenosis 11 (n.s.). seminarium geobotanicum, 6: 1-24. kaźmierczakowa, r. & zarzycki, k. 2002: polska czerwona księga roślin. paprotniki i rośliny kwiatowe. instytut botaniki im. w. szafera pan, instytut ochrony przyrody pan, kraków. 664 p. kojs, p. & kojs, r. 2004: educational spaces in botanical gardens. bulletin of botanical gardens, 13: 125-132. kollmann, j. 2000: dispersal of fleshy-fruited species: a matter of spatial scale? perspectives in plant ecology, evolution and systematics, 3/1: 29–51. křivánek, m. & pyšek, p. 2008: forestry and horticulture as pathways of plant invasions: a database of alien woody plants in the czech republic. 21-38. in: tokarska-guzik b., brock j.h., brundu g., child l., daehler c.c. & pyšek p. (eds), plant invasions: human perception, ecological impacts and management. backhuys publishers, leiden, netherlands. liu, k.; eastwood, r.j.; flynn, s.; turner, r.m. & stuppy, w.h. 2008: seed information database (release 7.1, may 2008). http://www.kew.org/data/sid (accessed 22.02.2011). mizianty, m. 1995: trawy – grupa, która odniosła ewolucyjny sukces. wiadomości botaniczne, 29 (1-2): 59-70. lindacher, r. (ed.). 1995: phanart. datenbank der gefässpflanzen mitteleuropas, erklärung der kennzahlen, aufbau und inhalt. veröffentlichungen des geobotanischen institutes der eth stiftung rübel, 125 heft. zürich. pyšek, p. 1997: compositae as invaders: better than the others? preslia, 69(1): 9-22. reichard, s.h. & white, p. 2001. horticulture as a pathway of invasive plant introductions in the united states. bioscience, 51: 103-113. rybczyński, j.j.; mikuła, a. & fiuk, a. 2004: endangered species model plants for experimental botany and biotechnology. bulletin of botanical gardens, 13: 59-63. rhymer, j. m. & simberloff, d. 1996: extinction by hybridization and introgression. annual review of ecology and systematics, 27: 83–109. seneta, w. & dolatowski, j. 2000: dendrologia. wyd. naukowe pwn, warszawa. biologica nyssana 3 (1)  september 2012: 01-10 bomanowska, a. et al.  floristic diversity of plants spontaneously… 10 sharma, g.p.; singh, j.s. & raghubanshi, a.s. 2005: plant invasions: emerging trends and future implications. current science, 88(5):726734. smith, r.m.; thompson, k.; hodgson, j.g.; warren, p.h. & gaston, k.j. 2006: urban domestic gardens (ix): composition and richness of the vascular plant flora, and implications for native biodiversity. biological conservation, 129: 312-322. soons, m.b. & ozinga, w.a. 2000: how important is long-distance seed dispersal for the regional survival of plant species. diversity and distributions, 11: 165-172. stefaniak, a. & bomanowska, a. 2011: plant collections in the teaching and experimental botanical garden of the university of lodz (poland). ulmus,7: 28-35. sudnik-wójcikowska, b. & galera, h. 2005: floristic differences in some anthropogenic habitats in warsaw. annals botanici fennici, 42: 185-193. tokarska-guzik, b. 2005: the establishment and spread of alien plant species (kenophytes) in the flora of poland. wydawnictwo uniwersytetu śląskiego, katowice, 192 p. van der pijl, l. 1972: principles of dispersal in higher plants. springer-verlag, berlinheidelberg-new york. virtue, j.g.; spencer, r.d.; weiss, j.e. & reichard, s.e. 2008: australia’s botanic gardens weed risk assessment procedure. plant protection quarterly, 23(4): 166-178. vittoz, p. & engler, r. 2007: seed dispersal distances: a typology based on dispersal modes and plant traits. botanica helvenica, 117: 109124. wyse jackson, p.s. & sutherland, l.a. 2000: international agenda for botanic gardens in conservation. botanic gardens conservation international, u.k. zając, a. 1979: pochodzenie archeofitów występujących w polsce. , 29: 1-130. zarzycki, k.; trzcińska-tacik, h.; różański, w.; szeląg, z.; wołek, j. & korzeniak, u. 2002: ecological indicator values of vascular plants of poland. w. szafer insitute of botany, polish academy of sciences. kraków. 183 p. đekić, v., milivojević, j., branković, s.: the interaction of genotype and environment on yield and quality components in triticale. biologica nyssana, 9 (1). september, 2018: 45-53. biologica nyssana 9 (1) ⚫ september 2018: 45-53 đekić et al. ⚫ the interaction of genotype and environment on yield… 45 original article received: 29 january 2018 revised: 29 mart 2018 accepted: 27 june 2018 the interaction of genotype and environment on yield and quality components in triticale vera đekić1, jelena milivojević1, snežana branković2 1small grains research centre, save kovačevića 31, 34000 kragujevac, serbia 2university of kragujevac, faculty of science, institute of biology and ecology, radoje domanović 12, kragujevac, serbia * e-mail: verarajicic@yahoo.com abstract: đekić, v., milivojević, j., branković, s.: the interaction of genotype and environment on yield and quality components in triticale. biologica nyssana, 9 (1). september, 2018: 45-53. the experiment was established at the small grains research centre in kragujevac, where two varieties of triticale were investigated. the highest three-year average of grain yield (4.910 t/ha), 1000-kernel weight (42.67 g) and test weight (69.85 kg/hl) were obtained by kg 20 variety. the highest three-year average value of protein content was found in the variety trijumf (13.157%). highly significant influence of the year on grain yield, 1000-kernel weight and protein content was established for investigated winter triticale cultivars by variance analysis, and significant influence of the year on the test weight. the influence of the cultivar on grain yield, 1000-kernel weight, test weight and protein content was not statistically significant. environmental conditions have had a significant effect on grain yield and quality in triticale. grain yield shows a tendency to increase in the years having a higher total amount and better distribution of rainfall during critical plant development stages. key words: grain yield, protein, test weight, triticale apstrakt: đekić, v., milivojević, j., branković, s.: interakcija genotipa i vegetacione sezone na prinos i kvalitet komponenti prinosa tritikalea. biologica nyssana, 9 (1). septembar, 2018: 45-53. eksperiment je izveden na oglednom polju centra za strna žita u kragujevcu, a istraživane su dve ozime sorte tritikalea. najveći trogodišnji prinos zrna (4.910 t/ha), masa 1000 zrna (42.67 g) i hektolitarska masa (69.85 kg/hl) ustanovljeni su kod sorte kg 20. najveća trogodišnja prosečna vrednost sadržaja proteina dobijena je kod sorte trijumf (13,157%). analizom varijanse ustanovljen je visoko značajan uticaj godine na prinos, masu 1000 zrna i sadržaj proteina i značajan za hektolitarsku masu. uticaj sorte na prinos, masu 1000 zrna, hektolitarsku masu i sadržaj proteina statistički nije bio značajan. vegetaciona sezona značajno je uticala na prinos i kvalitet prinosa zrna kod ispitivanih sorti tritikalea. prinos zrna pokazuje tendenciju povećanja u 9 (1) • september 2018: 45-53 doi: 10.5281/zenodo.1470850 biologica nyssana 9 (1) ⚫ september 2018: 45-53 đekić et al. ⚫ the interaction of genotype and environment on yield… 46 godinama koje imaju veću ukupnu količinu padavina i bolju distribuciju padavina tokom kritičnih faza razvoja biljaka. ključne reči: prinos zrna, sadržaj proteina, hektolitarska masa, tritikale introduction the yield per unit area is the result of the action of varieties fertility in interaction with environmental factors. therefore, the yield is relative term and is determined by the variety, environmental conditions and the level of applied technology. yield is largely dependent on the genetic potential, which could be defined as yield of variety, grown in conditions on which it had adapted, with adequate amounts of water and nutrients and efficient control of pests, diseases, weeds and other stresses (milovanovic et al., 1998; đekić et al., 2014). yields vary considerably primarily as a result of agro-ecological conditions during the growing season (tomasović, 2005; glamočlija et al., 2010; đekić et al., 2010; biberdžić et al., 2012; kondić et al., 2012; milovanović et al., 2012; janušauskaitė, 2013; kendal et al., 2014; đurić et al., 2016). the 1000-kernel weight is a direct component of grain yield and changes under the influence of environmental factors, but depends primarily on varietal characteristics. it indicates the grain size and is an important criterion in triticale breeding. milovanović et al. (2014) consider that the 1000 kernel weight in addition to the number of grains per grain heads is the most important criterion in breeding barley for yield increase. the 1000-grain weight, however, is not only a component of yield, but also a very important component of quality of grains (milovanović et al., 2011; đekić et al., 2016; kendal et al., 2016b). test weight is the weight of one hectoliter grain expressed in kilograms and is the oldest and still the most widely used method of evaluating the quality of the grains, because its assessment is quick and easy. test weight is based on physical measures of size and grain shape, which measures weight in the volume mass density. larger hectoliter means higher content of starch and protein in relation to the content of fiber and a space filled with air (milovanovic et al., 2006; đekić et al., 2014; kendal and sayar, 2016a; kendal et al., 2016b). the chemical composition and nutritive characteristics of triticale are result of huge number of varieties with a huge spectrum of characteristics (đekić et al., 2009; djekic et al., 2011; janušauskaitė, 2013; kendal et al., 2016b). milovanović (1993), emphasizes that different winter triticale genotypes have fluctuated within the two year period from 13.44 up to 16.42%. since the early 1970s, university of florida has conducted a triticalebreeding program. three spring-type cultivars that were selected from the cimmyt (international maize and wheat improvement center) nurseries have been released (barnett et al., 1999). considerable improvements in increasing grain yield and test weight have been made. the protein content of the three released cultivars is about 12% with 0.45% lysine on a dry matter basis. within the four year average values, milovanović et al. (1995), recorded protein content from 14.32% to 16.29% in different winter triticale genotypes, while for the winter wheat varieties, value of 13.70% was recorded. đekić et al. (2009) reported the protein content of 12.24% for the triticale variety kg 20, while variety favorit had 12.55% of dry matter. perišić et al. (2008) reported that another winter triticale variety, general, had 14.6% of crude proteins. within six year averages, djekic et al. (2011) recorded proteins from 11.23 to 13.87% in different winter triticale genotypes. in previous studies, grain protein content in triticale genotypes was reported by many researchers from 11.2% to 16.4% (milovanović et al., 1995; janušauskaitė, 2013; kendal and sayar, 2016a; kendal et al., 2016b). the triticale is considered a prospective crop for conditions of serbia, especially from the point of the global climate change which has not avoided the agricultural regions of serbia. the aim of this study was to determine the influence of semi-arid ecological environmental factors of central serbia on the winter triticale varieties and on their differences in stability and adaptability in regard to the grain yield and parameters of grain chemical quality, as well as specificity of explored varieties in regard to growing conditions. material and methods materials and field trials during the 2009/10, 2010/11 and 2011/12 growing seasons, two varieties of winter triticale were investigated (kg 20 and trijumf) at the small grains research centre in kragujevac (in central serbia). the experiments were conducted in randomized block systems, with a plot size of 50 m2 (5 m x 10 m) in five replications. the sowing was carried out using a machine with row spacing of 12.5 cm. the soil on biologica nyssana 9 (1) ⚫ september 2018: 45-53 đekić et al. ⚫ the interaction of genotype and environment on yield… 47 which the trial was conducted was uniform and well prepared. the amount of seed per square meter corresponded to 400-450 viable seeds, depending on the characteristics of varieties. it was sown in the third decade of october, with 400 kg/ha of fertilizer npk 15:15:15, which was added in the fall, while during the spring, fertilization soil was supplemented with 300 kg/ha (kan 27%n). the following properties were analyzed: grain yield (t/ha), 1000-kernel weight (g), test weight (kg/hl) and chemical quality of grain. grain yields were measured for each plot and converted to yield tons per hectare on the basis of 14% grain moisture. the total protein content in the grain was determined by infrared spectroscopy technique on the apparatus perten da 7000 (nir/vis spectro-photometer) employing non-destructive method. soil conditions the trial at the small grains research centre in kragujevac (in central serbia), was performed on soil that is characterized as smonitsa in process of degradation. the physical properties of the soil are very unfavorable, and it belongs to the type of heavy clays. according to the analysis, this is a soil of medium acidity, poor in humus (2.65%), with a substitution and total hydrolytic acidity that were quite high (ph in h2o=5.99, in kcl=4.91). the soil was moderately provided with total nitrogen (0.14% of n), it is poor in easily accessible phosphorus (p2o5≤12 mg/100 g of soil), a medium level of easily accessible potassium (k2o=14-23 mg/100 g of soil) was recorded. meteorological conditions this study was conducted over a three-year period in the šumadija region, central serbia, on a vertisol soil, at kragujevac location, 173-220 m a.s.l. (44° 22′ n, 20° 56′ e), in a temperate continental climate having an average annual temperature of 11.5 °c, typical for šumadija districts in serbia and a rainfall amount of about 550 mm. kragujevac area is characterized by a moderate continental climate, which in general is characterized by uneven distribution of rainfall by month. the data in tab. 1 for the investigated period (2009-2012) clearly indicate that the years in which the researches were conducted differed from the typical multi-year average for kragujevac region regarding the meteorological conditions. the data presented in tab. 1 for the analyzed triticale growing season (20092012) clearly suggest differences in weather conditions between the years of the study and the long-term mean for the region. the average air temperatures were by 1.26 °c, 1.5 °c and 2.1 °c lower in 2009/10, 2010/11 and 2011/12, respectively, as compared to the long-term mean, whereas the sums of rainfall were by 612.7 mm and 12.5 mm higher in 2009/10 and 2011/12 years and lower by 86.2 in 2010/11 year as compared to the long-term mean. compared to the long-term mean, total rainfall values, especially in the first year, second and third year, were considerably higher in february, april and may, whereas total rainfall in april 2010/11 decreased by 31.1 mm. given the high importance of sufficient rainfall amount during the spring months, particularly april and may, for triticale production, the distribution and amount of rainfall over the growing season 2010/11 were considerably more favorable, resulting in increased yields in this year. apart from the rainfall deficiency during the spring months and the non-uniform distribution of rainfall across months, an increase in average air temperatures was also observed. based on the fact that sufficient amounts of rainfall in these months are very important for the table 1. mean monthly air temperatures and precipitation in kragujevac, serbia (2009-2012), in relation to many years average (1960-2009) months interval x xi xii i ii iii iv v vi average mean monthly air temperature (oc) 2009/10 11.7 8.8 2.6 0.9 3.2 7.2 12.1 16.5 20.2 9.24 2010/11 10.2 11.4 2.4 0.9 0.5 7.2 12.0 15.8 20.9 9.0 2011/12 10.4 3.1 4.6 0.7 -3.7 8.1 12.9 16.1 23.0 8.4 average 12.5 6.9 1.9 0.5 2.4 7.1 11.6 16.9 20.0 10.5 the amount of precipitation (mm) 2009/10 102.6 77.5 194.2 57.0 150.5 43.3 142.2 116.7 196.7 1080.7 2010/11 86.9 27.9 50.1 29.1 48.5 20.4 20.8 65.8 32.3 381.8 2011/12 33.3 1.3 43.3 117.2 60.1 5.7 74.5 87.3 57.8 480.5 average 45.4 48.9 56.6 58.2 46.6 32.4 51.9 57.6 70.4 468.0 biologica nyssana 9 (1) ⚫ september 2018: 45-53 đekić et al. ⚫ the interaction of genotype and environment on yield… 48 successful production of cereal crops it can be concluded that the years in which the researches were conducted were not favorable for the triticale growing. statistical analysis on the basis of achieved research results, the usual variational statistical indicators, average values and standard deviations, were calculated. experimental data were analyzed by descriptive and analytical statistics using the statistics module analyst program sas/stat (sas institute, 2000) for windows. all evaluations of significance were made on the basis of the anova test at 5% and 1% significance levels. relative dependence was defined through correlation analysis (pearson's correlation coefficient), and the obtained coefficients were tested at the 5% and 1% levels of significance. results grain yield and chemical characteristics average values of grain yield, 1000-kernel weight, test weight and protein content of investigated kragujevac winter triticale varieties are presented in tab 2. the grain yield of triticale significantly varied across years, from 4.059 t/ha in 2009/10 to 5.615 t/ha in 2010/11 (tab. 2). in the first investigation year (2009/10), variety kg 20 achieved higher yield of grain (4.104 t/ha), while trijumf variety yielded 4.014 t/ha. in the second year (2010/11), the grain yield of variety kg 20 was the highest with 5.868 t/ha, while the slightly lower yield was realized by variety trijumf (5.362 t/ha). in the third year of research (2011/12), the grain yield of variety kg 20 was 4.757 t/ha, which was 320 kg more than the yield obtained by the variety trijumf. the thousand kernel weight of winter triticale grain was variable, depending on environmental conditions. thousand kernel weight in the test period was the highest in 2010/11 (45.34 g) but had significantly lower values in 2009/10 (by 5.72 g or 12.62%) and also significantly decreased in 2011/12 (by 3.91 g or 9%). average thousand kernel weight observed in the three-year period was the highest in kg 20 variety (42.67 g), while the trijumf cultivar had lower yield (41.59 g). tab. 2 presents average values for grain test weight across years and varieties. the average values for test weight in both cultivars were as follows: 70.34 kg/hl in 2009/10, 70.84 kg/hl in 2010/11 and 68.02 kg/hl in 2011/12, suggesting significant variations in test weight during the three years of the study. the average crude protein content of triticale significantly varied across years, from 12.345% in 2010/11 to 13.796% in 2010/11 (tab. 2). during all three years of investigation (2009/10, 2010/11 and 2011/12), the variety trijumf achieved higher average protein content (14.010%, 12.386% and 13.073%). table 2. average values of characteristics of investigated triticale varieties varieties 2009/10 2010/11 2011/12 average x s x s x s x s grain yield, (t/ha) kg 20 4.104 0.674 5.868 0.373 4.757 0.527 4.910 0.904 trijumf 4.014 0.732 5.362 0.669 4.544 0.437 4.640 0.815 average 4.059 0.665 5.615 0.576 4.651 0.470 4.775 0.857 1000-kernel weight, (g) kg 20 40.02 1.154 45.88 0.460 42.10 1.557 42.67 2.727 trijumf 39.22 0.549 44.80 1.000 40.76 3.038 41.59 2.990 average 39.62 0.951 45.34 0.929 41.43 2.383 42.13 2.864 test weight, (kg/hl) kg 20 70.11 3.171 70.89 1.699 68.56 2.533 69.85 2.557 trijumf 70.57 2.834 70.80 1.724 67.48 1.583 69.62 2.513 average 70.34 2.846 70.84 1.615 68.02 2.071 69.73 2.494 proteins content, (%) kg 20 13.581 0.361 12.300 0.454 12.919 0.637 12.935 0.709 trijumf 14.010 0.528 12.386 0.846 13.073 0.331 13.157 0.889 average 13.796 0.483 12.345 0.642 12.996 0.485 13.046 0.798 biologica nyssana 9 (1) ⚫ september 2018: 45-53 đekić et al. ⚫ the interaction of genotype and environment on yield… 49 analysis of variance between observed traits of triticale the analysis of yield variance of 1000-kernel weight, test weight and protein content of the tested winter triticale varieties grown at investigated in kragujevac during three growing seasons 2009/10, 2010/11 and 2011/12, are shown in tab. 3. based on the analysis of variance, it can be concluded that there were highly significant differences in grain yield in regard to the year of investigation (fexp=18.58659 **), while among the investigated triticale varieties the differences were not significant. analysis of variance found highly significant effect of year on the 1000-kernel weight (fexp=34.44302 **), protein content (fexp=18.00073 **), and significant effect on the test weight (fexp=4.54129 *). based on the analysis of variance, it can be concluded that there were no significant differences in grain yield, 1000-kernel weight, test weight and protein content in investigated triticale varieties relative to the cultivar of investigation (tab. 3). the interaction of the investigated factors (y x g) exhibited no statistically significant effect on grain yield, 1000-kernel weight, test weight and protein content (p > 0.05). discussion in regard to the tested winter triticale genotypes, variety kg 20 achieved higher yield of grain (4.910 t/ha). during the research, the values obtained in the second investigation year were significantly different from the first and third year. in year 2009/10, the lowest yield was achieved (4.059 t/ha), while the highest yield was achieved in year 2010/11 (5.615 t/ha). however, determined grain yields of triticale genotypes reported by kendal and sayar (2016a) ranged between 3.680 t/ha to 9.250 t/ha. in another study of kendal et al. (2016b), grain yield of genotypes ranged from 2.92 t/ha in 2013/14 to 6.61 t/ha in 2012/13 season (normal season). barnett et al. (2006) examined 22 genotypes of the winter and spring triticale in four geographically different locations (quincy, fl, usa; plains, ga, usa; bozeman, mt usa and aberdeen, id, usa) during two vegetation seasons. the same authors have also established the genetic diversity of yields of the tested triticale genotypes at different locations. the grain yield ranged from 4.709 t/ha in california to 6.133 t/ha in georgia. in the first year of the study (1997/98), grain yield of the tested triticale genotypes at different locations ranged from 2.507 t/ha to 7.245 t/ha, while in the second year of the research (1998/99) yield varied in the range of 2.341-8.033 t/ha. in these studies, a significantly higher yield of triticale in northern locations (aberdeen, bozeman) was found in relation to the southern (quincy, plains). the average grain yield values differed statistically significantly between the investigated triticale genotypes, between the investigated varieties and between the investigated sites (barnett et al., 2006). kendal et al. (2016b) have indicated that the yield of triticale genotypes in the 2012/13 season have ranged from 4.66 t/ha to 7.67 t/ha. the average grain yield was found to be 6.61 t/ha in this season. milovanovic et al. (2001) suggested that spring triticale is more prone to the negative impact of table 3. anova values of characteristics in investigated triticale varieties effect of year on the traits analyzed traits mean sqr effect mean sqr error f (2,27) p-level grain yield (t/ha) 6.167350 0.331817 18.58659** 0.000008 1000-kernel weight (g) 85.47100 2.481519 34.44302** 0.000000 test weight (kg/hl) 22.69675 4.997861 4.54129* 0.019948 protein content (%) 5.280500 0.293349 18.00073** 0.000011 effect of cultivar on the traits analyzed traits mean sqr effect mean sqr error f (2,27) p-level grain yield (t/ha) 0.545940 0.740994 0.736768 0.397987 1000-kernel weight (g) 8.640333 8.189382 1.055066 0.313136 test weight (kg/hl) 0.420083 6.425560 0.065377 0.800060 protein content (%) 0.369408 0.646858 0.571080 0.456141 effect of the year x cultivar interaction traits mean sqr effect mean sqr error f (2,24) p-level grain yield (t/ha) 0.114417 0.341012 0.335523 0.718264 1000-kernel weight (g) 0.182333 2.416500 0.075453 0.927542 test weight (kg/hl) 1.522583 5.478209 0.277935 0.759751 protein content (%) 0.083670 0.307654 0.271961 0.764201 biologica nyssana 9 (1) ⚫ september 2018: 45-53 đekić et al. ⚫ the interaction of genotype and environment on yield… 50 abiotic stress than winter triticale. milovanović et al. (2011) have observed significantly (p < 0.05) higher yield stability of hybrids compared to lines for triticale crops. the enhanced yield stability represents a major step forward, facilitating coping with the increasing abiotic stress expected from the predicted climate change. achieved statistically significantly higher yields in 2010/11 were primarily the result of heavy rainfall and their good distribution as well as favorable air temperatures during the vegetation period (tab. 1). đekić et al. (2016) stated in their research that the air temperatures and the rainfall amount and distribution during the triticale growing season have the greatest impact on high yields and grain quality. the significant difference in triticale grain yield was also indicated by barnett et al. (2006), janušauskaitė (2013) and kendal et al. (2016a). for the southeastern anatolia region, the results of the study of kendal et al. (2014), demonstrated that the triticale grain yield is higher than those of durum and bread wheat varieties under more extreme growing conditions of the kiziltepe region. milovanovic et al. (2001) stated that in the domestic production conditions, higher yields are achieved by varieties with the shorter growing season because they manage to form the largest part of the yield before the incidence of high temperatures. in this study, in both years, the triticale was not exposed to extremely high temperatures so early growth did not come into its own. variety kg 20, compared to the other genotype trijumf, in the three-year period, had significantly higher value for the thousand kernel weight (42.67 g). the thousand kernel weight of triticale significantly varied across years, from 39.62 g in 2009/10 to 45.34 g in 2010/11. highly significant influence of the year on 1000-kernel weight was established in investigated winter triticale varieties by variance analysis. milovanović (1993) stated that the 1000 grain weight, however, is not only a component of yield but also a very important component of quality of grains. milovanovic et al. (1998) reported that good quality grain needs to have the 1000 grain weight of 45-50 g. kendal et al. (2014) have found that the thousand kernel weight in the tested triticale varieties ranged from 32.9 to 42.7 g in 2010/11. the same authors indicated that the new spring varieties are more suitable than wheat for more extreme growing conditions of southeastern anatolia region. heavier grains will have more starch and less protein (filipčev et al., 2005). a number of authors (đekić et al., 2010; milovanović et al., 2012; janušauskaitė, 2013; kendal et al., 2014, 2016b; đurić et al., 2016) have underlined that 1000-grain weight is a cultivarspecific trait, with considerably higher variations being observed among genotypes than among treatments or environmental factors. previously, many researchers have reported that the thousand kernel weight values of triticale genotypes have ranged from 23.9 g to 54.9 g. (janušauskaitė, 2013; đekić et al., 2014; kendal et al., 2014, 2016b; kendal and sayar, 2016a). test weight depends on many factors such as grain filling and shape, chemical composition, the content of impurities and moisture. low test weight may be due to pre-harvest sprouting (milovanović, 1993). higher test weight means the higher content of starch and protein in relation to the content of fiber and a space filled with air (milovanović et al. 1995; filipčev et al. 2005). the genotype kg 20 had an extreme value (70.89 kg/hl) in the second year (2010/11). all three years are different in regard to the test weight. particularly in the third year (68.02 kg/hl), genotypes had significantly lower test weight compared to other years. based on the analysis of variance, it can be concluded that there were significant differences in test weight in regard to the year of investigation, which is in accordance with the results obtained by đekić et al. (2016). kendal et al. (2016b) recorded an average value of test weight between 73.0 up to 78.6 kg/hl during two growing seasons (2012/13-2013/14) in 25 genotypes, including 20 advanced lines (from cimmyt), three triticale varieties (presto, tacettinbey and karma) and two candidate lines (g15 and g10). barnett et al. (2006) have analyzed the yield and hectoliter mass of the 22 triticale genotypes in four ecologically diverse geographical locations (quincy, fl, usa (agc)=30on84ow, approximate elevation (ae)=58 m), plains, ga, usa (agc=32on84ow, ae=76 m), bozeman, mt usa (agc=45on 111ow, ae=1458 m), and aberdeen, id, usa (agc=42on 112ow, aberdeen, id, usa (agc=42on 112ow, ae=1360 m). field trials were carried out with the winter triticale, which was sowed during a two year period, 1997 and 1998, while the spring sowing of triticale was carried out in 1998 and 1999. the average value of the hectoliter mass in these studies ranged from 68.3 kg/hl to 75.0 kg/hl in the florida area. winter forms of triticale had higher average test weight (75.7 kg/hl) compared to the spring forms (73.4 kg/hl). statistically significant differences between the tested triticale genotypes and investigated sites were determined by analyzing variance of average values of the test weight. in the study reported here, considerable genetic diversity in triticale for test weight at diverse geographic areas was found indicating that selection for specific environments would be possible. generally, the test weight of triticale ranged from 63 to 72 kg/hl, while in dehulled triticale values were up to 80 kg/hl (barnett et al., biologica nyssana 9 (1) ⚫ september 2018: 45-53 đekić et al. ⚫ the interaction of genotype and environment on yield… 51 2006; janušauskaitė, 2013; đekić et al., 2014; kendal et al., 2014; kendal and sayar, 2016a). protein content differed very significantly between years and the average for all genotypes was higher in 2009/10 (13.796%) compared to 2010/11 (12.345%). kendal et al. (2014) have recorded an average value of protein content between 13.3 and 14.7% in the three different triticale genotypes (tacettinbey, karma 2000 and presto), during the growing season 2010/11. perišić et al. (2008) reported 14.6% of crude proteins in winter triticale. djekic et al. (2011) recorded values ranging from 11.23 to 13.87% of proteins in different winter triticale genotypes. in different spring triticale genotypes, kendal et al. (2016b) recorded two year (2012/13 and 2013/14) average from 12.9 to 18.4% of proteins. during the 2012/13 season, which was considered a normal season, protein content ranged from 12.0% to 14.9%. during the 2013/14 season, the protein content ranged from 16.3% to 20.8%. the protein content in triticale genotypes, as reported by researchers kendal and sayar (2016a), ranged from 12.7% to 14.7% and by researcher janušauskaitė (2013), from 11.4% to 14.3% proteins. many authors have pointed that spring triticale varieties have more proteins compared to winter varieties (djekic et al., 2011a; kendal and sayar, 2016a; kendal et al., 2014, 2016b). humid 2010/11 was more favorable year for protein synthesis. our results are consistent with the results obtained by đekić et al. (2016) and milovanovic et al. (2001), where the authors stated that the growing conditions in the observed years had a very significant impact on protein content. analysis of variance showed highly significant effect of year on the protein content (p<0.01). realized average values for these characteristics in the study were slightly higher than the values obtained by djekic et al. (2011b). janušauskaitė (2013) has recorded an increase of the yield of triticale grain achieved by fertilization by 35.7%, compared with the unfertilized control treatment. the analysis of variance of the average grain yield over the four years showed that year significantly influenced the grain yield. the influence of n rate on the grain yield was significant in basic fertilization almost in all the cases; however, the influence of n splitting regime on the yield was insignificant. utilization of fertilizers and certain supplements on extremely acid soils in certain years, particularly those less favorable for production, almost certainly had different effect on grain filling, resulting in diverse relationships between productive and qualitative traits (biberdžić et al., 2012; đekić et al., 2014; janušauskaitė, 2013). presented results confirm the opinion of many authors that the traits analyzed are genetically determined, but strongly modified by the nutrient status of the environment as well as weather conditions (tomasović, 2005; glamočlija et al., 2010; kondić et al., 2012; milovanović et al., 2012; đurić et al., 2016; kendal et al., 2016a,b). the study results indicate that under the influence of the year, highly significant differences are recorded in all of the investigated traits. also, kendal and sayar (2016a) and kendal et al. (2016b) have indicated that in the assessment of genotypes more locations and years could increase the reliability of plant breeding programs and they have drawn attention to the fact that multi-location trials were more important than multi-year trials on the same location in determining performance of tested genotypes in terms of investigated traits in plant breeding programs. conclusion based on the results obtained during the three-year investigation of the two winter triticale varieties, it can be concluded that the highest three-year average and the best adaptability in semi-arid climate of central serbia for properties grain yield and 1000kernel weight were recorded for the variety kg 20. highly significant influence of the year on grain yield, 1000-kernel weight, test weight and protein content was established in investigated winter triticale varieties by variance analysis, while genotype influence on grain yield, 1000-kernel weight, test weight and protein content was not statistically significant. acknowledgements. investigations presented in this paper are part of the project tp 31054 "development of new cereals cultivation technologies on acid soils by usage of modern biotechnology", financed by the ministry of education and science of republic of serbia. references barnett, r.d., blount, a.r., pfahler, p.l. 1999: benefits of the uf/ifas small grain breeding program to florida and the southeastern united states. soil and crop science society of florida proceedings, 58: 20-23. barnett, r.d., blount, a.r., pfahler, p.l., bruckner, p.l., wesenberg, d.m., johnson, j.w. 2006: environmental stability and heritability estimates for grain yield and test weight in triticale. journal of applied genetics, 47: 207-213. biberdžić, m., jelić, m., deletić, m., barać, s., stojković, s. 2012: effects of agroklimatic conditions at trial locations and fertilization on grain yield of triticale. research journal of agricultural science, 44(1): 3-8. biologica nyssana 9 (1) ⚫ september 2018: 45-53 đekić et al. ⚫ the interaction of genotype and environment on yield… 52 glamočlija, đ., staletić, m., ikanović, j., spasić, m., đekić, v., davidović, m. 2010: possibilities alternative grain production in the highlands area of central serbia. economics of agriculturemultifunctional agriculture and rural development (v), belgrade, ep, 57(si-2): 71-77. đekić, v., glamočlija, đ., staletić, m., perišić, v. 2009: variability of grain yield and yield components of kg winter triticale cultivars. journal of scientific agricultural research, 70(3): 55-60. đekić, v., milovanović, m., glamočlija, đ., staletić, m. 2010: influence of variety and year on grain yield and quality of triticale varieties of kragujevac. 45 rd croatian and 5 rd international symposium on agriculture, 15.-19. february 2010, opatija, croatia, 707-711. djekic, v., mitrovic, s., milovanovic, m., djuric, n., kresovic, b., tapanarova, a., djermanovic, v., mitrovic, m. 2011: implementation of triticale in nutrition of non-ruminant animals. african journal of biotechnology, nigeria, afrika, 10 (30): 5697-5704. đekić, v., milovanović, m., popović, v., milivojević, j., staletić, m., jelić, m., perišić, v. 2014: effects of fertilization on yield and grain quality in winter triticale. romanian agricultural research, dii 2067-5720 rar 2012-255, 31: 175-183. đekić, v., milivojević, j., perišić, v., luković, k., jelić, m., janjić, s., terzić, d. 2016: maximum profitability of triticale to optimizing nutrition. correlations between the triticale yield of mineral nutrition. proceedings, xx international ecoconference ® 2016, 9th eco-conference ® on safe food, 28-30. september 2016, novi sad, 251260. đurić, n., cvijanović, g., matković, m., dozet, g., đekić, v., trkulja, v. 2016: effect of the year on grain yield of some winter triticale varieties. pkb agroekonomik, 22(1-2): 37-42. filipčev, b., mastilović, j., bodroža-solarov, m. 2005: hemical composition of triticale and winter barley varieties in 2002-2004, cereal bread, 32(3): 85-88. janušauskaitė, d. 2013: spring triticale yield formation and nitrogen use efficiencyas affected by nitrogen rate and its splitting. zemdirbysteagriculture, 100(4): 383‒392. kendal, e., sayar, m.s. 2016a: the stability of some spring triticale genotypes using biplot analysis. the journal of animal & plant sciences, 26(3): 754-765. kendal, e., sayar, m.s., tekdal, s., aktas, h., karaman, m. 2016b: assessment of the impact of ecological factors on yield and quality parameters in triticale using gge biplot and ammi analysis. pakistan journal of botany, 48(5): 1903-1913. kendal, e., tekdal, s. aktas, h., karaman, m. 2014: determınation compliance abilities of some triticale varieties and comparıson with wheat in southeastern anatolia condıtıons of turkey. communications in agricultural and applıed biologıcal science, 79(4): 192-200. kondić, d., knežević, d., paunović, a. 2012: grain weight of genotypes of triticale (x triticosecale wittmack) in agroekological conditions of banja luka. genetics, 44(2): 419-428. milovanović, m. 1993: investigation of yield and technological traits of grain of the intergenus hybrids triticale (x triticosecale wittmack). rewiew of research work at the faculty of agriculture, zemun-belgrade, 38(2): 71-82. milovanović, m., kuburović, m., stojanović, s., ognjanović, r. 1995: some recent results of winter triticale breeding in kragujevac. proceedings of 1 st balkan symposium “breeding and cultivation of wheat, sunflower and legume crops”, albena-iws, bulgaria, 125-129. milovanovic, m., kuburovic, m. stojanovic, s., kovacevic, b. 1998: some results of investigations and winter 6x triticale breeding in kragujevac. proceedings, internernational symposium “breeding of small grains”, kragujevac, yugoslavia, 225-231. milovanovic, s.m., rigin, v.b., xynias, n.i. 2001: genetic and breeding studies on triticale (x triticosecale wittmack). in: genetics and breeding of small grains. ari serbia, belgrade, 235-298. milovanovic, m.., perisic, v., staletic, m. 2006: favorit-new vinter triticale cultivar for intensive growing conditions. a collection of articles from the technical vocational college in pozarevac, 1-2: 93-97. milovanović, m., staletić, m., đekić, v., nikolić, o., luković, k. 2011: seed production and contribution of kg varieties to biodiversity of small grains in the period 2006-2010. 01.-02. december 2011, beograd, economics of agriculture, book ii, 58(cб/si-1): 103-111. milovanović, m, perišić, v., staletić, m., cvijanović, d., đekić, v. 2012: recent results of triticale breeding in kragujevac. proceedings, 47 th croatian and 7 th international symposium on agriculture, 13.-17. februar, opatija, croatia, 294-298. milovanovic, m., perishic, v., staletic, m., đekic, v., nikolic, o., prodanovic, s., lukovic, k. 2014: diallel analysis of grain number per spike in biologica nyssana 9 (1) ⚫ september 2018: 45-53 đekić et al. ⚫ the interaction of genotype and environment on yield… 53 triticale. bulgarian journal of agricultural science, 20(5): 1109-1115. perišić, v., milovanović, m., bratković, k. 2008: new cultivar of winter triticale-general. a collection of articles from the technical vocational college in pozarevac, 1-2, 59-64. tomasović, s. 2005: a newly registered winter triticale developed by the zagreb bc institute. annual wheat newsletter, 51: 23-24, kansas state university, usa. grdovć' et al., 2019, biologica nyssana 10(2) 10 (2) december 2019: 99-103 doi: 10.5281/zenodo.3600181 bryophyte collection beou – a neglected national research treasure original article svetlana grdović faculty of veterinary medicine, university of belgrade, 11000 belgrade, serbia cecag@vet.bg.ac.rs (corresponding author) jovana pantović university of belgrade, faculty of biology, institute of botany and botanical garden, takovska 43, 11000 belgrade, serbia jpantovic@bio.bg.ac.rs milan veljić university of belgrade, faculty of biology, institute of botany and botanical garden, takovska 43, 11000 belgrade, serbia veljicm@bio.bg.ac.rs marko sabovljević university of belgrade, faculty of biology, institute of botany and botanical garden, takovska 43, 11000 belgrade, serbia marko@bio.bg.ac.rs received: october 13, 2019 revised: november 07, 2019 accepted: december 19, 2019 abstract: considering recent progress of bryology in serbia digitisation of bryophyte collection within beou (bryo beou) was initiated. it is the most important and the largest collection of the bryophytes in the country, and represent a significant basis for all further research and analysis of bryophyte flora of serbia. this collection also keeps relevant material from neighbouring countries that are considered poorly investigated (e.g. n. macedonia, albania, bosnia and herzegovina). here an overview of the specimens kept in the collection, data-based up to 2019, is given. however, work on the organisation and systematisation of this growing collection is ongoing. key words: bryophytes, herbarium, collection, digitisation, serbia apstract: kolekcija briofita beou zanemareno nacionalno istraživačko blago sa razvojem briologije u srbiji, koja je decenijama bila zapostavljena, započeta je digitalizacija kolekcije briofita u okviru beou herbarijuma (bryo beou). ova kolekcija je najznačajnija i najveća kolekcija briofita u zemlji, i predstavlja važnu osnovu za sva buduća istraživanja i analize flore briofita u srbiji. zbirka sadrži i značajan materijal iz drugih zemalja koje se smatraju slabo ili nedovoljno briološki istraženim (npr. severna makedonija, albanija, bosna i hercegovina). u radu je dat trenutni pregled briološke zbirke, sa presekom digitalizacije do 2019. godine. rad na organizaciji i sistematizaciji ove kolekcije je i dalje u toku. ključne reči: briofite, herbarijum, zbirka, digitalizacija, srbija the value of herbarium collections herbaria document the world’s flora and provide a record of botanical diversity. historically, the primary importance of herbarium specimens was related to the questions of taxonomy, which includes identification, nomenclature, and classification of taxa (e. g. janković et al., 2014; niketić, 2014; di pietro et al., 2017). beyond this, herbarium collections have become crucial for a wide array of studies. primarily, they ensure a continuous record of species distributions (buzurović et al., 2013), and even its predictions (loiselle et al., 2008; wollan et al., 2008; feeley & silman, 2011). hence, historical plant collections are of great value for studying changes in time of flora and vegetation due to the global climate change or degradation of habitats. herbarium collections can also be used to monitor the spread of invasive taxa (delisle et al., 2003; crawford & hoagland, 2009; jenačković et al., 2015), population trends and traits of rare and endangered taxa (christenson, 1994), and to identify priority sites for conservation. historical herbarium specimens are also an important source of information for the conservation of rare or even extinct taxa (kricsfalusy & trevisan, 2014), but also for (phylo)genetic and evolutionary studies. the collections represent an important educational resource for researchers and students. herbarium beou one of the most important and largest herbarium collections in southeastern europe is located at the institute of botany and botanical garden “jevremovac” of the faculty of biology, university of belgrade. it was founded almost 160 years ago, in 1860, when the famous serbian botanist josif pančić gave his collection of pressed plants to the great school, cur© 2019 grdović et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 99 13th symposium on the flora of southeastern serbia and neighboring regions rent university of belgrade (vukojičić et al., 2011). at that time, the collection numbered 20.000 specimens of 6.000 different species that he was gathering during his field trips, but also as an exchange with the european herbaria such as germany, france, italy, spain and russia. pančić’s collection (herbarium pancicianum) is of exceptional scientific, historical and cultural value and importance. in addition to pančić, many other serbian botanists of the 19th and 20th centuries have contributed to the enrichment of the herbarium, such as s. petrović, p. jurišić, đ. ilić, đ. ničić, s. pelivanović, n. košanin, t. soška, l. adamović, v. blečić, i. rudski, p. černjavski, b. tatić, m.m. janković, j. blaženčić and many others. over the past 30 years, researchers of the recent generations (v. stevanović, d. lakušić, s. jovanović, g. tomović, s. vukojičić, and others) have contributed significantly to the herbarium collection, with over 60.000 herbarium specimens collected mostly from the balkan peninsula. herbarium of the university of belgrade today numbers more than 180.000 specimens of vascular plants, bryophytes (bryo/beou) and algae. it is included in the global directory of registered herbaria (index herbariorum), under the herbarium code beou. bryophyte collections beou bryology in serbia started in the middle of the 19th century. during the so-called early period of exploration (pantović & sabovljević, 2017), many serbian botanists and teachers from pančić, katić, jurišić, simić and soška, to košanin, rudski, černjavski and grebenščikov in addition to the vascular flora also collected bryophytes. bryophyte material collected by the first researchers is kept at the natural history museum in belgrade (pavletić, 1955), which is also included in the index herbariorum under code beo. material from this collection is from localities from all over former yugoslavia and the balkan peninsula. although historically important, this collection was forgotten and remained neglected with unnamed specimens for a long time. according to the catalogue, it numbers over 800 specimens. a far greater collection of bryophytes has been kept at the beou herbarium, which has been intensively increasing over the last twenty years. it is estimated to have over 18.000 specimens (vukojičić at al., 2011), of which 8.000 are identified and data-based, and about 10.000 undetermined specimens. the collection was established in the 1990s, within the two chairs of the institute of botany. at the department of plant ecology and phytogeography, professor vladimir stevanović and his associates collected bryophytes while exploring the flora and vegetation of the durmitor mountain in montenegro. then svetlana grdović enriched the collection with bryophytes from belgrade and its surroundings (e. g. grdović, 2005; grdović & stevanović, 2006;). from 2000 until today, professor marko sabovljević had the highest importance for the enrichment of the herbarium collection. together with his foreign and local coworkers (beata papp, erszebet szurdoki and jovana pantović), or by himself, he has collected an impressive number of herbarium specimens of mosses and liverworts from serbia and balkan countries (e. g. 100 biologica nyssana ● 10 (2) december 2019: 99-103 grdović et al. ● bryophyte collection beou – a neglected national research treasure fig. 1. labelled and digitised moss specimen in the bryophyte collection beou fig. 2. stored boxes of bryophyte collection in the beou herbarium all original data on taxa, locality, collector, date, locality, habitat and ecology and identifier, were entered into excel spreadsheet. specimens have been identified to at least genus level, although the majority was identified to a species or lower level. specimens are kept in paper envelops, labelled (fig. 1) and stored in card boxes (fig. 2) in controlled conditions. the collection of bryophytes at the beou herbarium is of great scientific and cultural value today. bryophyte specimens originate from 6 different continents: europe, asia, africa, north america, australia and antarctica, from 46 different countries. however, the majority of the specimens are from europe (fig. 3). the largest number was collected on the territory of serbia, altogether 4696 bryophyte specimens, followed by montenegro 544, slovenia 327, bosnia and herzegovina 295, italy 241 and so on (fig 4). however, these numbers refer only to identified specimens. until the 1990s mosses were sporadically collected, hence only 613 records in the bryo beou collection were collected before 1990. from 1990 onwards, bryophyte flora has been intensively studied and all other records originate from this so-called modern period (pantović & sabovljević, 2017) of exploration. by years (fig. 5), largest number of 4696 544 327 295 241 236 236 204 130 127 116 105 102 sabovljević & cvetić, 2003; papp & sabovljević, 2010; sabovljević et al., 2010), but also from other european countries (e. g. papp & sabovljević, 2009; papp et al., 2016). at the same time in the 1990s, at the department of plant morphology and systematics, professor milan veljić has started his collection of bryophytes, which is now part of the bryo beou. he collected material from various sites in serbia (e. g. veljić et al., 2001, 2008). part of this bryo beou collection is valuable material from nature reserves and protected areas in serbia, collected and published afterwards by mića popović in the middle of the last century (popović, 1966). the modern period of the bryo beou collection considering recent progress of bryology in serbia a comprehensive data summarisation on the distribution of bryophytes in serbia was necessary to be able to critically assess certain taxa and to update the country list of the species. for that purpose, bryo database was established (pantović & sabovljević, 2017), and besides aggregation of literature data, digitisation of bryophyte herbarium specimens within beou was initiated. 8084 specimens have been digitised so far, but a large part of the collection has not been identified nor digitised yet. biologica nyssana ● 10 (2) december 2019: 99-103 101 fig. 2. map indicating the number of specimens in bryo beou collected from different european countries fig. 4. countries with more than 100 specimens in the bryophyte collection grdović et al. ● bryophyte collection beou – a neglected national research treasure identified herbarium specimens was collected in 2000 and 2001 (951 and 676), followed by over 500 specimens in 2013, 2016 and 2018, and more than 400 specimens collected in 1997, 1998, 2002, 2014 and 2015. more than half (66%) of the specimens, a total of 5112, was collected by marko sabovljević and his coworkers. analysis of the period of collecting showed that the majority of the field trips were done in the spring and summer months (fig. 6). largest number of bryophyte specimens was collected in may (1334) and july (1240) while the least records were collected in january (79) and december (166). all digitised bryophytes can be grouped into 797 different taxa and 294 genera. bryo beou collection and its digitised data rep0 100 200 300 400 500 600 700 800 19 91 19 93 19 94 19 95 19 96 19 97 19 98 19 99 20 00 20 03 20 04 20 05 20 06 20 07 20 08 20 09 20 10 20 11 20 12 20 13 20 14 20 15 20 16 20 17 20 18 102 resent an important basis for all further research and analysis of bryophyte flora of serbia. this collection also keeps important material, although not yet completely identified and digitised, from neighbouring countries that are considered poorly investigated (e.g. macedonia, albania, bosnia and herzegovina). work on the organisation and systematisation of this growing collection is ongoing. references buzurović, u., stevanović, v., niketić, m., jakov-ljević, k., tomović, g. 2013: on the distribution of goniolimon tataricum (plumbaginaceae) in serbia. botanica serbica, 37(2): 167-172. christenson, e. a. 1994: significant collections of orchidaceae conserved in herbarium hamburgense (hbg). brittonia, 46(4): 344-354. crawford, p. h., hoagland, b. w. 2009: can herbarium records be used to map alien species invasion and native species expansion over the past 100 years?. journal of biogeography, 36(4): 651-661. delisle, f., lavoie, c., jean, m., lachance, d. 2003: reconstructing the spread of invasive plants: taking into account biases associated with herbarium specimens. journal of biogeography, 30(7): 1033-1042. di pietro, r., kuzmanović, n., iamonico, d., lakušić, d. 2017: nomenclatural and taxonomic notes on sesleria sect. argenteae (poaceae). phytotaxa, 309(2): 101-117. feeley, k. j., silman, m. r. 2011: keep collecting: accurate species distribution modelling requires more collections than previously thought. diversity and distributions, 17(6): 1132-1140. grdović, s. 2005: mahovine šireg područja beograda i njihov bioindikatorski značaj, zadužbina andrejević, beograd. 130 p. grdović, s., stevanović, v. 2006: the moss flora in the central urban area of belgrade. archives of biological sciences 58(1): 55-59. janković, i., kuzmanović, n., clementi, m., lakušić, d. 2014: lectotypification of campanula secundiflora vis. & pančić (campanulaceae), a species of european concern. botanica serbica, 38(2): 269-271. biologica nyssana ● 10 (2) december 2019: 99-103 fig. 5. number of collected specimens per year collected only during the modern period of investigation (since 1990 onwards) fig. 6. number of collected specimens per each calendar month grdović et al. ● bryophyte collection beou – a neglected national research treasure 79 546 858 748 1334 924 1240 820 330 301 479 166 103 jenačković, d., miljković, m., mitrović, d., ranđelović, v. 2015: contribution to the knowledge of distribution of certain macrophytes, invasive and threatened species in serbia. biologica nyssana, 6(2): 59-65. kricsfalusy, v. v., trevisan, n. 2014: prioritizing regionally rare plant species for conservation using herbarium data. biodiversity and conservation, 23(1): 39-61. loiselle, b. a., jørgensen, p. m., consiglio, t., jiménez, i., blake, j. g., lohmann, l. g., montiel, o. m. 2008: predicting species distributions from herbarium collections: does climate bias in collection sampling influence model outcomes?. journal of biogeography, 35(1): 105-116. niketić, m. 2014: nomenclature review of the plants published by josif pančić (nomenclator pancicianus novus). botanica serbica, 38(2): 209-236. pantović, j., sabovljević, m. 2017: overview of bryophyte flora research in serbia with presentation of the serbian bryo database. botanica serbica, 41(2): 153-162. papp, b., pantović, j., szurdoki, e., sabovljević, m. s. 2016: new bryophyte records for the republic of macedonia. journal of bryology, 38(2): 168-171. papp, b., sabovljević, m. 2009: notes on some new and interesting bryophyte records from croatia. journal of bryology, 31(4): 272-275. papp, b., sabovljević, m. 2010: contribution to the bryophyte flora of the vršačke planine mts., serbia. botanica serbica, 34(2): 107-110. pavletić, z. 1955: prodromus flore briofita jugoslavije. jugoslavenska akademija znanosti i umetnosti. 578 p. popović, m. 1966: prilog poznavanju mahovina u rezervatima i zaštićenim područjima u srbiji. zaštita prirode, 33: 219-228. sabovljević, m., cvetić, t. 2003: bryophyte flora of avala mt. (c. serbia, yugoslavia). lindbergia, 28: 90-96. sabovljević, m., papp, b., szurdoki, e. 2010: new bryophyte records to some countries of the southeastern europe. cryptogamie, bryologie, 31(3): 289-292. veljić, m., marin, p., boža, p., petković, b. 2001: bryoflora of some well-springs of the dinaric alps and carpathian karst in serbia. bocconea, 13: 343351. vukojičić, s., lakušić, d., jovanović, s., marin, p.d., tomović, g., sabovljević, m., šinžarsekulić, j., veljić, m., cvijan, m., blaženčić, j., stevanović, v. 2011: university of belgrade herbarium treasury of data and challenges for future research. on the occasion of the 150th anniversary of university of belgrade herbarium (18602010). botanica serbica, 35:163-178. wollan, a. k., bakkestuen, v., kauserud, h., gulden, g., halvorsen, r. 2008: modelling and predicting fungal distribution patterns using herbarium data. journal of biogeography, 35(12): 2298-2310. biologica nyssana ● 10 (2) december 2019: 99-103 grdović et al. ● bryophyte collection beou – a neglected national research treasure šabanović et al, 2019, biologica nyssana 10(2) 10 (2) december 2019: 143-153 doi: 10.5281/zenodo.3600557 the genus orchis tourn. ex l. and its related genera in the zenica-doboj canton (bosnia and herzegovina): diversity, distribution and conservation original article elvedin šabanović public institution “regional museum“ visoko, ul. alije izetbegovića 29, visoko, bosnia and herzegovina sabanovic.elvedin@gmail.com (corresponding author) aldin boškailo university “džemal bijedić“ in mostar, teachers faculty, univerzitetski kampus bb, mostar, bosnia and herzegovina aldinboskailo@hotmail.com šemso šarić jp špd-zdk d.o.o zavidovići, alije izetbegovića 25, ba-72220 zavidovići, bosnia and herzegovina semsosumar@gmail.com sanida bektić university of tuzla, faculty of sciences and mathematics, department of biology, tuzla, bosnia and herzegovina sanida.osmanovic@untz.ba vladimir ranđelović university of niš, faculty of sciences and mathematics, department of biology and ecology, niš, serbia vladar@pmf.ni.ac.rs received: september 29, 2019 revised: december 23, 2019 accepted: december 25, 2019 abstract: this paper represents the results of the distribution study focused on the genera anacamptis, neotinea, orchis and related species belonging to orchidaceae family. the investigation was conducted in the zenica-doboj canton, during the period of four years (2016-2019). the presence of 12 species and two subspecies was noticed. all of the populations turned out to be very vulnerable, consisting of a reduced number of individuals. the most endangered populations are orchis militaris, o. pallens and o. spitzelii. key words: orchidaceae, anacamptis, neotinea, orchis apstract: rod orchis tourn. ex l. i njemu srodni rodovi u zeničko-dobojskom kantonu (bosna i hercegovina): diverzitet, rasprostranjenje i konzervacija u vom radu su dati rezultati istraživanja distribucije vrsta iz rodova anacamptis, neotinea i orchis iz familije orchidaceae na području zeničkodobojskog kantona u periodu od 2016. do 2019. godine. utvrđeno je prisustvo 12 vrsta i dve podvrste. populacije svih vrsta su izrazito malobrojne, što ih čini veoma ugroženim. najugroženije su populacije vrsta orchis militaris, o. pallens i o. spitzelii. ključne reči: orchidaceae, anacamptis, neotinea, orchis introduction the genus orchis belongs to orchidaceae family (chase, 2005; stroh, 2016), the family with the highest diversity, which comprises about 28,000 species sorted in 736 genera (christenhusz & byng, 2016). the genus is affiliated to the subtribus orchidinae, the tribus orchideae and the subfamily orchidoideae. moreover, the rest of the genera from the central part of the balkan peninsula anacamptis, dactylorhiza, gymnadenia, herminium, himantoglossum, neotinea, ophrys, platanthera, pseudorchis and traunsteinera (djordjevic, 2018) – are having the same taxonomic status. according to the flora europaea (soó, 1980), the genus orchis also included some species, recently relocated within the anacamptis and neotinea genera, as a consequence of phylogenetic analysis, based on nuclear its sequences (bateman et al., 1997). in bosnia and herzegovina, the orchidaceae family is represented by 23 genera and 69 species (šilić, 2008) – among them, 8 species belong to the genus orchis (o. italica, o. mascula, o. militaris, o. pallens, o. provincialis, o. purpurea, o. simia and o. spitzelii), 5 to the genus anacamptis (a. pyramidalis, a. coriophora, a. morio, a, laxiflora and a. palustris) and 2 to the genus neotinea (n. tridentata and n. ustullata) (back managetta, 1903; šoljan © 2019 sabanovic et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 143 13th symposium on the flora of southeastern serbia and neighboring regions et al., 2014; bucalo, 2015). “the red list of flora of the federation of bosnia and herzegovina” (đug et al., 2013) contains 4 species from the previously mentioned genera: orchis purpurea, o. simia, o. spitzelii and anacamptis pyramidalis. the same species can be found in “the red list of flora of bosnia and herzegovina“ šilić (1996). “the red list of flora and fauna of republika srpska“ (http://www. nasljedje.org/docs/crvenalista/vascular_flore.pdf), is highlighting the importance of the orchid flora, comprising all of the species from the mentioned genera. the orchid flora of the zenica-doboj canton, hadn’t been investigated previously. the data about their distribution are seriously restricted; on the one hand, most of the literature data, including 11 species from the genera orchis, anacamptis and neotinea, date from the second half of the 19th and the first half of the 20th century (kummer & sendtner, 1849; fiala, 1896, protic , 1898, beck mannagetta, 1903; maly, 1908; plavsic, 1940); on the other hand, more recent data have been reduced to individual citations (ritter-studnička, 1970; abadžić, 2012; šabanović & bektić, 2017). the morphological similarity, followed by the phylogenetic status of the genera orchis, anacamptis and neotinea, as well as the fact that species from the genera anacamptis (with a. pyramidalis as an exception) and neotinea are even nowadays represented as the members of the genus orchis (šoljan et al., 2014) in bosnia and herzegovina, are indicating the need of the thorough investigation. hence, the aim of this paper was to determine the diversity of all three genera in the area of zenica-doboj canton, to show the distribution and condition of their populations and to point out the need for conservation of habitats as the only way of preserving populations. material and methods study area the study was focused on the zenica-doboj canton (fig. 1), the territory located in the central part of bosnia and herzegovina, more precisely, bosna river basin. it covers an area of 3345 km2, between 17°44’38’’ and 18°50’11’’ longitude, and 43°54’13’’ and 44°43’41’’ latitude. it is a distinctly mountainous area, with a huge geomorphological diversity and altitudes ranging from 160 to 1472 m. the relief is characterized by three specific areas: usore river valley with its plains and hilly areas in the north, hilly-mountainous terrain in the central part and hilly relief in the south. the central area is 144 biologica nyssana ● 10 (2) december 2019: 143-153 šabanović et al. ● the genus orchis tourn. ex l. and its related genera in the zenica-doboj canton (bosnia and herzegovina) fig. 1. geographical position of investigated area al., 2019). black pine forests (erico-pinetum nigrae serpentinicum stef. 1962) and other communities preferring serpentinite represent a special feature to the vegetation (ritter-studnička, 1970a). field work the diversity and geographical distribution of the taxa from the genera orchis, anacamptis and neotinea were investigated in the period from 2016 to 2019. gps and utm (10×10 km) coordinates are determined for each population. population status was defined by counting flowering and non-flowering individuals in the square areas 50×50 m. at the same time, potential factors in taxa endangerment have been reported. the collected floristic material was deposited in the museum „visoko“ in the visoko city. considering the high degree of threatened taxa, a rich photodocumenting material was created in order to avoid the collection of the plant material. photographs are also stored in the museum. data collection and analysis in addition to the field data, literature and herbarium data (sara herbarium of the national museum of bosnia and herzegovina) were included. the identification of the taxa was performed by using keys from modern literature (soó, 1980; delforge, 2006). the nomenclature of the taxa is consistent with the euro+med plantbase (http:// ww2.bgbm.org/europlusmed/results.asp). the distribution of the taxa is shown on the grid map and zone s4t with squares of 10×10 km, based on the universal transverse mercator (utm) projection (lampinen, 2001). the different types of the data are presented as follows: the white dot the field data, the black dot the literature data, the black-white dot the field and literature data, and the grey dot the herbarium data. the affiliation to an appropriate chorological type and floral element was determined on the basis of its geographical range. the determination of the chorological type is based on the coincidence of the geographical range with the corresponding phytochoria (stevanović, 1992). the abbreviations of the chorological type, as defined in ranđelović, zlatković (2010) and đorđević (2018), are given as listed: ea eurasian, ce central european, msm mediterranean-sub-mediterranean and sem south european mountainous. the floral elements were determined according to meusel (meusel et al., 1965) modified by stevanović (1992). the endangerment of the taxa wase based on the iucn criteria. the iucn categories are presented as follows: cr critically endangered, en endangered, vu vulnerable and lc least the largest, extending between the rivers of bosnia and krivaya and consisting of the mountain massifs ravan planina, konjuh, smolin, zvijezda and čemerska planina. the highest mountain peak of karasanovina (1472 m asl) is located on zvijezda mountain (goletić, ed. 2016). from the aspect of the geological substrates, the study area is quite diverse, including silicates, carbonates (goletić, ed. 2016) and, in particular, serpentinite (ritter-studnička, 1930). the entire area is characterized by a large number of river flows belonging to the bosna river basin. the lowland and hilly parts of the canton are characterized by a continental to a temperate continental climate, while with the altitude increases, the influence of the mountain climate is getting more pronounced (goletić, ed. 2016). in a domain of phytogeography, zenica-doboj canton belongs to the illyrian subregion of the central european region and holarctic floristic kingdom (horvat et al., 1974). this area is characterized by high diversity of flora, including endemic species, e.g. daphne blagayana, hesperis dinarica, viola beckiana, euphorbia gregersenii, e. montenegrina, dianthus croaticus and others. many plant species are declared as endangered, such as halacsya sendtneri, notolaena maranthe, eranthis hiemalis, kitaibela vitifolia and others. the orchid species contained in this study are included in the group of highly sensitive species. this area is characterised by the pronounced vegetation diversity as well, unfortunately enormously degraded due to the anthropogenic factor. on the banks of rivers and streams riparian gallery forests (classis alno glutinosae-populetea albae p. fukarek et fabijanić 1968) have been developed. the mountain belt is overgrown with the oak-hornbeam and mesic oak forests (ordo carpinetalia betuli p. fukarek 1968). the climatogenic vegetation of this area is represented with the sessile oak and the common hornbeam forest (querco-carpinetum illyricum ht. et al. 1974), whereas the sessile oak forests (quercetum montanum čer. et jov. 1968) prefer thermophilic habitats. forest vegetation of the mountain belt is seriously degraded, giving a place to the mountain grasslands as a secondary type of vegetation. above this belt, at higher altitudes, beech forests (fagetum illyricum ht. 1938) gradually turn into mixed deciduous and coniferous forests (abieti-fagetum illyricum ht. 1938). coniferous spruce forests (piceetum illyricum montanum ht. 1950) are growing on the small areas of the mountains zvijezda and ravna planina (horvat et al. 1974, fukarek, jovanović, ed., 1983). in surrounding of vareš, forests with peat moss and spruce (sphagno-piceetum montanum stefanović 1964) are developed in the depressions of the mountain belt (barudanović et 145 biologica nyssana ● 10 (2) december 2019: 143-153 šabanović et al. ● the genus orchis tourn. ex l. and its related genera in the zenica-doboj canton (bosnia and herzegovina) 146 biologica nyssana ● 10 (2) december 2019: 143-153 šabanović et al. ● the genus orchis tourn. ex l. and its related genera in the zenica-doboj canton (bosnia and herzegovina) fig. 2. investigated species: 1. anacamptis coriophora, 2. a. morio, 3. a. palustris, 4. a. pyramidalis, 5. neotinea tridentata, 6. n. ustulata, 7. orchis mascula, 8. o. militaris, 9. o. pallens, 11. o. purpurea, 11. o. simia, 12. o. spitzelii 147 fig. 3. distribution of investigated species: 1. anacamptis coriophora, 2. a. morio, 3. a. palustris, 4. a. pyramidalis, 5. neotinea tridentata, 6. n. ustulata, 7. orchis mascula, 8. o. militaris, 9. o. pallens, 11. o. purpurea, 11. o. simia, 12. o. spitzelii biologica nyssana ● 10 (2) december 2019: 143-153 šabanović et al. ● the genus orchis tourn. ex l. and its related genera in the zenica-doboj canton (bosnia and herzegovina) concern. relative frequency, as defined in vuković et al. (2011), was given as listed: a abundant, f frequent, r rare, rr extremely rare. the species and subspecies with the corresponding data (chorological type, iucn category, relative frequency, localities and habitats if available) are presented alphabetically in the list as follows: name of species chorological type/floral element iucn category/abundance distribution: literature and herbarium data: locality, altitude, utm 10×10, habitat (reference or collector, dd.mm.yyyy, herbarium). field data: locality, altitude, utm 10x10, habitat (collector, dd.mm.yyyy). results the presence of 12 orchid species and 2 subspecies from genera anacamptis (4 species, 1 subspecies), neotinea (2 species) and orchis (6 species, 1 subspecies) has been identified in the zenica-doboj canton. list of species anacamptis coriophora (l.) r. m. bateman, pridgeon & m. w. chase (fig. 2-1) ea/central european-mediterranean-sub-mediterranean-southwest asian vu/f (small populations, only the population near village porječani is more than 10 individuals) distribution (fig. 3-1): literature and herbarium data: oko vareša, bp89 (protić, 1898); oko vareša, bp89 (beck, 1903); pajtov han, bp88 (plavšić, 11/6/1940, sara). field data: stipin han, 350 m, bq91, mesophilous grassland (šabanović, 10/5/2018); prhinje, 465 m, bp77, hygrophilous grassland by the mining pit lake (šabanović, 27/5/2018); visočica, 690 m, bp77, mesophilous grassland from alliance arrhenatherion elatius (šabanović, 27/5/2018); porječani, 445 m, bp78, mesophilous grassland from alliance arrhenatherion elatius (šabanović, 3/6/2019); jelaške, bq90 (šarić, 14/5/2016; 17/5/2018); milankovići, cp09 (šarić, 6/6/2016); smailbegovići, breza, bp87 (šarić, 29/5/2018); drecelj, olovo, cp19 (šarić, 8/6/2017); ajdinovići, cp08, (šarić, 30/6/2017). anacamptis morio (l.) r.m.bateman, pridgeon & m.v.chase (fig. 2-2) ea/european-mediterranean-west asian lc/a distribution (fig 3-2): literature and herbarium data: borovica, bp79; vranduk, yk30 (kummer & sendtner, 1849); in grassland around pobrin han, bp89 (protić, 1898); around vranduk, yk30; between žepče and golubinje, yk31; near visoko, bp77; near vareš, bp89 (beck, 1903); kruškovica, bp99 (plavšić, 19/6/1940, sara); kondžilo, bp68 (plavšić, 9/6/1939, sara); visočica, bp77 (plavšić, 25/6/1939, sara). field data: sokolina, 870 m, bq90, mesophilous pasture in a belt of mixed deciduous and coniferous forests of the abieti-fagion type (šabanović, ranđelović, šarić, 30/4/2018); župa-jelaške, 585 m, bq90, mesophilous grassland (šabanović, ranđelović, šarić, 30/4/2018); visočica, 670 m, 685 m, bp77, mesophilous grassland (šabanović, 13/5/2018); visočica, 640 m, bp77, mesophilous grassland from alliance arrhenatherion elatius (šabanović, 11/5/2018); visočica, 710 m, bp77, dry grassland festuco-brometea type (šabanović, 11/5/2018); visočica, 790 m, bp77, mesophilous grassland from alliance arrhenatherion elatius (šabanović, 11/5/2018); dvor, 585 m, bp76, mesophilous grassland (šabanović, 9/5/2018); ribnica, 350 m, bq91, mesophilous grassland from alliance arrhenatherion elatius (šabanović, 12/5/2018); pogar, 1055 m, bp89, mesophilous pasture (šabanović, 12/5/2018); gorani, 635 m, bp77, thermophilic rocky meadows (šabanović, 13/5/2018); dvor, 515 m, bp76, mesophilous grassland (šabanović, 17/5/2018); porječani, 445 m, bp78, mesophilous grassland from alliance arrhenatherion elatius (šabanović, 3/6/2019); karići, 1190 m, bp98 (šabanović, 18/5/2019); žepče, 220 m, bq62, mesophilous grassland from alliance molinion caeruleae (šabanović, 25/5/2019); perun, 1330 m, bp88, mesophilous pasture (šabanović, 9/6/2019); brezik, zavidovići, bq82 (šarić, 21/4/2016); jelaške, bq90 (šarić, 21/4/2016; 25/4/2018); dištica, zavidovići, bq90 (šarić, 27/4/2018); bukov dol, bq90 (šarić, 29/5/2019). anacamptis palustris (jacq.) r.m. bateman, pridgeon & m.w. chase (fig. 2-3) ea/atlantic-european-euxinian-caucasianpontian-irano-turian vu/r (population near lupoglav is very rich and counts over 500 individuals, while other populations are with few individuals) distribution (fig 3-3): literature and herbarium data: između trbuka i maglaja, (beck, 1903); tajan, bq70 (maly, 22/5/1898; 24/6/1906; 21/5/1908; 30/5/1912; 148 biologica nyssana ● 10 (2) december 2019: 143-153 šabanović et al. ● the genus orchis tourn. ex l. and its related genera in the zenica-doboj canton (bosnia and herzegovina) 29/6/1912); tajan, bq70 (maly, 6/6/1921, sara); ljeskovica, bq72 (maly, 29/5/1930, sara); klek-zavidovići, bq72 (fiala, 16/6/1892, sara). a. palustris subsp. elegans (heuff.) r.m. bateman, pridgeon & m.w. chase distribution (fig. 3-3): field data: stošnica, 260 m, bq81, swamp meadow (šabanović, 30/5/2018); lupoglav, 230 m, bq62, swamp meadow (šabanović, 4/6/2019); stošnica, bq81 (šarić, 12/5/2016; 29/5/2018); žepče, bq62 (šarić, 5/6/2019); vozuća, bq81 (šarić, 2/6/2016). anacamptis pyramidalis (l.) richard (fig. 2-4) msm/mediterranean-sub-mediterranean-central european-pontian-euxinian-caucasian-iranian vu/f (populations are with few individuals) distribution (fig. 3-4): literature and herbarium data: in grasslands near pobrin han, bp89 (protić, 1898); between tolović and zenica, yj29; near vareš, bp89 (beck, 1903); raspotočje, yj39 (ritter, 26/6/1939, sara); sutjeska, bp78 (plavšić, 5/7/1939, sara); visoko, bp77 (laschnigg, 28/5/1930, sara); dabravine, bp88 (maly, 8/6/1930, sara); kula banjer, bp77 (maly, 8/6/1930, sara); vardište, bp88 (bjelčić, 24/6/1907, sara). field data: kula banjer, 570 m, bp77, thermophilic shrub (šabanović, 23/5/2018); prhinje, 510 m, 520 m, 530 m, bp77, thermophilic shrub and grassland (šabanović, 27/5/2018); porječani, 560 m, bp78, mesophilous grassland (šabanović, 1/6/2018); lipnica, 1210 m, bp79, mesophilous pasture (šabanović, 15/7/2018); bajrići, maglaj, 300 m, bq64 (šabanović, 25/5/2019); klupe-stog zavidovići, bq81 (šarić, 1/6/2016, serpentin); potoci near vareš, bp89 (šarić, 28/7/2016); lovnica, zavidovići, bq71 (šarić, 11/6/2016); magulica, bq90 (šarić, 13/6/2016); ćude, olovo, cp19 (šarić, 8/6/2017); smailbegovići, breza, bp87 (šarić, 27/5/2018). neotinea tridentata (scop.) r.m. bateman, pridgeon & m.v. chase (fig. 2-5) msm/mediterranean-sub-mediterranean-central european-euxinian-caucasian-pontian en/f (populations are with few individuals, a number rarely exceeding 10 individuals) distribution (fig. 3-5): literature and herbarium data: osova, bq62 (fiala, 1896); around vranduk, yk30; around vareš, bp89 (beck, 1903); on the mt. macat near vradište, bp88 (maly, 1912); visoko, bp77 (plavšić, 23.5.1939, sara). field data: magulica, near the franciscan church, 830 m, bq90, by the mountain road (šabanović, ranđelović, šarić, 30/4/2018); dvor, 590 m, bp76, mesophilous grassland (šabanović, 9/5/2018); visočica, 640 m, 650m, 670m, 680m, bp77, mesophilous grassland (šabanović, 11/5/2018); visočica, 790 m, bp77, mesophilous to thermophilous grassland (šabanović, 13/5/2018); gorani, 630 m, bp77, thermophilous rocky grassland (šabanović, 13/5/2018); prhinje, 530 m, bp77, dry grassland (šabanović, 27/5/2018); ajdinovići, 870 m, cp08, thermophilous grassland (šabanović, 5/6/2019); perun, 845 m, bp88, mesophilous grassland (šabanović, 9/6/2019); perun, 1340 m, bp88, mesophilous pasture (šabanović, 9/6/2019); ćude near olovo, cp19 (šarić, 12/5/2016); crni potok, bp99 (šarić, 19/5/2018); magulica, bq90 (šarić, 17/5/2016); bukov dol, bq90 (šarić, 29/5/2019). neotinea ustulata (l.) r.m. bateman, pridgeon & m.v. chase (fig. 2-6) ce/central european-pontian-south sibirian lc/f (populations are with few individuals, a number rarely exceeding 10 individuals) distribution (3-6): literature and herbarium data: borovica, bp79 (kummer & sendtner, 1849); around osova, bq62 (fiala, 1896); near pobrin han, bp89; on the slopes above vareš, bp89 (protić, 1898); between tolović and zenica, yj29; vareš, bp89; osova, bq62 (beck, 1903); banjer near visoko, bp77 (maly, 1933b); swamp bistrik, haljinići, bp78 (abadžić, 2012); čatići kod kaknja, bp68 (brandis, 19/7/1896, sara); vranjkovci, bp89 (plavšić, 19/6/1940; 7/7/1940, sara). field data: jelaške, 500 m, bq90, ruderal habitat (šabanović, ranđelović, šarić, 30/4/2018); visočica, 640 m, bp77, mesophilous grassland (šabanović, 11/5/2018); ribnica, 350 m, bq91, mesophilous grassland (šabanović, 12/5/2018); visočica, 685 m, bp77, mesophilous grassland (šabanović, 13/5/2018); ginje, 540 m, bp76, mesophilous grassland (šabanović, 16/5/2018); lipnica, 840 m, bp79, mesophilous grassland in a belt of mixed deciduous and coniferous forests of the abieti-fagion type (šabanović, 15/7/2018); brezik, zavidovići, 250 m, bq73, mesophilous grassland (šabanović, 25/5/2019); near borik resort, maglaj, 215 m, bq63, mesophilous grassland (šabanović, 25/5/2019); 149 biologica nyssana ● 10 (2) december 2019: 143-153 šabanović et al. ● the genus orchis tourn. ex l. and its related genera in the zenica-doboj canton (bosnia and herzegovina) lipnica, 860 m, bp79, mesophilous grassland (šabanović, 26/5/2019); perun, 1310 m, bp88, mesophilous pastures (šabanović, 9/6/2019); jelaške, bq90 (šarić, 21/4/2016; 21/5/2016; 7/5/2017; 13/5/2017; 29/5/2017; 23/4/2018; 28/4/2018; 14/5/2019; 29/5/2019); ribnica, zavidovići, bq91 (šarić, 7/5/2018); vozuća near zavidovići, bq81 (šarić, 14/5/2016). orchis mascula (l.) l. (fig. 2-7) ea/mediterranean-sub-mediterranean-central european-euxinian-caucasian-iranian vu/f (populations are with few individuals, a number rarely exceeding 20 individuals) distribution (fig. 3-7) literature and herbarium data: around pajtov han, bp88 (protić, 1898); around vareš, bp89; near osova, bq62 (beck, 1903). orchis mascula subsp. speciosa (vest) soo literature and herbarium data: tajan, 1296 m, bq70 (hilda ritter-studnička, 1970); sutjeska, bp78 (plavšić, 19/6/1940, sara). field data: jelaške 610 m, bq90, mesophilous grassland (šabanović, 10/5/2018); duboštica, 770 m, bq80, mesophilous grassland (šabanović, 12/5/2018); ponijeri, 1090 m, bp79, mesophilous pasture (šabanović, 20/5/2018); from ponijeri to lipnica, 830 m, bp79, mesophilous grassland (šabanović, 26/5/2019); lipnica, 815 m, bp79, mesophilous grassland (šabanović, 26/5/2019); perun, 1436 m, bp88, mesophilous grassland (šabanović, 9/6/2019); duboštica, bq80 (šarić, 19/5/2016; 13/5/2018); jelaške, bq90 (šarić, 14/5/2017); kopališta in vareš, bp89 (šarić, 23/5/2016); ponijeri, bp79 (šarić, 28/5/2017). orchis militaris l. (fig. 2-8) ea/central european-caucasian-pontian-south sibirian en-cr/rr (occurs single or with 2 to 3 individuals) distribution (fig. 3-8) literature and herbarium data: near pobrin han and village potoci near vareš, bp89 (beck, 1903); vardište, 900 m, bp88 (maly, 30/5/1918, sara). field data: duboštica, 770 m, bq80, mesophilous grassland (šabanović, 12/5/2018); duboštica, bq80 (šarić, 12/5/2018). orchis pallens l. (fig. 2-9) ce/central european-caucasian-pontiansouthwest sibirian en-cr/rr (occurs single or with 2 to 3 individuals) distribution (fig. 3-9) literature and herbarium data: on the hill bogoš, bp88 (protić, 1898); on the hill bogoš near vareš, bp88 (beck, 1903); visočica, bp77 (plavšić, 8/5/1939, sara); vardište, bp88 (maly, 30/5/1911, sara). field data: jelaške, stojčići, 600 m, bq90, roadside (šabanović, 28/04/2019); vranduk, yk30 (šarić, 29/10/2016); jelaške, bq90 (šarić, 29/10/2016; 27/04/2017; 23/4/2018); stojčići, bq90 (šarić, 14/5/2019). orchis purpurea huds. (fig.2-10) ce/central european-mediterranean-submediterranean-caucasian-pontian vu/f (small populations) distribution (fig. 3-10) literature and herbarium data: oko sela potoci prema pobrinom hanu, bp89 (protić, 1898); between tolovići and zenica, yj29; around vareš, bp89 (beck, 1903). field data: kamensko, 915 m, bq90, in mixed deciduous and coniferous forests of the abietifagion type (šabanović, ranđelović, šarić, 30/4/2018); perun, 990 m, bp88, in mixed deciduous and coniferous forests of the abietifagion type (šabanović, 9/6/2019); kamensko, bq90 (šarić, 29/5/2017); sokolina, bq90 (šarić, 17/5/2016); jelaške, bq90 (šarić, 14/5/2019). orchis simia lam. (fig. 2-11) msm/mediterranean-sub-mediterraneancaucasian-crimean vu/f (small populations) distribution (fig. 3-11) literature and herbarium data: kod zenice, yj39 (beck, 1903); oko vareša, bp89 (beck, 1903); oko vareša, bp89 (protić, 1898); visočica, bp77 (plavšić, 8/5/1939, sara); sutjeska, bp78 (plavšić, 22/5/1940, sara). field data: visočica, 660 m, bp77, forest carpino-quercetum petreae, roadside (šabanović, 11/5/2018); pobrin han, 1105 m, bp89, coniferous forests, roadside (šabanović, 12/5/2018); gorani, 630 m, bp77, forest carpinoquercetum petreae, roadside (šabanović, 13/5/2018); lipnica, 820 m, bp79, mesophilous grassland (šabanović, 26/5/2019); bobovac, 780 m, bp89, mesophilous grassland (šabanović, 8/6/2019); road from dabravine to kariće, 660 m, bp88, roadside (šabanović, 18/05/2019); boganovići, ćude, cp19 (šarić, 12/5/2018); bukov dol near olovo, bq90 (šarić, 28/4/2018; 29/5/2019); milankovići, cp09 (šarić, 150 biologica nyssana ● 10 (2) december 2019: 143-153 šabanović et al. ● the genus orchis tourn. ex l. and its related genera in the zenica-doboj canton (bosnia and herzegovina) 21/5/2017); jelaške, bq90 (šarić, 17/5/2018); žeravice, olovo, cp29 (šarić, 6/6/2016). orchis spitzelii saut. ex w.d.j.koch. (fig. 2-12) sem/pyrenean-alpine-apennine-dinaric-balkancarpathian-anatolian en-cr/rr (occurs single or with 2 to 3 individuals) distribution (fig. 3-12) literature and herbarium data: zavidovići (ritter studnička, 1970); visočica, bp77 (goul, 30/4/1936, sara); vardište, 900 m. bp88 (maly, 30/5/1911, sara). field data: kamensko, 900 m, bq90, šume bukve, jele i smrče (šabanović, ranđelović, šarić, 30/4/2018); vareš, bp89 (šarić, 13/5/2016); sokolina, kamensko, bq90 (šarić, 1/5/2018). discussion the study revealed the presence of 12 species and one subspecies of the genera anacamptis, neotinea and orchis. compared to the data available in the literature (protic, 1898; back mannagetta, 1903 and other), one of the subspecies found, anacamptis palustris ssp. elegans, is new to the zenica-doboj canton. additionally, after the close check of the all potential localities listed in the literature sources, the presence of orchis mascula, described as the type species for this area, has not been confirmed. since the type species wasn’t recorded in the neighboring sarajevo canton, but its mentioned subspecies under the name orchis ovalis (šoljan et al, 2014), it‘s almost certain that subspecies o. mascula ssp. speciosa (which has been found in several localities) is the only one growing in the investigated region. compared to the neighboring sarajevo canton (šoljan et al., 2014), it‘s just the subspecies anacamptis morio ssp. picta (loisel.) jacquet & scappat that is lacking. on the other hand, the study area is characterized by the species that are not recorded in sarajevo canton, including anacamptis palustris, orchis militaris and o. spitzelii. the wild orchids are protected by cites convention (appendice ii), which is implying the additional importance of this study. considering the species diversity in the investigated region as well, the necessity to propose the continuous monitoring system of all the recorded populations (including the remaining genera from the orchidaceae family throughout the canton) is obvious. according to the iucn red list of threatened species, anacamptis palustris categorized as the least concern (lc) represents the only one globally threatened species among all the others included in this study. a. coriophora, a. pyramidalis, neotinea tridentata, orchis mascula, o. militaris, o. pallens, o. purpurea and o. simia are having the same status at the european level. anacamptis morio and orchis spitzelii are categorized as near threatened (nt) species in europe. the proper conservation mechanism of the investigated genera is still lacking from the territory of bosnia and herzegovina. more precisely, “the red list of flora of bosnia and herzegovina“ (đug et al., 2013) contains only 4 species of these genera: orchis spitzelii as critically endangered (cr), orchis purpurea and o. simia as vulnerable (vu) and anacamptis pyramidalis with the near threatened (nt) status. the same species were mentioned by šilić (1996) in his list of plant species for the red book of bosnia and herzegovina, but all defined as vulnerable (vu). surprisingly, both of these lists do not contain anacamptis palustris (present on the iucn red list of threatened species as global endangered) and all the species from the european red list. consequently, the red list of flora of bosnia and herzegovina requires a thorough revision in order to ensure the adequate red list categories and criteria for all the representatives of the orchidaceae family, followed by defining measures for their conservation. references abadžić, s. 2012: stanje biodiverziteta prirodnog močvarnog područja bistrik. kakanj. barudanović, s., mašić, e., macanović, a., hatibović, e. 2019: state of peatland ecosystems in bosnia and herzegovina. fondeco science, sarajevo, 1(1): 48-60. bateman, r.m., pridgeon, a.m., chase, m.w. 1997: phylogenetics of subtribe orchidinae (orchidoideae, orchidaceae) based on nuclear its sequences. 2. infrageneric relationships and reclassification to achieve monophyly of orchis sensu stricto. lindleyana, 12: 113–141 beck mannagetta, g. 1903: flora bosne, hercegovine i novopazarskog sandžaka. gymnospermae i monocotyledones. glasnik zemaljskog muzeja bosne i hercegovine 15 (2). bucalo, v. 2015: biljne vrste iz fam. orchidaceae na planini jadovnik u zapadnoj bosni. šumarstvo, 67 (2-2): 83-94. 151 biologica nyssana ● 10 (2) december 2019: 143-153 šabanović et al. ● the genus orchis tourn. ex l. and its related genera in the zenica-doboj canton (bosnia and herzegovina) chase, m.w. 2005: classification of orchidaceae in the age of dna data. curtis’s botanical magazine, 22 (1): 3-7. christenhusz, m.j., byng, j.w. 2016: the number of known plants species in the world and its annual increase. phytotaxa, 261 (3): 202-217. cites, 2019: the convention on international trade in endangered species, appendices i, ii and iii: https://www.cites.org/eng/app/appendices.php. delforge p. 2006: orchids of europe, north africa and middle east. 3rd edition, a&c black ltd. publishers, london. đorđević, v. 2018: prostorna distribucija i ekologija orhideja (orchidaceae) zapadne srbije. doktorska disertacija. biološki fakultet, univerzitet u beogradu. 709 str. đug, s., muratović, e., drešković, n., boškailo, a., dudević, s. 2013: crvena lista flore federacije bosne i hercegovine. federalno ministarstvo okoliša i turizma. sarajevo. fiala (1896): prilozi flori bosne i hercegovine. glasnik zemaljskog muzeja u bosni i hercegovini, 8(1): 293-324. freyn j., brandis e. 1888: beitrage zur flora von bosnien und der angrenzenden hercegovina (nach den von p. erich brandis gesammelten pflanzen). verhandlungen des zoologisch-botanischen vereins, 38: 577-644, wien. fukarek, p., jovanović, b., ed. 1983: karta prirodne potencijalne vegetacije sfr jugoslavije. naučno veće vegetacijske karte jugoslavije, skoplje. goletić, š. (ed.) 2016: kantonalni ekološki akcioni plan zeničko-dobojskog kantona za period 20172025. univerzitet u zenici, metalurški institut „kemal kapetanović“, zenica. 210 str. horvat, i., glavač, v., ellenberg, h. 1974: vegetation södosteuropas. gustav fischer verlag, stuttgart. 768 p. iucn red list of threatened species: https://www. iucnredlist.org/ kummer, p., sendtner, o., 1849: enumeratio plantarum in itinere sendtneriano in bosnia lectarum, cum definitionibus novarum specierum et adumbrationibus obscurarum varietatumque. flora, 32(1): 2-10. lampinen r. 2001: universal transverse mercator (utm) and military grid reference system (mgrs). retrieved october, 21, 2015. maly (1908): nabrajanje sakupljenih biljaka u bosni i hercegovini od članova međunarodnog kongresa u godini 1905. glasnik zemaljskog muzeja u bosni i hercegovini, 20 (4): 558-567. maly k. 1928: prilozi za floru bosne i hercegovine 10. glasnik zemaljskog muzeja bosne i hercegovine, 40: 107-166. maly k. 1940: notizen zur flora von bosnien-herzegovina. glasnik zemaljskog muzeja nezavisne države hrvatske u bosni i hercegovini, 52 (2): 2246. maly k. 1952: grundlagen zur kenntnis der flora von travnik (posthumno!). godišnjak biološkog instituta univerziteta u sarajevu, 5(2-2): 52-121. meussel, h., jager, e., weinert, e. 1965: vergleinchende chorologie der zentraleuropaischen flora. veb. gustav fischer verlag, 1. jena. milanović đ., brujić j., stupar v., bucalo v., travar j., cvjetićanin r. 2015: vaskularna flora planine klekovače u bosni i hercegovini. glasnik šumarskog fakulteta univerziteta u banjoj luci 23: 15-83. plavšić, s. 1940: uber neue und seltene pflanzenformen aus mittelbosnien. glasnik zemaljskog muzeja nezavisne države hrvatske u bosni i hercegovini. prirodne nauke: 13-20. protić, đ. 1898: prilog k poznavanju flore okoline vareša u bosni. glasnik zemaljskog muzeja bosne i hercegovine, 10(1): 93-102. ranđelović, v., zlatković, b. 2010: flora i vegetacija vlasinske visoravni. prirodnomatematički fakultet, univerzitet u nišu. 448 str. riter-studnička, h. 1956: flora i vegetacija na dolomitima bosne i hercegovine. godišnjak biološkog instituta univerziteta u sarajevu, 9 (2-2): 73-122. riter-studnička h. 1957: flora i vegetacija na dolomitima bosne i hercegovine 3-3: dalja okolina konjica, kompleks drvara i 2 manja nalazišta u bosni. godišnjak biološkog instituta univerziteta u sarajevu, 10 (2-2): 129-161. ritter-studnicka, h. 1970: die flora der serpentinvorkommen in bosnien. bibliotheca botanica, 130: 2-100. ritter-studnička, h. 1970a: die vegetation der serpentinvorkommen in bosnien. vegetatio, 21 (1/3): 75-156. soó, r. 1980: orchis l. in: tutin, t.g., heywood, v.h., burges, n.a., moore, d.m., valentine, d.h., walters s.m., webb d.a. (eds.): flora europaea, v: 337-342. cambridge university press. 452 p. 152 biologica nyssana ● 10 (2) december 2019: 143-153 šabanović et al. ● the genus orchis tourn. ex l. and its related genera in the zenica-doboj canton (bosnia and herzegovina) stevanović, v. 1992: floristička podela teritorije srbije sa pregledom viših horiona i odgovarajućih flornih elemenata. in sarić, m. (ed.): flora srbije, i. (drugo izdanje). sanu. beograd. 49-70. stroh, p.a. 2016. orchis militaris l. military orchid. species account. botanical society of britain and ireland. šabanović, e., bektić, s. 2017: ljekovite biljne vrste na visočici. dobra knjiga d.o.o. sarajevo. sarajevo. šilić, č. 1996: spisak biljnih vrsta (pteridophyta i spermatophyta) za “crvenu knjigu“ bosne i hercegovine. glasnik zemaljskog muzeja bosne i hercegovine, 31: 323-367. šilić, č. 2008: diverzitet vrsta. in: redžić, s., barudanović, s., radević, m. (eds.): bosna i hercegovina – zemlja raznolikosti. izvješće bosne i hercegovine za konvenciju o biološkoj raznolikosti. federalno ministarstvo okoliša i turizma. sarajevo. šoljan, d., muratović, e., abadžić, s. 2014: orhideje planina oko sarajeva. dobra knjiga d.o.o. sarajevo. sarajevo. tutin t.g., heywood v.h., burges n.a., moore d.m., valentine d.h., walters s.m., webb d.a. (eds.), 1980: flora europaea, v. cambridge university press. west-bosnien. österreische botanische zeitschrift 55(11): 424-438. vuković n, brana s, mitić b. 2011: orchid diversity of the cape of kamenjak (istria, croatia). acta botanica croatica, 70(1): 23-40. 153 biologica nyssana ● 10 (2) december 2019: 143-153 šabanović et al. ● the genus orchis tourn. ex l. and its related genera in the zenica-doboj canton (bosnia and herzegovina) mitrović, a.. bogdanović pristov, j.  maternal effect of continuous light … biologica nyssana 6 (1)  september 2015: 11-16 mitrović, a.. bogdanović pristov, j.  maternal effect of continuous light … 11 original article received: 28 july 2015 revised: 28 august 2015 accepted: 20 september 2015 maternal effect of continuous light on seed properties in a short day plant chenopodium rubrum l. (chenopodiaceae) aleksandra mitrović*, jelena bogdanović pristov institute for multidisciplinary research, university of belgrade, kneza višeslava 1, belgrade, serbia * e-mail: mita@imsi.rs abstract: mitrović, a., bogdanović pristov, j.: maternal effect of continuous light on seed properties in a short day plant chenopodium rubrum l. (chenopodiaceae). biologica nyssana, 6 (1), september 2015: 11-16. environmental effects on morphological and physiological properties of offspring which occurs during development of mother plant are called maternal environmental effects. photoperiod is one of the crucial environmental factors according to which plants modify numerous physiological processes. maternal effect of photoperiod in a short day plant chenopodium rubrum extends through the whole life cycle of offspring and persist to the second generation, photoperiod during induction and evocation of flowering of mother plants showing the key influence. here we show that also non-inductive photoperiod preceding flowering induction of mother plants shows its maternal effect on offspring properties: seed size, seed germination and seed protein composition. presented data argues in favor of earlier suggested that relative amounts of seed proteins represent an “archive“ of photoperiods experienced by mother plants during their lives. key words: chenopodium rubrum, maternal effect of photoperiod, seed proteins, seed weight, seed germination apstrakt: mitrović, a., bogdanović pristov, j.: materinski efekat neprekidne svetlosti na karakteristike semena kratkodnevne biljke chenopodium rubrum l. (chenopodiaceae). biologica nyssana, 6 (1), septembar 2015: 11-16. faktori spoljašnje sredine koji utiču na morfološke i fiziološke karakteristike potomstva nazivaju se materinski efekti spoljašnje sredine. fotoperiod je jedan od ključnih faktora spoljašnje sredine prema kojem biljke modifikuju mnogobrojne fiziološke procese. materinski efekat fotoperioda proteže se kroz čitav životni ciklus potomstva i održava se i u sledećoj generaciji, pri čemu ključni uticaj ima fotoperiod kome su majke biljke bile izložene tokom indukcije i evokacije cvetanja. ovde pokazujemo da i neindukcioni fotoperiod koji prethodi indukciji cvetanja materinskih biljaka pokazuje materinski efekat na potomstvo: veličinu semena, klijanje semena i sastav proteina semena. prikazani rezultati govore u prilog ranije predloženog: relativna količina proteina semena predstavlja “arhivu” fotoperioda koju je majka biljka iskusila tokom svog životnog ciklusa. key words: chenopodium rubrum, materinski efekat fotoperioda, proteini semena, masa semena, klijanje semena 6 (1) • september 2015: 11-16 biologica nyssana 6 (1)  september 2015: 11-16 mitrović, a.. bogdanović pristov, j.  maternal effect of continuous light … 12 introduction annual plants experience a single environment during their life cycle, but they could be exposed to different environmental conditions through environmental changes. environmental effects on morphological and physiological properties of offspring which occurs during development of mother plant are called maternal environmental effects (g u t t e r m a n & e v e n a r i , 1972; g a l l o w a y , 2005). their expression depends on the offspring environment, they are expressed throughout the life cycle of the offspring and may persist for several generations. maternal environmental effects could be provoked by different environmental factors such as soil nutrients (s t r a t o n , 1989), temperature (l a c e y et al., 1997), photoperiod (c o o k , 1975; g u t t e r m a n , 1978; b e r t t e r o et al., 1999; m i t r o v i ć et al., 2010), co2 levels (s t e i n g e r et al., 2000). the day length (night length), i.e. photoperiod, is one of the most crucial environmental factors according to which plants modify numerous physiological processes, the transition to flowering being one of the most important turning points in mother plant life cycle in order to provide the success of its offspring. in species with strong maternal effects, maternal plants could be grown under appropriate environmental conditions to promote successful establishment of new populations. for example, in the case of assisted dispersal to habitats with longer day length, maternal plants can be grown under the appropriate day length so maternal effects are adaptive (s c h u l e r & o r r o c k , 2012). on the other hand, if the seeds disperse into a different habitat, maternal environmental effects may reduce the offspring fitness, but only for a single generation, since the response to the new habitat will induce new maternal effects (g a l l o w a y , 2005). chenopodium rubrum l. sel. 184 is a qualitative short day (sd) weedy annual (c u m m i n g , 1967) with well defined photoperiodic sensitivity. it is sensitive to the small changes in day length, with defined critical night length of 8 h (t s u c h i a & i s h i g u r i , 1981). c. rubrum is sensitive to photoperiodic stimulus for flowering as early as at cotyledonary stage (s e i d l o v á & o p a t r n á , 1978), when 6 adequate photoperiodic cycles are sufficient for photoperiodic flower induction. it is an early flowering species (c u m m i n g , 1967) which makes it a suitable model plant for studies of ontogenesis. under the suitable photoperiodic conditions in vitro it produces seeds in 10 weeks (m i t r o v i ć et al., 2007). c. rubrum plants modify their vegetative and reproductive development, in accordance with the photoperiod they are exposed to (c o o k , 1975; m i t r o v i ć et al., 2007). its response to photoperiod extends to the offspring and persists to the second generation, the suggested mechanism of maternal effect of photoperiod being through seed protein composition (m i t r o v i ć et al., 2010). in c. rubrum, as a sd plant able to receive photoperiodic flowering induction as early as at about 5 days of age, most of the data concerning maternal effect of photoperiod on offspring deal with photoperiods inductive for flowering and its alterations on the plants of the same age and early during their life (c o o k , 1975; m i t r o v i ć et al., 2010). hence, maternal effect of different photoperiods inductive for flowering on different phases of offspring ontogenesis (m i t r o v i ć et al., 2010) is defined. c. rubrum seed number and seed weight is determined by photoperiod mother plants are exposed to during induction and evocation of flowering (c o o k , 1975; m i t r o v i ć et al., 2007). seed germination and offspring growth is determined by photoperiod during flowering induction of mother plants, offspring flowering and seed maturation is determined by photoperiod their mothers experienced during induction and evocation of flowering, while photoperiod after the evocation of flowering of mother plants does not affect offspring ontogenesis significantly (m i t r o v i ć et al., 2010). on the other hand, in nature, c. rubrum plants flowers when summer days become shorter. this means that they receive photoperiodic induction for flowering later during their life and thus under the longest photoperiods physiologically possible (c o o k , 1975). in other words, in nature, transition from vegetative to reproductive development (flowering induction) occurs when night length becomes longer than its critical night length of 8 h (t s u c h i a & i s h i g u r i , 1981). in this regard, the information about timing in offspring life when photoperiod inductive for flowering is to be expected could be of great importance for offspring success. therefore, it is to be expected that besides photoperiod during induction and evocation of flowering of mother plants, also non-inductive photoperiod preceding flowering induction of mother plants and its duration shows its (maternal) effect on offspring. c u m m i n g (1967) observed significant decrease in seed size if sd plants from genus chenopodium were grown under non-inductive photoperiod preceding flowering induction compared to those grown continuously under inductive short days. but there is no more precise data of the effect of noninductive photoperiod preceding flowering induction on offspring properties. biologica nyssana 6 (1)  september 2015: 11-16 mitrović, a.. bogdanović pristov, j.  maternal effect of continuous light … 13 here we present preliminary data on maternal effect of non-inductive photoperiod preceding flowering induction on seed properties: seed size, seed germination and variation in seed protein pattern. in vitro culture of intact plants was selected because it enables precise control over environmental factors, as it was shown that other environmental factors, such as temperature, significantly affects maternal effect of photoperiod on different chenopodium species (b e r t e r o et al., 1999; m i t r o v i ć et al., 2007). as non-inductive photoperiod for obligatory sd plant c. rubrum, continuous light (cl) was selected as extreme long day (c u m m i n g , 1967; m i t r o v i ć et al., 2007). the duration of non-inductive photoperiod preceding flowering induction was chosen to be longer than the period necessary for c. rubrum plants to reach full flowering under inductive 14 h /10 h photoperiod in vitro (ž i v a n o v i ć et al., 1995). finally for photoperiod inductive for flowering 14 h /10 h was used, established as inductive in c. rubrum flowering research (k r e k u l e & s e i d l o v a , 1976; s e i d l o v á & o p a t r n á , 1978; ž i v a n o v i ć et al., 1995). material and methods plants in vitro intact c. rubrum plants were grown in vitro on ms (m u r a s h i g e & s k o o g , 1962) medium, as described in m i t r o v i ć et al. (2007). seedlings, 5 days old, were exposed to two different photoperiodic treatments: 65 days of a 14 h/10 h photoperiod or 17 days of continuous light followed by 43 days of 14 h/10 h (m i t r o v i ć et al., 2008). irradiance was about 70 μmol m-2 s-1. temperature in the growth chambers was 25 ± 2 ºc. seed collection matured seed were collected, dried for 1 month at room temperature, measured (4 replicates of 100 seeds) and capped on +8c until use for determination of seed protein content, for separation of seed proteins on sodium dodecylsulfate polyacrylamide gel electrophoresis (page), and for testing the effect of maternal photoperiod on germination. seed extraction samples of 0.03 g of dry seeds were imbibed 2.5 h in darkness at 32 c, and powdered in liquid nitrogen. proteins were extracted for 30 min at 4 ºc with 0.5 ml 0.05 m tris buffer (ph 7.4) containing 0.25 m sucrose and 1 mm edta, and centrifuged (4 ºc, 10000 × g 10 min). seed protein concentration protein concentration in seed samples derived from mother plants grown under different photoperiodic conditions was determined by b r a d f o r d (1976) method with bovine serum albumin as the standard. separation of seed proteins protein separation, in seed samples derived from mother plants grown under different photoperiodic conditions, was performed on sodium dodecilsulfate poliacrilamide gel electrophoresis (sds-page) (l a e m m l i , 1970). polyacrylamide gel electrophoresis was carried out under non/denaturing conditions in gels containing 10% polyacrylamide with a 4% stacking gel. a constant current of 25 (15) ma per gel was applied. equal volumes of all samples were loaded into the gels. proteins were visualized by coomassie blue staining. for sds-page separation all samples were run in triplicate. relative values of seed protein band intensities were determined in image master totallab 1.11. seed germination seeds were sown on moistened filter paper (5 ml distilled water) in petri dishes. germination was tested during 4 days (24 h dark at 32 °c, 24h dark at 10 °c and 48 h white light at 32 °c). every 24 h, 4 replicates of 100 seeds per treatment of mother plant were scored for germination. as a criterion of germination, radical protrusion by more than 2 mm was used. results and discussion for an obligatory sd plant, such as c. rubrum, extreme long day (cl) is non-inductive photoperiod, under which it grows continuously vegetative (c u m m i n g , 1967; m i t r o v i ć et al., 2007). analyzing literature concerning the effect of cl on plant growth and development s y s o e v a et al. (2010) showed that there are reports of cl both increasing plant developmental rate and inhibiting it. plant developmental response to cl depends on many factors including photoperiodic sensitivity, developmental stage and environmental conditions. in most long-day plants cl accelerated the reproductive cycle, while sdps responded to cl differently. on the other hand growing plants under cl is a way of economical crop production. it also provides better understanding of plant adaptations to the arctic polar 24 h photoperiod (s y s o e v a et al., 2010). biologica nyssana 6 (1)  september 2015: 11-16 mitrović, a.. bogdanović pristov, j.  maternal effect of continuous light … 14 maternal effect of non-inductive extreme long days, cl, preceding c. rubrum flowering induction on seed weight in previous work (m i t r o v i ć & b o g d a n o v i ć , 2008), we showed that about 5 times more seeds were collected from plants in which 17 non-inductive extreme long days (cl) preceded flowering induction by 14 h /10 h photoperiod, compared to those grown continuously under inductive 14 h /10 h photoperiod (m i t r o v i ć & b o g d a n o v i ć , 2008). as opposed, seed weight is for one third lower if collected from mother plants in which cl preceded flowering induction (fig. 1a). hence, we show significant difference in weight of seeds collected from mother plants induced for flowering under the same 14 h /10 h photoperiod, but with the time span of 17 days in their life cycle. this confirms c u m m i n g (1967) observation that sd plants from the genus chenopodium, grown under long days or cl before flowering induction, produces smaller seeds compared to seeds collected from plants grown continuously under the short days. on the other hand, on the basis of the experiments in which c. rubrum mother plants were grown continuously under different inductive photoperiods, it was shown that seed number and seed weight is determined by photoperiod mother plants experienced during induction and evocation of flowering (c o o k , 1975; m i t r o v i ć et al., 2007). from the abovementioned arises that seed size and weight is determined not only by the photoperiod under which induction and evocation of fig. 1. maternal effect of 17 days of continuous light (cl), extreme non-inductive photoperiod, preceding flowering induction on offspring properties in a qualitative short day plant chenopodium rubrum on: a) seed weight, b) seed germination, c) total seed protein content, d) width and intensity of seed protein bands (coomassie blue stained sds-page gel, rf values are marked for seed protein bands for which the difference in width and intensity are noticed between seed samples collected from mother plants grown continuously under inductive 14 h /10 h photoperiod and those in which 17 days of non-inductive cl preceded flowering induction by 14 h /10 h photoperiod), and e) relative intensities of seed protein bands (determined by image master totallab 1.11) biologica nyssana 6 (1)  september 2015: 11-16 mitrović, a.. bogdanović pristov, j.  maternal effect of continuous light … 15 flowering of mother plants occurred (c o o k 1975; m i t r o v i ć et al., 2007), it is also significantly affected by non-inductive photoperiod preceding flowering induction of mother plants. maternal effect of non-inductive extreme long days, cl, preceding c. rubrum flowering induction on seed germination fig. 1b shows that cl preceding flowering induction of mother plants also affect seed germination. germination of small seeds collected from mother plants in which 17 days of cl preceded flowering induction, is delayed and synchronized compared to germination of seeds collected from mother plants grown continuously under inductive 14 h /10 h photoperiod (fig. 1b). also, c u m m i n g (1967) showed that in small seeds, obtained from mother plants grown under non-inductive long days or cl before flowering induction were more dormant, the delay in germination happens due to thicker seed integument. at the same time, germination of seeds collected from mother plants grown continuously under different inductive photoperiods was determined by photoperiod experienced during flowering induction of mother plants (m i t r o v i ć et al., 2010). so, the same as seed size and weight, seed germination is, as well, determined not only by the photoperiod under which induction and evocation of flowering of mother plants occurred, but also is significantly affected by photoperiod preceding flowering induction of mother plants. maternal effect of non-inductive extreme long days, cl, preceding c. rubrum flowering induction, on seed proteins although seed storage proteins in chenopodium species are localized mostly in endosperm and embryo (p r e g o et al., 1998), while the difference in seed size are due to proportional difference in both the size of the embryo and endosperm in (c o o k , 1975), protein content (fig. 1c) was nearly twice higher in smaller seeds (fig. 1a) collected from mother plants in witch flowering induction was preceded by 17 days of cl. we suggested earlier that the mechanism of maternal effect of photoperiod can be through relative seed protein composition representing an “archive“ of photoperiods experienced by mother plants during their lives (m i t r o v i ć et al., 2010). b h a r g a v a et al. (2005) showed the difference in number, width and intensity of seed protein bands on sds-page gel, not only in seeds samples of different chenopodium species, but also in seed samples of the same species collected from widely separated localities (characterized by highly variable environmental factors). fig. 1d shows the presence of 33 protein bands on sds-page gel in both c. rubrum seed samples. the same number of protein bands was previously obtained (m i t r o v i ć et al., 2010) in seed samples collected from mother plants grown continuously under different inductive photoperiods in vitro. this confirmes that the difference in quantities of those 33 seed protein bands is only the result of different photoperiods mother plants were exposed to during their life cycle, as grown under precisely controlled environment in vitro. for 6 of them (fig. 1e), significant differences in width and intensity were noticed. protein bands with rf values 0.58, 0.64, 0.66, 0.80 and 0.83 showed about twice higher relative intensities in small seeds collected from mother plants in which flowering induction was preceded by 17 days of cl (fig. 1e). hence, in seeds collected from mother plants in witch flowering induction was preceded by cl, higher protein content (fig. 1c) is a result of increased amounts of those protein bands (with rf values 0.58, 0.64, 0.66, 0.80 and 0.83). at the same time, for those protein bands we showed earlier high correlations with the day length mother plants experienced during induction and evocation of flowering (m i t r o v i ć et al., 2010). therefore, this is the additional confirmation that relative amounts of those specific seed proteins are dependent on photoperiods experienced by mother plants during their lives. conclusion c. rubrum is a species with strong maternal effects. besides photoperiod during induction and evocation of flowering of mother plants (m i t r o v i ć et al., 2010), here we show that also non-inductive photoperiod preceding flowering induction of mother plants shows its maternal effect on offspring properties. maternal effect of noninductive, extreme long days, cl, preceding flowering induction of c. rubrum mother plants results in lowering seed size, enhancing seed dormancy, and increase in the amounts of some specific seed proteins. this goes in favor of earlier suggested that seed proteins represent an “archive“ of photoperiods experienced by mother plants during their lives. detailed analysis of altering noninductive photoperiods and its duration preceding c. rubrum flowering induction, as well as identification of seed proteins we found dependant on photoperiods experienced by mother plants, would contribute understanding the mechanism of maternal environmental effects. biologica nyssana 6 (1)  september 2015: 11-16 mitrović, a.. bogdanović pristov, j.  maternal effect of continuous light … 16 acknowledgements. this work was financed by the grants on173017 and iii43010 from the ministry of the education and science of the republic of serbia. references bertero, h.d., king, r.w., hall, a.j. 1999: photoperiod-sensitive development phases in quinoa (chenopodium quinoa willd.). field crops research, 60: 231-243. bradford, m.m. 1976: a rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principles of protein-dye binding. analitical biochemistry, 72: 248-254. cook, r.e. 1975: the photoinductive control of seed weight in chenopodium rubrum l. american journal of botany, 62: 427-431. cumming, b.g. 1967. early flowering plants. in: f.h. will and n.k. wesselss, thomas y. cromwell, (eds.), methods in developmental biology: 277-299, new york. galloway, l.f. 2005: maternal effects provide phenotipic adaptation to local environmental conditions. new phytologist, 166: 93-100. gutterman, y., evenari, m. 1972: the influence of day length on seed coat colour, an index of water permeability of the desert annual ononis sicula guss. the journal of ecology, 60: 713–719. gutterman, y. 1978: germinability of seeds as a function of the maternal environment. acta horticulturae, 83: 49-56. krekule, j., seidlova, j. 1976: effects of exogenous cytokinins on flowering of the short-day plant chenopodium rubrum l. biologia plantarum, 18: 142-149. lacey, e.p., smith, s., case, a.l. 1997: parental effects on seed mass: seed coat but not embryo/endosperm effects. american journal of botany, 84: 1617-1620. mitrović a, giba z., ćulafić lj. 2007: the photoperiodic control of growth and development of chenopodium rubrum l. plants in vitro. archives of biological sciences, 59: 203-208. mitrović, a., bogdanović, j. 2008: activities of antioxidative enzymes during chenopodium rubrum l. ontogenesis in vitro. archives of biological sciences, 60: 223-231. mitrović, a., bogdanović, j., giba, z., ćulafić, lj. 2010: effect of photoperiod during growth of chenopodium rubrum mother plants on properties of offspring. biologia plantarum, 54: 735-739. murashige, t., skoog, f. 1962: a revised medium for rapid growth and bioassays with tobacco tissue cultures. physiologia plantarum, 15: 473497. prego, i., maldonado, s., otegui, m. 1998: seed structure and localization of reserves in chenopodium quinoa. annals of botany, 82: 481-488. schuler, m.s, orrock, j.l. 2012: the maladaptive significance of maternal effects for plants in anthropogenically modified environments. evolutionary ecology, 26:475–481. seidlová, f., opatrná, j. 1978: change of growth correlation in the shoot meristem as the cause of dependance of flowering. zeitschrift fur planzenphysiologie, 89: 377-392. steinger, t.b. 2000: maternal and direct effects of elevated co2 on seed provisioning, germination and seedling growth in bromus erectus. oecologia, 123: 475-480. straton, d.a. 1989: competition prolongs expression of matenal effects in seedlings of erigeron annuus (asteraceae). american journal of botany, 76: 1646-1653. sysoeva, m., markovskaya, e. f., shibaeva, t.g. 2010: plants under continuous light: a review. plant stress, 4: 5-17. tsuchiya, t., ishiguri, y. 1981: role of the quality of light in the photoperiodic flowering response in four latitudinal ecotypes of chenopodium rubrum l. plant and cell physiology, 22: 525532. živanović, b., ćulafić, lj., filipović, a. 1995: the effects of hormones and saccharides on growth and flowering of green and herbicides-treated chenopodium rubrum l. plants. biologia plantarum, 37: 257-264. antioxidant activity of oregano essential oil (origanum vulgare l.) biologica nyssana 7 (2)  december 2016: 131-139 stanojević, lj.p. et al.  antioxidant activity of oregano essential oil… 131 original article received: 30 june 2016 revised: 31 october 2016 accepted: 24 november 2016 antioxidant activity of oregano essential oil (origanum vulgare l.) ljiljana p. stanojević*, jelena s. stanojević, dragan j. cvetković, dušica p. ilić faculty of technology, university of niš, bulevar oslobodenja 124, 16000 leskovac, serbia * e-mail: stanojevic@tf.ni.ac.rs abstract: stanojević, lj.p., stanojević, j.s., cvetković, d.j., ilić, d.p.: antioxidant activity of oregano essential oil (origanum vulgare l.). biologica nyssana, 7 (2), december 2016: 131-139. essential oil obtained from oregano (origanum vulgare l.) by clevenger-type hydrodistillation and hydromodulus 1:10 m/v during 180 minutes, has been investigated in this work. qualitative and quantitative composition of the oil was determined by gc-ms and gc-fid spectrometry. antioxidant activity of the obtained oil was examined spectrophotometrically by dpph test (after 20, 30, 45 and 60 minutes of incubation) and tba-mda assay. the yield of essential oil was 4.1 ml/100 g of plant material. seven components were identified: α-thujene, myrcene, α-terpinene, o-cymene, γ-terpinene, thymol and carvacrol. the major components were thymol (45%) and carvacrol (37.4%). oil incubated for 60 minutes has shown the best antioxidant activity according to dpph test. the concentrations of essential oil, required for neutralization of 50% of initial dpph radical concentration (ec50), were 0.761, 0.590, 0.360 and 0.326 mg/ml, after 20, 30, 45 and 60 minutes of incubation, respectively. lipid peroxidation inhibition of 92.3% was achieved by 1.35 mg/ml essential oil concentration. the results obtained indicate that oregano essential oil is a good source of natural antioxidants with potential application in food and pharmaceutical industries, as a safer alternative to the synthetic antioxidants. key words: origanum vulgare l., essential oil, antioxidant activity, gc-ms analysis apstrakt: stanojević, lj.p., stanojević, j.s., cvetković, d.j., ilić, d.p.: antioksidativna aktivnost etarskog ulja vranilove trave (origanum vulgare l.). biologica nyssana, 7 (2), december 2016: 131-139. u ovom radu je ispitivano etarsko ulje vranilove trave (origanum vulgare l.) dobijeno clevenger hidrodestilacijom pri hidromodulu 1:10 m/v, u toku 180 minuta. kvalitativni i kvantitativni sastav dobijenog etarskog ulja određen je gc-ms i gc-fid spektrometrijom. antioksidativna aktivnost ulja je određena spektrofotometrijski, dpph testom (posle 20,30, 45 i 60 minuta inkubacije) i tba-mda testom. prinos etarskog ulja bio je 4.1 ml/100 g biljnog materijala. identifikovano je sedam komponenti: α-tujen, mircen, αterpinen, o-cimen, γ-terpinen, timol i karvakrol. kao glavne komponente identifikovane su timol (45%) i karvakrol (37,4%). najbolju dpph-antioksidativnu aktivnost pokazalo je ulje nakon 60 min inkubacije. koncentracije etarskog ulja neophodne za neutralizaciju 50% od početne koncentracije dpph radikala (ec50 vrednosti) bile su 0,761; 0,590; 0,360 i 0,326 mg/ml, nakon 20, 30, 45 i 60 min inkubacije, respektivno. inhibicija lipidne peroksidacije od 92,3% je postignuta sa 1,35 mg/ml eterskog ulja. dobijeni rezultati sugerišu da dobijeno etarsko ulje vranilove trave predstavlja dobar izvor prirodnih antioksidanasa sa potencijalnom primenom u prehrambenoj i farmaceutskoj industriji kao bezbednija alternartiva sintetskim antioksidansima. ključne reči: origanum vulgare l., etarsko ulje, antioksidativna aktivnost, gc-ms analiza 7 (2) • december 2016: 131-139 12th sfses • 16-19 june 2016, kopaonik mt doi: 10.5281/zenodo.200410 biologica nyssana 7 (2)  december 2016: 131-139 stanojević, lj.p. et al.  antioxidant activity of oregano essential oil… 132 introduction the oxidation process is one of the major causes of food spoilage, which results in rancidity and deterioration of the nutritional quality, color, flavor, texture, and safety of foods (b e t a i e b et al., 2010). a significant number of herbs is used as natural preservatives in food industry. besides being used to achieve the proper flavor and to intensify the flavors some spices and herbs exhibit antioxidant effects which is of great importance for food industry (s t a n k o v i ć & s t a n o j e v i ć , 2014; t o n g n u a n c h a n et al., 2014). plants rich in antioxidant compounds are present in food industry (b e t a i e b et al., 2010). there are evident requirements for increasing application of natural antioxidants obtained from plant material. undesirable side effects of synthetic antioxidants, such as butyl hydroxy anisole (bha) and butylated hydroxytoluene (bht), which are associated with their toxic and carcinogenic effects (m a e s t r i et al., 2006), are the reason of such requirements. essential oils are very heterogeneous group of complex mixtures of secondary plant metabolites. composition of essential oil may be different between different species or varieties, related to different cultivation, origin, vegetative stage and growing seasons of the plant (v a z i r i a n et al., 2015). beside aroma, odor and fragrance of many of the oils, some of them have also been confirmed to possess antioxidant activities (b e t a i e b et al., 2010; v a z i r i a n et al., 2015). essential oils are also widely used in perfumes, cosmetics, aromatherapy and nutrition (b u r t , 2004; b a k k a l i et al., 2008). earlier studies have been shown that, among the herbs and spices which are extensively studied, the plants from the lamiaceae (labiatae) family possess a significant antioxidant activity (t s i m i d o u & b o s k o u , 1994; l a g o u r i et al., 1993). within this family oregano (origanum vulgare l.) is probably one of most widely used aromatic plant which essential oils are particularly rich in monoand sesquiterpenes (d e f a l c o et al., 2013). the genus origanum includes around 38 species, most of which are indigenous to the mediterranean, euro-siberian and irano-siberian regions (s a h i n et al., 2004). origanum vulgare l. is one of the most widely among all the species within the genus which distributed all over the europe, west and central asia up to taiwan (s a h i n et al., 2004; r a d u š i e n e et al, 2008). it is one of the most important culinary herbs in the world. leaves and flowers of oregano are traditionally used to cure cough and sore throats and for relieve of gastrointestinal disorders. origanum vulgare is a source of essential oils and phenolic metabolites (r a d u š i e n e et al, 2008). the main bioactive components of oregano are phenolic components (s e g e i t k u j a w a et al., 1990; k u l e v a n o v a et al., 2001; l e u n g & f o s t e r , 2003; r a d u š i e n ė et al., 2008) and essential oil (l e u n g & f o s t e r , 2003). origanum vulgare contains 0.1-1.0% of essential oil composed of thymol, carvacrol, -bisabolene, caryophyllene, pcimene, borneol, linalool, linalyl acetate, geranyl acetate, -pinene, -pinene, -terpinene, with highly variable relative proportion, depending on source (l e u n g & f o s t e r , 2003). oregano essential oils have been shown to possess antioxidant, antibacterial, antifungal, diaphoretic, carminative, antispasmodic and analgesic activities (d e f a l c o et al., 2013). thymol and carvacrol, usually the major phenols present in oregano, have strong fungicidal, anthelmintic, irritant, and other properties (l e u n g & f o s t e r , 2003). based on numerous studies it was established that oregano essential oil, rich in thymol and carvacrol, has a significant antioxidant activity in the process of the lard oxidation (l a g o u r i et al., 1993; t s i m i d o u & b o s k o u ,1994). y a n i s h l i e v a and m a r i n o v a (1995) examined the antioxidant activity of hexane extracts from oregano grown in bulgaria, and the mechanism of action of pure thymol and carvacrol (y a n i s h l i e v a et al.,1999), while kulišić and coworker presented different methods for antioxidant activity of oregano essential oil testing (k u l i š i ć et al., 2004). the aim of the present study was to examine the antioxidant properties of oregano essential oil, originated from serbia, by using two different methods, namely, dpph test, and tba-mda assay. material and methods plant material the commercial sample of aerial parts of origanum vulgare l. (origani herba) was purchased from the local health food store in leskovac, serbia. according to the declaration, the material used for investigations originates from serbia (packed by: malina-impex d.o.o. popučke b.b., valjevo) chemicals and reagent ethanol, 96% (centrochem, zemun, serbia), 1,1diphenyl-2-picrylhydrazyl (dpph radical), butylated hydroxy toluene (bht), thiobarbituric acid (tba), 2,2'-azobis (2-methylpropionamidine) dihydrochloride (aaph) (sigma chemical company, st. louis, usa), trichloroacetic acid (tca) (j.t. baker, biologica nyssana 7 (2)  december 2016: 131-139 stanojević, lj.p. et al.  antioxidant activity of oregano essential oil… 133 va deventer, netherlands). phospholipids (phospholipon® 90; pl90) were a gift from phospholipid gmbh (cologne, germany). according to the declaration pl90 mixture consisting of phosphatidylcholine 94.6%, lyso-phosphatidylcholine 1.3%; fatty acid: palmitic acid 12 ± 2%, stearic acid 3 ± 1%, oleic acid 10 ± 3%, linoleic acid 66 ± 5%, linoleic acid 5% ± 2; peroxide number 1.4. isolation of essential oil essential oil from aerial parts of o. vulgare was isolated by classic clevenger-type hydrodistillation according to ph. jug. v (2000). plant material (50 g) was immersed in 500 ml of water in round bottom flask, and the oil was isolated using a clevenger-type apparatus for 180 min. the resulting essential oil was dried over anhydrous sodium sulfate, filtered and stored at +4 °c in a well-filled, airtight container, protected from light, until the analysis. gc-ms and gc-fid analysis gc-ms analysis of the oregano essential oil was performed on agilent technologies 7890b gas chromatograph, equipped with weakly polar, silica capillary column, hp-5ms (5% diphenyland 95% dimethyl-polysiloxane, 30 m x 0.25 mm, 0.25 μm film thickness; agilent technologies, usa) and coupled with inert, selective 5977a mass detector of the same company. one μl of the sample dissolved in diethyl ether in the concentration of 1000 ppm was injected in 20:1 split mode. helium was used as the carrier gas, at a constant flow rate of 1 ml/min. the oven temperature was programmed from 50 °c for 2.25 minutes and then increased to 290 °c at the rate of 4 °c/min. temperatures of the msd transfer line, ion source and quadruple mass analyzer were set at 300 °c, 230 °c and 150 °c, respectively. the ionization voltage was 70 ev and mass range m/z 35650. gc-fid analysis was carried out under identical experimental conditions as gc-ms. the temperature of the flame-ionization detector (fid) was set at 300°c. data processing was performed using msd chemstation, masshunter qualitative analysis and amdis 32 softwares (agilent technologies, usa). retention indices of the components from the analyzed samples were experimentally determined using a homologous series of n-alkanes from c8-c20 as standards. compounds identification was based on the comparison of their retention indices (riexp – tab. 1) with those available in literature (adams, 2007) (rilit – tab. 1), as well as their mass spectra with those from willey, nist and rtlpest libraries. the percentage composition of particular components in the essential oil was determined on the basis of automatically integrated peak areas of the gc-fid signal. antioxidant activity dpph assay antioxidant activity of oregano essential oil was determined by dpph test (a q u i n o et al., 2002; c h o i et al., 2002; s a n c h e z m o r e n o , 2002). the essential oil was dissolved in ethanol (96%) and a series of different concentration solutions were prepared (0.098 to 12.5 mg/ml). the ethanol solution of dpph radical (1 ml, 3×10-4 mol/l) was added to 2.5 ml of each essential oil solutions. absorbance of one sample was immediately measured at 517 nm, while the other samples were incubated at room temperature in the dark, for 20, 30, 45 and 60 minutes, and the absorbance was also measured at 517 nm (as). the absorbance at 517 nm was measured for pure ethanol solution of dpph radical prepared as described above – 1 ml of the dpph radical (3×10-4 mol/l) diluted with 2.5 ml of ethanol, ac), as well as for the essential oil before treatment with dpph radical (2.5 ml of essential oil diluted with 1 ml of ethanol, ab). free radical scavenging capacity was calculated by the eq. 1 (s t a n o j e v i ć et al., 2015): dpph rsc (%) =          c bs a aa 100 100 ......... (1) rscradicals scavenging capacity ec50 value was defined as essential oil concentration needed for the neutralization of 50% of the initial dpph radical concentration. this value was determined by interpolation from the linear regression analysis in the concentration range between 0.098 and 0.391 mg/ml of essential oil added to the reaction mixture. bht was used as the reference compound. tba-mda assay thiobarbituric acid – malondialdehyde (tba-mda) test is one of the most commonly used tests for lipid peroxidation process monitoring in vitro. it’s based on heating of sample with tba in acidic environment, i.e. on tba and mda reaction. mda is end-product of lipid peroxidation formed during hydroperoxide degradation which build pink chromogen ([tba]2-malondialdehyde adduct) with absorption maximum at 532 nm. colored complex is formed by condensation of 2 moles of tba and 1 mole of mda only from the fatty acid chains that contain at least three double bonds (h a l l i w e l l & c h i r i c o , 1993; l a g u e r r e et al., 2007). the antioxidant activity of oregano essential oil was determined by the method developed for caroteinoid, biologica nyssana 7 (2)  december 2016: 131-139 stanojević, lj.p. et al.  antioxidant activity of oregano essential oil… 134 flavonoids, extracts and some potential antioxidants (c v e t k o v i c & m a r k o v i c , 2008; c v e t k o v i c et al., 2011; z v e z d a n o v i ć et al., 2014; s t a n o j e v i ć et al., 2015a), with some modifications. "sample" contains 0.3 cm3 of pl90 methanolic solution (1·10-2 mol/l), and essential oil ethanolic solution (0.042-5.375 mg/ml) in 2:1 (v/v) ratio. lipid peroxidation was initiated by addition of 0.2 cm3 of aqueous solution of thermal azo-initiator aaph (2.2·10-2 mol/l) during 3 h at a temperature of 40 °c, protected from light. after incubation, 1 ml of aqueous solution of tca (5.5%), 0.5 ml of methanolic solution of bht (1∙10-3 mol/l) and 0.5 ml of tba (4.2∙10-2 mol/l in 5∙10-2 mol/l naoh) were added to the reaction mixture. the mixture was incubated for 10 min at 65 °c and then centrifuged for 5 min at 13800 rpm. the increase in absorbance of the supernatant at 532 nm represents an absorption maximum of generated tba-mda complex. the absorbance of pl90 solution, where lipid peroxidation is initiated with aaph, treated with tba (”control“), as well as of pl90 solution, without lipid peroxidation initiation, but also treated with tba (”blank“). lipid peroxidation inhibition was calculated by the eq. 2 (stanojević et al., 2015a):   100 bc sc aa ) a(a lpi (%) ............................. (2) lpiinhibitionxidationlipid pero where ac – represents the absorbance of ”control“; as – absorbance of ”sample“; ab – absorbance of ”blank“. bht (0.0125-0.05 mg/ml) was used as the reference compound. all experiments were carried out in three replications. data were expressed as mean ± standard deviation. the obtained data were analyzed by microsoft excel 2007 and origin 7 trial. results and discussion hydrodistillation kinetics and oregano essential oil composition figure 1 shows the influence of hydrodistillation time on the essential oil yield. maximal essential oil yield of 4.1 ml/100g of plant material was achieved after 180 minutes. the hydrodistillation curve shows that there are two different periods of hydrodistillation (fig. 1). the essential oil was evaporated out from the surface from the cells of plant material in the first period (the fast oil hydrodistillation). in the second, slow oil hydrodistillation period, a slow molecular diffusion of the essential oil from internal part from cells of plants material occurred. fig. 1. hydrodistillation kinetics of oregano essential oil in the tab. 1 and on fig. 2 the results of gc– ms analysis of oregano essential oil are presented. seven components were identified in essential oil: αthujene, myrcene, α-terpinene, o-cymene, γterpinene, thymol and carvacrol. the major components were thymol (45%) and carvacrol (37.4%). the content of thymol and carvacrol in the oregano essential oil from various regions was different: 35% and 32% of thymol and carvacrol, respectively, from dalmacia (k u l i š i ć et al., 2004), 0.8 and 0.6% respectively, from turkey (s a h i n et al., 2004), 1.1 and 14.3% respectively, from southern italy (d e f a l c o et al., 2013), 37.1 and 9.6 respectively, from iran (v a z i r i a n et al., 2015), 31.8 and 0.2%, respectively from portugal (g a l e g o et al, 2008). other investigators reported a lower content of carvacrol in oregano essential oil (less than 35%) compared to the oregano from serbia. these changes of thymol and carvacrol content in the oregano essential oil, and changes in the compositions of the oil might arise from several environmental reasons (climatic, seasonal, geographical), used drying method of plant material and genetic differences of the plant material (f a b e r et al., 1997; c a l l a n et al., 2007; g h a s s e m i g o l e z a n i et al., 2008; f i g i e l et al, 2010; s t a n o j e v i ć et al., 2011; d e f a l c o et al, 2013). significantly higher content of this two bioactive components with an array of different biological activities is especially important (u l t e e et al., 2002; n o s t r o et al., 2007; w a n g et al., 2009; m a t h e l a et al., 2010; m e h d i et al., 2011). the major components are believed to be mainly responsible for the biological activity of this b a k k a l i , 2008). based on the results obtained in biologica nyssana 7 (2)  december 2016: 131-139 stanojević, lj.p. et al.  antioxidant activity of oregano essential oil… 135 particular essential oil (b a i l e r et al., 2001; our investigation, the oregano essential oil from serbia could be a potential natural source of thymol and carvacrol. antioxidant activity dpph test is most commonly in vitro method for antioxidant activities determination of plant extracts and essential oils. it is very suitable and useful for the antioxidant activity determination since it’s fast and sufficiently sensitive (m o l y n e u x , 2004). this method is based on hydrogen atoms or electrons exchange between antioxidant molecules and dpph radicals in the solution (s a n c h e z m o r e n o et al., 2002). dpph radical scavenging activity of isolated oregano essential oil was shown on fig. 3, while the ec50 values are listed in tab. 2. degree of dpph radical neutralization depends on oil concentration as well as the incubation time it increases with the concentration increase which is the lowest for non incubated samples (fig. 3). the highest antioxidant activity (about 95%) has been measured after 60 minutes of incubation. the ec50 (dpph) value of fig. 2. gc-fid chromatogram of oregano essential oil table 1. chemical composition of origanum vulgare essential oil no. tret., min compound ri exp rilit method of identification content, % monoterpene hydrocarbons (14.4%) 1 9.78 α-thujene 925 924 ri, ms 0.4 2 11.99 myrcene 987 988 ri, ms 1.0 3 12.93 α-terpinene 1014 1014 ri, ms 1.1 4 13.22 o-cymene 1022 1022 ri, ms 4.4 5 14.46 γ-terpinene 1057 1054 ri, ms 7.5 phenols (82.4%) 6 22.59 thymol 1292 1289 ri, ms 45.0 7 22.92 carvacrol 1300 1298 ri, ms 37.4 total 96.8% tret.: retention time; ri lit a,b-retention indices from literature (adams, 2007), respectively; riexp: experimentally determined retention indices using a homologous series of n-alkanes (c8-c20) on the hp-5ms column. ms: constituent identified by mass-spectra comparison; ri: constituent identified by retention index matching. biologica nyssana 7 (2)  december 2016: 131-139 stanojević, lj.p. et al.  antioxidant activity of oregano essential oil… 136 synthetic antioxidant bht was 0.021 mg/ml, indicating better antioxidant activity compared to the tested essential oil. mentioned antioxidant is the most commonly used synthetic antioxidant, but with harmful effects in human body (i t o et al., 1986). so, presented results suggest that oregano essential oil can be potentially used as a safer alternative to synthetic antioxidants in pharmaceutical and food industries. fig. 3. dpph antioxidant activity of oregano essential oil k u l i š i ć et al. (2004) and s a h i n et al. (s a h i n et al., 2004) have come to similar results in their investigations. in earlier reports, thymol and carvacrol, in particular, were found to be main antioxidant components of essential oil from different origanum species (b a r r a t a et al., 1998; r u b e r t o et al., 2002; p u e r t e s -m e j i a et al., 2002). because the main components of the oregano essential oil in our investigations are phenolics thymol and carvacrol, they are probably mostly responsible for the high degree of dpph radical neutralization (y a n i s h l i e v a & m a r i n o v a , 1995; k u l i š i ć et al., 2004; s a h i n et al., 2004). the tba method is sensitive and achieves reproducible results. this method is preferable in order to obtain useful data in an environment similar to the real-life situation (k u l i š i ć et al., 2004). fig. 4 shows degree of lipid peroxidation inhibition by investigated oregano essential oil, while tab. 2 shows the ec50 value (tba-mda) of the oil. fig. 4. inhibition of lipid peroxidation by oregano essential oil (tba-mda test) based on the presented results it can be concluded that isolated essential oil from oregano shows good concentration dependent antioxidant activity. the highest degree of lipid peroxidation inhibition was achieved by essential oil in concentration of 3.0 mg/ml. the ec50 (tba-mda) value of synthetic antioxidant bht was 0.0185 mg/ml. k u l i š i ć et al. (2004) reported antioxidant activity of oregano essential oil from dalmacia and its fractions, measured by tba method. in addition, c a p e c k a et al. (2005) found that o. vulgare showed the strongest inhibition of linolenic acid peroxidation. our results of antioxidant activity are in accordance with s a h i n et al. (2004) who reported that essential oil of origanum vulgare ssp. vulgare possesses antioxidant compounds, and therefore can be used as a natural preservative in food and/or pharmaceutical industry. presented results confirm that oregano essential oil possesses remarkable antioxidant activities as assessed by two different methods. this biological effect is probably due to the presence of thymol and carvacrol, but possible synergistic effect table 2. ec50 values of oregano essential oil ec50 (dpph), mg/ml 20 min 30 min 45 min 60 min 0.761  0.003 0.590  0.005 0.360  0.004 0.326  0.002 ec50 (tba-mda), mg/ml 0.455  0.004 biologica nyssana 7 (2)  december 2016: 131-139 stanojević, lj.p. et al.  antioxidant activity of oregano essential oil… 137 among other compounds in the oil can be also suggested. conclusion the presented results indicate that the oregano essential oil could be used as potential source of natural antioxidants for the food, pharmaceutical and chemical industry. therefore, oregano essential oil represents the alternative to synthetic additives that exhibit toxic and carcinogenic effects. so, it is interesting to investigate its application as natural antioxidant additive in some final food and pharmaceutical products, for preservation and/or extension the shelf-life of raw and processed foods as well as pharmaceuticals. in addition, the results of antioxidant activity in the present study suggested that use of oregano is not just reasonable but it should be even favored in the traditional serbian cuisine. acknowledgements. this work is part of the research project "plant and synthetic bioactive products of new generation", no. tr 34012, financed by the ministry of education, science and technological development of republic of serbia. references aquino, r., morelli, s., tomaino, a., pellegrino, m., saija, a., grumetto, l., puglia, c., ventura d., bonina, f., grumetto, l. 2002: antioxidant and photoprotective activity of a crude extract of culcitium reflexum h.b.k. leaves and their major flavonoids. journal of ethnopharmacology, 79: 183–191. bailer, j., aichinger, t., hackl, g., hueber, k., dachler, m. 2001: essential oil content and composition in commercially available dill cultivars in comparison to caraway. industrial crops and products,14: 229–239. bakkali, f., averbeck, s., averbeck, d., idaomar, m. 2008: biological effects of essential oils – a review. food and chemical toxicology, 46: 446– 475. barrata, m.d.s., dorman, h.j.d., deans,s.g., figueiredo, a.c., barroso, j.g., ruberto,g. 1998: chemical composition, antimicrobial and antioxidative activity of laurel, sage, rosemary, oregano and coriander essential oils. journal of essential oil research, 10: 618–627. bettaieb, i., bourgou, o., wannes, w. a., hamrouni, i., limam, f. , marzouk, b. 2010: essential oils, phenolics, and antioxidant activities of different parts of cumin (cuminum cyminum l.). journal of agricultural and food chemistry, 58: 10410– 10418. burt, s. 2004: essential oils: their antibacterial properties and potential applications in foods – a review. international journal of food microbiology, 94: 223–253. callan, n.w.,, johnson, d. l., westcott, m.p., welty, l.e. 2007: herb and oil composition of dill (anethum graveolens l.): effects of crop maturity and plant density. industrial crops and products, 25: 282–287. capecka, e., mareczek, a., leja, m. 2005: antioxidant activity of fresh and dry herbs of lamiaceae species. food chemistry, 93(2): 223– 226. choi, w.c., kim, c.s., hwang, s.s., choi, k.b., ahn, j.h., lee, y.m., park. h.s., kim, k.s., lee, y.m. 2002: antioxidant activity and free radical scavenging capacity between korean medicinal plants and flavonoids by assay-guided comparison. plant science, 163: 1161–1168. cvetković, d., marković, d. 2008: uv-induced changes in anti-oxidant capacities of selected carotenoids toward lecithin in aqueous solution. radiation physics and chemistry, 77: 34–41. cvetković, d., marković, d., cvetković, d., radovanović, b. 2011: effects of continuous uvirradiation on the antioxidant activities of quercetin and rutin in solution in the presence of lecithin as the protective target. journal of the serbian chemical society, 76: 973–985. de falco, e., mancini, e., roscigno, g., mignola, e., taglialatela-scafati, o., senatore, f. 2013: chemical composition and biological activity of essential oils of origanum vulgare l. subsp. vulgare l. under different growth conditions. molecules, 18: 14948–14960. faber, b., bangert, k., mosandl, a., 1997: gc-irms and enantioselective analysis in biochemical studies in dill (anethum graveolens l.). flavour and fragrance journal, 12: 305–314. figiel, a., szumny, a., gutiérrez-ortíz, a., carbonell-barrachina, a.a. 2010: composition of oregano essential oil (origanum vulgare) as affected by drying method. journal of food engineering, 98: 240–247. galego, l., almeida, v., gonçalves, v., costa, m., monteiro, i., matos, f., miguel, g. 2008: antioxidant activity of the essential oils of thymbra capitata, origanum vulgare, thymus mastichina and calamintha baetica. acta horticulture, 765:325-34. ghassemi-golezani, k., andalibi, b., zehtabsalmasi, s., saba, j. 2008: effects of water stress during vegetative and reproductive stages on seed yield and essential oil content of dill (anethum graveolens l.). journal of food, agriculture and environment, 6: 282–284. https://www.google.rs/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&cad=rja&uact=8&ved=0ahukewifsfufic_nahvhorqkhclhdj0qfggamaa&url=http%3a%2f%2fpubs.acs.org%2fjournal%2fjafcau&usg=afqjcnh5paftp8g3y2wt0pd14ztu8pqzeg&sig2=2r5ovv3il1oeynbo5m-cbg&bvm=bv.125801520,d.bgs https://www.google.rs/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&cad=rja&uact=8&ved=0ahukewifsfufic_nahvhorqkhclhdj0qfggamaa&url=http%3a%2f%2fpubs.acs.org%2fjournal%2fjafcau&usg=afqjcnh5paftp8g3y2wt0pd14ztu8pqzeg&sig2=2r5ovv3il1oeynbo5m-cbg&bvm=bv.125801520,d.bgs biologica nyssana 7 (2)  december 2016: 131-139 stanojević, lj.p. et al.  antioxidant activity of oregano essential oil… 138 halliwell, b., chirico, s. 1993: lipid peroxidation: its mechanism measurement, and significance. american journal of clinical nutrition, 57: 715s725s. ito, n., hirose, m., fukushima, h., tsuda, t., shirai, t., tatenatsu, m. 1986: studies on antioxidants: their carcinogenic and modifying effects on chemical carcinogens. food and chemical toxicology, 24:1071–1092. kulevanova, s., stefova, m., stefkov, g., stafilov. t. 2001: identification, isolation, and determination of flavones in origanum vulgare from macedonian flora. journal of liquid chromatography & related technologies, 24(4): 589–600. kulišić, t., radonić, a., katalinić, v., miloš, m. 2004: use of different methods for testing antioxidative activity of oregano essential oil. food chemistry, 85: 633–640. lagouri, v., blekas, g., tsimidou, m., kokkini, s., boskou, d. 1993: composition and antioxidant activity of essential oils from oregano plants grown wild in greece. zeitschrift für lebensmittel-untersuchung und -forschung, 197: 20–23. laguerre, m., lecomte, j., villeneuve, p. 2007: evaluation of the ability of antioxidants to counteract lipid oxidation: existing methods, new trends and challenges. progress in lipid research, 46: 244–282. leung, a.y., foster, s. 2003: encyclopedia of common natural ingredients (used in food, drugs, and cosmetics), second edition, a john wiley & sons, inc., hoboken, new jersey. 398–399. maestri, d.m., nepote, v., lamarque, a.l., zygadlo, j.a. 2006. natural products as antioxidants. in: imperato f. (ed.), phytochemistry: advances in research, research signopost: 105–135, kerala, india. mathela, c. s., singh, k. k., gupta, v. k. 2010: syntesis and in vitro antibacterijal activity of thymol and carvacrol derivates. acta poloniae pharmaceutican drug research, 67(4): 375–380. mehdi, s.j., ahmad, a., irshad, m., manzoor, n., rizvi, m.m.a. 2011: cytotoxic effect of carvacrol on human cervical cancer cells. biology and medicine, 3(2): 307–312. molyneux, p., 2004: the use of the stable free radical diphenylpicrylhydrazyl (dpph) for estimating antioxidant activity. songklanakarin journal of science and technology, 26(2): 211–219. nostro, a., sudano roccaro,a., bisignano, g., marino, a., cannatelli, m.a., pizzimenti, f.c., cioni, p.l., procopio. f. and blanco, a.r. 2007: effects of oregano, carvacrol and thymol on staphylococcus aureus and staphylococcus epidermidis biofilms. journal of medical microbiology, 56: 519–523. pharmacopoeia jugoslavica v, 2000: savremena administracija, beograd (in serbian), vol. 1: 118. puertes-mejia, m., hillebrand,s., stashenko,e., winterhalter, p. 2002: in vitro radical scavenging activity of essential oils of columbian plants and fractions from oregano (origanum vulgare l.) essential oil. flavour and fragrance journal, 17: 380–384. radušienė, j., ivanauskas, l., janulis, v., jakštas, v. 2008: composition and variability of phenolic compounds in origanum vulgare from lithuania. biologia, 54(1): 45–49. ruberto, g., barrata, m.t., sari, m., kaabexhe, m. 2002: chemical composition and antioxidant activity of essential oils from algerian origanum glandulosum desf. flavour and fragrance journal, 17: 251–254. sahin, f., gulluce, m., daferera, d., sokmen, a., sokmen, m., polissiou, m., agar, g., ozer, h. 2004: biological activities of the essential oils and methanol extract of origanum vulgare ssp. vulgare in the eastern anatolia region of turkey. food control, 15: 549–557. sanchez-moreno, c. 2002: methods used to evaluate the free radical scavenging activity in foods and biological systems. food science and technology international, 8(3), 121–137. segeit–kujawa, e., michalowska, a. 1990: determination of flavonoids in hb. origani. herba polonica, 36(3): 79–82. stanković, m.z., stanojević, lj.p., tehnologija lekovitog i začinskog bilja, tehnološki fakultet, leskovac, 2014. 113 p. stanojević, j.s., stanojević, lj.p., cvetković, d.j., danilović b.r., 2015: chemical composition, antioxidant and antimicrobial activity of the turmeric essential oil (curcuma longa l.). advanced technologies, 4(2): 19–25. stanojević, lj.p., stanojević, j.s., cvetković, d. j., cakić, m.d., ilić, d.p. 2015a: antioksidativna aktivnost etanolnog ekstrakta lista gajene jagode (fragariae folium). hemijska industrija, 69(5): 567–576. stanojević, lj., stanković, m., cakić, m., nikolić, v., nikolić, lj., ilić, d., radulović, n. 2011: the effect of hydrodistillation techniques on yield, kinetics, composition and antimicrobial activity of essential oils from flowers of lavandula officinalis l. hemijska industrija, 65: 455–463. tongnuanchan, p., benjakul, s. 2014: essential oils: extraction, bioactivities, and their uses for food preservation. journal of food science, 79(7): r1231–r1249. biologica nyssana 7 (2)  december 2016: 131-139 stanojević, lj.p. et al.  antioxidant activity of oregano essential oil… 139 tsimidou, m., boskou, d. 1994: antioxidant activity of essential oils from the plants of the lamiaceae family. in: g. charalambous, spices, herbs and edible fungi, 273–284, amsterdam: elsevier. ultee, a., bennik, m. h. j., moezelaar, r. 2002: the phenolic hydroxyl group of carvacrol is essential for action against the food-borne pathogen bacillus cereus. applied and environmental microbiology, 68(4):1561–1568. vazirian, m., mohammadi, m., farzaei, m.h., amin, g., amanzadeh, y. 2015: chemical composition and antioxidant activity of origanum vulgare subsp. vulgare essential oil from iran. research journal of pharmacognosy, 2(1): 41–46. wang, q., gong, j., huang, x., yu, h., xue. f. 2009: in vitro evaluation of the activity of microencapsulated carvacrol against escherichia coli with k88 pil. journal of applied microbiology 1, 107(6): 1781–178. yanishlieva, n.v., marinova, e.m., gordon, m.h., raneva, v.g. 1999): antioxidant activity and mechanism of action of thymol and carvacrol in two lipid systems. food chemistry, 64: 59–66. yanishlieva, n.v., marinova, e.m. 1995: antioxidant activity of selected species of the family lamiaceae grown in bulgaria. die nahrung, 39: 458–463. zvezdanović, j., daskalova, l., yancheva, d., cvetković, d., marković, d., anderluh, m., šmelcerović, a. 2014: 2-amino-5alkylidenethiazol-4-ones as promising lipid peroxidation inhibitors. monatshefte fur chemie,145: 945–952. sarajli' et al., 2019, biologica nyssana 10(2) 10 (2) december 2019: 135-142 doi: 10.5281/zenodo.3600191 spontaneous flora of the vraca memorial park (sarajevo, bosnia and herzegovina) original article nermina sarajlić ornithological society „naše ptice“, semira frašte 6, 71000 sarajevo, bosnia and herzegovina nermina.sarajlic@ptice.ba (corresponding author) nejc jogan university of ljubljana, biotechnical faculty, department of biology, večna pot 111, 1000 ljubljana, slovenia jernej.jogan@bf.uni-lj.si senad murtić department of plant production, faculty of agricultural and food sciences, university of sarajevo, zmaja od bosne 8, 71000 sarajevo, bosnia and herzegovina murticsenad@hotmail.com vladimir ranđelović university of niš, faculty of sciences and mathematics, department of biology and ecology, niš, serbia vladar@pmf.ni.ac.rs received: october 12, 2019 revised: november 19, 2019 accepted: november 24, 2019 abstract: the vraca memorial park was built in 1980-1981 around the old austrohungarian fortress located above the city of sarajevo, on northwestern slopes of trebević mountain slightly above 600 m asl. it covers an area of approximately 8 ha. the southwestern part of the park is mostly covered by semi-natural forest, the central part is mostly paved, whereas the northeastern part is partly covered by ornamental forest and some grassland patches. despite being declared a national monument of bosnia and herzegovina in 2005, the park is neglected and ruined, which allowed diverse subspontaneous vegetation to develop. the paper presents the results of systematic research and analysis of the spontaneous vascular flora of the vraca memorial park. a total of 280 species of 182 genera and 67 families were recorded. with 37 species, poaceae are the most abundant, followed by compositae with 29, and fabaceae and rosaceae (23 species each). numerous seedlings of shrubs and trees planted for ornamental purposes were observed, as well as the presence of two protected orchidaceae species. key words: sarajevo, urban flora, urban habitats, alien flora, indicators of anthropogenic changes apstract: spontana flora spomen-parka vraca (sarajevo, bosna i hercegovina) spomen-park vraca izgrađen je u periodu 1980-1981. godine oko stare austrougarske tvrđave koja se nalazi iznad grada sarajeva, na severozapadnim padinama planine trebević, malo iznad 600 m nadmorske visine. prostire se na površini od oko 8 ha. jugozapadni deo parka obrastao je poluprirodnom šumom, centralni deo je popločan, a severoistočni prekriven travnatum površinama i ukrasnim drvećem i grmljem. iako je 2005. proglašen nacionalnim spomenikom bosne i hercegovine, park je u velikoj meri zapušten, što je omogućilo razvoj raznovrsne subspontane vegetacije. u radu su prikazani rezultati sistematskog istraživanja i analize spontane vaskularne flore spomenparka vraca, gde je zabeleženo ukupno 280 vrsta iz 182 roda i 67 porodica, od kojih su najbrojnije poaceae sa 37 vrsta, a slede compositae sa 29 i fabaceae i rosaceae (svaka sa 23 vrste). u parku je zabeležen veliki broj klijanaca ukrasnog drveća i grmlja, kao i dve zaštićene vrste iz porodice orchidaceae. ključne reči: sarajevo, urbana flora, urbana staništa, strana flora, indikatori antropogenih promena introduction parks are open urban spaces generally reserved for public use. they are often larger than other urban green spaces and tend to consist of different habitats with a number of native and introduced plant species, combined to satisfy recreational and other requirements of urban green areas. nielsen et al. (2014) reviewed 62 papers on biodiversity of parks, and concluded that the findings consistently showed that parks are among the most species rich types of urban green spaces. they also noted that most papers indicated large share of non-native species in parks. the same was noted for park of lužnica manor © 2019 sarajlić et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 135 13th symposium on the flora of southeastern serbia and neighboring regions near zaprešić (hršak et al., 2015) and large public park in tirana (mesiti & dinga, 2016). changes in management of urban parks were found to have significant effect on changes in composition of flora (bianco et al., 2003). despite being declared a national monument of bosnia and herzegovina in 2005 (komisija za očuvanje nacionalnih spomenika, 2005), the vraca memorial park, which was built around the old austro-hungarian fortress located above the city of sarajevo, and open to the public on 25. november 1981 (sulejmanagić, 2017), is neglected and ruined, which allowed diverse subspontaneous vegetation to develop. the paper presents the results of systematic research and an analysis of the spontaneous vascular flora of the vraca memorial park. materials and methods study area the vraca memorial park is located above the city of sarajevo, on the top of a small range of northwestern slopes of trebević mountain reaching slightly above 600 m asl. it covers an area of approximately 8 ha. the southwestern part is mostly covered by semi-natural forest, central part is mostly paved by various types of stones, and northeastern part is covered by partly ornamental forest and with some grassland patches. the area is under influence of mid-european continental climate from the north, and the mediterranean from the south. field investigation the field survey was performed from the summer of 2015 to the spring of 2019. a complete list of taxa developed in three “sub-habitat” types (walls/ pavements, grassland and forest) of the vraca memorial park was made. ornamental species were listed only if specimens developed in a site where there were obviously not deliberately planted (e.g. in cracks of the wall or pavement) were observed. data analysis the nomenclature of plants follows euro+med plantbase and the international organization for plant information database. the analyses of plant life forms (phanerophyte ph, chamaephyte ch, hemicryptophyte h, geophyte g, hydrophyte hy and therophyte t), as well as origin of plants are given. life forms were given according to raunkiaer (1934), and degree of naturalization according to pyšek et al. (2002), mosyakin & yavorska (2003) and ignatieva & konechnaya (2004). the indicators of anthropogenic changes in the flora of vraca memorial park were calculated according to jackowiak (1990, 2006), as stated by witosławski & bomanowska (2009) (tab. 1). results and discussion during this research, a total of 280 species of 182 genera and 67 families were recorded. the most abundant families were poaceae (37 taxa, 13.21%), compositae (29 taxa, 10.36%), fabaceae and rosaceae (23 taxa, 8.21% each) (tab. 2). poaceae and compositae also were the dominant families in the “grand park of tirana” in albania (mesiti & dinga, 2016) and park of lužnica manor in croatia (hršak et al., 2015). 136 table 1. indicators of anthropogenic changes indicator of total anthropophytization iant = an/(sp+an) ×100% indicator of permanent anthropophytization ianp = mt/(sp+mt) ×100% indicator of total archaeophytization iart = ar/(sp+an) ×100% indicator of permanent archaeophytization iarp = ar/(sp+mt) ×100% indicator of total kenophytization iknt = kn/(sp+an) ×100% indicator of permanent kenophytization iknp = kn/(sp+mt) ×100% indicator of modernization im = kn/mt ×100% indicator of fluctuation changes if = df/(sp+an) ×100% an – total number of alien species, sp – number of native species, mt – number of permanently established alien species (ar + kn df), ar – number of archaeophytes, kn – number of kenophytes (neophytes), df – number of diaphytes (casual aliens). table 2. the most abundant families in the flora of vraca memorial park family no. of taxa % of total park flora (280) poaceae 37 13.21 compositae 29 10.36 rosaceae 23 8.21 fabaceae 23 8.21 lamiaceae 14 5 caryophyllaceae 11 3.93 brassicaceae 10 3.57 table 3. number of taxa in different habitats of vraca memorial park habitat no. of taxa % of total park flora (280) walls/pavement 174 62.14 forest 196 70 grassland 116 41.43 biologica nyssana ● 10 (2) december 2019: 135-142 sarajlić et al. ● spontaneous flora of the vraca memorial park (sarajevo, bosnia and herzegovina) least numerous group (2.87%), in grassland it was the phanerophytes (2.59%) and in forested parts of the park, chamaephytes (4.59%) (tab. 4). the alien flora of the vraca memorial park consists of 24 taxa (8.57%), 7 of which (29.17%) are archaeophytes, and 17 (70.83%) neophytes. according to del tredici (2010), the ratio of neophytes to archaeophytes rises in direct relation to the intensity of human disturbance. this is due to the fact that archaeophytes are typically associated with traditional rural environments or intermediate levels of anthropogenic activities (preston et al., 2004), and neophytes are more common in highly disturbed anthropogenous habitats, which provide distinctive environmental conditions that favor the establishment of species from warmer and drier areas (pyšek, 1998; pyšek et al., 2002; godefroid & koedam, 2007; la sorte et al., 2007). of three habitats in the vraca memorial park, most alien plant species were registered in cracks of walls and pavements, followed by forested parts of the park and only 6 alien species were recorded in grasslands (tab. 5). indicators of anthropogenic changes values in the flora of vraca memorial park are presented in tab. 6. the indicators of anthropophytization (iant = 8.57%; ianp = 7.25%) showed the considerable anthropogenic influence on the total flora of vraca memorial park, but were not as high as in savica park area near zagreb (iant = 28.78%; ianp = 26.76%) calculatmost taxa (196, or 70% of the total number of taxa of the park) were recorded in forested part of the vraca memorial park, and the least (116, or 41.43%) in the grassland patches in the northeast (tab. 3). the analysis of life forms in the entire park showed the domination of hemicryptophytes with 130 taxa (46.43%), followed by therophytes (68 taxa, 24.28%), phanerophytes (47 taxa, 16.78%) geophytes (20 taxa, 7.14%), and chamaephytes (15 taxa, 5.36%). hemicryptophytes also represented the major part of taxa in all three habitats of the park (44.83% in walls/pavements, 46.94% in forested part of the park and 65.52% in grassland). therophytes were the second most numerous group in walls/ pavements (27.01%) and grasslands (20.69%). the domination of therophytes and hemicryptopytes in the wall flora was also observed by altay et al. (2010). in forested part of the park, phanerophytes were the second most numerous group (21.43%). in walls/pavement habitats, the geophytes were the 137 table 4. distribution of life forms in vraca memorial park, per habitat types life form habitat ch g h ph t no. % no. % no. % no. % no. % walls/pavement 10 5.75 5 2.87 78 44.83 31 17.82 47 27.01 forest 9 4.59 15 7.65 92 46.94 42 21.43 38 19.39 grassland 4 3.45 9 7.76 76 65.52 3 2.59 24 20.69 entire park 15 5.36 20 7.14 130 46.43 47 16.78 68 24.28 table 5. alien flora of vraca memorial park, per habitat types alien (total) naturalized invasive archaeophytes neophytes walls/pavement 17 13 10 4 13 forest 15 7 4 7 8 grassland 6 4 3 3 3 entire park 24 16 11 7 17 table 6. indicators of anthropogenic changes in the vraca memorial park indicator (%) walls/pavement forest grassland entire park iant 9.77 7.65 5.17 8.57 ianp 8.72 6.22 5.17 7.25 iart 2.3 3.06 2.59 2.14 iarp 2.33 3.11 2.59 2.17 iknt 6.32 3.06 2.59 5 iknp 6.39 3.11 2.59 5.07 im 73.33 50 50 70 if 1.15 1.53 0 1.43 biologica nyssana ● 10 (2) december 2019: 135-142 sarajlić et al. ● spontaneous flora of the vraca memorial park (sarajevo, bosnia and herzegovina) ed by alegro et al. (2013) or rzeszów foothills in poland (iant = 21.3%) by jaźwa & stadnicka-futoma (2015). this is probably due to the fact that very small number of aliens in comparison to the native plants was registered in forested area and grassland. higher indicator values of kenophytization (iknt = 5%; iknp = 5.07%) showed that the flora of the park is more influenced by neophytes than by archaeophytes (iart = 2.14%; iarp = 2.17%). in both archaeophytes and neophytes, the values of total and permanent indicators were similar, showing that alien flora is well established, which was confirmed by the low value of the indicator of fluctuating changes (if = 4.45%). the forested part of the vraca memorial park is characterized by numerous seedlings of shrubs and trees planted for ornamental purposes, notably aesculus hippocastanum l., catalpa bignonioides walter, quercus rubra l., parthenocissus quinquefolia (l.) planchon, and mahonia aquifolium (pursh.) nutt. were observed, as well as the presence of two orchidaceae species included in the red list of flora of federation of bosnia and herzegovina: cephalanthera damasonium (mill.) druce (nt) and cephalanthera longifolia (l.) fritsch (vu) (đug et al., 2013). the flora developing in the vertical parts of walls was characterized by the presence of widespread and ruderal plants typical for rocky places and uncultivated lands, heliophilous, droughtresistant taxa with shallow root system, capable of growing on a thin soil layer. in pavements, tops and bottoms of the walls, where the soil layer is thicker, shade-tolerant species, some of which require higher humidity are developed. the same was noted by pavlova & tonkov (2005) and nedelcheva (2011). numerous seedlings of spiraea japonica l. were observed in cracks of the pavement in vraca memorial park. the vegetation of the park is similar to the vegetation of trampled and paved habitat types in sarajevo, and developed partly as a result of early naturalization of planted ornamental species. conclusion current neglected state of the vraca memorial park led to the development of diverse subspontaneous vegetation, similar to the vegetation of trampled and paved habitat types in sarajevo. the field survey performed in an area of 8 km2, in three “sub-habitat” types (walls/pavements, grassland and forest) of the vraca memorial park. a total of 280 species of 182 genera and 67 families were recorded. most taxa were recorded in forested part of the memorial park, and the least in the grassland patches in the northeast. the park’s flora is dominated by hemicryptophytes and native plants are more numerous in relation to 138 alien taxa, but the indicators of anthropophytization still showed the considerable anthropogenic influence on the total flora of vraca memorial park. the presence of two orchidaceae species included in the red list of flora of federation of bosnia and herzegovina indicates the potential importance of this area for conservation of native species. references alegro, a., bogdanović, s., rešetnik, i., boršić, i., cigić, p., nikolić, t. 2013: flora of the seminatural marshland savica, part of the (sub)urban flora of the city of zagreb (croatia). natura croatica, 22 (1): 111–134. altay, v., özyigit, i., yarci, c. 2010: urban ecological characteristics and vascular wall flora on the anatolian side of istanbul, turkey. maejo international journal of science and technology 4(03): 483-495. bianco p.m., fanelli g., tescarollo p., pignatti s. 2003: ruderalization in a roman park as a result of changing management. urban habitats, 1(1): 87104. del tredici, p. 2010: spontaneous urban vegetation reflections of change in a globalized world. nature and culture, 5 (3): 299-315. đug, s., muratović, e., drešković, e., boškailo, a., dudević, s. 2013: crvena lista flore federacije bosne i hercegovine. nacrt izvještaja – prijedlog. projekat šumskih i planinskih zaštićenih područja, „nvo green way”, sarajevo. pp. 1347. euro+med 2006-: euro+med plantbase the information resource for euro-mediterranean plant diversity. published on the internet http://ww2.bgbm. org/europlusmed/ [accessed september 2019] godefroid, s., koedam, n. 2007: urban plants species patterns are highly driven by density and function of built– up areas. landscape ecology, 22: 1227–1239. hršak, v., šegota, v., irić-šironja, s., sedlar, z. 2015: spontaneous and ornamental flora of the park of lužnica manor near zaprešić (northwestern croatia). natura croatica, 24 (1) 37–57. ignatieva, m., konechnaya, g. 2004: floristic investigations of historical parks in st. petersburg, russia. urban habitats, 2 (1): 174-216. jackowiak, b. 1990: antropogeniczne przemiany flory roślin naczyniowych poznania. wyd. naukowe uniwersytetu a. mickiewicza w poznaniu, ser. biol. 42: 1-208. jackowiak, b. 2006: methodological proposals for biologica nyssana ● 10 (2) december 2019: 135-142 sarajlić et al. ● spontaneous flora of the vraca memorial park (sarajevo, bosnia and herzegovina) 139 studies on the structure and dynamics of urban flora. polish botanical studie. 22: 251-260. jaźwa, m., stadnicka-futoma, a. 2015: the alien flora of the rzeszów foothills. biodiversity, 38 (1): 25-36. komisija za očuvanje nacionalnih spomenika 2005: odluka o proglašenju graditeljske cjeline – spomen-park vraca nacionalnim spomenikom bosne i hercegovine. službeni glasnik bih, 12/06. la sorte, f., mckinney, m., pyšek, p. 2007: compositional similarity among urban floras within and across continents: biogeographical consequences of human-mediated biotic interchange. global change biology, 13 (4): 913–921. mesiti, a., dinga, l. 2016: floristic investigation of the “grand park of tirana” with regard to urban indicators. biologica nyssana, 7 (2): 113-124. mosyakin, s.l., yavorska, o.g. 2003: the nonnative flora of kiev (kyiv) urban area, ukraine: a checklist and brief analysis. urban habitats, 1: 4565. nedelcheva, a. 2011: observations on the wall flora of kyustendil (bulgaria). eurasian journal of bioscience 5: 80-90. nielsen, a.b., van den bosch, m., maruthaveeran, s., van den bosch c. k. 2014: species richness in urban parks and its drivers: a review of empirical evidence. urban ecosystems, 17(1): 305-327. pavlova, d., tonkov, s. 2005: the wall flora of the nebet tepe architectural reserve in the city of plovdiv (bulgaria). acta botanica croatica 64: 357368. preston, c.d., pearman, d.a., hall, a.r. 2004: archaeophytes in britain. botanical journal of the linnean society, 145: 257–294. pyšek, p. 1998: alien and native species in central european urban floras: a quantitative comparison. journal of biogeography, 25, 155-163. pyšek, p., sáldo, j., mandák, b. 2002: catalogue of alien plants of the czech republic. preslia, 74: 97-186. raunkiaer, c. 1934: the life forms of plants and statical plant geography. clarendon press, oxford. sulejmanagić, a. 2017: spomen-park vraca. zbornik radova historijskog muzeja bosne i hercegovine 12: 82-84. the international organization for plant information 1996-2007 http://ww2.bgbm.org/iopi/gpc/default. asp [accessed september 2019] witosławski, p., bomanowska, a. 2009: southern european species in the flora of towns in the central poland. botanica serbica 33 (2): 115-129. biologica nyssana ● 10 (2) december 2019: 135-142 sarajlić et al. ● spontaneous flora of the vraca memorial park (sarajevo, bosnia and herzegovina) n o. taxon w al ls /p av em en t g ra ss la nd fo re st 1 acer campestre l. + + 2 acer negundo l. + 3 acer platanoides l. + + 4 acer pseudoplatanus l. + + 5 acer tataricum l. + + 6 achillea millefolium l. + + + 7 aegopodium podagraria l. + 8 aesculus hippocastanum l. + + 9 agrimonia eupatoria l. + 10 agrostis gigantea roth + 11 agrostis stolonifera l. + + + 12 ailanthus altissima (mill.) swingle + 13 ajuga genevensis l. + + + 14 ajuga reptans l. + + + 15 alliaria petiolata (m. bieb.) cavara et grande + + + 16 allium scorodoprasum l. + 17 ambrosia artemisiifolia l. + 18 amelanchier ovalis medik. + 19 anisantha sterilis (l.) nevski + + + 20 anthriscus sylvestris (l.) hoffm. + + 21 apera spica-venti (l.) p.beauv. + + 22 arabis hirsuta (l.) scop. + 23 arctium lappa l. + + 24 arctium tomentosum mill. + 25 aremonia agrimonoides (l.) dc. + + 26 arenaria serpyllifolia l. + + 27 arrhenatherum elatius (l.) p.beauv. ex j.presl et c.presl + + + 28 artemisia vulgaris l. + + + 29 arum maculatum l. + 30 asplenium ruta-muraria l. + 31 asplenium trichomanes l. + 32 atriplex patula l. + annex 1. overview of flora of vraca monumental park 140 33 ballota nigra l. + 34 barbarea vulgaris r. br. + 35 bellis perennis l. + + + 36 betula pendula roth + + 37 brachypodium sylvaticum (huds.) p.beauv. + + 38 bromopsis benekenii (lange) holub + 39 bromopsis erecta (huds.) fourr. + + 40 bromopsis inermis (leyss.) holub + 41 bromus commutatus schrad. + + + 42 bromus hordeaceus l. + + + 43 bromus squarrosus l. + 44 calystegia sepium (l.) r. br. + + + 45 calystegia silvatica (kit.) griseb. + 46 campanula patula l. + + 47 campanula rapunculus l. + 48 capsella bursa-pastoris (l.) medik. + + + 49 cardamine hirsuta l. + + 50 carex hirta l. + + 51 carex spicata huds. + + 52 carex sylvatica huds. + 53 carpinus betulus l. + 54 catalpa bignonioides walter + + 55 centaurea jacea l. + 56 centaurea scabiosa l. + 57 centaurea stoebe l. + 58 centaurium pulchellum (sw.) druce + 59 cephalanthera damasonium (mill.) druce + 60 cephalanthera longifolia (l.) fritsch + 61 cerastium arvense l. + 62 cerastium fontanum baumg. + + + 63 cerastium glomeratum thuill. + + + 64 chelidonium majus l. + + 65 cichorium intybus l. + + + 66 cirsium arvense (l.) scop. + 67 cirsium vulgare (savi) ten. + + 68 clematis vitalba l. + + + 69 clinopodium acinos (l.) kuntze + + 70 clinopodium vulgare l. + + + 71 convolvulus arvensis l. + + + 72 cornus sanguinea l. + + 73 corylus avellana l. + 74 cotoneaster horizontalis decne. + 75 crataegus monogyna jacq. + 76 crepis biennis l. + 77 cruciata laevipes opiz + + 78 cymbalaria muralis p.gaertn., b.mey. et scherb. + 79 cynosurus cristatus l. + + 80 dactylis glomerata l. + + + 81 daucus carota l. + + + 82 dianthus barbatus l. + 83 dioscorea communis (l.) caddick & wilkin + 84 diplotaxis muralis (l.) dc + 85 dipsacus fullonum l. + 86 dipsacus laciniatus l. + + + 87 echinochloa crus-galli (l.) p.beauv. + 88 echium vulgare l. + 89 elytrigia repens (l.) nevski + + + 90 epilobium roseum schreber + 91 equisetum arvense l. + 92 equisetum telmateia ehrh. + 93 eragrostis minor host + 94 erigeron annuus (l.) pers. + + + 95 erigeron canadensis l. + 96 erodium cicutarium (l.) ľ hér. + + 97 euphorbia amygdaloides l. + 98 euphorbia cyparissias l. + + 99 euphorbia esula l. + 100 euphorbia helioscopia l. + 101 euphorbia platyphyllos l. + 102 festuca heterophylla lam. + 103 festuca rubra l. + + 104 festulolium loliaceum (huds.) p.fourn + 105 ficaria verna huds. + 106 filipendula ulmaria (l.) maxim. + 107 filipendula vulgaris moench + + 108 fragaria vesca l. + + 109 fraxinus excelsior l. + + 110 fraxinus ornus l. + + + 111 galium aparine l. + + + 112 galium mollugo l. + + + 113 galium verum l. + + + 114 geranium columbinum l. + 115 geranium molle l. + + 116 geranium pusillum burm. f. + + 117 geranium robertianum l. + + + 118 geum urbanum l. + + + 119 glechoma hederacea l. + + + 120 hedera helix l. + + 121 heracleum sphondylium l. + + 122 holcus lanatus l. + + + 123 hordeum murinum l. + + + 124 hypericum perforatum l. + + + 125 juglans regia l. + + 126 juncus articulatus l. + 127 juncus bufonius l. + 128 juncus compressus jacq. + 129 juncus tenuis willd. + 130 knautia arvensis (l.) dc. + 131 lactuca muralis (l.) gaertn. + + 132 lactuca serriola l. + + biologica nyssana ● 10 (2) december 2019: 135-142 sarajlić et al. ● spontaneous flora of the vraca memorial park (sarajevo, bosnia and herzegovina) 141 133 lamium maculatum l. + 134 lamium purpureum l. + 135 lapsana communis l. + + 136 lathyrus latifolius l. + + 137 lathyrus nissolia l. + + 138 lathyrus pratensis l. + 139 lathyrus tuberosus l. + + 140 leontodon hispidus l. + 141 lepidium campestre (l.) r. br. + + 142 lepidium draba l. + 143 leucanthemum vulgare lam. + 144 ligustrum vulgare l. + 145 linaria vulgaris mill. + + 146 lolium perenne l. + + + 147 lonicera caprifolium l. + + 148 lonicera nitida wilson + 149 lotus corniculatus l. + + + 150 lysimachia nummularia l. + + 151 lythrum salicaria l. + + + 152 mahonia aquifolium (pursh.) nutt. + 153 malva moschata l. + 154 matricaria discoidea dc. + 155 medicago falcata l. + + + 156 medicago lupulina l. + + + 157 medicago sativa l. + + 158 melica ciliata l. + + 159 melilotus albus medik. + 160 melilotus officinalis (l.) lam. + + 161 mentha longifolia (l.) l. + + + 162 microrrhinum minus (l.) fourr. + 163 myosotis arvensis (l.) hill + + + 164 myosotis ramosissima rochel + 165 myosotis sylvatica hoffm. + 166 ochlopoa annua (l.) h. scholz + + + 167 origanum vulgare l. + 168 ornithogalum pyrenaicum l. + 169 oxalis corniculata l. + + 170 oxalis fontana bunge + 171 parthenocissus quinquefolia (l.) planchon + + 172 pastinaca sativa l. + 173 persicaria lapathifolia (l.) delarbre + 174 petrorhagia saxifraga (l.) link + 175 phleum pratense l. + 176 physalis alkekengi l. + + 177 picea abies (l.) h. karst. + 178 picris hieracioides l. + + 179 pimpinella saxifraga l. + 180 plantago lanceolata l. + + + 181 plantago major l. + + + 182 plantago media l. + + + 183 poa angustifolia l. + 184 poa bulbosa l. + 185 poa compressa l. + + 186 poa nemoralis l. + + 187 poa pratensis l. + 188 poa trivialis l. + + + 189 polygonum aviculare l. + 190 populus nigra l. + 191 potentilla argentea l. + 192 potentilla micrantha ramond ex dc. + 193 potentilla reptans l. + + + 194 primula acaulis (l.) l. + 195 prunella vulgaris l. + + + 196 prunus avium l. + 197 prunus cerasifera ehrh. + + + 198 prunus spinosa l. + 199 pteridium aquilinum (l.) kuhn + 200 quercus robur l. + + 201 quercus rubra l. + 202 ranunculus acris l. + + 203 reseda lutea l. + 204 rhinanthus rumelicus velen. + 205 robinia pseudoacacia l. + + 206 rorippa sylvestris (l.) besser + + 207 rosa arvensis huds. + + 208 rosa canina l. + + 209 rubus caesius l. + + 210 rubus plicatus weihe et nees + + 211 rumex acetosa l. + 212 rumex crispus l. + + + 213 rumex obtusifolius l. + 214 rumex patientia l. + + 215 rumex sanguineus l. + 216 sagina apetala ard. + 217 sagina procumbens l. + 218 salix alba l. + 219 salix caprea l. + 220 sambucus ebulus l. + + 221 sambucus nigra l. + + 222 sanguisorba minor scop. + + + 223 sanguisorba minor subsp. balearica (nyman) muñoz garm. & c. navarro + 224 schedonorus arundinaceus (schreb.) dumort. + + + 225 schedonorus pratensis (huds.) p. beauv. + + 226 sedum album l. + 227 sedum rupestre l. + 228 sedum sexangulare l. + 229 senecio vulgaris l. + 230 setaria viridis (l.) p.beauv. + 231 silene vulgaris (moench) garcke + 232 sisymbrium officinale (l.) scop. + 233 smyrnium perfoliatum l. + biologica nyssana ● 10 (2) december 2019: 135-142 sarajlić et al. ● spontaneous flora of the vraca memorial park (sarajevo, bosnia and herzegovina) 142 234 solanum nigrum l. + 235 solidago gigantea aiton + + 236 sonchus asper (l.) hill + 237 sonchus oleraceus l. + + 238 sorbus aucuparia l. + 239 spiraea japonica l. f. + 240 stachys sylvatica l. + 241 stellaria graminea l. 242 stellaria media (l.) vill. + 243 syringa vulgaris l. + 244 taraxacum sect. taraxacum f. h. wigg. + + + 245 taxus baccata l. + 246 teucrium chamaedrys l. + 247 thymus pulegioides l. + + + 248 tilia cordata mill. + 249 tilia platyphyllos scop. + + 250 torilis arvensis (huds.) link + 251 torilis japonica (houtt.) dc. + + 252 trifolium aureum pollich + + 253 trifolium campestre schreber + + 254 trifolium dubium sibth. + 255 trifolium hybridum l. + 256 trifolium medium l. + 257 trifolium pratense l. + + + 258 trifolium repens l. + + + 259 tripleurospermum inodorum (l.) sch. bip. + + 260 trisetum flavescens (l.) p.beauv. + + + 261 tropaeolum majus l. + 262 tussilago farfara l. + + 263 ulmus minor mill. + + 264 urtica dioica l. + + + 265 verbascum thapsus l. + 266 verbena officinalis l. + + + 267 veronica arvensis l. + + + 268 veronica chamaedrys l. + 269 veronica hederifolia l. + 270 veronica persica poir. + + 271 viburnum lantana l. + 272 viburnum opulus l. + 273 vicia cracca l. + + 274 vicia hirsuta (l.) gray + 275 vicia sativa l. + + 276 vicia tetrasperma (l.) schreber + + 277 vicia villosa roth ssp. varia (host) corb. + 278 vinca minor l. + 279 viscum album l. + 280 vulpia myuros (l.) c.c.gmel. + + biologica nyssana ● 10 (2) december 2019: 135-142 sarajlić et al. ● spontaneous flora of the vraca memorial park (sarajevo, bosnia and herzegovina) živković et al. 2020, biologica nyssana 11(1) 11 (1) september 2020: 59-64 doi: 10.5281/zenodo.4060302 yield of the tomato farmed by organic principles in the greenhouse with the application of retort beach charcoal original article sanja živković agriculture faculty, kruševac, university of niš, serbia gajicsanja43@gmail.com (corresponding author) tanja vasić agriculture faculty, kruševac, university of niš, serbia tanjavasic82@gmail.com biljana mihajlović trayal korporacija ad, kruševac, serbia biljanamih037@gmail.com snežana andjelković institue for forage crops, kruševac, serbia snezana.andjelkovic@ikbks.com biljana anđelić agriculture faculty, kruševac, university of niš, serbia andjelic.biljana@ni.ac.rs ivana stanojević agriculture faculty, kruševac, university of niš, serbia stanojevic.ivana85@gmail.com sonja filipović agriculture faculty, kruševac, university of niš, serbia sonjafilipovic86@yahoo.com received: december 06, 2019 revised: february 04, 2020 accepted: march 23, 2020 abstract: the experiment was established in the greenhouse designed by random block system with tomato genotype optima, treated with cow manure and retort beech charcoal, while the control treatment comprised of cow manure only. the aim was to determine the influence of the applied material on the number of fruits per plant and the weight of the fruit directly affecting the yield in the organic growing system. in the treatment with retort beech charcoal, the optima genotype had an average yield 23.88% higher than the control plants. key words: fruit, organic cultivation, retort beach charcoal, tomato, yield apstract: prinos paradajza uzgajanog po organskim principima u stakleniku uz primenu retortnog bukovog uglja eksperimentalni ogled bio je postavljen po dizajnu slučajni blok sistem u plasteniku sa sortom paradajza optima, tretmanom sa kravljim stajnjakom i retortnim bukovim ugljem i kontrolom sa kravljim stajnjakom bez retortnog bukovog uglja. cilj je bio da se utvrdi uticaj retortnog bukovog uglja na broj ploda po biljci i masu ploda koji direktno utiču na prinos u organskom sistemu gajenja. u tretmanu, sorta optima je u proseku imala 23,88% veći prinos u odnosu na biljke iz kontrole. ključne reči: plod, organsko gajenje, retortni bukov ugalj, prinos introduction tomato (lycopersicon esculentum mill.) is the most widely grown vegetable in the world, with a very wide range of distribution. it is grown on 4,725,416 ha with a yield of about 35 t ha-1 (fao, 2013). the world’s largest producers of tomatoes are china, india, the united states, turkey, egypt, russia, italy and mexico. in 2018, 8,629 ha were sown under these vegetables in serbia, with an average yield of 15.3 t ha-1 (webrzs.stat.gov.rs, 2018). tomato accounts for 11.6% of total vegetable consumption in serbia 15.2 kg per capita per year (vlahović & puškarić, 2012). tomato is one of the most used and widespread vegetable species used as a fresh vegetable, ripe fruit and in the form of wide-range products (de sousa et al., 2008). annually, over 40 million tons of tomatoes are processed worldwide to produce canned tomatoes, ketchup, tomato juice, sauce and many other products (wptc, 2015). in particular, the consummation of tomato and its products has been shown to be associated with a reduced risk of prostate, lung, and gastric cancer (hwang & bowen, 2005; palozza et al., 2011; yang et al., 2013). depending on the type of growth, tomato can be produced in different ways and used for different © 2020 živković et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 59 purposes. seeds and planting material, especially in the vegetable industry, represent an important factor for high quality production (popović et al., 2015). organic farming combines tradition, innovation and science in order to produce healthy product and keep the environment protected. the use of activated carbons in organic production is one of the possibilities to preserve and raise the soil quality and therefore the yield of the cultivated plants (lehman et al., 2005; biederman & harpole, 2013). soil biochar amendment is based on two thousand years old experience, which in recent decades has been renewed because of proven multiple benefits (chan et al., 2007). this importance is largely long-term, but also reveals the short-term effects (mann, 2005). both biochar and activated carbon are pyrogenic carbonaceous materials (pcm). they are produced by thermochemical conversion of carbonaceous feedstock (pyrolysis or/and activation). biochar is produced from sustainably sourced biomass and is used for non-oxidative applications in agriculture (e.g., in the soil) and is also discussed as a raw material for industrial processes. by definition, it is used for carbon sequestration. hence, if “biochar” is used as a fuel, it is burned and the carbon is transformed (oxidized) into co2, it is actually classified as charcoal. activated carbon is produced from any carbon source (fossil, waste or renewable) and engineered to be used as sorbent to remove contaminants from both gases and liquids. both materials have their distinct history, widely separated scientific communities and separated bodies of literature. unfortunately, a generally accepted terminology and definition is lacking (hagemann et al., 2018). however, as the proposed applications of biochar and activated carbon increasingly overlap, awareness of the “other” domain in each case can be beneficial. nowadays both biochar and activated carbon are used for soil remediation, which before has been solely an application of activated carbon. when the activated carbon is not removed after the application and if this activated carbon was produced from renewable feedstock and is complying to further specifications, it can be considered as biochar (laird 2008; woolf et al., 2010; hagemann et al., 2018; yadav et al., 2018). many studies confirmed that soil incorporated with biochars can improve plant growing (šeremešić et al., 2015; tian et al., 2018; yadav et al., 2018). according to tian et al. (2018) biochar incorporation induces soil alkalization which can increase soil nitrification and nitrogen (n) levels. increases in soil ph are likely to affect electrical conductivity (ec), cation exchange capacity (cec) and increase alkaline metal (mg2+, ca2+ and k+) oxides. likewise, it reduces soluble forms of aluminum, which is suggested as the most significant biochar factor affecting p solubility (de luca et al., 2009; tian et al., 2018; yadav et al., 2018). beneficial effects of biochar have been elaborated in studies word wide. however, there is a lack of experimental confirmation of the biochar application in our agricultural science. researches of biochar use have been mainly conducted on soils under tropical and humid climatic conditions, which are more degraded and have a lack of soil organic carbon (šeremešić et al., 2015; tian et al., 2018; yadav et al., 2018). therefore, the aim of this study is to research the charcoal application in vegetable farming under organic conditions. the parameter that is followed is tomato yield under temperate climatic conditions. for the organic production, it is very important to choose the right genotype to be grown according to ecological principles (vasić, 2016). the total area under certification (taking into account the organic status of the plots and plots in the conversion period) in serbia is 7,998 ha, plus meadows and pastures of 1,549 ha. in 2014, vegetables accounted for only 2% of the certified plant species. material and methods the experiment was set up at trmčare locality, kruševac municipality in a greenhouse according to a random block system in 2 replicates, planting 23 plants treated with cow manure and retort beech charcoal and 23 plants treated only with cow manure as a control. the row spacing was 70 cm, while the plant spacing in one row was 40 cm. the production of tomato optima genotype seedlings has been started on 20 march, 2019 in the glasshouse, and the seedling has been done on 20 may, 2019 in the greenhouse. before sowing, the soil was prepared by adding cow manure in the amount of 200 kg per 60 m2 at the depth of the sowing layer. seven days before planting, a mixture of cow manure in the amount of 200 g/plant and 200 g/plant of retort beech charcoal was introduced at the depth of the sowing layer. the control was not treated with retorted beech charcoal, but only with cow manure in the amount of 200 g/ plant. the activated charcoal used for this purpose was produced from natural raw materials and obtained by carbonation of beech, selected according to strictly defined technical requirements by activation of steam in a static furnace. due to its organic origin and production method, charcoal has a certain degree of activity (iodine number of 233-750 mg/g), which allows it to retain water reserves and thus provide the moisture needed by the plant. the use of activated carbon in organic production is very useful because of the introduc60 biologica nyssana ● 11 (1) september 2020: 59-64 živković et al. ● yield of the tomato farmed by organic principles in the greenhouse with the application of retort beach charcoal 61 tion of n, p, k, trace elements (mg/kg: ca-8590, mg-1260, k-7400, p-380, s-350 mn-32, fe-230, zn-20, cu-23), organic substances, humic acids and amino acids that help the plant’s level and improve their health. the granulometric range of the material ensures that the soil is loose because it does not dissolve in the soil. activated carbon granulometry 0-2.5 mm was used in this experiment. the ashes of the material thus obtained have a high level of elements such as oxides of potassium, calcium and magnesium. also, activated charcoal contains a higher percentage of charcoal so it has phosphates in ash, which is an excellent source of this microelement for the plant. due to the origin and content of alkali metals, the ph of this material is 9-11. tomatoes were grown by a support (pillar) on a single tree. the following parameters were monitored in the experiment: fruit yield per plant and number of fruits per plant. analysis of variance (anova) in statistica 8 statistical program was used to examine differences in the measured characteristics between treated and untreated tomato plants and their interaction, using the student’s ttest of significance level 0.05. the results were presented in a graph and table. results the yield of treated plants averaged 3,535.65 g per plant and in the control plants averaged 2,690.87 g (fig. 1). fruit mass is a genotype characteristic and is one of the factors that determine its purpose. analysis of variance (anova) for fruit yield (grams per plant) indicated a statistically significant difference between plants treated with retorted beech charcoal and non-treated plants as well as their interactions. specifically, it was found that the plants from the treatment had a statistically significantly higher fruit yield (in grams per plant) than the plants from the control. it was also found that there was a statistically significant difference between the total fruit yield and the number of fruits on the tomato plants under treatment compared to the control plants (fig. 2 and 3, tab. 1 and 2). treatment and control varied, both in total fruit table 1. t-test for dependent samples marked differences are significant at p<0.05 for fruit yield and fruit size (in grams per plant) t-test for dependent samples marked differences are significant at p<0,05000 variable mean std. dv. n diff. std. dv. diff. t df p test 242,6221 38,4676 test 242,6221 38,4676 172 -0,0000 0,00000 0,00000 171 1,00000 test 239,1909 36,8420 cont. 316,2818 123,1446 110 -77,0909 120,4985 -6,70993 109 0,00000 cont. 316,2818 123,1446 test 239,1909 36,8420 110 77,0909 120,4985 6,70993 109 0,00000 cont. 310,4830 118,5543 cont. 310,4830 118,5543 147 -0,0000 0,00000 0,00000 146 1,00000 * the statistically significant differences in treatment and control are indicated in red. fig. 1. effect of treatment and control on yield of the friut (grams per plant) fig. 2. total fruit yield (grams) of treated and untreated tomato plants with retorted beech charcoal biologica nyssana ● 11 (1) september 2020: 59-64 živković et al. ● yield of the tomato farmed by organic principles in the greenhouse with the application of retort beach charcoal 62 yield per plant, harvest time and fruit yield over time. in plants treated with charcoal the first fruits were harvested on june 24, 2019 and the first harvest for plants in control was on july 7, 2019. it was also observed that the plants in the treatment had more even distribution of fruits for harvest than the plants in the control. plants treated with activated charcoal began to bear fruits earlier than plants in control. it was also found that the plants under treatment had a larger number of smaller and uniform fruit sizes compared to control plants that had a smaller number of larger fruits, of unequal size (tab. 1, fig. 3). the differences in yield between treatment and control within each individual harvest were statistically significant, with the smallest difference within the last harvest. on average, in treatment and control, the highest yield was recorded at the last harvest (tab. 1 and 2). the total fruit yield for the tomato plants under treatment was 81,320 g and for the control plants 61,890 g. we also monitored the dimension of the fruits and after statistical data processing it was observed that the fruits from the treatment were smaller than the fruits from the control (tab. 1, fig. 4). discussion tomato yield is positively correlated with the number of fruits per plant and the weight of the fruit (popović et al., 2015). the yield of the treated and control plants ranged from 3,535.62 g/plant and 2,690.87 g/plant, respectively (tab. 1). today, there are genotypes of large (120 g 250 g), medium (80 g 120 g), small fruits (60 g 80 g), and more recently genotypes of cocktail type (30 g 50 g) and mini (cherry) tomatoes (10 g 30 g) (đurovka et al., 2006). optimal temperatures and brightness in the early stages of development determine the yield and quality of the fruit (rylski et al., 1994). in this study, it was found that plants treated with beech retort charcoal had a higher total fruit yield per plant as well as compared to control plants. thus, for the plants from the treatment the first harvest was already on june 24, while for the plants from the control the first harvest was on july 7, 2019. fruit yield is conditioned primarily by genetic polygenic factors, but is also dependent on the external environment (zhuchenko, 1973). it is clear from this study that beech retort charcoal has a positive effect on tomato yield in the greenhouse. by growing tomatoes on five or six floors or growing on two trees, the yields would be higher by about 30-35%. there are no data in the literature on the effect of retorted beech charcoal on tomato yield. positive crop and biomass yield was found for biochar produced from wood, paper pulp, wood chips and poultry litter. yadav et al. (2018) reviewed published data from 59 pot experiments and 57 field experiments from 21 countries and found crop productivity increased by 11% on average. also, yadav et al. (2018) found benefits at field application rates typically below 30 t ha-1 field application and biologica nyssana ● 11 (1) september 2020: 59-64 živković et al. ● yield of the tomato farmed by organic principles in the greenhouse with the application of retort beach charcoal fig. 3. effect of treatment and control on total number of fruits fig. 4. effect of treatment and control on the dimension of tomato fruit effect degr. of freedom test test test test control control control control intercept 1 6293352 6293352 4636,567 0,00 11003761 11003761 725,6221 0,00 error 109 147949 1357 1652940 15165 total 109 147949 1652940 table 2. analysis of variance (anova) for fruit yield (in grams per plant) reported that increases in crop productivity varied with crop type with greater increases for legume crops (30%), vegetables (29%), and grasses (14%) compared to cereal crops corn (8%), wheat (11%), and rice (7%). these data are consistent with the results of this paper. according to yamato et al. (2006) maize production was significantly increased after the application of bark charcoal under a fertilized condition in an infertile soil environment. a positive effect of biochar addition on maize dry biomass could be ascribed to higher soil n-retention that was observed by baronti et al. (2010). these are only preliminary results. further detailed investigations should be undertaken in order to find the most optimal amount and time of application of beech retort charcoal in crops under the climatic conditions of serbia. conclusion the highest fruit yield was achieved on activated carbon plants, while the yield on control plants was 23.88% lower. this study indicates the positive impact of retorted beech charcoal on the yield of tomato plants in the greenhouse. references baronti, s., alberti, g., vedove, g.d., gennaro, f.d., fellet, g., genesio, l, miglietta, f., peressotti, a., vaccari, f.p. 2010: the biochar option to improve plant yields: first results from some field and pot experiments in italy, italian journal of agronomy. 5: 3–12. biederman, l.a., harpole, w.s. 2013: biochar and its effects on plant productivity and nutrient cycling: a meta-analysis, gcb bioenergy, 5: 202– 214. chan, k.y., van zwieten, l., meszaros, i, downie, a., joseph, s. 2007: agronomic values of greenwaste biochar as a soil amendment, australian journal of soil research, 45: 629–634. de luca, t.h., mac kenzie, m.d., gundale, m.j. 2009: biochar effects on soil nutrient transformation. in: j lehmann, s joseph (ed.), biochar for environmental management science and technology. earthscan, london, pp 251–280. de sousa, a.s., borges, s.v., magalhaes, n.f., ricardo, h.v., azevedo, a. d. (2008): spraydried tomato powder: reconstitution properties and colour. brazilian archives of biology and technology, 51(4): 807-814. đurovka, m., lazić, b., bajkin, a., potkonjak, a., marković, v., ilin, ž., todorović, v. 2006: proizvodnja povrća i cveća u zaštićenom prostoru. univerzitet u novom sadu. poljoprivredni fakultet, novi sad, 207. hagemann, n., kurt spokas, k.., schmidt, h.p., kägi, r., böhler, m.a. and thomas, d. bucheli, t.d. 2018: activated carbon, biochar and charcoal: linkages and synergies across pyrogenic carbon’s abcs. water, 10, 182. http://webrzs.stat.gov.rs (pristupljeno novembra 2019.). http://faostat.fao.org h t t p : / / w w w. w p t c . t o / r e l e a s e s w p t c . p h p hwang, e.s. and bowen, p.e. 2005: effects of tomato paste extracts on cell proliferation, cellcycle arrest and apoptosis in lncap human prostate cancer cells. biofactors, 23: 75-84. laird, d. 2008: the charcoal vision: a winwin-win scenario for simultaneously producing bioenergy, permanently sequestering carbon, while improving soil and water quality. agronomy journal,100: 178–181. lehmann, j., gaunt, j., rondon, m. 2005: biochar sequestration in terrestrial ecosystems–a review. mitigation and adaptation strategies for global change, 11: 403–427. mann, c.c. 2005: new revelations of the americas before columbus. vintage and anchor books, new york pp. 1–576. vlahović, b., puškarić, a. 2012: obeležja potrošnje povrća u republici srbiji. xvii savetovanje o biotehnologiji. zbornik radova, 17. univerzitet u kragujevcu. poljoprivredni fakultet, čačak, 125129. palozza, p., simone, r.e., catalano, a., mele, m.c. 2011: tomato lycopene and lung cancer prevention: from experimental to human studies. cancers, 3: 2333-2357. popović, v., takač, a., glogovac, s., medić pap, s., červenski, j. 2015: prinos semena i ploda kod indeterminantnih genotipova paradajza gajenih na četiri etaže. selekcija i semenarstvo, 21(1): 43-56. rylski, i., aloni, b., karni, l., zaidman, z. 1994: flowering, fruit set, fruit development and fruit quality under different environmental conditions in tomato and pepper crops. acta horticulturae, 366: 45-56. tian, x., li, c., zhang, m., wan, y., xie, z., chen, b. 2018: biochar derived from corn straw affected availability and distribution of soil nutrients and cotton yield. plos one 13(1): e0189924. biologica nyssana ● 11 (1) september 2020: 59-64 živković et al. ● yield of the tomato farmed by organic principles in the greenhouse with the application of retort beach charcoal vasić, m. 2016: sorte pasulјa u različitim sistemima gajenja. 50. savetovanje agronoma i poljoprivrednika, 24.01. – 30.01.2016., zlatibor. institut za ratarstvo i povrtarstvo, novi sad. woolf, d., amonette, j.e., street-perrott, f.a., lehmann, j., joseph, s. 2010: sustainable biochar to mitigate global climate change. nature communications, 1: 56. yadav, n.k., vijay, k., sharma, k.r., choudhary, r.s., butter, t.s., singh, g., kumar, m., kumar, r. 2018: biochar and their impacts on soil properties and crop productivity: a review. journal of pharmacognosy and phytochemistry, 7(4): 49-54. yamato, m., okimori, y., wibowo, i.f., anshori, s., ogawa, m. 2006: effects of the application of charred bark in acacia mangium on the yield of 64 biologica nyssana ● 11 (1) september 2020: 59-64 živković et al. ● yield of the tomato farmed by organic principles in the greenhouse with the application of retort beach charcoal maize, cowpea, peanut and soil chemical properties in south sumatra, indonesia. soil science and plant nutrition, 52: 489–495. yang, t., yang, x., wang, x., wang, y., song, z. 2013: the role of tomato products and lycopene in the prevention of gastric cancer: a meta-analysis of epidemiologic studies. medical hypotheses, 80: 383-388. žučenko, a.a. 1973: genetika tomatov. štiinca. kišinev. šeremešić, s., živanov, m., milošev, d., vasin, j., ćirić, v., vasiljević, m., ujić, n. 2015: effects of biochar application on morphological traits in maize and soybean. zbornik matice srpske za prirodne nauke, n 129: 17-25. anticancer compounds from medicinal plants biologica nyssana 3 (2)  december 2012: 53-60 mitrović, t. et al.  bioindication of heavy metal pollution … 53 original article bioindication of heavy metal pollution in the area of southeastern serbia by using epiphytic lichen flavoparmelia caperata (l.) hale tatjana mitrović 1 , slaviša stamenković 1 , vladimir cvetković 1 *, tatjana đekić 2 , rada baošić 3 , jelena mutić 3 , tatjana anđelković 4 , aleksandar bojić 4 1 university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia 2 university of niš, faculty of sciences and mathematics, department of geography, višegradska 33, 18000 niš, serbia 3 university of belgrade, faculty of chemistry, studentski trg 12-16, 11158 belgrade, serbia 4 university of niš, faculty of sciences and mathematics, department of chemistry, višegradska 33, 18000 niš, serbia * e-mail: biovlada@yahoo.com abstract: mitrović, t., stamenković, s., cvetković, v., đekić, t., baošić, r., mutić, j., anđelković, t., bojić, a.: bioindication of heavy metal pollution in the area of southeastern serbia by using epiphytic lichen flavoparmelia caperata (l.) hale. biologica nyssana, 3 (2), december 2012: 53-60. the content and distribution of 21 metals in the central and peripheral parts of the foliose epiphytic lichen flavoparmelia caperata (l.) hale, collected in the area of southeastern serbia, were analysed in terms of biological monitoring. inductively coupled plasma atomic emission spectrometry revealed higher concentrations of as, b, ba, cd, ga, pb, se, cr, cu, fe, in, li and/or ni in peripheral, younger, parts and ba, k, tl, mg, na and/or zn in central, older parts of lichens. principal component analysis and hierarchical cluster analysis were used to identify the relationship among metals in samples and their possible sources. significant correlations were found among ni-cr, cd-ga-in-as-se, zn-ba, cu-pb-b, suggesting a common source of pollution. given the location of sampling, these findings probably reflect airborne metal pollution in relation to the main wind directions and vicinity of the roads and industrial complexes. the importance of this study is the evidence that the special nature reserve jelašnička gorge is influenced by pollution sources in the area. flavoparmelia caperata could be effective as an early indicator of environmental changes of the studied area. key words: air pollution, bioindication, flavoparmelia caperata, heavy metals, lichens introduction lichens have been the most widely accepted sensors of enviromental pollution since the 19th century (g r i n d o n , 1859). lack of waxy cuticle and associated stomatas, parenchymal nature, slow growth and long duration of lichens result in the absorption of pollutants (metals, nonmetals, radionuclides, etc) across entire thallus surface and thus allow long-term biomonitoring of persistent pollutants in the ecosystem. lichens depend on mineral nutrients in the form of soluble salts and particles from wet atmospheric deposition (precipitation and occult precipitation, like fog and dew) and dry atmospheric deposition (sedimentation, impaction and gaseous absorption). they are very efficient accumulators of heavy metals by: ion exchange at specific cation exchange 3 (2) • december 2012: 53-60 biologica nyssana 3 (2)  december 2012: 53-60 mitrović, t. et al.  bioindication of heavy metal pollution … 54 sites, intracellular uptake of metal solution, and entrapment of airborne, metal-rich particles (size 0.5 to 1.0 μm) (n a s h i i i , 2008). lichens are capable of accumulating heavy metals to a concentration that vastly exceeds their physiological requirements by sequestering them extracellulary as insoluble oxalates or lichen acid complexes (b e e b y , 2001). the presence of particulate materials on the lichen surface which are trapped between the hyphae of medulla is shown by electron microprobe studies (b a r g a g l i , 1998). metal accumulation is in correlation with the environmental levels of particulate materials (b a r i et al., 2001; b a r g a g l i et al., 2002 ; l o p p i et al., 2004) and spatialand/or temporaldeposition patterns of trace metals are demonstrated (z s c h a u et al., 2003). the concentration of sequestered trace metals in lichen really reflects ambient level of airborn trace metals in terms that after the withdrawal of the source of contamination from the ecosystem (closures of industries, iron-steel factories, mines, waste treatment plants, roads and railroads, etc) a dramatic decrease of trace metals concentration in lichen thalli is observed (w a l t h e r et al., 1990). n i m i s et al. (2001) discussed the importance of intraand interspecific variability in metal accumulation and relationship between metal content and the age of the thalli. in this study we investigated heavy metal pollution using flavoparmelia caperata (l.) hale as bioindicator and bioaccumulator. the study was performed at 3 different locations: northwest (near the village of cerje), southeast (near the village of vlase) and northeast (jelašnička gorge) of the biggest urban and industrial center of southeastern serbia niš (approximately 350 000 inhabitants). we would like to emphasize the importance of one location jelašnička gorge. this area, situated 15 km away from niš and 3 km away from niška spa, is a protected natural area having the status of a special nature reserve. one of the reasons to investigate this part of serbia is the fact that a similar study had never been undertaken previously, although the area is densely populated with large urban-industrial infrastructure, which thus has an important anthropogenic impact on surrounding nature and ecosystems. lichen samples were analyzed for metal content. composition data have been treated with figure 1. study area (jelašnica gorge, vlase and cerje in respect to the city of niš) withwind roses for the area of niš (data from republic hydrometeorological service of serbia) biologica nyssana 3 (2)  december 2012: 53-60 mitrović, t. et al.  bioindication of heavy metal pollution … 55 various statistical multivariate techniques, primarily principal component analysis and cluster analysis. material and methods study area the study was performed in the subrural and rural area of southeastern serbia. the collection sites were: jelašnička gorge (330 m altitude), vlase (350 m altitude) and cerje (600 m altitude) in the vicinity of roadside (2m, 500 m and 2000 m, respectively) (figure 1). the nearest urban and industrial area is the city of niš (approximately 350 000 inhabitants) (figure 1). the climate is moderate continental with mean annual rainfall ranging over 543.3 mm, a mean temperature of 11.5 °c, and a mean annual relative humidity of approximately 69 %. prevailing winds are northwesterly in winter and northeasterly and easterly in summer. wind roses are shown in figure 1. lichen material foliose lichen flavoparmelia caperata (l.) hale (syn. parmelia caperata (l.) ach.; common name: greenshield lichen) was collected in april 2009. lichen specimens were obtained from a height of 1.5-2 m above the ground, at the side of trunks of prunus domestica and salix sp. not affected by stemflow. the material from each location was sorted into two samples corresponding to peripheral and central parts of lichen. the samples were air-dried, grinded, homogenized and further analyzed. the determination of lichens was performed by using several standard keys (boqueras, 2000; dobson, 2005; wirth, 1995). lichen samples were deposited in the lichenological herbarium of the department of biology and ecology, faculty of sciences and mathematics, university of niš. reagents and chemicals all chemicals were of analytical grade and were supplied by merck (darmstadt, germany). all glassware was soaked in 4 mol/l hno3 for a minimum of 12 hours and rinsed well with distilled water. ultra-pure water was prepared by passing doubly de-ionized water through milli-q system (millipore simplicity 185 system incorporating dual uv filters (185 and 254 nm) to remove carbon contamination). multi-element stock solution containing 1.000 g/l of each elements was used to prepare intermediate multi-element standard solutions. sodium borhydride solutions were stabilized with sodium hydroxide (merck, germany) for hydride generation. table 1. instrument operating conditions for determination of heavy metals in lichen samples spectrometar icap 6500 (thermo scientific) nebulizer concentric spray chamber cyclonic radio frequency power (w) 1150 principal argon flow rate (l/min) 12 auxiliary argon flow rate (l/min) 0.5 nebulizer flow rate (l/min) 0.5 sample flow rate (ml/min) 1.0 detector cid86 table 2. selected emission lines element λ [nm] ag 328.0 as 193.7 b 249.6 ba 455.4 cd 228.8 co 228.6 cr 283.5 cu 324.7 fe 259.9 ga 294.3 in 230.6 k 766.4 li 670.7 mg 280.2 mn 257.8 na 588.9 ni 231.6 pb 220.3 sr 215.0 tl 276.7 zn 213.8 hg 253.6 se 196.0 instrumentation all the measurements were made with a inductively coupled atomic emission spectrometer model 6500 duo (thermo scientific, united kingdom) equipped with a cid86 chip detector. the system is equipped with an integrated unit for biologica nyssana 3 (2)  december 2012: 53-60 mitrović, t. et al.  bioindication of heavy metal pollution … 56 hydride generation. this instrument operates sequentially with both radial and axial torch configurations. the entire system is controlled by iteva software. instrument operating conditions for the determination of heavy metals in lichen samples and selected emission lines are shown in table 1 and table 2, respectively. microwave digestion was performed in a pressurized microwave (ethos 1, advanced microwave digestion system, milestone, italy) equipped with a rotor holding 10 polytetrafluoroethylene (ptfe) cuvettes. procedure for microwave digestion samples (0.5 g) were transfered into ptfe cuvette, 7 ml of concentrated hno3 and 1 ml 30% h2o2 were added. digestion was performed under the following programme: warmed up for 10 mins to 200° c and held for 10 mins at that temperature. after the cool off period samples were quantitatively transferred into a volumetric flask (25 ml). statistical analysis to identify the relationship among metals in samples and their possible sources, pearson’s correlation coefficient analysis, principal component analysis (pca) and cluster analysis (ca) were performed using pls toolbox version 5.2.2 (eigenvector research) for the matlab version 7.4.0.287 (r2007a) (mathworks, natick, ma, usa). the principal component analysis was performed using varimax normalized rotation. cluster analysis was performed on the data sets using the between-groups linkage based on correlation coefficients (pearson coefficient) by pair-wise deletion. normalized data set was analyzed by ward’s method using squared euclidean distances as a measure of similarity between metal concentrations. results and discussion the concentrations of 21 metals in lichen flavoparmelia caperata, collected at 3 different localities in southeastern serbia (jelašnička gorge, cerje and vlase), are given in table 3. the determination of metals was performed on both central and peripheral parts of lichens. central parts are inner, older parts of thalli and they have therefore been exposed to pollutants longer. peripheral parts comprise outmost 3-4 mm of the thalli, with highest physiological activity and the maximum age of 1 year (fisher & proctor, 1978; loppi et al., 1997). thus, peripheral parts represent recent changes of the environment. our data indicated differences in the metal content of central and peripheral parts of lichens. central parts tend to give higher concentrations of a number of metals as table 3. concentration of metals in central and peripheral parts of the thalli of flavoparmelia caperata ( g/g dry wt.) elements flavoparmelia caperata from jelašnička gorge flavoparmelia caperata from cerje flavoparmelia caperata from vlase center (sample 1) perifery (sample 2) center (sample 3) perifery (sample 4) center (sample 5) perifery (sample 6) as 0.0037 0.0038 0.0036 0.0033 0.0031 0.0027 b 19.4303 9.5051 6.1341 6.9682 5.8576 6.7637 ba 27.2333 21.5575 9.5615 24.5084 13.8145 15.2969 cd 0.2558 0.1942 0.1681 0.1864 0.1310 0.1546 co 0.1512 0.0822 1.0894 2.1865 0.0970 0.0856 cr 1.9119 1.4572 3.7140 1.4670 1.7550 1.4879 cu 8.3806 5.9573 15.3721 5.7307 6.1412 6.5179 fe 476.6209 374.5863 645.2168 397.9597 493.2096 399.2086 ga 0.1069 0.0000 0.0413 0.0000 0.0346 0.0177 in 0.0000 0.0000 0.0802 0.0341 0.0810 0.0000 k 2608.8040 2852.2720 2300.1670 2706.2540 2506.7160 3156.7050 li 0.6932 0.5035 0.7566 0.4607 0.5617 0.4582 mg 251.8673 315.6654 274.1127 313.7536 287.8818 373.2304 mn 14.1271 13.5242 13.6761 12.4049 13.9091 15.6287 na 71.1085 98.1346 89.5684 103.6953 78.8976 110.4994 ni 1.2703 1.0977 1.5682 1.1916 1.4227 1.3011 pb 22.3011 10.0742 12.0318 7.3955 9.8771 7.4223 se 0.0043 0.0045 0.0042 0.0039 0.0036 0.0031 sr 31.4610 23.8918 16.8445 11.3996 40.6104 32.6951 tl 0.0000 0.6093 0.0000 0.9164 0.0000 1.5415 zn 17.6053 17.0019 18.0832 19.5534 20.7641 24.2578 biologica nyssana 3 (2)  december 2012: 53-60 mitrović, t. et al.  bioindication of heavy metal pollution … 57 previously reported in the studies of nimis et al. (2001). pearson’s correlation coefficients for metals in lichen samples are shown in table 4. determined correlations between metals provided interesting information on the sources and pathways of the metals. the correlation matrix was created from the values of the variables of all 21 metals in 6 samples. the pair-wise method was employed for the missing values. the results showed that these metals were strongly interrelated (p<0.01), with correction coefficients ranging from -0.790 to 0.990 at the 99% confidence level. b, cd, ga and pb evidently displayed significant positive correlations with each other (see: table 3), which indicates their association in analyzed samples. other metals, like cr, cu, fe, li and ni, showed significant correlations, too. the exceptions were elemental pairs as-zn and se-zn with significant negative correlations and as, mn and zn without any correlations observed. in order to better describe the relationship among metals and/or samples, principal component analysis (pca) was performed. analytical data were represented in a multidimensional space with variables defining the axes, and projected into a few principal components (pcs) that were linear combinations of the original variables and described the maximum variation within the data (brereton, 2003). obtained results clearly showed that the co, mn and sr had no impact. this was in accordance with results of metals determination (table 3) due to small differences in theirs values in different samples. therefore, pca analysis was done without these metals resulting in a three-component model explaining 91.78% of the data variation. the first pc comprised 49.75% of the total data variability, and the cumulative variance explained by the first two components was 81.09%. the addition of more pcs did not significantly change the classification of the analytes described below. score values for the samples, i.e. their mutual projections, for the first two pcs are shown in figure 2. additionally, score values for the samples and metals, for the first two pcs are shown in figure 3. the first pc distinguished two separate groups of samples according to metals content and age of parts of the lichen thalli sampled. samples 1, 3 and 5 are central (older) parts of the thalli from different areas while 2, 4 and 6 are peripheral (younger) parts of the thalli. the first pc distinguished two separate groups with characteristic patterns of metals accumulation (samples 2, 4 and 6 with ba, k, mg, na, tl and zn figure 2. score values of the first and the second pcs for the samples additionally, score values for the samples and metals, for the first two pcs are shown in figure 3. figure 3. biplot of the first and the second pcs for the samples and metals and samples 1, 3 and 5 with as, b, cd, cr, cu, fe, ga, in, li, ni, pb and se). these results clearly defined the highest accumulated metals characteristic for every sample. the following metals were characteristic of sample 1: as, b, cd, ga, pb and se. higher concentration of ba, k and tl were noticed in samples 2 and 4. besides, ba was located on the border that defined a pc and it appeared characteristic of sample 1, too. the accumulation of cr, cu, fe, in, li and ni were characteristic for samples 3 and 5. increased concentrations of mg, na and zn were observed in sample 6. compared to location of sampling, it biologica nyssana 3 (2)  december 2012: 53-60 mitrović, t. et al.  bioindication of heavy metal pollution … 58 biologica nyssana 3 (2)  december 2012: 53-60 mitrović, t. et al.  bioindication of heavy metal pollution … 59 seems that the special nature reserve jelašnička gorge from which sample 1 and 2 originated, is becoming polluted due to vicinity of the road (2m) and towns (niš and niška spa, 15 km and 3 km, respectively). evidence for this could be found in the increased concentration of cd, pb and se as the levels of these elements are similar to literature values for high-density traffic areas (mendil et al., 2009). the other two locations of lichen sampling, cerje and vlase, with samples 3-6, showed richness in litophile elements and macronutrients such as: cr, cu, fe, in, k, li, mg and na. this could imply a specific composition of geological substrate, as well as the influence of vegetation, i.e. substrate from which lichen samples were collected. moreover, there is a certain amount of anthropogenic pressure and it is manifested by pollution originating from traffic origin. finally, metal concentration data were submitted to cluster analysis (ca) using correlation coefficient as similarity measure and weighted pair group as clustering algorithm. this method is the most appropriate for the validation of the correlation between variables and is often used in environmental studies. the obtained dendrogram is shown in figure 4. figure 4. dendrogram derived from the hierarchical cluster analysis of samples nevertheless the cluster analysis organized the metals (without macroconstituents) in such a way that within-group similarity was maximized and among-group similarity was minimized. the ca results for the heavy metals studied are shown in figure 5 as a dendrogram. figure 5 displays four clusters: (1) ni-cr; (2) cd-ga-in-as-se; (3) zn-ba; (4) cu-pb-b in agreement with the pca results. it is observed, however, that clusters 1 and 2 as well as clusters 3 and 4 join together at a relatively higher level, possibly implying a common source. fossil fuel emits ni, cd, cr, cu, pb, zn during combustion (aslan et al., 2011). besides, the wear of car tires, degradation of parts, peeling paints and greases and metals in catalysts could be sources of pollutants (pecheyran et al., 2000). although vehicles with unleaded petrol prevail, the high density traffic of old vehicles using leaded petrol and diesel oil is still present in serbian roads. zn, as well as cd, is well known as an atmophile element subject to longdistance transport (loppi & pirintsos, 2003). a part of zn could be obtained from supporting trees since higher plants are known to release 20 % of total zn coming from natural sources (nriagu, 1979). also, flavoparmelia caperata is known for its higher capacity for zn and cd uptake (nimis et al., 2001). figure 5. dendrogram derived from the hierarchical cluster analysis of metals content interestingly, cluster 4 indicates pollution caused by exhaust gases due to traffic, but having in mind the poor frequency of traffic at the roadsides in close range of the location of lichen sampling, one could wonder about the origin of cu, pb and b detected. this is probably due to the specific wind roses (see figure 1) which put this area under the indirect impact of metal industry, situated 10-15 km northwesterly (in the outskirt of the city of niš). further research and monitoring should be performed in order to obtain the right conclusions. conclusion our study confirms the metal accumulation capacity of lichen flavoparmelia caperata and its potential for biomonitoring. this lichen species could be effective as an early warning system for detecting changes in the environment. peripheral parts of lichen samples allow annual changes to be detected. detected accumulation of heavy metals in lichen samples is probably a result of the frequency of traffic and types of engines on the roads in this biologica nyssana 3 (2)  december 2012: 53-60 mitrović, t. et al.  bioindication of heavy metal pollution … 60 area, the activity of industrial complexes in and around the city of niš, soil and substrate compositions and predominant wind directions. further research and biomonitoring surveys of air pollution in the studied area should contribute to the amelioration of air quality and environment in southeastern serbia. acknowledgment. this research was supported by the ministry of science and education of the republic serbia during activities on the projects iii41018, oi 171025 and tr 34008. references aslan, a., çiçek, a., yazici, k., karagöz, y., turan, m., akku, f., yildirim, o.s. 2011: the assessment of lichens as bioindicator of heavy metal pollution from motor vehicles activites. african journal of agricultural research, 6 (7): 1698-1706. bargagli, r. 1998: trace elements in terrestrial plants: an ecophysiological approach to biomonitoring and biorecovery. springer verlag. berlin. bargagli, r., monaci, f., borghini, f., bravi, f., agnorelli, c. 2002: mosses and lichens as biomonitors of trace metals. a comparison study on hypnum cupressiforme and parmelia caperata in a former mining district in italy. environmental pollution, 116 (2): 279-287. bari, a., rosso, a., minciardi, m.r., troiani, f., piervittori, r. 2001: analysis of heavy metals in atmospheric particulates in relation to their bioaccumulation in explanted pseudevernia furfuracea thalli. environmental monitoring and assessment, 69 (3): 205-220. beeby, a. 2001: what do sentinels stand for? environmental pollution, 112 (2): 285-298. boqueras, m. 2000: líquens epífits i fongs liquenícoles del sud de catalunya: flora i comunitats. institut d'estudis catalans. barcelona. brereton, r.g. 2003: chemometrics: data analysis for the laboratory and chemical plant. john wiley & sons inc. chichester. dobson, f.s. 2005: lichens. the richmond publishing co. ltd. richmond. fisher, p., proctor, m. 1978: observations on a season's growth in parmelia caperata and p. sulcata in south devon. the lichenologist, 10 (01): 81-89. grindon, l.h. 1859: the manchester flora. white. london. loppi, s., pirintsos, s.a. 2003: epiphytic lichens as sentinels for heavy metal pollution at forest ecosystems (central italy). environmental pollution, 121 (3): 327-332. loppi, s., frati, l., paoli, l., bigagli, v., rossetti, c., bruscoli, c., corsini, a. 2004: biodiversity of epiphytic lichens and heavy metal contents of flavoparmelia caperata thalli as indicators of temporal variations of air pollution in the town of montecatini terme (central italy). science of the total environment, 326 (1-3): 113-122. loppi, s., nelli, l., ancora, s., bargagli, r. 1997: accumulation of trace elements in the peripheral and central parts of a foliose lichen thallus. bryologist, 100 (2): 251-253. mendil, d., çelik, f., tuzen, m., soylak, m. 2009: assessment of trace metal levels in some moss and lichen samples collected from near the motorway in turkey. journal of hazardous materials, 166 (2-3): 1344-1350. nash iii, t.h. 2008: lichen biology. cambridge university press. cambridge. nimis, p., andreussi, s., pittao, e. 2001: the performance of two lichen species as bioaccumulators of trace metals. science of the total environment, 275 (1-3): 43-51. nriagu, j.o. 1979: global inventory of natural and anthropogenic emissions of trace metals to the atmosphere. nature, 279: 409-411. pécheyran, c., lalère, b., donard, o.f.x. 2000: volatile metal and metalloid species (pb, hg, se) in a european urban atmosphere (bordeaux, france). environmental science & technology, 34 (1): 27-32. walther, d.a., ramelow, g.j., beck, j.n., young, j.c., callahan, j.d., marcon, m.f. 1990: temporal changes in metal levels of the lichens parmatrema praesoredious and ramalina stenospora southwest louisiana. water, air and soil pollution, 53: 189-200. wirth, v. 1995: die flechten baden-württembergs, teil 1 & 2. eugen ulmer & co. gmbh. stuttgart. zschau, t., getty, s., gries, c., ameron, y., zambrano, a., nash, t. 2003: historical and current atmospheric deposition to the epilithic lichen xanthoparmelia in maricopa county, arizona. environmental pollution, 125 (1): 2130. rajković, vračarić 2020, biologica nyssana 11(2) 11 (2) december 2020: 103-107 doi: 10.5281/zenodo.4393959 breeding range extension of the peregrine falcon (falco peregrinus l.) in the pannonian plain original article draženko rajković šumadijska 18/30, 21000 novi sad, seerbia strix.draze@gmail.com (corresponding author) miroslav vračarić novi sad, serbia miroslav.vracaric@gmail.com received: may 21, 2020 revised: september 18, 2020 accepted: november 30, 2020 abstract: during three consecutive winter/spring seasons in the period 2018-2020, the breeding territory of peregrine falcon was monitored at an abandoned quarry on fruška gora mt., (utm dr 00; 45.165527 n, 19.803396 e) at vojvodina province, northern serbia. described record represents the first confirmed nesting of peregrine falcon in this part of the country and provides evidence of the further expansion of breeding distribution range of this species in the pannonian plain. key words: falco peregrinus, fruška gora, nesting, peregrine falcon, vojvodina province apstract: širenje areala gnežđenja sivog sokola (falco peregrimus l.) u panonskoj niziji u periodu zima/proleće tokom tri uzastopne godine (2018-2020), praćeno je gnežđenje sivog sokola u napuštenom kamenolomu na fruškoj gori (utm dr 00; 45.165527 n, 19.803396 e) u vojvodini (severna srbija). opisani nalaz predstavlja prvi potvrđen slučaj gnežđenja sivog sokola u ovom delu zemlje koji pripada panonskoj niziji, a koji ukazuje na dalje širenje gnezdećeg areala ove vrste. ključne reči: falco peregrinus, fruška gora, sivi soko, vojvodina, gnežđenje introduction the peregrine falcon (falco peregrinus) has a widespread, nearly a cosmopolitan distribution, except extreme polar and tropical regions including new zealand as well as high mountain ranges and many oceanic islands (cade, 1982; ferguson-lees & christie, 2001; white et al., 2013). depending on geographical location and environmental conditions, it establishes their eyries in a variety type of habitats including cliffs, rocky outcrops, trees, ground mounds and, in a recent time artificial structures (cade, 1982; ratcliffe, 1993; banks et al., 2003). since the dramatic decline after world war ii caused by the widespread use of organochlorine pesticides, its populations has recovered in most parts of former distribution range (white et al., 2002; ratcliffe, 1993; rizzolli et al., 2005; prommer & bagyura, 2018). the current european nesting population is composed of three subspecies: f. p. calidus in remote tundra areas of north-eastern europe, f. p. peregrinus in most parts of the continent, including the british islands and f. p. brookei situated mostly in the mediterranean, including interior balkan peninsula (white, 1994; ferguson-lees & christie, 2001). however, there were no clear boundaries between f. p. peregrinus and f. p. brookei and in some areas, hybridisation could occur (zuberogoitia © 2020 rajković, vračarić. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 103 et al., 2009; wink, 2018). according to the most recent data, the european population extent lies between 14,900 and 28,800 breeding pairs with an increasing trend (birdlife international, 2015). in serbia, during the first half of the 20th century, peregrine falcon sporadically bred in the rocky areas, mainly in the western part of the country (matvejev, 1950, 1964). the similar situation persisted during the rest of 20th century with slight changes in population abundance and density (marinković & grubač, 2000; puzović et al., 2003; grubač, 2018). historically, in northern serbia (vojvodina province), there are no evidence-based breeding records, although few authors reported occasional breeding in low numbers (marčetić & medaković, 1954; marčetić, 1957; antal et al., 1971), but with insufficient information and lack of irrefutable proofs (pelle et al., 1977; mikuska, 1982; pelle, 1990; šćiban et al., 2015; grubač, 2018). in the recent period, peregrine falcon breeds in serbia only in hilly and mountain regions with cliffs and gorges south of danube and sava rivers (grubač, 2018). the population is estimated to 51-63 breeding pairs with a moderate decline (puzović et al., 2015). results since 2010, we have been visiting central and eastern parts of fruška gora mt. near novi sad (vojvodina province, northern serbia) for birds mapping. on 4th march 2019, central parts of fruška gora mt. was surveyed, more precisely, the area between main ridge road (partizanski put) and stari ledinci village. near one forest clearing, in close vicinity of abandoned trachyte quarry “srebro” (utm dr 00; 45.165527 n, 19.803396 e) one specimen of peregrine falcon, was unintentionally disturbed by our appearance. the peregrine perched on the lateral branch of european beech (fagus sylvatica) close to the quarry edge and feeding on a feral pigeon (columba livia f. domestica). the abandoned trachyte quarry “srebro” is situated around 1 km south from village stari ledinci (petrovaradin municipality) in the territory of national park “fruška gora” at approximately 274 m above sea level. the quarry has an erratic shape; most resemble an ellipse with approximate dimensions 450 × 140 m. the height of the vertical cliffs varies from 15 to 80 m. deep-water cover the bottom of the quarry, forming the ledinačko lake (fig. 1). the observed individual had a dark cap, whitish breast and bluishgrey mantle, which are characteristics of an adult bird. shortly after, another larger individual of peregrine falcon was repeatedly calling from the opposite, south-faced cliff. dark brown plumage and profoundly marked breast were showed that this specimen was in their second year. the bird was sitting on the protruding rock in the middle of the cliff, around 40 m high from the water surface. the observed peregrines were probably a pair in suitable habitat within the breeding season. thus, in the following weeks, the pair was monitored using a spotting scope and binoculars. these visits were conducted every 3 to 10 days, usually in the morning hours until the end of may. the observations have been obtained from a distance of 250-400 m away from peregrines to avoid disturbance. the pair spent most of their time on the western part of a quarry where the vertical cliffs are broadest and highest. both individuals were usually sitting and preening or occasionally aggressively chasing away the common buzzards (buteo buteo) and common ravens (corvus corax) from the vicinity of the quarry. between 8th and 17th march of 2018, a peregrine’s copulation was observed on nine occasions. the copulation took 8.5 sec on average (ranged 6-12 sec). the act of copulation in 66% of cases took place on the lateral branch of a partially dead, around 10 m tall tree, which grew out from one of the cracks between cliffs. during our visit on 10th april, the female of peregrine falcon was observed sitting low on a cliff shelf. therefore, we suspected that the female was incubating, although we did not observe eggs. at the same time, male patrolled in soaring flight above the quarry and defended eyrie from one specimen of common buzzard and two 104 fig. 1. trachyte quarry “srebro” near village stari ledinci – breeding eyrie of peregrine falcon (falco peregrinus). the yellow circle indicates the position of the nest from 2020. biologica nyssana ● 11 (2) december 2020: 103-107 rajković, vračarić ● breeding range extension of the peregrine falcon (falco peregrinus l.) in the pannonian plain 105 common ravens. the height of the cliff with potential nest ledge was around 60 m above the lake level. the scrape was approximately 20 m from the clifftop with estimated dimensions of 30 × 20 cm. the cliff with potential nest had a southwest aspect and with an angle of approximately 80°. during visits in april, it seemed that potential nesting attempt had failed apparently due to frequent human disturbance or less likely that no eggs were laid at all. however, the scrape was empty with only a few body feathers from peregrine’s female. in 2018, the pair of peregrine falcons was noticed the last time on 28th april. meanwhile, due to significant improvement of weather conditions from april onwards, approximate 50-100 visitors were regularly hiking, camping and even burning fire in the proximity of potential nesting scrape. as the peregrine falcon is a strictly protected species in serbia, the provincial institute for nature conservation was informed about the situation. unfortunately, no conservation measures have been made for the restriction of visitors since march of 2020. during two short visits in late may, the specimens of peregrine falcon were not observed inside the quarry or in its surroundings. during two our visits in march and three visits in april of 2019, only adult male was observed on the described location. the fate of the immature female remains unknown. proven eggs laying and incubation on the abandoned quarry finally happened in 2020. on 7th march, peregrines were sitting close to each other, watching the surrounding and periodically preening. both birds clearly showed adult morphological features. from time to time, they showed aggressive behaviour towards the pair of common raven who, in the end, left the quarry. later in the same morning, the male brought a small passerine bird as prey to the female. that was the start of courtship behaviour with many display flights, which continued at least 45 minutes and culminated in copulation on four occasions. copulations lasted from seven to 13 seconds and occurred on the tree at the western part of the quarry. shortly after that, the female took off from the copulation tree and landed on abandoned raven’s nest, after a circular flight in the northern slopes of the quarry. it was the first sign that a pair of peregrines had selected an old stick nest for laying eggs instead of cliff crevice or crag. the position of the nest was in the shade on the northern cliffface, below rock vault, around 22-25 m from water level. one week later, on 14th march, a female was observed on incubation posture at the same raven’s nest (fig. 2). when the female was briefly absent from the nest, it was possible to see at least two eggs inside the nest. hence, it was assumed that she laid the first egg between 8th and 10th march. on 17th april, the female was sitting on the old raven’s nest. however, she was not in such a low posture like at the beginning of the incubation period. therefore, we supposed that the chicks had already hatched, which was confirmed an hour later when the female shortly left the nest to pick up prey from the male. three chicks, 4-6 days old, were observed. accordingly, the incubation lasted approximately 29-31 days. after taking a prey (feral pigeon) female landed on the nest and fed the chicks and herself around 15 minutes. shortly after that, she continued brooding on puny chicks. at the same time, male spent most of its time outside of the quarry. however, between 25th april and 3rd may, the nesting attempt had failed, but reason stays unclear. the nest depression was empty, and no adult birds were observed. during the study period, specimens of peregrine falcon were observed hunting a different prey, mostly small or medium-sized birds. however, because of long-distance and unfavourable environmental conditions (fog and intense sunlight), the prey was rarely identified to the genus or species level. in three occasions that was a feral pigeon, in two occasions common blackbird (turdus merula) and once the prey was starling (sturnus vulgaris), common chaffinch (fringilla coelebs), song thrush (turdus philomelos) and european green woodpecker (picus viridis). according to the moustache, breast colour and spot density, it appears fig. 2. incubating peregrine falcon (falco peregrinus) female in the occupied raven nest in northern cliffs of trachyte quarry “srebro” during march of 2020 biologica nyssana ● 11 (2) december 2020: 103-107 rajković, vračarić ● breeding range extension of the peregrine falcon (falco peregrinus l.) in the pannonian plain 106 that all three observed specimens of peregrine falcon phenotypically corresponded to the nominal subspecies falco peregrinus peregrinus, which is typical subspecies in most parts of central europe. discussion the findings described in this article present the first documented evidence of a peregrine falcon nesting in vojvodina province and at the same time the northernmost breeding site of this species in serbia. consequently, the territory defending and breeding attempts at the same locality in three consecutive years indicated presumably satisfactory habitat quality and suggested site fidelity. the colonisation of the fruška gora mt. by breeding individuals of peregrine falcon has been expected for more than one decade (stojnić & puzović, 2008), but without concrete evidence until 2018. thus, these nesting attempt(s) represent evidence of extension of the species nesting range in the pannonian basin and serbia. the described nesting site is approximately 140 km east from slavonski brod in croatia (budinski, 2013), 130-160 km south from pairs in the baranya county in hungary (https://www.pecsiujsag.hu), and around 100 km from the breeding location near valjevo in central serbia (stojnić & puzović, 2008). in hungary, the first breeding pair was confirmed 23 years ago, after at least 33 years of absence (bagyura 1997). in croatia, single data was collected in 2009, and few pairs are breeding near the capital city of zagreb (budinski, 2013). recently, there are no new published data from continental parts of croatia. however, the situation of the peregrine falcon in hungary is much better, and recently has more than 70 active eyries (prommer & bagyura, 2018). there is no breeding evidence from the romanian part of the pannonian plain, although some pairs nest at the border of the area at apuseni mt. (komáromi et al., 2009). the laying dates, landscape, location and other notes presented in this paper generally coincide with previous data reported across pannonian plain and europe (ratcliffe, 1993; carlier, 1995; moore et al., 1997; prommer & bagyura, 2018). dimensions of potential nesting crag from the year 2018 are significantly smaller than reported earlier (ratcliffe, 1993). this female choice may be related to the insufficient supply of crags and crevices, which is the case at this quarry. although the diet sample is small, the combined observations from different years suggest notable flexibility and wide trophic niche by peregrines on the fruška gora mt. as peregrine falcon established first pair and fruška gora mt. and also regularly hosts floaters from increasing central european stock (prommer & bagyura, 2018), we expect that breeding population would continue to rise in the future and expand its range through other suitable locations on fruška gora and rest of the vojvodina province (e.g. titel hills, vršac mt). together with the discovery of a new breeding site, it is necessary to implement strict conservation measures to protect its breeding territory. acknowledgements. authors are warmly grateful to ivan đorđević, dragomir damnjanović, nikola stanojević and jovana koturov for their help during field visits. mátyás prommer kindly provided references from hungary. additionally, we are grateful for all comments and suggestions of two anonymous reviewers and editor-inchief of the journal. references antal, l., fernbach, j., mikuska, j., pelle, i., szlivka, l. 1971: namenverzeichnis der vögel der autonomen provinz vojvodina. (mit einer historischen übersicht von der andrás keve). larus, 23: 73-127. banks, a.n., coombes, r.h., crick, h.q.p. 2003: the peregrine falcon breeding population of the uk & isle of man in 2002. research report 330. british trust for ornithology, thetford, uk. birdlife international 2015: european red list of birds. office for official publications of the european communities, luxembourg eu. budinski, i. 2013: sivi sokol falco peregrinus. str. 191-192 u: tutiš, v., kralj, j., radović, d., ćiković, d., barišić, s. (ur.) crvena knjiga ptica hrvatske. ministarstvo zaštite okoliša i prirode i državni zavod za zaštitu prirode, zagreb. cade, t.j. 1982: the falcons of the world. cornell university press, new york, us. carlier, p. 1995: vocal communication in peregrine falcons falco peregrinus during breeding. ibis, 137(4): 582-585. doi: 10.1111/j.1474-919x.1995. tb03269.x ferguson-lees, j., christie, d.a. 2001: raptors of the world. christopher helm publishing, london. grubač, b. 2018: the peregrine falcon falco peregrinus (tunstall, 1771) in serbia. zaštita prirode, 68(1-2): 67-75. komáromi, i., kovács i., hegyeli, z., kiss, r.b. 2009: the status of peregrine falcon in romania. in: sielicki j., mizera t. peregrine falcon populations – status and perspectives in the 21st century. turul & poznan university of life sciences press, poznan, pl. marčetić, m., medaković, k. 1954: prilog poznavanju ornitofaune vojvodine falconiformes biologica nyssana ● 11 (2) december 2020: 103-107 rajković, vračarić ● breeding range extension of the peregrine falcon (falco peregrinus l.) in the pannonian plain – grabljivice. zbornik matice srpske za prirodne nauke, 1-23: 88-110. marčetić, m. 1957: peregrine, falco peregrinus, on the territory of vojvodina and its nesting on buildings. larus, 9/10: 139-141. marinković, s., grubač, b. 2000: sivi soko/ peregrine falcon falco peregrinus. in: puzović s. [ed.] atlas ptica grabljivica srbije / atlas of birds of prey of serbia, beograd: zavod za zaštitu prirode srbije, pp. 177-182 (in serbian and english summary). matvejev, s.d. 1950: rasprostranjenje i život ptica u srbiji. san, knjiga br 3, beograd, 362 pp. matvejev, s.d. 1964: višegodišnje i sezonske promene brojnosti ptica grabljivica u srbiji. arhiv bioloških nauka, 15(3-4): 127-147. mikuska, j. 1982: the birds of prey in yugoslavia. wwgbp bulletin, 1: 1-10. moore, n.p., kelly, p.f., lang, f.a., lynch, j.m., langton, s.d. 1997: the peregrine falco peregrinus in quarries: current status and factors influencing occupancy in the republic of ireland. bird study, 44(2): 176-181. pelle, i. 1990: sivi soko, falco peregrinus, u zrenjaninu. ciconia, 2: 95-96. pelle, i., ham, i., rašajski, j., gavrilov, t. 1977: pregled gnezdarica vojvodine. larus, 29/30: 171197. prommer, m., bagyura, j. 2018: review of the development of the peregrine falcon (falco peregrinus) population in hungary between 1997 and 2018. ornis hungarica, 26(2): 2-11. doi: 10.1515/orhu-2018-0011 puzović, s., simić, d., saveljić, d., gergelj, j., tucakov, m., stojnić, n., hullo, i., ham, i., vizi, o., šćiban, m., ružić, m., vučanović, m., jovanović, t. 2003: ptice srbije i crne gore – veličine gnezdilišnih populacija i trendovi: 19902002. ciconia, 12: 35-120. puzović, s., radišić, d., ružić, m., rajković, d., radaković, m., pantović, u., janković, m., stojnić, n., šćiban, m., tucakov, m., gergelj, j., sekulić, g., agošton, a. & raković, м. 2015: ptice srbije: procena veličinа populacija i trendova gnezdarica 2008–2013. društvo za zaštitu i proučavanje ptica srbije & prirodno-matematički fakultet, departman za biologiju i ekologiju, univerzitet unovom sadu, novi sad. ratcliffe, d. 1993: the peregrine falcon. 2nd edition. poyser, london, uk. rizolli, f., sergio, f., marchesi, l., pedrini, p. 2005: density, productivity, diet and population status of the peregrine falcon falco peregrinus in the italian alps. bird study, 52(2):188–192. šćiban, m., rajković, d., radišić, d., vasić, v., pantović, u. 2015: ptice srbije – kritički spisak vrsta. pokrajinski zavod za zaštitu prirode i društvo za zaštitu i proučavanje ptica srbije, novi sad. white, c.m. 1994: family falconidae. in del hoyo, j.; elliot, a.; sargatal, j. (eds.). handbook of birds of the world: new world vultures to guinea fowl. 2. barcelona: lynx edicions. pp. 216–275, plates 24–28. white, c.m., cade, t.j., enderson, j.h. 2013: peregrine falcons of the world. lynx edicions, barcelona. white, c.m., clum, n.j., cade, t.j., hunt, w.g. 2002: peregrine falcon (falco peregrinus), version 2.0. in the birds of north america (a. f. poole and f. b. gill, editors). cornell lab of ornithology, ithaca, ny, usa. wink, m. 2018: phylogeny of falconidae and phylogeography of peregrine falcons. ornis hungarica 26(2): 27–37. zuberogoitia, i., azkona, a., zabala, j., astorkia, l., castillo, i., iraeta, a., martínez, j.a., martínez, j.e. 2009: phenotypic variations of peregrine falcon in subspecies distribution border in: sielicki j., mizera t. (eds.). peregrine falcon populations – status and perspectives in the 21st century. turul & poznan university of life sciences press, poznan, pl. h t t p s : / / w w w. p e c s i u j s a g . h u / p e c s / h i r / h e l y i hireink/tiz-fiokat-neveltek-fel-a-vandorsolymok baranyaban?fbclid=iwar20zgp-mk4lyc-l353ed m711xreo2apznkliv2ka7yknjkljlr1sepq1dm 107 biologica nyssana ● 11 (2) december 2020: 103-107 rajković, vračarić ● breeding range extension of the peregrine falcon (falco peregrinus l.) in the pannonian plain the ecological and floristic characteristics of natural population of micromeria juliana (l.) benth. ex rchb. in bulgaria biologica nyssana 7 (2)  december 2016: 91-99 aneva, i. et al.  the ecological and floristic characteristics… 91 original article received: 12 october 2016 revised: 08 november 2016 accepted: 29 november 2016 the ecological and floristic characteristics of natural population of micromeria juliana (l.) benth. ex rchb. in bulgaria ina aneva1*, petar zhelev2, milena nikolova1, ivan evtimov2 1institute of biodiversity and ecosystem research, bulgarian academy of sciences, 1113 sofia, bulgaria 2university of forestry, 10 kliment ohridsky blvd., 1797 sofia, bulgaria * e-mail: ina.aneva@abv.bg abstract: aneva, i., zhelev, p., nikolova, m., evtimov, i.: the ecological and floristic characteristics of natural population of micromeria juliana (l.) benth. ex rchb. in bulgaria. biologica nyssana, 7 (2), december 2016: 91-99. micromeria juliana is a rare species in bulgaria. it is included in the red data book of the country with conservation status “endangered” and is protected by the biodiversity act. the present report focuses to the study of natural populations and plant communities of m. juliana in the two regions of its occurrence in bulgaria – eastern rhodopes and the valley of mesta river. in the first locality the species grows on steep stony slopes and on walls of a medieval fortress, and the second one – on a steep calcareous slope, and on abandoned agricultural land. we present results of a survey on the species composition of plant communities of m. juliana together with analyses of floristic elements and ecological forms. the floristic composition indicates that the participation ratio of elements with mediterranean origin is high. the ecological factors that have the highest impact on the floristic composition are intensive light, air temperature and humidity. the area occupied by the species is limited due to its very specific requirements and low competition ability. key words: medicinal plant, species composition, floristic elements apstrakt: aneva, i., zhelev, p., nikolova, m., evtimov, i.: ekološke i florističke karakteristike prirodnih populacija vrste micromeria juliana (l.) benth. ex rchb. u bugarskoj. biologica nyssana, 7 (2), decembar 2016: 9199. micromeria juliana je retka vrsta u bugarskoj. nalazi se u crvenoj knjizi ove zemlje sa konzervacionim statusom “ugrožena” i zaštićena je zakonom o biodiverzitetu. ovde prezentovano istraživanje je bilo usmereno na proučavanje prirodnih populacija i biljnih zajednica vrste m. juliana unutar dva regiona u kojima se ova vrsta pojavljuje u bugarskoj – istočnim rodopima i dolini reke mesta. na prvom lokalitetu vrsta raste na strmim kamenitim padinama, kao i na zidinama srednjevekovne tvrđave, dok na drugom – na strmim krečnjačkim padinama i napuštenim poljoprivrednim zemljištima. mi smo prezentovali rezultate pregleda sastava vrsta biljnih zajednica m. juliana, zajedno sa analizom florističkih elemenata i ekoloških formi. floristički sastav ukazuje da je udeo mediteranskih elemenata visok. intenzivna svetlost, temperature vazduha 7 (2) • december 2016: 91-99 12th sfses • 16-19 june 2016, kopaonik mt doi: 10.5281/zenodo.200405 biologica nyssana 7 (2)  december 2016: 91-99 aneva, i. et al.  the ecological and floristic characteristics… 92 i vlažnost su ekološki faktori koji imaju najznačajniji uticaj na floristički sastav. oblast koju zauzima vrsta je ograničena zbog veoma specifičnih prohteva i niske kompetitivne sposobnosti vrste. ključne reči: lekovite biljke, sastav vrsta, florni elementi introduction genus micromeria benth. (lamiaceae) is represented in bulgaria by four species (a n č e v , 1989), two of them (m. juliana (l.) benth. ex rchb. and m. cristata (hampe) griseb.) belonging to section micromeria and two (m. dalmatica benth. and m. frivaldszkyana (degen) velen.) – to section pseudomelissa benth. the species of the latter section were transferred to genus clinopodium based on the evidence of recent phylogenetic studies (b r ä u c h l e r et al., 2005, 2006; r y d i n g 2006). according to b r ä u c h l e r et al. (2008) there are still a lot of taxonomic problems to be solved, despite of the success achieved using modern cladistic analyses based on molecular data (w a g s t a f f et al., 1995; b r ä u c h l e r et al., 2005; see b r ä u c h l e r et al., 2008, for synopsis and review). many species of the genus possess substantial interest as medicinal and aromatic plants. there were some studies on the cytology, morphology and chemical composition of the essential oil of some micromeria representatives in balkans and at least part of them addressed also the question of antibacterial and antifungal activity of the species (m a r i n k o v i c et al., 2002; s l a v k o v s k a et al., 2005; k o s t a d i n o v a et al., 2007; m a r t i n et al., 2011; k a r o u s o u et al., 2012; k r e m e r et al., 2014, m a r i n et al., 2015). bulgarian species of the genus are considered important as medicinal and aromatic plants and also from conservation point of view. two of them – m. juliana (fig. 1) and m. frivaldszkyana – are listed in the different editions of the red data book of bulgaria with the category “endangered” (a n č e v , 1984; a p o s t o l o v a , 2015; s t o y a n o v , 2015). even though not an endemic, m. juliana is a rare species and occurs only in two small localities in the eastern rhodopes (a n č e v , 1984, 1989; s t o y a n o v , 2015). however, t a s h e v (2015) reported another occurrence in the valley of river mesta. the information about biological peculiarities, habitat requirements and natural resources of micromeria species is very important for designing of proper strategy for their conservation and sustainable use. therefore, the objective of the present study was to characterize the natural localities of micromeria juliana as a part of a program for conservation and sustainable use of the species of genus micromeria in bulgaria. material and methods the objects of the study included the two known localities of the species in bulgaria. the first one is located in the eastern rhodopes, locality called “kaleto”, close to gugutka village, ivaylovgrad district (41°23'52.60", 25°57'17.96"), which will be referred to hereafter as rhodopes. the second one is in the valley of river mesta, locality called “dimanska stena”, close to the village godeshevo (41°27'57.41", 24° 3'31.53") and will be referred to hereafter as mesta. the field observations took place in the period april-july 2016. full floristic inventory was performed in the two natural localities of the species. taxonomic treatment followed a s s y o v & p e t r o v a (eds., 2012). phytogeographic affinity of the species was determined according to w a l t e r (1985), summarized by a s s y o v & p e t r o v a (eds., 2012), and the life forms – according to r a u n k i a e r (1934). fig. 1. micromeria juliana biologica nyssana 7 (2)  december 2016: 91-99 aneva, i. et al.  the ecological and floristic characteristics… 93 results and discussion in the locality “kaleto”, eastern rhodopes, m. juliana was found mostly on the wall of the medieval fortress (fig. 2), and also one small spot was found on the road ledge close to the fortress. the populations of m. juliana are represented by small number of individuals. the main reason can be traced in its ecological requirements to specific type of habitats – stony and rocky places or open soils free of turf grasses. usually on balkans m. juliana occurs in the plant communities known as aegean phrygana (eunis 2016), and these habitats are characterized by open stony and sandy spots. however, even though the habitats in the studied locality resemble the phrygana, they are not identical. the potential vegetation on the place could be classified as quercus pubescens forests (b o n d e v , 1991), and these forests, even rather dry, often form dense herbaceous cover, thus preventing the establishment of m. juliana individuals. therefore, the most visible occurrence of the species was on the walls of the fortress. it occurred also on the rocky places and on the slopes free of turf grasses. occurrence of the species on walls is not unprecedented, as reported by l a g i o u et al. (1998). m. juliana is heat demanding species and is therefore found predominantly on eastern and southern expositions. the area occupied by the species in rhodopes is about 0.1 ha and the population size is about 40 individuals. further studies are necessary to reveal whether there are more spots of this rare species in the rocky slopes of byala reka river. in the second locality, in the valley of river mesta, the species was found on a steep (3040°) rocky slope in the place called “dimanska stena”, but also few isolated and smaller spots were found in the adjacent abandoned arable land (fig. 3). all these spots were bulked together when analyzing the locality. the area occupied by the species in the valley of mesta river is about 0.4 ha. the population size is larger and consists of about 90 individuals, whose dimensions tend to be larger than those in rhodopes. m. juliana is not collected by people, and direct anthropogenic pressure cannot be considered as a threat, with the exception of some potential incidental fire, as suggested by s t o y a n o v (2015). total 124 species were recorded in the two localities – 47 species in the eastern rhodopes and 89 species in the valley of mesta river. it should be noted that these values represent a minimum species number, because some short-lived spring annuals and/or geophytes could have terminated their vegetation at the time of the field study. also, some species with insufficiently developed diagnostic traits at that moment could have been overlooked or misidentified. the higher number of species recorded in the locality mesta is due to its larger occupied area on the rocky slope and on the abandoned arable land. there were 4 species of ferns, one gymnosperm, and 119 angiosperm species – 107 dicots and 12 monocots (tab. 1). the species recorded belong to 39 families and 103 genera. the most numerous family is asteraceae – 13 genera and 14 species, followed by lamiaceae – 10 genera and 13 species, poaceae – 9 genera and 11 species, and rosaceae – 7 genera and 9 species. eighteen families are represented by only one species. the flora in these two localities had not been studied in detail previously. t a s h e v (2015) mentioned few species occurring in the community of mesta locality together with m. juliana, like asyneuma limonifolium (l.) janch,, inula aschersoniana janka, teucrium polium l., euphorbia myrsinites l., and dichanthium ischaemum (l.) roberty, all of them recorded during our field survey. classification of species regarding their light dependence, or luminosity of the environment, fig. 2. locality 1 (eastern rhodopes) – the medieval fortress fig. 3. locality 2 (mesta) biologica nyssana 7 (2)  december 2016: 91-99 aneva, i. et al.  the ecological and floristic characteristics… 94 fig. 4. (see table 1 for legend.) a. species distribution according to their light dependence b. species distribution according to their dependence on humidity c. species distribution according to their biological type d. species distribution according to their life form fig. 5. distribution of the floristic elements. see table 1 for legend. biologica nyssana 7 (2)  december 2016: 91-99 aneva, i. et al.  the ecological and floristic characteristics… 95 revealed that 119 species (96%) are heliophytes, only one can be classified as sciophyte and four species are of transitional character (fig. 4a). xerophytes represent the most numerous type regarding dependence on water availability (104 species, 84%). the other species are classified as mesophytes (7 species), xero-mesophytes (8 species) and 5 species belong to other categories (fig. 4b). perennial species predominate – 65 (52%) followed by annuals – 26 (21%), plus 3 species (2%), which are of transitional type annual-biennial. thirteen species (10%) are shrubs and 9 species (7%) are trees (fig. 4c). this fact reflects the potential vegetation type of the locality, which can be defined as quercus pubescens forests, like in eastern rhodopes. currently, trees and shrubs are represented by a few individuals and even though they do not shape the physiognomy of the plant community, they show the natural trend towards restoring the indigenous vegetation. classification by life forms could be predicted by the biological type. hemi-cryptophytes are the predominating type – 63 species (52%), which is expected because most perennial plants fall into this category (fig. 4d). the second most numerous group is the one of therophytes – 32 (26%) and the third one – of phanerophytes – 18 (15%). distribution of floristic elements reflects the mediterranean character of the flora. the predominant elements are: sub-mediterranean – 29 species (23%), euro-mediterranean – 18 (14.5%), typical mediterranean – 11 (9%), and euro-asian – 10 (8%). however, if all elements having mediterranean component are considered (for example, eur-med, eur-submed, med, med-as, pont-med etc.) they would amount to 80 species (65%). the trends are the same in the two studied localities, in spite of some minor differences (fig. 5). the results of the study underline the submediterranean character of the flora and vegetation of the two localities (p a v l o v a et al., 2004; p e t r o v a , 2004; g o r a n o v a et al., 2013). conclusion species composition and characteristics of the two natural localities of m. juliana in bulgaria revealed that the plant communities the species is part of are typical for the dray and hot habitats in the zone of strong mediterranean influence. the locality in the valley of mesta river is larger both in terms of area occupied and population size. the status of the species can be evaluated as relatively stable. it is practically not affected by the human activities and only some incidental fire could pose a threat. acknowledgements. the authors are grateful to the financial support provided by program for career development of young scientists, bulgarian academy of sciences– grant № dfnp-67_a1. references ančev, m. 1984: micromeria juliana. in: velchev v. (ed.), red data book of the people’s republic of bulgaria. vol. 1. plants: 318. publishing house bulg. acad. sci., sofia. (in bulgarian). ančev, m. 1989: micromeria. in: velčev, v. (ed.), fl. republ. popularis bulgaricae. vol. 9. in aedibus acad. sci. bulgaricae, serdicae: 356– 362. (in bulgarian). apostolova, i. 2015: micromeria frivaldszkyana (degen) velen. in: peev d. (ed.) red data book of bulgaria, volume 1 – plants and fungi. iber – bas and moew: 550. assyov, b., petrova, a. (eds.) 2012: conspectus of the bulgarian vascular flora. distribution maps and floristic elements. fourth edition, bulgarian biodiversity foundation, sofia, 489 p. bondev, i., 1991. the vegetation of bulgaria. a map in scale 1:600000 with explanatory text. university of sofia “st. kliment ohridski” publ. house, 183 p. bräuchler, c., meimberg, h., abele, t., heubl, g. 2005: polyphyly of the genus micromeria benth. (lamiaceae): evidence from cpdna sequence data. taxon, 54 (3): 639-650. bräuchler, c., meimberg h., heubl, g., 2006: new names in old world clinopodium – the transfer of the species of micromeria sect. pseudomelissa to clinopodium. taxon, 55 (4): 977-981. bräuchler, c., ryding, o., heubl, g., 2008: the genus micromeria (lamiaceae), a synoptical update. willdenowia, 38 (2): 363-410. eunis, 2016: eunis habitat classification (http://eunis.eea.europa.eu/habitats/114). retrieved 29.11.2016. goranova, v., vassilev, k., pedashenko, h., 2013: vascular flora of the valley of mesta river floristic region, sw bulgaria. phytologia balcanica, 19 (1): 89-114. karousou, r., hanlidou, e., lazari, d., 2012: essential oils of micromeria dalmatica benth., a balkan endemic species of section pseudomelissa. chemistry & biodiversity, 9 (12): 27752783. kostadinova, e., alipieva, k., stefova, m., stafilov, t., antonova, d., evstatieva, l., matevski, v., kulevanova, s., stefkov, g., bankova, v., 2007: chemical composition of the essential oils of three micromeria species growing in macedonia biologica nyssana 7 (2)  december 2016: 91-99 aneva, i. et al.  the ecological and floristic characteristics… 96 and bulgaria. macedonian journal of chemistry and chemical engineering, 26 (1): 3-7. kremer, d., dunkić v., stešević, d., kosalec, i., ballian, d., bogunić, f., bezić, n., stabentheiner, e., 2014: micromorphological traits and essential oil of micromeria ongipedunculata bräuchler (lamiaceae). central european journal of biology, 9 (5): 559-568. lagiou, e., krigas, n., hanlidou, e., kokkini, s., 1998: the vascular flora of the walls of thessaloniki (n greece). in: tsekos i., moustakas m. (eds.). progress in botanical research: proceedings of the 1st balkan botanical congress. springer science + business media, dordrecht, the netherlands: 81-84. marin, m.a., novaković, m.m., tešević, v.v., kolarević, s.m., vuković-gačić, b.s., 2015: antimicrobial activity of the essential oil of wildgrowing micromeria thymifolia (scop.) fritsch. journal of bioscience and biotechnology, 4 (1): 29-31. marinković, b., marin, p.d., knezević-vukcević, j., soković, m.d., brkić, d., 2002: activity of essential oils of three micromeria species (lamiaceae) against micromycetes and bacteria. phytotherapy research, 16 (4): 336-339. martın, e., cetın, o., dırmencı, t., ay, h., 2011: karyological studies of clinopodium l. (sect. pseudomelissa) and micromeria benth. s. str. (lamiaceae) from turkey. caryologia, 64 (4): 398-404. pavlova, d., dimitrov, d., kozuharova, e., 2004: flora of the serpentine complexes in eastern rhodopes (bulgaria). in: beron p., popov a. (eds.) biodiversity of eastern rhodopes (bulgaria and greece). pensoft publishers and national museum of natural history: 119-129. petrova, a., 2004: flora of the eastern rhodopes (bulgaria) and its conservation significance. in: beron p., popov a. (eds.) biodiversity of eastern rhodopes (bulgaria and greece). pensoft publishers and national museum of natural history: 53-118. raunkiaer c., 1934. the life-forms of plants and their bearing on geography. in: the life forms of plants and statistical plant geography. the collected papers of c. raunkiaer. clarendon press, oxford: 2-104. slavkovska, v., couladis, m., bojovic, s., tzakou, o., pavlovic, m., lakusic, b., jancic, r., 2005: essential oil and its systematic significance in species of micromeria bentham from serbia & montenegro. plant systematics and evolution, 255 (1): 1-15. stoyanov, s., 2015: micromeria juliana (l.) rchb. in: peev d. (ed.) red data book of bulgaria, volume 1 – plants and fungi. iber – bas and moew: 551. tashev a., 2015. reports 237-241. in: vladimirov v., dane f., tan k. (compilers). floristic records on the balkans: 26. phytologia balcanica, 21 (1): 83-84. wagstaff, s.j., olmstead, r.g., cantino, p.d., 1995: parsimony analysis of cpdna restriction site variation in subfamily nepetoideae (labiatae). american journal of botany, 82 (7): 886-892. walter, h., 1985: vegetation of the earth and ecological systems of the geo-biosphere. (english translation), springer-verlag, berlinheidelberg, 318 p. biologica nyssana 7 (2)  december 2016: 91-99 aneva, i. et al.  the ecological and floristic characteristics… 97 table 1. list of plant taxa recorded in the two localities, together with their characteristics ( er-eastern rhodopes, mr-mesta river valley) taxon light dependence humidity dependence biological type life form floristic element er mr polypodiophyta polypodiopsida aspleniaceae asplenium adianthum-nigrum l. sc m/hg p h subboreal + asplenium ruta-muraria l. h x p h boreal + + ceterach officinarum dc. h x p h submed + magnoliophyta hypolepidaceae pteridium aquilinum (l.) kuhn h m p h kos + pinophytina pinopsida cupressaceae juniperus deltoides r.p. adams h x s ph eur-med + + magnoliophytina magnoliopsida aceraceae acer campestre l. h x/m t ph eur-ot + acer monspessulanum l. h x/m t ph submed + anacardiaceae pistacia terebinthus l. h x t ph pont-med + rhus coriaria l. h x t ph med-as + apiaceae eryngium campestre l. h m/x p h pont-med + scandix pecten-veneris h x a th eur-as + asteraceae carlina vulgaris l. h m/x a th eur-med + + centaurea stoebel. h x p h submed + centaurea salonitana vis. h x p h pont-med + chondrilla juncea l. h x a th eur-sib + cirsiumligulareboiss. h x a-b th med + crepis biennisl. h x a th submed + crupina vulgariscass. h x a th submed + inula aschersonianajanka h x p h bal-anat + jurinea consanguinea dc. h x p h submed-sib + leontodon hispidusl. h x p h eur-med logfia arvensis (l.) holub h x a th eur-med + picnomon acarna (l.) cass. h x p h med + scorzonera laciniata l. h x p h med + tragopogon pratensis l. h x a th eur-med + betulaceae carpinus betulusl. h x/m t ph eur-submed + carpinus orientalis miller h x/m t ph submed + boraginaceae buglossoides purpurocaerulea (l.) i. m. johnst. h x p h eur-as + myosotis arvensis (l.) hill h x a th eur-as + + onosma echioides l. h x p h med + brassicaceae aethionema saxatile (l.) r. br. h x p h submed + alyssum alyssoides (l.) l. h x p h eur-med + alyssum murale waldst. & kit. h x p h eur-submed + clypeola jonthlaspi l. h x a th med + erysimum diffusum ehrh. h x/m a-b h/th eur + campanulaceae asyneuma limonifolium (l.) janchen h x p cr ap-bal + caryophyllaceae cerastium bulgaricum uechtr. h x a th bul + herniaria incana lam. h x a th eur-med + minuartia caespitosa (ehrh.) degen h x p h eur-med + minuartia hirsuta (m. bieb.) hand.mazz. h x p h submed + scleranthus perennis roch. ex baumg. h x p h eur-med + silene conica l. h x a th submed-as + + biologica nyssana 7 (2)  december 2016: 91-99 aneva, i. et al.  the ecological and floristic characteristics… silene italica (l.) pers. h/sc m/x p h eur-med + stellaria graminea l. h x p h eur-as + cistaceae cistus incanus l. h x s ch med + fumana procumbens (dunal) gren. &godr. h x s ch pont-med + + helianthemum numularium (l.) mill. h x s ch alp-med + rhodax canus (l.) fuss h x s ch pont + convolvulaceae convolvulus cantabrica l. h/sc x p h pont + crassulaceae sedum anopetalum dc. h x p ch submed + sedum hispanicum l. h x p h eur-med + dipsacaceae scabiosa rotata m. bieb. h x p h med + euphorbiaceae euphorbia cyparissias l. h/sc m p h eur + euphorbia myrsinites l. h x p h submed + euphorbia plathyphyllos l. h x a th eur-med + fabaceae astragalus onobrychis l. h x p h eur-as + coronilla cretica l. h x p h med coronilla scorpioides (l.) c. koch h x a th submed dorycnium herbaceum vill. h x p h eur-med + lathyrus nissolia l. h x p h eur-submed + lotus aegaeus (griseb.) boiss. h x p h med + medicago lupulina l. h x p h eur-as + medicago minima (l.) bartal. h x a th eur-as + + onobrychis arenaria (kit.) dc. h x p h pont + ononis pusilla l. h x a th submed + vicia dalmatica a. kern. h x p h submed + fagaceae quercus pubescens willd. h x/m t ph eur-submed + geraniaceae erodium cicutarium (l.) l'hér. h x a th subboreal + geranium robertianum l. h x a th subboreal + + geranium rotundifolium l. h x a th eur-as + + hypericaceae hypericum cerastoides (spach) n.k.b. roloson. h m p h submed + hypericum olympicum l. h x/m p h submed + lamiaceae acinos suaveolens (sm.) don h x p h submed + + ajuga chamaepitys (l.) schreber h x a-b h/th pont-med + calamintha nepeta (l.) savi h x p h eur-med + clinopodium vulgare l. h x p h subboreal + micromeria dalmatica bentham ssp. bulgarica (velen.) guinea h x p h bal + micromeria juliana (l.) benth. ex rchb. h x p h med + satureja coerulea janka h x s ch submed + satureja cuneifolia ten. h x s ch submed + sideritis montana l. h x a th submed + stachys germanica l. h x p h eur-submed + teucrium chamaedrys l. h/ sc x/m p h submed + teucrium polium l. h x p h pont-med + + thymus atticus čelak. h x s ch bal-anat + linaceae linum hologynum rchb. h x p h submed + moraceae ficus carica l. h x t ph adv (med) + oleaceae fraxinus ornus l. h x t ph submed + jasminum fruticans l. h x s ph pont-cas + plantaginaceae plantago lanceolata l. h m p h kos + primulaceae anagalis arvensis l. h x a th kos + biologica nyssana 7 (2)  december 2016: 91-99 aneva, i. et al.  the ecological and floristic characteristics… 99 ranunculaceae clematis vitalba l. h x s ph eur + resedaceae reseda lutea l. h x p h subboreal + rhamnaceae paliurus spina-christi mill. h x s ph eur-as + rosaceae agrimonia eupatoria l. h m p h eur-med + crataegus monogyna jacq. h x s ph subboreal + potentilla argentea l. h x p h pont + potentilla sulphurea lam. h x p h submed + prunus spinosa l. h x s ph pont + rosa myriacantha dc.ex lam. & dc. h x s ph submed + rubus discolor weihe & nees h x s ph submed + rubus sanguineus friv. h x s ph pont-med + + sanguisorba minor scop. h x p h subboreal + + rubiaceae cruciata pedemontana (bellardi) ehrend h x a th med-as + + galium pseudoaristatum schur. h x p h pann-bal + santalaceae thesium linophyllon l. h m p h submed + scrophulariaceae linaria vulgaris mill. h m p h eur-sib + misopates orontium (l.) raf. h x a th eur-med + verbascum densiflorum bertol. h x a th submed + veronica triphyllos l. h x a th eur-med + urticaceae parietaria lusitanica l. h x a th med-as + valerianaceae valerianella carinata loisel. h x a th eur-med + liliopsida cyperaceae carex otrubae podp. h m/x p h eur + poaceae aegilops neglecta req. ex bertol. h x a th submed + bromus squarrosus l. h x a th submed + bromus sterilis l. h x a th boreal + chrysopogon gryllus (l.) trin. h x p h pont-med + dichanthium ischaemum (l.) roberty h x p h submed-as + echinaria capitata (l.) desf. h x a th med + festuca valesiaca scheicher ex gaudin h x p h pont + koeleria nitidula velen. h x p h pont + melica ciliata l. h x p h eur-submed + poa bulbosa l. h x p h eur-as + + poa pratensis l. h x p h kos + legend: light dependence: h heliophyte; h/sc – helio-sciophyte; sc/h – scio-heliophyte; sc sciophyte. humidity dependence: x xerophyte; m mesophyte; m/x – meso-xerophyte; x/m – xero-mesophyte; m/hg – meso-hygrophyte. biological type: a – annual; a-b – annual to biennial; b – biennial; p – perennial; s – shrub; t – tree. life form: th therophyte; н hemicryptophyte; ch chamaephyte; ph – phanerophyte. floristic element: adv – adventive; anat – anatolian; ap – appenine; as – asian;bal – balkan; boreal; bul – bulgarian; eur – european; kos – cosmopolitan; med– mediterranean; ot – oriental-turanian; pann – pannonian; pont – pontic; sib – siberian; additional abbreviation: s – southern; e – eastern; w – western; n – northern; c – central. biologica nyssana 7 (2)  december 2016: 91-99 aneva, i. et al.  the ecological and floristic characteristics… stamenković et al. 2021, biologica nyssana 12(1) 12 (1) september 2021: 63-70 doi: 10.5281/zenodo.5523027 benthic macroinvertebrate community structure in batušinac ponds (serbia) relative to the distance from a river original article olivera stamenković department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, niš, serbia olivera.stamenkovic@pmf.edu.rs (corresponding author) vladica simić institute of biology and ecology, faculty of science, university of kragujevac, radoja domanovića 12, 34000 kragujevac, serbia vladica.simic@pmf.kg.ac.rs djuradj milošević department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, niš, serbia djuradj@pmf.ni.ac.rs ana petrović institute of biology and ecology, faculty of science, university of kragujevac, radoja domanovića 12, 34000 kragujevac, serbia ana.petrovic@pmf.kg.ac.rs milica stojković piperac department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, niš, serbia milicas@pmf.ni.ac.rs received: december 04, 2020 revised: march 30, 2021 accepted: april 02, 2021 abstract: batušinac ponds are situated in south-eastern serbia, near the južna morava river. in this study we compared benthic macroinvertebrate community structures of the river and three ponds located at contrasting distances from the river in order to assess the impact of the river proximity on benthic macroinvertebrate community structures in batušinac ponds. only 7 out of 43 taxa were common for the river and the study ponds. based on the nmds analysis, the community structures of the river and the study pond situated closest to the river were the most similar. the results of an anosim analysis showed that community structures of the river and all study ponds were significantly different (r=0.901; p=0.003). given the observed differences between the river and pond macroinvertebrate communities, it can be concluded that batušinac ponds contribute significantly to freshwater diversity of the study area. key words: macrozoobenthos, species composition, lotic taxa, lentic taxa, spatial proximity apstract: struktura zajednice bentosnih makroinvertebrata batušinačkih bara (srbija) u odnosu na udaljenost od reke batušinačke bare su smeštene u jugoistočnoj srbiji, u blizini reke južne morave. u ovoj studiji smo upoređivali strukturu zajednica bentosnih makroinvertebrata reke i tri bare smeštene na različitoj udaljenosti od reke, s ciljem da procenimo uticaj blizine reke na strukturu zajednica bentosnih makroinvertebrata u batušinačkim barama. od ukupno 43 taksona, svega je 7 bilo zajedničko za reku i istraživane bare. rezultati nmds analize su pokazali da su zajednice reke i bare smeštene najbliže reci najsličnije po strukturi. rezultati anosim analize su pokazali da se strukture zajednica reke i svih bara međusobno značajno razlikuju (r=0.901; p=0.003). uzimajući u obzir uočene razlike između zajednica makroinvertebrata reke i bara, može se zaključiti da batušinačke bare značajno doprinose slatkovodnom diverzitetu istraživanog područja. ključne reči: makrozoobentos, sastav vrsta, lotički taksoni, lentički taksoni, prostorna bliskost introduction ponds are small and shallow (surface area 1 m2–2 ha; max depth 8 m), natural or man-made water bodies that can be permanently or seasonally filled with standing water (biggs et al., 2005). despite their small size, ponds represent a valuable source of freshwater biodiversity, since they host more rare and threatened species than other freshwater ecosystems (williams et al., 2004; davies et al., 2008). ponds contribute disproportionately to regional biodiversity due to their high β diversity, which is related to the existence of a wide variety of pond types that differ in their features (e.g., length of their hydroperiod, size, or physical and chemical properties), even on a small spatial scale (de meester et al., 2005). macroinvertebrates constitute an important part of animal diversity in small water bodies, such as ponds (davies et al., 2008). it has been recognized that community structure (species composition and © 2021 stamenković et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 63 abundances) of macroinvertebrate communities in ponds is strongly driven by local environmental and biotic factors (e.g., water permanence, water chemistry, macrophyte cover, and fish predation) (trigal et al., 2007; hentges & stewart, 2010; walker et al., 2013; gleason & rooney, 2018). in addition, evidence has accumulated that macroinvertebrate communities in ponds are structured by dispersal processes (leibold et al., 2004; van de meutter et al., 2007). therefore, besides local, regional factors are also of huge importance for structuring macroinvertebrate communities in ponds. among structuring processes that operate at regional level, connectivity between habitats (e.g., small distance among habitats) is of huge importance for macroinvertebrate communities in ponds (oertli et al., 2008). in this study we compared the community structures of benthic macroinvertebrates between a part of the river and three ponds situated at contrasting distances from the river. we aimed to assess the impact of the river proximity on benthic macroinvertebrate community structures in ponds. we hypothesised that a pond situated closest to the river and the river have the most similar benthic macroinvertebrate community structures. materials and methods study area batušinac ponds are located in south-eastern serbia, about 10 km west of the city of niš. study area encompasses three permanent ponds situated on the left bank of the južna morava river, at distances of 17.5 m, 212 m, and 386 m (fig. 1). study ponds are the remains of the former river channel that were isolated, as a result of the redirection of the river flow during the construction of a nearby highway (fig. 1). the ponds are connected to the current riverbed both by groundwater supplies and by river flooding (ranđelović et al., 2007). pond 1 is the smallest (surface area of 429.42 m2; maximum depth 120 cm). it is situated nearest to the river (fig. 1), and it is consequently the most intensively flooded. pond 2 has a surface area of 6340.88 m2 and maximum depth of 250 cm. due to their spatial proximity pond 1 and pond 2 become a connected system during the flood events (fig. 1). pond 3 is the largest (surface area of 14206.05 m2; maximum depth 200 cm) and it is farthest from the river (fig. 1). in addition, pond 3 is isolated from the rest of the two study ponds by the nearby highway (fig. 1). sampling macroinvertebrate sampling was performed in september 2016. within each pond, we developed three sampling transects (study sites) starting from the shore towards the deeper water. sampling transects were distributed across the ponds to cover all mesohabitats within a pond. one sampling transect was developed within the river, encompassing river banks and a midstream. the part of the river investigated represents the river’s middle rich. benthic macroinvertebrates were sampled using a kick-net of 250 µm mesh size. three benthic subsamples were taken from the most common substrate types along each transect. all three benthic subsamples were merged into a single sample. the material collected was sorted out of sediment and preserved in 70% ethanol. macroinvertebrates were identified to the lowest possible taxonomic level using relevant taxonomic keys (elliot et al., 1988; wallace et al., 1990; edington & hildrew, 1995; nilsson, 1997; waringer & graf, 1997; gerken & sternberg, 1999; timm, 1999; pfleger, 2000; bauernfeind & humpesch, 2001; glöer, 2002; eiseler, 2005; elliot & humpesch, 2010). most of the taxa were identified to species or genus level, except the families whose individuals were damaged: aphelocheiridae (hemiptera), heptageniidae (ephemeroptera), hydrophilidae, elmidae (coleoptera), or whose identification requires taxonomic expertise, such as chironomidae (diptera). data analysis family chironomidae was present at all study sites, and due to its ubiquitous nature, was excluded from the analyses. 64 biologica nyssana ● 12 (1) september 2021: 63-70 stamenković et al. ● benthic macroinvertebrate community structure in batušinac ponds (serbia) relative to the distance from a river fig. 1. map of the study area with the position of the study sites 65 biologica nyssana ● 12 (1) september 2021: 63-70 stamenković et al. ● benthic macroinvertebrate community structure in batušinac ponds (serbia) relative to the distance from a river taxon abbreviation p1a p1b p1c p2a p2b p2c p3a p3b p3c river radix auricularia (linnaeus, 1758) raur x x x x x x radix labiata (rossmässler, 1835) rlab x x physella acuta (draparnaud, 1805) pacu x x x x x x x gyraulus sp. gyr x acroloxus lacustris (linnaeus, 1758) alac x viviparus acerosus (bourguignat, 1862) vace x unio pictorum (linnaeus, 1758) upic x limnodrilus hoffmeisteri claparède, 1862 lhoff x x x x x limnodrilus claparedeanus ratzel, 1868 lcla x x limnodrilus undekemianus claparède, 1862 lund x tubifex tubifex (müller, 1774) ttub x x x x x erpobdella octoculata (linnaeus, 1758) eoct x x helobdella stagnalis (linnaeus, 1758) hsta x haementeria costata (fr. müller, 1846) hcos x heamopis sanguisuga (linnaeus, 1758) hsan x gammarus balcanicus schäferna, 1922 gbal x pelocoris sp. pel x aphelocheiridae aph x platycnemis pennipes (pallas, 1771) ppen x x x x x coenagrion puella (linnaeus, 1758) cpue x x x ischnura elegans (vander linder, 1820) iele x x pyrrhosoma nymphula (sulzer) pnym x x x sympetrum fonscolombii (selys, 1840) sfon x x x x anax imperator leach, 1815 aimp x calopteryx splendens (harris, 1780) cspl x table 1. list of benthic macroinvertebrate taxa recorded across the study sites. x indicates the presence of a taxon 66 to visualise differences/similarities in the benthic macroinvertebrate community structures of study ponds and the river and spatial patterns of benthic macroinvertebrate taxa we used non-metric multidimensional scaling (nmds) with a braycurtis dissimilarity matrix. to test the statistical significance of the nmds results on benthic macroinvertebrate community structures we used an analysis of similarities (anosim). prior to analyses abundance data were square-root transformed. all analyses were performed using primer version 6 (clarke & gorley, 2006). results overall, 43 taxa (including the family chironomidae) were recorded across the study sites (tab. 1). a total of 31 taxa were recorded across the study ponds, of which 9 taxa were recorded in pond 1; 20 taxa in pond 2, and 17 taxa in pond 3. the recorded taxa belonged to 22 families, 10 orders, 3 classes and 3 phyla. benthic macroinvertebrate community of the river was represented with 20 taxa, belonging to 18 families, 12 orders, 5 classes and 3 phyla. insects were the taxonomically dominant class, both in the river and the study ponds, of which the order ephemeroptera was the taxonomically dominant in the river (represented with 4 taxa), while the order odonata was the taxonomically dominant in the study ponds (represented with 6 taxa). limnodrilus hoffmeisteri claparède, 1862 (annelida, oligochaeta) was the most abundant species both in the river and in pond 1. the most abundant species in pond 2 was radix auricularia (linnaeus, 1758) (mollusca, gastropoda), followed by pyrrhosoma nymphula (sulzer) (insecta, odonata) (fig. 2). pyrrhosoma nymphula was the most abundant species in pond 3 (fig. 2). overall, 23 taxa were recorded exclusively in the study ponds, while 13 taxa were recorded exclusively in the river (tab. 1; fig. 3). besides the chironomidae family, common taxa of both pond and the river were: radix labiata (rossmässler, 1835) (mollusca, gastropoda); caenis sp. (insecta, ephemeroptera); culicoides sp. (insecta, diptera); l. hoffmeisteri, tubifex tubifex (müller, 1774) (annelida, oligochaeta), and erpobdella octoculata (linnaeus, 1758) (annelida, hirudinea) (tab. 1; fig. 3). the study sites were grouped in the nmds plot in two dimensions based on the similarities of their benthic macroinvertebrate community structures biologica nyssana ● 12 (1) september 2021: 63-70 stamenković et al. ● benthic macroinvertebrate community structure in batušinac ponds (serbia) relative to the distance from a river gomphus vulgatissimus (linnaeus, 1758) gvul x onychogomphus forcipatus (linnaeus, 1758) ofor x ecnomus sp. ecn x athripsodes aterrimus (stephens, 1836) aate x leptocerus sp. lep x hydropsyche contubernalis mclachlan, 1865 hcon x caenis sp. cae x x x x cloeon dipterum cdip x baetis rhodani (pictet, 1843) brho x heptageniidae hep x potamanthus luteus (linnaeus, 1767) plut x hydrophilidae hydr peltodytes caesus (duftschmid, 1805) pcae x dytiscus sp. dyt x elmidae elm x culex sp. cule x culicoides sp. culic x x x x x x chironomidae x x x x x x x x x x with stress value 0.06 (fig. 4). the study sites from pond 1 and the river were grouped in the same cluster, while sites form pond 2 and pond 3 were clearly separated from the river and pond 1 (fig. 4). the results of the anosim analysis showed that benthic macroinvertebrate structures of the river and all three ponds were significantly different (r = 0.901; p = 0.003). discussion as previously reported in the literature, ponds and rivers, as distinct freshwater habitats, usually support distinct invertebrate assemblages, even at the genusfamily level (céréghino et al., 2008). this was the case in our study, since only 7 out of 43 recorded taxa were common for the river and the study ponds. moreover, our results are similar to previous research showing that rivers make a higher contribution than ponds to the taxonomic richness of ephemeroptera, trichoptera, plecoptera, and mollusca, while ponds make a higher contribution to the taxa richness of odonata, heteroptera, and coleoptera (céréghino et al., 2008). it has been previously shown that the species we recorded both in the river and in the ponds are generalists, inhabiting both lotic and lentic ecosystems. for instance, e. octoculata is usually dominant among leeches in both lotic and lentic habitats (kubová et al., 2013). the snail r. labiata is adapted to inhabit both lotic and lentic environments by having larger and wider but shorter (globular) shells in lotic versus lentic habitats (lam & calow, 1988). oligochaetes, such as l. hoffmeisteri and t. tubifex inhabit a wide variety of freshwater body types, and the latter one is known to tolerate pollution and can be found in very different conditions (timm, 1999). similarly, the biting midges from the genus culicoides inhabit various aquatic, but also semiaquatic habitats (mullen & murphree, 2019). on the other hand, most of the species we recorded exclusively in the river or in the study 67 biologica nyssana ● 12 (1) september 2021: 63-70 stamenković et al. ● benthic macroinvertebrate community structure in batušinac ponds (serbia) relative to the distance from a river fig. 2. partitioning of the taxa recorded at the study ponds and the river by their abundances. for taxa name abbreviations see tab. 1 ponds have been previously found to prefer to a certain extent either lotic or lentic aquatic habitats. this is especially true for dragonflies and damselflies (odonata) whose clear preferences for specific habitat types are mainly determined by the presence of water flow, water regime, vegetation structure, and physicochemical water properties (corbet, 1999). species that were recorded in ponds in our study, such as: coenagrion puella (linnaeus, 1758), ischnura elegans (vander linder, 1820) (coenagrionidae), sympetrum fonscolombii (selys, 1840) (libellulidae) and anax imperator leach, 1815 (aeshnidae) typically inhabit standing waters (rajkov, 2013). in contrast, the species we recorded in the river, such as: calopterix splendens (harris, 1780) (calopterigidae), gomphus vulgatisimus (linnaeus, 1758) and onychogomphus forcipatus (linnaeus, 1758) (gomphidae) almost exclusively inhabit running waters (rajkov, 2013). similarly, the mayfly species (ephemeroptera) that we recorded in the river, such as baetis rhodani (pictet, 1843) and potamanthus luteus (linnaeus, 1767), are typical lotic species whose larval development is restricted to running waters (eliot et al., 1988). the similar situation is with caddisflies (trichoptera) recorded in our study. namely, hydropsyche contubernalis mclachlan, 1865, the species we recorded in the river, is regarded as a species of the large rivers (edington & hildrew, 1995). on the other hand, the distribution of the species we recorded in the ponds, such as athripsodes aterrimus (stephens, 1836), is determined by slow flowing water, fine sediments and presence of macrophytes, and it is typical for standing waterbodies (wallace et al., 1990; previšić et al., 2010). although l. hoffmeisteri has low dispersal capacity, a possible reason for high abundance of this species both in the river and pond 1 could be its passive dispersion via river floods. it has been shown that an increase in water exchange by flooding events can increase exchange of macroinvertebrates (siziba et al., 2011), and that passive dispersers can spread by direct water connections in flooded areas (van leeuwen et al., 2013). on the other hand, spatial proximity and connectivity of the ponds in our study were not crucial for structuring their benthic macroinvertebrate communities. the observed differences between macroinvertebrate communities of pond 1 and pond 2, despite their connectedness and spatial proximity, suggest that local factors were more important for structuring benthic macroinvertebrates in the study ponds. for instance, similar depth or water quality could be the reasons for the observed similarity of benthic macroinvertebrate communities between pond 2 and pond 3, even though these ponds are separated by the nearby highway. overall, by taking into account that benthic macroinvertebrate communities of the river and all study ponds were statistically significantly different, it may be concluded that river proximity does not affect macroinvertebrate 68 biologica nyssana ● 12 (1) september 2021: 63-70 stamenković et al. ● benthic macroinvertebrate community structure in batušinac ponds (serbia) relative to the distance from a river fig. 3. non-metric multidimensional scaling (nmds) plot showing spatial patterns of benthic macroinvertebrates from the river and study ponds. p and r denote pond and river respectively. for taxa name abbreviations see tab. 1 fig. 4. non-metric multidimensional scaling (nmds) plot showing similarities among the study sites based on their benthic macroinvertebrate community structures communities in the study ponds and that batušinac ponds contribute to overall freshwater biodiversity of the study area by supporting unique macroinvertebrate fauna. acknowledgements. this study was supported by the serbian ministry of education, science and technological development (contract number 451-03-9/202114/200124) and a bilateral cooperation scientific project between serbia and croatia funded by the serbian ministry of education, science and technological development and croatian ministry of science and education. references bauernfeind, e., humpesch, u. 2001: die eintagsfliegen zentraleuropas (insecta: ephemeroptera): bestimmung und ӧkologie. verlag des naturhistorischen museums, wien, 239 p. biggs, j., williams, p., whitfield, p.n., weatherby, a. 2005: 15 years of pond assessment in britain: results and lessons learned from the work of pond conservation. aquatic conservation: marine and freshwater ecosystems 15: 693–714. céréghino r., ruggiero, a., marty, p., angélibert, s. 2008: biodiversity and distribution patterns of freshwater invertebrates in farm ponds of a south-western french agricultural landscape. hydrobiologia 597: 43–51. clarke, k., gorley, r. 2006: primer v6: user manual/ tutorial. primer-e, plymouth. corbet, p.s. 1999: dragonflies: behaviour and ecology of odonata. cornell university press, ithaca, new york. davies, b., biggs, j., williams, p., whitfield, m., nicolet, p., sear, d., bray, s., maund, s. 2008: comparative biodiversity of aquatic habitats in the european agricultural landscape. agriculture, ecosystems and environment 125(1–4): 1–8. de meester, l., declerck, s., stoks, r., louette, g., van de meutter, f., de bie, t., michels, e., brendonck, l. 2005: ponds and pools as model systems in conservation biology, ecology and evolutionary biology. aquatic conservation: marine and freshwater ecosystems 15: 715–725. edington, j.m., hildrew, a.g. 1995: caseless caddis larvae of the british isles. a key with ecological notes. freshwater biological association, scientific publication 53, ambleside, cumbria, 134 p. eiseler, b. 2005: bildbestimmungsschlussel für die eintagsfliegenlarven der deutschen mittelgebirge und des tieflandes. identification key to the mayfly larvae of the german highlands und lowlands. lauterbornia 53: 1–112. elliot, j., humpesch, u., macan, t. 1988: larvae of the british ephemeroptera: a key with ecological notes. fba scientific publication, 145 p. elliot, j., humpesch, u. 2010: mayfly larvae (ephemeroptera) of britain and ireland: keys and review of their ecology. freshwater biological association, ambleside, 152 p. gerken, b., sternberg, k. 1999: die exuvien europäischer libellen (insecta, odonata). the exuviae of european dragonflies. arnika & eisvogel, höxter, jena, 354 p. gleason, j.e., rooney, r.c. 2018: pond permanence is a key determinant of aquatic macroinvertebrate community structure in wetlands. freshwater biology 63(3) 246–277. glöer, p. 2002: die süßwassergastropoden nordund mitteleuropas. bestimmungsschlüssel, lebensweise, verbreitung. zbirka die tierwelt deutschlands. založba conchbooks, bonn, 327 p. hentges, v.a., stewart, t.w. 2010: macroinvertebrate assemblages in iowa prairie pothole wetlands and relation to environmental features. wetlands 30: 501–511. kubová, n., schenková, j., horsák, m. 2013: environmental determinant of leech assemblage patterns in lotic and lentic habitats. limnologica 43(6): 516–524. lam, p.k.s., calow, p. 1988: differences in the shell shape of lymnea peregra (müller) (gastropoda: pulmonata) from lotic and lentic habitats; environmental or genetic variance? journal of molluscan studies 54(2): 197–207. leibold, m.a., holyoak, m., mouquet, n., amarasekare, p., chase, j.m., hoopes, m.f., holt, r.d., shurin, j.b., law, r., tilman, d., loreau, m., gonzalez, a. 2004: the metacommunity concept: a framework for multi-scale community ecology. ecology letters 7(7): 601–613. mullen, g.r., murphree, c.s. 2019: biting midges (ceratopogonidae). in: mullen, g.r., durden, l.a., (eds.) medical and veterinary entomology. 3rd ed. san diego: academic press. pp. 213–236. nilsson, a. 1997: aquatic insects of north europe. a taxonomic handbook. odonata diptera. volume 2. apollo books, stenstrup, 440 p. oertli, b., indermuehle, n., angélibert, s., hinden, h., stoll, a. 2008: macroinvertebrate assemblages in 25 high alpine ponds of the swiss national park (cirque of macun) and relation to 69 biologica nyssana ● 12 (1) september 2021: 63-70 stamenković et al. ● benthic macroinvertebrate community structure in batušinac ponds (serbia) relative to the distance from a river environmental variables. hydrobiologia 597: 29–41. pfleger, v. 2000: a field guide in colour to molluscs. uk edition. silverdale books, 216 p. previšić, a., graf, w., kučinić, m. 2010: caddisfly (trichoptera) of the plitvice lakes national park (croatia). denisia 29: 287–294. rajkov, s. 2013: pregled staništa akvatičnih stadijuma odonata vojvodine sa ključem za identifikaciju larvi. univerzitet u novom sadu, prirodno-matematički fakultet, departman za biologiju i ekologiju, novi sad. ranđelović, v., matejić, j., zlatković, b. 2007: flora i vegetacija batušinačkih bara kod niša. in: proceedings of 9th symposium on flora of southeastern serbia and neighbouring regions, 1940. siziba, n., chimbari, m.j., masundire, h., mosepele, k. 2011: spatial and temporal variations of macroinvertebrates across temporary floodplains of the lower okavango delta, botswana. physics and chemistry of the earth, parts a/b/c 36(14–15): 939–948. timm, t. 1999: eestirõngusside (annelida) määraja. a guide to the estonian annelidae. estonian academy publishers, tartu-tallinn, 208 p. trigal, c., garcía-criado, f., aláes, c.f. 2007: macroinvertebrate communities of mediterranean ponds (north iberian plateau): importance of natural and human-induced variability. freshwater biology 52(10): 2042–2055. van de meutter, f., de meester, l., stoks, r. 2007: metacommunity structure of pond macroinvertebrates: effects of dispersal mode and generation time. ecology 88(7): 1687–1695. van leeuwen, c.h.a., huig, n., van der velde, g., van alen, t.a., wagemaker, c.a.m., sherman, c.d.h., klaassen, m., figuerola, j. 2013: how did this snail get here? several dispersal vectors inferred for an aquatic invasive species. freshwater biology 58(1): 88–99. walker, p.d., wijnhoven, s., van der velde, g. 2013: macrophyte presence and growth form influence macroinvertebrate community structure. aquatic botany 104: 80–87. wallace, i.d., wallace, b., philipson, g.n. 1990: a key to the case-bearing caddis larvae of britain and ireland. freshwater biological association scientific publication 51, ambleside, cumbria., 237 p. waringer, j., graf, w. 1997: atlas der ӧsterreichischen köcherfliegenlarven: unter einschluss der angrenzenden gebiete. facultas universitätsverlag, wien, 288 p. williams, p., whitfield, m., biggs, j., bray, s., fox, g., nicolet, p., sear, d. 2004: comparative biodiversity of rivers, streams, ditches and ponds in an agricultural landscape in southern england. biological conservation 115(2): 329–341. biologica nyssana ● 12 (1) september 2021: 63-70 70 stamenković et al. ● benthic macroinvertebrate community structure in batušinac ponds (serbia) relative to the distance from a river bogdanović et al. 2022, biologica nyssana 13(2) 13 (2) december 2022: 153-156 doi: 10.5281/zenodo.7437286 antimicrobial activity of evernia prunastri (оakmoss) resinoids original article svetlana bogdanović toplicka academy of applied studies, department of agricultural and food studies, prokuplje, serbia celebogdanovic@gmail.com (corresponding author) ivana zlatković toplicka academy of applied studies, department of agricultural and food studies, prokuplje, serbia dobrila ranđelović toplicka academy of applied studies, department of agricultural and food studies, prokuplje, serbia received: september 14, 2022 revised: november 04, 2022 accepted: november 15, 2022 abstract: infections caused by strains of bacteria that show resistance to a large number of antibiotics represent one of the leading problems today. the aim of this work is to examine new, natural resources with potential antimicrobial effects. the antibacterial activity of lichen resinoids (evernia prunastri) was tested against reference strains staphylococcus aureus atcc 25923, staphylococcus epidermidis atcc 12228, pseudomonas aeruginosa atcc 27853, escherichia coli atcc 25922 and clinical isolates of bacteria s. aureus, s. epidermidis, p. aeruginosa and e. coli, by the disc-diffusion method. each 10 µl of resinoid, of different concentrations, was applied to sterile discs with a diameter of 6 mm. the tested resinoid exhibited the most significant antibacterial activity against pseudomonas aeruginosa atcc 27853, while it was the weakest against the clinical isolate of escherichia coli. the results indicate that the tested oakmoss resinoid shows moderate antimicrobial activity against the tested strains. key words: antimicrobial activity, resinoid, lichen, bacteria apstrakt: antimikrobna aktivnost rezinoida vrste evernia prunastri (hrastova mahovina) infekcije izazvane sojevima bakterija koji pokazuju rezistenciju na veći broj antibiotika predstavljaju jedan od vodećih problema današnjice. cilj ovog rada je ispitivanje novih, prirodnih resursa sa potencijalnim antimikrobnim dejstvom. antibakterijska aktivnost rezinoida hrastove mahovine (evernia prunastri) ispitivana je na referentne sojeve staphylococcus aureus atcc 25923, staphylococcus epidermidis atcc 12228, pseudomonas aeruginosa atcc 27853, escherichia coli atcc 25922 i kliničke izolate bakterija s. aureus, s. epidermidis, p. aeruginosa i e. coli, disk-difuzionom metodom. po 10 µl rezinoida, različitih koncentracija nanošeno je na sterilne diskove prečnika 6 mm. ispitivani rezinoid ispoljio je najznačajniju antibakterijsku aktivnost prema pseudomonas aeruginosa atcc 27853, dok je najslabiji prema kliničkom izolatu escherichia coli. rezultati ukazuju da ispitivani rezinoid hrastove mahovine pokazuje umerenu antimikrobnu aktivnost na ispitivane sojeve. ključne reči: antimikrobna aktivnost, rezinoid, hrastova mahovina, bakterije introduction the introduction of antimicrobial agents in the treatment of infectious diseases saved millions of lives. antimicrobial agents have the ability to eliminate microorganisms by acting on various metabolic and structural targets such as interfering with cell wall synthesis, with nucleic acid synthesis or by other ways of inhibiting (tenover, 2006; bobbarala, 2012). the production of the first antibiotics represents the greatest achievement in medicine. however, bacterial resistance to certain antibiotics increases significantly with age (grundmann et al., 2011). tenover (2006) cites as known cases of resistance, resistance of escherichia coli to third generation cephalosporins, emergence of vancomycin-resistant staphylococcus aureus and multiresistance of pseudomonas aeruginosa, which is why there is a constant need to find new antimicrobial agents. according to data from the world health organization (2020), not a single new class of antibiotics has been developed since 1987. until now, the most clinically important classes of antibiotics are derived from molds, but we can except that basis for the identification of new antimicrobial compounds can be lichen. lichens represent a community formed by algae or cyanobacteria and fungi, developing a mutually beneficial relationship or symbiosis (calcott et al., © 2022 bogdanović et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 153 14th symposium on the flora of southeastern serbia and neighboring regions 2018). in this symbiosis, algae or cyanobacteria represent the photobiont, while fungi represent the mycobiont. nash (2008) considers lichen evernia prunastri (l.) ach. „vegetation pioneers” because of their high stress tolerance. most lichen are slowgrowing and long-lived, which indicates that they retain an unchanged morphology for a long period of time (nash, 2008). evernia prunastri (l.) ach. is widely distributed in the northern hemisphere. it belongs to the family parmeliaceae, genus evernia, and is popularly known as oak or plum moss. according to the type of structure, it belongs to foliose lichen. they have a dichotomously branched thallus, with more or less branches that are attached at one point. it reaches a width between 0.5 and 3 mm. the upper surface of the lichenis greenish-gray to pale green-yellow in color. the lower surface of the thallus in healthy lichen is the same or a shade lighter in color. if the lichen is damaged, its color becomes grayish and less intense. e. prunastri grows on the bark of deciduous trees, most often oak, while occasionally it can be found on conifers in areas of high humidity (shcherbakova et al., 2021). therefore, it is not surprising that this species is widely distributed on the territory of serbia. it likes moist, sunny areas at lower altitudes, and is very rarely found in areas higher than 1,675 m above sea level (nash, 2001). the species e. prunastriis acidophilic and sensitive to the presence of nitrogen, especially ammonia from the air. e. prunastri is characterized by usnic acid, atranorin and chloratranorin in the upper cortex, while evernic acid is found in the medulla. about 170 compounds were identified in the extract of this species, of which 47 are depsides, 25 are triterpenes and steroids (joulain & tabacchi, 2008). usnic acid is known as an acid with a broad spectrum of antimicrobial activity against gram-positive bacteria, such as streptococcus spp., bacillus spp., staphylococcus aureus (including methicillinresistant strains) and gram-negative bacteria, such as proteus vulgaris (shcherbakov et al., 2021). evernic acid, although studied to a lesser extent, has shown antimicrobial activity against bacillus mycoides and b. subtilis, escherichia coli and pseudomonas aeruginosa (shcherbakova et al., 2021). atranorin also shows some antimicrobial activity (studzinskasroka et al., 2017). according to the literature, the resinoid of e. prunastri contains monoterpenes, sesquiterpenes, diterpenes and various terpenoids. also, α-pinene, camphene, β-pinene, limonene, γ-terpinene, p-cymene, trans-pinocarveol, α-copene and α-muurolene are compounds characteristic of both the resinoids and the essential oil of e. prunastri (kahriman et al., 2011). the ancient greeks were still familiar with the healing properties of e. prunastri, from which they made salves for urogenital diseases. more recently, it has been used for diseases of the digestive tract and respiratory tract (crawford, 2015). medical use today is justified by the fact that lichens contain unique biologically active metabolites that have a wide variety of biological effects (antibacterial, analgesic, antiproliferative and cytotoxic) (kosanic et al., 2013). it is often used in research because it is easy to collect, transport and prepare for analysis. materials and methods samples of lichen e. prunastri (fig. 1) were collected in october 2021 in the village of prekopuce, at the foot of the jastrebac mountain. the thallus was dried in the air, in a shady place, at room temperature for 10 days. 154 biologica nyssana ● 13 (2) december 2022: 153-156 bogdanović et al. ● antimicrobial activity of evernia prunastri (оakmoss) resinoids fig. 1. photograph of evernia prunastri (l.) ach. extraction dried lichen (10 g) poured with 100 cm3 of 70% aqueous ethanol solution and left at a temperature of 25 °c. after a certain time, the extract was separated from the dried material by vacuum filtration. the solvent was evaporated in vacuo until a semisolid residue was obtained. the resulting semisolid residue was dried at a temperature of 60 °c biologica nyssana ● 13 (2) december 2022: 153-156 bogdanović et al. ● antimicrobial activity of evernia prunastri (оakmoss) resinoids 155 to a constant mass. the resulting dry residue is the resinoid, that is, the total alcoholic extract. disc-diffusion method to determine the antimicrobial activity of lichen resinoids, laboratory reference cultures from the american type culture collection (atcc) staphylococcus aureus atcc 25923, staphylococcus epidermidis atcc 12228, pseudomonas aeruginosa atcc 27853, escherichia coli atcc 25922 and clinical isolates of bacteria s. aureus, s. epidermidis, p. aeruginosa and e. coli, by the disc-diffusion method. clinical isolates were isolated from swabs of patients’ skin wounds. a turbidity suspension of 0.5 mcfarland containing 1.5x108 cfu/ml was made from overnight cultures of tested strains of microorganisms grown on nutrient agar (nccls national committee for clinical laboratory standards, 2003). on sterile müller-hinton agar (torlak) substrates, 0.1 ml of all prepared microorganism suspensions were inoculated. sterile cellulose discs with a diameter of 6 mm (himedia, milano, italy) were placed on the inoculated surface of the agar plate. the disks were impregnated with 10 µl of different solutions (1, 1/2 and 1/5 v/v) of resinoid in ethanol. a disc with gentamicin (10 μg/ml) was used as a positive control. petri dishes were incubated at 37 °c for 24 hours. after that, the zone of inhibition was read in mm. all growth inhibition assays were performed in triplicate. results and discussion e. prunastri resinoid inhibited the growth of all tested microorganisms; however, their antimicrobial potential was lower than the applied positive control, gentamicin. the results of the disc-diffusion method showed antimicrobial activity against reference strains p. aeruginosa atcc 27853 (depending on the resinoid concentration, the zone of inhibition was from 18 mm to 10 mm), while a significantly weaker antimicrobial activity was shown against s. epidermidis atcc 12228 (depending on the resinoid concentration, the zone of inhibition was from 15 mm to 7 mm). inhibition zone s. aureus atcc 25923 ranged from 14 mm to 9 mm. the weakest antimicrobial effect was shown by e. coli atcc 25922 with an inhibition zone ranging from 10 mm to 8 mm. it is necessary to emphasize that 1/5 v/v ethanol solution of resinoid according to s. aureus atcc 25923 and e. coli atcc 25922 did not show antimicrobial activity (tab. 1). the resinoid showed the most significant antimicrobial activity against the clinical isolate of p. aeruginosa, whose zone of inhibition ranged from 17 mm to 13 mm. it showed moderate antimicrobial activity according to s. epidermidis and s. aureus, whose inhibition zones ranged from 13 mm to 8 mm, that is, from 10 mm to 7 mm. the weakest antimicrobial activity was against e. coli, whose zone of inhibition was from 9 mm to 7 mm (tab. table 1. antimicrobial activity of e. prunastri resinoids bacterial strains gentamicin resinoid e. prunastri 10 µl 1 1/2 1/5 staphylococcus aureus atcc 25923 22 14 9 0 staphylococcus epidermidis atcc 12228 20 15 9 7 pseudomonas aeruginosa atcc 27853 28 18 16 10 escherichia coli atcc 25922 12 10 8 0 staphylococcus aureus isolate from the wound 19 13 8 0 pseudomonas aeruginosa isolate from the wound 26 17 13 0 escherichia coli isolate from the wound 10 9 7 0 156 biologica nyssana ● 13 (2) december 2022: 153-156 bogdanović et al. ● antimicrobial activity of evernia prunastri (оakmoss) resinoids 1). it is important to note that the 1/5 v/v ethanol solution of resinoid did not show antimicrobial activity against any of the tested clinical isolate, but reference strain staphylococcus epidermidis atcc 12228 had inhibition zone of 7 mm and inhibition zone of pseudomonas aeruginosa atcc 27853 was 10 mm. by comparing reference and clinical strains, it is concluded that the highest antimicrobial activity of resinoid in all concentrations is against p. aeruginosa atcc 27853, while it is the lowest against the clinical isolate of e. coli. in this study, the tested e. prunastri resinoid showed relatively low antimicrobial activity. the obtained results are in accordance with the results of testing the essential oil of e. prunastri from algeria, which showed a small effect against e.coli and a moderate antibacterial effect against p. aeruginosa (chahra et al., 2016). however, the obtained results are in contrast with the results of testing the antimicrobial activity of the essential oil of e. prunastri from turkey. in this study, the essential oil of e. prunastri did not show antimicrobial activity against p. aeruginosa, s. aureus, s. epidermidis (aslan et al., 2006). in a study conducted by kahriman et al. (2011), e. prunastri essential oil did not show antimicrobial activity against any tested bacteria. stojanović et al. (2013) showed the antimicrobial effect of e. prunastri against p. aeruginosa and e. coli. conclusions the results of the e. prunastri resinoid test indicate a relatively low antimicrobial potential. the tested microorganisms were not equally sensitive to the presence of lichen resinoids, which shows that the tested lichen possesses compounds with antimicrobial properties, and this requires further research to determine antimicrobial agents that could be used in new drugs for the treatment of infectious diseases in humans and animals, as well as diseases plants. references aslan, a., gulluce, m., sokmen, m., adiguzel, a., sahin, f., ozkan h. 2006: antioxidant and antimicrobial properties of the lichens cladonia foliacea, dermatocarpon miniatum, evernia divaricata, evernia prunastri and neofuscella pulla. pharmaceutical biology, 44: 247-252. bobbarala, v. 2012: antimicrobial agents. intech, isbn 978-953-51-0723-1. calcott, m.j., ackerley, d.f., knight, a. 2018: secondary metabolismin the lichen symbiosis. chemical societz reviews, 47: 1730-1760. chahra, d., ramdani, m., lograda, t., chalard, p., figueredo, g. 2016: chemical composition and antimicrobial activity of evernia prunastri and ramalina farinacea from algeria. biological sciences and pharmaceutical research, 4: 35-42. crawford, sd. 2015: lichens used in traditional medicine. in: ranković b. (ed.) lichen secondary metabolites: bioactive properties and pharmaceutical potential: 27-80, springer international publishing, cham. grundmann, h., de kraker, m., davey, p. 2011: clinical impact of antimicrobial resistance: design matters. the lancet infectious diseases, 11: 344. joulain, d., tabacchi, r. 2009: lichen extracts as raw materials in perfumery. part 1: oakmoss. flavour and fragrance journal, 24: 49-61. kahriman, n., yazici, k., arslan, t., aslan, a., karaoglu, s.a., yayli n. 2011: chemical composition and antimicrobial activity of the essential oils from evernia prunastri (l.) ach. and evernia divaricata (l.) ach. asian journal of chemistry, 23: 1937-1939. kosanić, m., manojlović, n., janković, s., stanojković, t., ranković, b. 2013: evernia prunastri and pseudoevernia furfuraceae lichens and their major metabolites as antioxidant, antimicrobial and anticancer agents. food and chemical toxicology, 53: 112-118. nash, th. 2008: lichen biology, 2nd ed. cambridge university press. cambridge. shcherbakova, a., strömstedt, a., göransson, u., gnezdilov, o., turanov, a., boldbaatar, d., kochkin, d., ulrich‑merzenich, g., koptina, a. 2021: antimicrobial and antioxidant activity of evernia prunastri extracts and their isolates. world journal of microbiology and biotechnology, 37: 129. stojanović, i., radulović, n., cvetković, v., mitrović, t., stamenković, s. 2013: antimicrobial activity of methanol extracts of four parmeliaceae lichen species. facta universitatis-series: physics, chemistry and technology, 11: 45-53. studzinska‑sroka, e., galanty, a., bylka, w. 2017: atranorin an interesting lichen secondary metabolite. mini-reviews medicinal chemistry, 17: 1633-1645. tenover, f.c. 2006: mechanisms of antimicrobial resistance in bacteria. american journal of infection control, 34. bogosavljević, s., zlatković, b.: report on the new floristic data from serbia ii. biologica nyssana, 9 (2). december, 2018 biologica nyssana 9 (2) ⚫ december 2018: 63-75 bogosavljević, zlatković ⚫ report on the new floristic data from serbia ii 63 original article received: 15 november 2018 revised: 29 november 2018 accepted: 12 december 2018 report on the new floristic data from serbia ii stefan bogosavljević1, bojan zlatković2 1lilly drogerie, health institution, trg oslobođenja 14, 19350 knjaževac, serbia 2 university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia * e-mail: stefan.bogosavljevic@gmail.com abstract: bogosavljević, s., zlatković, b.: report on the new floristic data from serbia ii. biologica nyssana, 9 (2). december, 2018: 63-75. this paper presents new chorological data for 16 taxa of vascular flora of serbia (records no. 1-16). these taxa belong to following families: amaryllidaceae (1-2), apiaceae (3), aristolochiaceae (4), aspleniaceae (5), asteraceae (6-9), iridaceae (11), juncaceae (12), ophioglossaceae (13), ranunculaceae (14), rutaceae (15) and urticaceae (16), also including the possibly first record of opuntia humifusa (raf.) raf., cactaceae (10) in serbia. key words: distribution, chorological data ii, serbia, vascular flora apstrakt: bogosavljević, s., zlatković, b.: izveštaj o novim florističkim podacima iz srbije ii. biologica nyssana, 9 (2). decembar, 2018: 63-75. prikazani su novi horološki podaci za 16 taksona vaskularne flore srbije (izveštaji br. 1-16). taksoni pripadaju sledećim familijama: amaryllidaceae (1-2), apiaceae (3), aristolochiaceae (4), aspleniaceae (5), asteraceae (6-9), iridaceae (11), juncaceae (12), ophioglossaceae (13), ranunculaceae (14), rutaceae (15) i urticaceae (16), uključujući i najverovatnije prvi podatak o prisustvu vrste opuntia humifusa (raf.) raf., cactaceae (10) u srbiji. ključne reči: distribucija, horološki podaci ii, srbija, vaskularna flora introduction this study represents a continuation of chorological studies of vascular flora of serbia. new distribution data are presented for 14 plant species and 2 subspecies, including representatives of rare, threatened and protected taxa as well as some invasive species in process of range increase. these data may provide additional information on distribution of taxa included in the edition “flora of 9 (2) • december 2018: 63-75 doi: 10.5281/zenodo.2538596 biologica nyssana 9 (2) ⚫ december 2018: 63-75 bogosavljević, zlatković ⚫ report on the new floristic data from serbia ii 64 sr serbia (i-x)” (josifović ed., 1970-1977; diklić, 1977; nikolić et al., 1986; sarić & diklić, 1986), as well as the taxa that will be included in the new edition “the flora of serbia” (stevanović ed., 1992, 2012). in addition, survey of existing literature yielded no previous data on appearance of the invasive species opuntia humifusa (raf.) raf. (cactaceae) in serbia, so we believe that this was probably the first scientific set of data on its presence in this country. material and methods identification of collected plant material was performed according to josifović ed. (1970-1977), stevanović, ed. (1992, 2012), tutin et al. (1964), nardi (1984), trinajstić (1990), majure et al. (2017) and pardo & alonso (2017). in addition, some of collected specimens (aristolochia lutea desf.) were compared to virtual collections at herbarium of institute of botany, university of vienna (wu) and herbarium of botanic garden and botanical museum berlin-dahlem (b) (http://herbarium.univie.ac.at/ database/search.php). the description of species opuntia humifusa was based on literature sources (petrova et al., 2013; majure et al., 2017) and combined with observations of collected material. plant material was preserved and stored at the herbarium of the faculty of sciences and mathematics, department of biology and ecology, university of niš (hmn), with acquisition numbers given in the brackets. invasiveness status was determined using the terminology according to lambdon et al. (2008). distribution of the studied taxa within the territory of serbia was determined and mapped in utm grid system (10 x 10 sq. km., utm zone 34t) (lampinen, 2001). the nomenclature and classification of taxa were matched with the plant list database (http://www.theplantlist.org/). in order to identify regions of the data occurrence in serbia we used a geographic regionalization according to marković (1970), modified by stevanović ed. (1999). data on population (subpopulation) vulnerability, number of individuals and habitats of the species were based on personal observations. results and discussion amaryllidaceae 1. allium guttatum subsp. dalmaticum (a. kern. ex janch.) stearn (east): surroundings of dimitrovgrad, gulenovci village, odorovačko polje, steppelike habitats in karst field, limestone, 690 m, fn47, 10.06.2016, b. zlatković & n. stanković (hmn 13818). after new literature data on topic of distribution of this critically endangered taxon in flora of serbia were published pertaining to central and southeastern serbia (tomović et al., 2009), the species was also recorded in eastern serbia. all previously known sites for this species in serbia were steppe-like, slightly salt-affected habitats of saltpans. this locality is the first in serbia where this species was recorded on limestone as a substrate lacking salt deposits, and it appears in a small population of 2030 individuals just as in all other localities. 2. galanthus elwesii hook.f. (east): surroundings of knjaževac, mt. tupižnica peak, shrubs and fragments of beech forests, limestone, 1140 m, ep93, 29.03.2017, m. ranđelović & s. bogosavljević (hmn 13819). according to jovanović et al. (2016), this species was present in serbia only in vicinity of niš and pirot in eastern serbia. the new locality is situated on a limestone plateau of mt. tupižnica, in the zone of beech forests, where individuals of this species appear in scrub vegetation, sometimes between stone blocks, as well as within various derivatives of disrupted forest vegetation, primarily in fragments of beech forests. the only species previously recorded in literature for the area of mt. tupižnica was g. nivalis l. (fritsch, 1909). apiaceae 3. trinia ramosissima (fisch. ex trevir.) w.d.j. koch (east): surroundings of knjaževac, podvis village, dry grasslands, 385 m, ep92, 09.06.2016, coll. s. bogosavljević & b. zlatković (hmn 13820). (east): surroundings of niš, kunovica village, ploče plateau, steppe-like grassland, limestone, 659 m, en99, 15.06.2016, b. zlatković (hmn 13822). (east): surroundings of niš, vlase village, mt. seličevica, silicate, 308 m, en78, 29.06.2005, b. zlatković (hmn 13821). in serbia, trinia ramosissima is relatively rare, with a status of protected species (anonymous, 2010). according to butorac et al. (1991) it was recorded in serbia in a small number of localities in vojvodina and central serbia. new research has shown that this species also appears in southwestern serbia (papović et al., 2014). in the localities http://herbarium.univie.ac.at/ biologica nyssana 9 (2) ⚫ december 2018: 63-75 bogosavljević, zlatković ⚫ report on the new floristic data from serbia ii 65 included in this study it was recorded in several small populations in eastern serbia, appearing together with other mostly steppe or steppe-mediterranean elements (chrysopogon gryllus, erysimum diffusum, galatella linosyris, linum tenuifolium, scabiosa micrantha, vinca herbacea). its habitats include dry pastures, edges of forests and forest glades within the thermophilic oak forests. aristolochiaceae 4. aristolochia lutea desf. (fig. 1a, b). (central): ibar river valley, surroundings of ušće, loose, rocky material in juniperus oxycedrus stand, ultramafite, 410 m, dp61, 22.05.2003, b. zlatković (hmn 13823). (central): brezna, brezanska river gorge, rocky places in oak forest, ultramafite, 350 m, dp72, 06. 2001, b. zlatković (hmn 13826); mt. stolovi, meljanica gorge, rocky places and thermophilous oak forest, ultramafite, 400 m, dp72, 05.2001, b. zlatković (hmn 13824). (central): mt. stolovi, čukar, screes and rocky places, serpentinit, 930 m, dp62, 06.2001, b. zlatković (hmn 13827). (western): mt. tara, brusnica gorge, ostryopinetum nigrae, limestone, cp66, 07.2001, b. zlatković (hmn 13825). (south): mt. rujan, orljak, thermophilous oak forest, silicate, em67, 06. 06. 2009, b. zlatković (hmn 13828). this species belongs to aggregate aristolochia pallida which includes following species: a. pallida, a. tyrrhena, a. lutea, a. elongata, a. macedonica, a. croatica and a. merxmuelleri (tatić, 1999). within the taxonomic revision of genus aristolochia l. by nardi (1984) it was determined that the name “aristolochia pallida willd.” should only apply to the taxon appearing in the western part of alps massif, with only a few localities outside of this region, while a. lutea is widely distributed in the regions to the east of the alps, the apennines and balkan peninsula, pannonia, the carpathian mountains and in the most of the asia minor. as a. pallida and a. lutea are morphologically very similar, it should be noted that their most important differential character is ratio between the length of perigone tube and the length of perigone limb. in a. pallida the perigone tube is of equal length or slightly shorter than the limb (which is also wider than the tube), while in a. lutea the perigone tube is significantly longer than the limb, which is of equal width or slightly narrower than the perigone tube (nardi, 1984; trinajstić, 1990) (fig. 1b). considering these facts we believe that hypothesis by tatić et al. (1995), that a. lutea is present in serbia but misidentified as a. pallida in floras, scientific papers and herbarium collections, is justified. this species was first recognized as a. pallida in flora of serbia by pančić (1874), citing its presence at two localities in šumadija. gajić (1970) considers it widespread in serbia but without citing precise localities. however, in the new edition of flora of serbia (gajić, 1992) it is stated that a. pallida does not live in serbia, while data by mayer & greuter (1985), stating that a. lutea is present in serbia, were not taken in consideration. according to our research and the literature references on distribution of a. pallida, which within the study area with high probability pertains to the present species (pančić, 1874; ilić, 1900; ranđelović & stamenković, 1979; ranđelović, 1979/1980; ranđelović & stamenković, 1984; stamenković, 1985; tatić et al., 1995; jotić et al., 2013), it may be concluded that a. lutea appears in several regions of serbia (fig. 1a), where it is represented by small populations isolated from each other. aspleniaceae 5. asplenium lepidum c. presl (east): surroundings of knjaževac, stogazovac village, ždrelo gorge, crevices in sheltered parts of rocks, 390 m, limestone ep93, 08.05.2016, coll. m. ranđelović & s. bogosavljević (hmn 13829). this species of fern inhabits specific protected shaded habitats in crevices of limestone rocks, often appearing in gorges and canyons of hill region (stevanović et al., 1991). due to its specific ecological demands it is relatively rare and localized in serbia, mostly in eastern and western part of state (stevanović et al., 1991). asteraceae 6. bidens connata muhl. ex willd. (northeast): the town of prahovo, prahovo port, banks of the danube, 40 m, fq20, 22.09.2016, coll. s. bogosavljević, m. ranđelović & b. zlatković (hmn 13830). (northeast): mihajlovac village, banks of the danube, 40 m, fq11, 22.09.2016, coll. s. bogosavljević, m. ranđelović & b. zlatković (hmn 13831). (northeast): the town of kladovo, banks of the danube, ruderal, 40 m, fq24, 29.10.2016, coll. m. ranđelović & s. bogosavljević (hmn 13832). bidens connata is characteristic for north america as an autochthonous species, however today biologica nyssana 9 (2) ⚫ december 2018: 63-75 bogosavljević, zlatković ⚫ report on the new floristic data from serbia ii 66 its presence is also recorded in 14 european countries, including serbia where it is considered an invasive naturalized species (bogosavljević & zlatković, 2015). new data on distribution of this species in serbia pertain to localities in north-eastern serbia along river danube, at the romanian border. in romania this species was recorded only in the southeastern part of country (anastasiu et al., 2007), but the results of our study indicate that it should be expected in southwestern romania as well as in northwestern parts of bulgaria. fig. 1. a distribution of aristolochia lutea in serbia; b a. lutea flower; c ophioglossum vulgatum general appearance; d distribution of o. vulgatum in serbia (legend: black circles literature data, red circles new data, white circles imprecise literature data). biologica nyssana 9 (2) ⚫ december 2018: 63-75 bogosavljević, zlatković ⚫ report on the new floristic data from serbia ii 67 7. bidens vulgata greene (fig. 2a). (northeast): surroundings of negotin, srbovo village, jasenička reka channel, nitrophilous, damp places along the forest roads, 50 m, fp39, 22.09.2016, coll. s. bogosavljević, m. ranđelović & b. zlatković (hmn 13833). the only known record of this species is from a single locality in northwestern serbia (tatić & žukovski, 1973). it originated in north america and was first introduced to europe in mid-20th century, to france and romania, while today it is present in a number of european countries (petrova & vladimirov, 2009). however, due to high morphological and ecological similarity to the widespread species b. frondosa, b. vulgata probably remained unnoticed in appropriate habitats in most of its artificial range. 8. erigeron sumatrensis retz. (syn. conyza sumatrensis (retz.) e. walker, c. albida spreng.) (northeast): the town of kladovo, banks of the danube, ruderal, 50 m, fq24, 29.10.2016, coll. m. ranđelović & s. bogosavljević (hmn 13835). (east): surroundings of knjaževac, rgošte village, ruderal, 250 m, ep92, 25.08.2016, coll. m. ranđelović & s. bogosavljević (hmn 13834). (šumadija): the town of ripanj, ruderal, 255 m, dq64, 15.09.2017, m. ranđelović & s. bogosavljević (field obs.). new data on distribution of e. sumatrensis in serbia indicate that the invasive character of this species is becoming more pronounced. although until recently it was known from central, south and eastern serbia (zlatković & bogosavljević, 2014) and belgrade (niketić & jovanović, 2002), the new data also indicate its presence in northeastern serbia. the locality in northeastern serbia is situated immediately next to the romanian border, so this species should be also expected in southwestern romania, in addition to known records from the southeastern part of that country (anastasiu & memedemin, 2012). this species reaches high abundance of individuals in all new localities in serbia, inhabiting various ruderal habitats within human settlements. 9. symphyotrichum novae-angliae (l.) g.l.nesom (syn. aster novae-angliae l.) (east): surroundings of knjaževac, rgošte village, banjica pool, arable fields, 235 m, ep92, 13.10.2013, coll. b. zlatković & s. bogosavljević (hmn 13836). new data on distribution of this invasive species indicate that it continues to spread through serbia, as expected due to its invasive potential. so far it has been recorded in eastern and western serbia as naturalized, while in many localities it was recorded as a cultivated species (lakušić & jovanović, 2012). the population recorded near knjaževac includes self-propagated individuals in wetlands along the riverbank and the peripheral parts of cultivated plots. monitoring of this species in the last several years (until 2017) has shown no significant increase in number of individuals or tendencies of range spread. cactaceae 10. opuntia humifusa (raf.) raf., med. fl. 2: 247. (1830) (fig. 2b, c, d) (southeast): pčinja valley, jablanica village, dry sand and rocky places, 475 m, em78, 09.07.2016, coll. b. zlatković & s. bogosavljević (hmn 13837). family cactaceae includes about 1810 species, with natural range in tropical and subtropical areas of america (essl & kobler, 2008). however, several representatives of this family are considered to be significant allochthonous elements distributed in other warmer parts of the world, while in europe their invasiveness is mostly connected to the mediterranean region (essl & kobler, 2008). there are 26-29 invasive species of cacti recorded in europe (lambdon et al., 2008; novoa et al., 2015). according to essl & kobler (2008) and novoa et al. (2015), most invasive species from family cactaceae, at both global (27) and european (20) level, belong to genus opuntia (l.) mill., which at the same time is also the richest genus of this family (essl & kobler, 2008). as a number of species of this genus is used in horticulture, in suitable conditions individuals often show subspontaneous, either reproductive or vegetative dispersal into natural ecosystems (tashev, 2012). presence of this species in serbia was presented in a study by tashev (2012), where it is mentioned as a naturalized species, and that author cites erre et al. (2009). however, the overview of original data (erre et al. 2009) indicates that this was an erroneous interpretation. the other literature data from serbia do not show presence of o. humifusa. opuntia humifusa is a succulent plant with strongly branching, often creeping and rooting stems. the vegetative shoots in this cactus include tightly connected cladodes. they form links of 1-4 segments, often with lateral branches at the top and at the base. the cladodes are elliptical to oval, dark green, fleshy, sometimes with ridged stripes, 3-17 cm long and 4-12 cm wide, with 4-5 diagonal areoles at the widest part. the glochids are numerous, biologica nyssana 9 (2) ⚫ december 2018: 63-75 bogosavljević, zlatković ⚫ report on the new floristic data from serbia ii 68 inconspicuous. spines are absent, which is one of the main characteristics setting this species apart from the other species of complex opuntia humifusa. the flowers are 5-9 cm in diameter, hermaphroditic, while outer layers of perianth are dark green to slightly greyish-green in color. there are 8-9 inner segments and they are bright yellow. the stamens are numerous, with yellow or yellowish-green filaments. the stigma is white, with 6-7 lobes. the fruit is a berry, 2.5-5 cm long, at first green but becoming red to orange-red as it matures, with areoles and glochids. there are numerous seeds, 4-5 mm wide and with thick coating. the number of chromosomes is 2n=44. fig. 2. a bidens vulgata infructescence; b opuntia humifusa flower, c general appearance, d mowed plants; e iris sibirica flower; f haplophyllum suaveolens general appearance; g distribution of h. suaveolens in serbia (legend as in fig.1). biologica nyssana 9 (2) ⚫ december 2018: 63-75 bogosavljević, zlatković ⚫ report on the new floristic data from serbia ii 69 genus opuntia shows high taxonomic complexity and most species are characterized by high infraspecific variability (fateryga & bagrikova, 2017). some of the species may also be represented by atypical forms within their secondary range, so there are objective problems in species identification (fateryga & bagrikova, 2017). during the last taxonomic revision there were 8 recognized species within the complex o. humifusa s.l. (majure et al., 2017). opuntia humifusa is morphologically most similar to species o. mesacantha raf., which is characterized by presence of spines at least in the terminal part of cladodes. parts of stem in o. mesacantha are more or less easily separable from each other, while there are just 3-4 areoles in the diagonal of the widest part of cladodes. population of o. humifusa in pčinja valley is present in dry grassland and stony areas, along the local road, most probably representing a garden escape. other plants recorded in the same habitat include: centaurea stoebe, chondrilla juncea, clinopodium alpinum subsp. hungaricum, cynodon dactylon, erodium cicutarium, filago arvensis, herniaria glabra, petrorhagia saxifraga and scabiosa argentea. the habitats described above are in direct contact with stands of association scabiosotrifolion dalmatici, as evidenced by the combination of weed and pontian-submediterranean elements developing under similar ecological conditions. in this phase of spread, the species is successfully reproducing by vegetative means. although certain individuals produce abundant flowers and fruits, it is not known whether other means of dispersion are present at this locality. population of o. humifusa in pčinja valley presently occupies space of several ares and is observed to a hundred individuals. this population is observed for several years, and we believe that introduction of this species took place a few decades ago. mowing was recorded at the locality, leading to partial hindrance to further spread of this species. analysis of existing data on distribution of this species in europe (fateryga & bagrikova, 2017) indicates that it is present in 10 countries including serbia. in half of these countries it is considered an invasive species, and in other half a naturalized species. taking into account the total abundance of population, the types of habitats where this species grows and the period of time elapsed since the first record in serbia, we conclude that o. humifusa should be considered to have the status of a casual alien species in our country. at this moment, the species does not pose any significant threat to autochthonous ecosystems and habitats in areas where it has been recorded. iridaceae 11. iris sibirica l. (fig. 2e). (east): surroundings of knjaževac, podvis village, reed and sedge beds, 385 m, ep92, 26.05.2016, coll. s. bogosavljević (hmn 13838). in serbia, iris sibirica is one of the strictly protected species (anonymous, 2010). according to kostrakiewicz (2007), it belongs to the euro-siberian element. its natural area includes central and eastern europe, from northeastern turkey, european part of russia and western siberia to northern italy, but presently it is rare throughout the range (kostrakiewicz-gierałt, 2013). in balkan peninsula this species is rare with a disjunctive range in central bosnia, bulgaria, montenegro and serbia. in serbia it was recorded at several localities in vojvodina, central and western part of country (stevanović et al., 1993). at the new locality its population includes a small number of individuals thriving in an area of only several square meters. the other species at the site include: anacamptis palustris, iris pseudacorus, ophioglossum vulgatum, salix caprea, scirpus silvaticus, typha latifolia etc. it is necessary to implement special protection measures, while the main negative factors threatening the remaining individuals of i. sibirica in this habitat include: drying and turning the natural habitats into cultivated land, succession of wet meadows and reed beds into shrub and forest vegetation, plantation farming of poplar trees etc. juncaceae 12. juncus capitatus weigel (east): surroundings of pirot, temska village, temštica river, wet, eroded sandy and rocky places, 500 m, fn39, 28.05.2016, coll. s. bogosavljević, m. ranđelović & b. zlatković (hmn 13839). this threatened taxon was until recently believed to have gone extinct from the territory of serbia (ranđelović, 1999), but this study has shown presence of a third known site (tomović et al., 2009; zlatković & bogosavljević, 2014). this species was first recorded in eastern serbia, on eroded wet areas along roads, on silicate substrate. ophioglossaceae 13. ophioglossum vulgatum l. (fig. 1c, d) (east): surroundings of knjaževac, podvis village, wet meadows, 385 m, ep92, biologica nyssana 9 (2) ⚫ december 2018: 63-75 bogosavljević, zlatković ⚫ report on the new floristic data from serbia ii 70 26.05.2016, coll. s. bogosavljević (hmn 13840). (east): surroundings of svrljig, lalinac village, lalinac river, wet meadows, 385 m, ep80, 29.04.2016, coll. s. bogosavljević, m. ranđelović & b. zlatković (hmn 13841). (east): surroundings of pirot, barje čiflik village, čeltaš, wet meadows, 390 m, fn27, 28.05.2016, coll. s. bogosavljević, m. ranđelović & b. zlatković (hmn 13842). (east): surroundings of knjaževac, gornja kamenica village, papratska river, wet meadows, 360 m, fp11, 02.06.2017, coll. s. bogosavljević, m. ranđelović & b. zlatković (hmn 13843). (east): surroundings of dimitrovgrad, mazgoš village, alluvium, 661 m, salicetum albofragilis, fn56, 22.06.2013, coll. b. zlatković & n. stanković (hmn 13844); protopopinci village, alluvium, 666 m, wet meadows, molinio-arrhenatheretea, fn56, 22.06.2013, coll. b. zlatković & n. stanković (hmn 13845). (east): sićevo gorge, dolac village, alluvium, 265 m, wet meadows, en98, 28.05.2016, coll. b. zlatković, s. bogosavljević & m. ranđelović (hmn 13846). according to krivošej et al. (2013), ophioglosum vulgatum is distributed from the northernmost areas in serbia, in bačka, across central, west, northeastern and eastern serbia, as well as kosovo and metohija. the new points of distribution indicate presence of a number of sites for this species in eastern and southeastern serbia. this species inhabits fragile habitat types such as wetland and marsh meadows and reed beds, but recorded abundance in new localities was never particularly high. the map of present distribution in serbia (stevanović ed., 1992; zlatković & ranđelović, 1993/4; mihajlović, 1995; ranđelović et al., 2000; zlatković, 2011; krivošej et al., 2013), including new data on range, is presented in fig. 2d. in serbia this species is included in the group of plant species protected by law (anonymous, 2010). ranunculaceae 14. eranthis hyemalis (l.) salisb. (fig. 3a, b, c, d, e) (east): surroundings of knjaževac, podvis village, thermophilous oak forests and edges, 325-395 m, n-ne, ep92, 02.03.2017, coll. m. ranđelović & s. bogosavljević, 31.03.2017, coll. m. ranđelović & s. bogosavljević and 11.04.2017 coll. m. ranđelović & s. bogosavljević (hmn 13848, 13847, 13858); orešac village, golemi kamen, thermophilous oak forests, 365 m, ep92, 05.02.2018, coll. m. ranđelović & s. bogosavljević (hmn 13851). (east): surroundings of knjaževac, orešac village, ploča, carpino orientalis-quercetum mixtum, 600 m, ep91, 13.03.2018, coll. m. ranđelović & s. bogosavljević (hmn 13852). (east): surroundings of zaječar, vratarnica village, zmijanac (provalina), oak forests, 235 m, fp04, 24.04.2017, coll. m. ranđelović & s. bogosavljević (hmn 13849). (east): the town of niš, gorica hill (tri lipe), forest fragments and lime plantations in suburban area of the city, 265 m, en79, 12.02.2017, coll. b. zlatković (hmn 13850). in serbia eranthis hyemalis is included in the category of strictly protected species (anonymous, 2010), and as a critically endangered taxon it was included in the red book of flora of serbia (budak, 1999). at the locality in eastern serbia (podvis), this species lives at the edges but also inside thermophilic oak forests developed on higher, warmer slopes of the gorge of svrljiški timok. at this locality, the population developed on the surface area of about 10 ha in form of seven smaller spatial fragments, each with 50-100 individuals. as part of this area is mostly inaccessible, we assume that this population includes a much greater number of individuals. our estimate of ratio of flowering/fruit-bearing vs. juvenile individuals in this population is 30:70. in addition to e. hyemalis, the habitat at podvis site also includes following herbaceous species: anemone apeninna, arabis procurrens, asplenium ceterach, a. trichomanes, cardamine graeca, corydalis solida, ficaria verna, fragaria vesca, glechoma hederacea, hedera helix, helleborus odorus, lamium purpureum, orchis purpurea, polypodium vulgare, potentilla micrantha, ruscus aculeatus, smirnium perfoliatum etc. approximation of population (subpopulation) size at a different locality within the same gorge (golemi kamen) has shown that number of e. hyemalis was even greater than in the first mentioned locality, considering more than a thousand individuals. as both localities are situated within the same utm square, the total number of individuals in the utm square is probably more than 1500. at the locality ploča there were only a few dozen recorded individuals within the stand of oriental hornbeam shrub. another group (100-150 individuals) of e. hyemalis was also recorded in anthropogenously altered forest stands and linden plantations at hill gorica (tri lipe) in immediate vicinity of niš. however, this population develops in an immediate vicinity of a human settlement and almost certainly is subspontaneous in origin. biologica nyssana 9 (2) ⚫ december 2018: 63-75 bogosavljević, zlatković ⚫ report on the new floristic data from serbia ii 71 the observe of number of individuals in other populations in eastern serbia, at localities (vratarnica, provalina) known from literature sources (petrović & lakusić, 2017), is at about several hundred individuals, but a surprisingly small number of reproductively mature individuals was recorded in field studies. in all known localities a high percentage of individuals were infected by a type of parasitic fungus. fig. 3. a eranthis hyemalis flower, b fruit, c general appearance, native habitat, d subspontaneous specimens; e distribution of e. hyemalis in serbia and observed number of individuals (legend: circles with black dots in center literature data, circles with white dots in center new data): white circle not observed, green circle above 10000 individuals, purple circle between 1500 and 10000 individuals, yellow circle between 100 and 1500 individuals, red circle under 100 individuals; black circle probably extinct population. biologica nyssana 9 (2) ⚫ december 2018: 63-75 bogosavljević, zlatković ⚫ report on the new floristic data from serbia ii 72 in light of new literature data (petrović & lakusić, 2017), and results of our own fieldwork, e. hyemalis should remain within the group of critically endangered (cr) taxa of flora in serbia, as it is represented by a small number of spatially distant natural populations with a relatively small number of individuals. however, it is important to note that the region of eastern serbia, according to observed population sizes in all known localities, contains a significant part of population of this species in serbia. localities in vicinity of knjaževac and zaječar are, after the locality near bačka palanka (panjković et al., 2015; kiš et al., 2016), certainly the richest habitats in serbia. therefore we believe that it is necessary to take conservation measures so this species would survive in these localities in eastern serbia. the locality near vratarnica is already enclosed within the nature park stara planina (anonymous, 2008), but the locality near knjaževac is not included in any form of spatial protection. the modified distribution map of e. hyemalis in serbia, with addition of new distribution points and data on reevaluated number of individuals in comparison to budak (1999) and petrović & lakusić (2017), is presented in fig. 3e. rutaceae 15. haplophyllum suaveolens ledeb. (fig. 2f, g) (east): surroundings of svrljig, plužina village, visoka stena, dry grasslands, 750 m, ep81, 18.05.2018, coll. m. ranđelović & s. bogosavljević (hmn 13853). (east): surroundings of dimitrovgrad, dry steppe-like habitats, 610 m, fn46, 15.05.2018, coll. s. bogosavljević, m. ranđelović & b. zlatković (hmn 13854). (east): surroundings of niš, sićevo village, rocky places, 360 m, en89, 20.05.2014, coll. b. zlatković (hmn 13855). (east): surroundings of niš, kamenica village, dry grasslands, 440 m, ep70, 23.05.2014, coll. b. zlatković (hmn 13856). the range of this steppe species reaches from southern siberia to asia minor on one side, and romania and the eastern part of balkan peninsula on the other side (mayer & wraber, 1974). within the balkan peninsula the species is present in bulgaria, macedonia, greece, albania and serbia (hayek, 1924; tutin et al., 1968). it inhabits dry, grassy, partially rocky terrain on limestone substrate. haplophyllum suaveolens is a strictly protected species in serbia (anonymous, 2010). population size is observed to a few hundred in vicinity of svrljig and only a handful of individuals in new localities in vicinity of niš and dimitrovgrad. the present distribution of this species in serbia is presented in fig. 2g, according to literature data (diklić, 1973; lakušić & niketić, 1988; ranđelović et al., 2000; ranđelović et al., 2007; pavlović et al., 2018) and results of our current research. urticaceae 16. parietaria lusitanica l. subsp. serbica (pančić) p.w.ball (east): surroundings of knjaževac, stogazovac village, ždrelo gorge, fissures of sheltered rocks, 390 m, limestone ep93, 08.05.2016, coll. m. ranđelović & s. bogosavljević (hmn 13857). this subendemic species inhabits limestone rock shelters, cave entrances and other similar habitats of southern carpathians, dobrogea and eastern part of balkan peninsula (stevanović et al., 2014). the new locality fits within the southwestern part of global range of this rare species. in serbia, parietaria lusitanica subsp. serbica is protected by law (anonymous, 2010). acknowledgements. the authors would like to thank milica ranđelović for help during the field work. the ministry of education, science and technological development of the republic of serbia (grant 173030) supported this research. references anastasiu, p., negrean, g., basnou, c., sîrbu, c., oprea, a. 2007: a preliminary study on the neophytes of wetlands in romania. in: rabitsch, w., essl f. & klingenstein, f. (eds.): biological invasions – from ecology to conservation. neobiota, 7: 181-192. anastasiu, p., memedemin, d. 2012: conyza sumatrensis: a new alien plant in romania. botanica serbica, 36 (1): 37-40. anonymous. 2008: uredba o utvrđivanju prostornog plana područja parka prirode i turističke regije stara planina. službeni glasnik republike srbije 115/08. anonymous. 2010: pravilnik o proglašenju i zaštiti strogo zaštićenih i zaštićenih divljih vrsta biljaka, životinja i gljiva. službeni glasnik republike srbije 36/09. bogosavljević, s., zlatković, b. 2015: two alien species of bidens (compositae), new to the flora of serbia. phytologia balcanica, 21 (2): 129-138. budak, v. 1999: eranthis hyemalis (l.) salisb. in: stevanović, v. (ed.), the red data book of flora of serbia, 1: 287-289, ministry of environment of the republic of serbia, faculty of biology, biologica nyssana 9 (2) ⚫ december 2018: 63-75 bogosavljević, zlatković ⚫ report on the new floristic data from serbia ii 73 university of belgrade, instution for protection of nature of the republic of serbia, belgrade. butorac, b., stojanović, s., pal, b., ranđelović, n. 1991: prilog horologiji roda trinia hoffm. na području srbije. zbornik radova, univerzitet u nišu, tehnološki fakultet u leskovcu, 1 (7-8): 69-72. diklić, n. 1973: haplophyllum a. juss in: josifović, m. (ed.), flora sr srbije, 5: 66-68, srpska akademija nauka i umetnosti. beograd. diklić, n. 1977: dopuna flori sr srbije novim podacima o rasprostranjenju biljnih vrsta in: josifović, m. (ed.), flora sr srbije, 9: 205-210, srpska akademija nauka i umetnosti. beograd. erre, p., chessa, i., nieddu, g., jones, p.g. 2009: diversity and spatial distribution of opuntia spp. in the mediterranean basin. journal of arid environments, 73 (12): 1058-1066. essl, f., kobler, j. 2008: spiny invaders – patterns and determinants of cacti invasion in europe. flora, 204 (7): 485-494. fateryga, v., bagrikova, n. 2017: invasion of opuntia humifusa and o. phaeacantha (cactaceae) into plant communities of the karadag nature reserve. nature conservation research, 2 (4): 26-39. fritsch, k. 1909: neue beiträge zur flora der balkanhalbinsel, insbesondere serbiens, bosniens und der herzegowina i. mitt. naturwiss. vereines steiermark 45: 131-183. gajić, m. 1970: aristolochia l. in: josifović, m. (ed.). flora sr srbije, 1: 194-196, srpska akademija nauka i umetnosti. beograd. gajić, m. 1992: aristolochia l. in: sarić, m. (ed.). flora srbije, 1: 257-259, srpska akademija nauka i umetnosti. beograd. hayek, a. 1924: prodromus florae peninsulae balcanicae 1. repertorium specierum novarum regni vegetabilis, beihefte 30 (1): 1-352. ilić, đ. 1900: prilog flori okruga vranjskog, 1: 1521, in: izveštaj 1899-1900 godine. gimnazija nemanjina u vranju. državna štamparija. beograd. josifović, m. (ed.) 1970-1977: flora sr srbije 1-9. srpska akademija nauka i umetnosti, beograd. jotić, b., miljković, m., marković, m., zlatković, b., ranđelović, v. 2013: the vascular flora of the tepoš plateau around pirot city. biologica nyssana, 4 (1-2): 19-33. jovanović, f., obratov-petković, d., niketić, m., vukojičić, s. 2016: distribution of the genus galanthus l. (amaryllidaceae) in serbia. botanica serbica, 40 (1): 69-81. kiš, a., panjković, b., galić, z., stojšić, v., plavšić, d., vujasinović, a., ponjarac, r. 2016: stanišne predispozicije i planska revitalizacija nalazišta kukurjaka (eranthis hyemalis) na prostoru srp “bagremara”. in proceedings, 2nd international symposium on nature conservation. novi sad, serbia. 383-391. kostrakiewicz, k. 2007: the effect of dominant species on numbers and age structure of iris sibirica l. population on blue moor-grass meadow in southern poland. acta societatis botanicorum poloniae, 76 (2): 165-173. kostrakiewicz-gierałt, k. 2013: the influence of neighbouring species on ecological variation of the selected subpopulations of iris sibirica l. biodiversity research and conservation, 32 (1): 45-52. krivošej, z., prodanović, d., lazarević, p., vasić, p. 2013: ophioglossum vulgatum l. (ophioglosaceae) – in the flora of kosovo and metohija (serbia). natura montenegrina, 12 (2): 395-404. lakušić, d., jovanović, s. 2012: symphyotrichum novae-angliae (compositae) new alien species in serbia. botanica serbica, 36 (1): 67-70. lakušić, d., niketić, m. 1988: novi podaci o rasprostranjenju biljaka u srbiji. biološko istraživačko društvo "josif pančić", zbornik radova. beograd. 43-57. lambdon, p.w., pyšek, p., basnou, c., hejda, m., arianoutsou, m., essl, f., jarošík, v., pergl, j., winter, m., anastasiu, p., andriopoulos, p., bazos, i., brundu, g., celesti-grapow, l., chassot, p., delipetrou, p., josefsson, m., kark, s., klotz, s., kokkoris, y., kühn, i., marchante, h., perglová, i., pino, j., vilà, m., zikos, a., roy, d., hulme, p.e. 2008: alien flora of europe: species diversity, temporal trends, geographical patterns and research needs. preslia, 80: 101–149. lampinen, r. 2001: universal transverse mercator (utm) and military grid reference system (mgrs). downloadable from http://www.luomus. fi/english/botany/afe/map/-utm.htm majure, l.c., judd, w.s., soltis, p.s., soltis, d.e. 2017: taxonomic revision of the opuntia humifusa complex (opuntieae: cactaceae) of the eastern united states. phytotaxa, 290 (1): 1-65. marković, j. 1970: geografske oblasti socijalističke federativne republike jugoslavije. zavod za izdavanje udžbenika i nastavna sredstva srbije, beograd. mayer, e., wraber, t. 1974. die gattung haplophyllum, juss. in jugoslawien. phyton, 16 (1-4): 117-125. mayer, e., greater, w. 1985: aristolochia merxmuelleri, ein neuer serpentin-endemit aus südwest-serbien. botanische jahrbucher fur systematik, pflanzengeschichte und pflanzengeographie, 107 (1-4): 321-327. biologica nyssana 9 (2) ⚫ december 2018: 63-75 bogosavljević, zlatković ⚫ report on the new floristic data from serbia ii 74 mihajlović, t. 1995: prilog poznavanju flore stare planine. zbornik radova x snirs-a. 73-95. nardi, e. 1984: the genus aristolochia l. (aristolochiaceae) in italy. webbia, 38 (1): 221 300. niketić, m., jovanović, s. 2002: conyza sumatrensis nova adventivna vrsta u flori srbije. vii simpozijum o flori jugoistočne srbije i susednih područja sa međunarodnim učešćem, zbornik rezimea, dimitrovgrad. 23. nikolić, v., sigunov, a., diklić, n. 1986: dopuna flori sr srbije novim podacima o rasprostranjenju biljnih vrsta. in: sarić, m., diklić, n. (eds.), flora sr srbije, 10: 259-336. srpska akademija nauka i umetnosti. beograd. novoa, a., le roux, j.j., robertson, m.p., wilson, j.r.u., richardson, d.m. 2015: introduced and invasive cactus species: a global review. aob plants, 7: 1-14. pančić, j. 1874: flora kneževine srbije. državna štamparija, beograd 798 p. panjković, b., stojšić, v., perić, r., rilak, s., kiš, a., vujasinović, a. 2015: mapping the extent of occurrence of winter aconite (eranthis hyemalis (l.) salisb.) in special nature reserve “bagremara” (serbia: vojvodina). book of abstracts. 6 th balkan botanical congress. rijeka. 97. papović, o., miljković, m., ranđelović, n., ranđelović, v. 2014: analysis of the flora of rogozna mountain in southwestern serbia. biologica nyssana, 5 (1): 17-30. pardo, f.m.v., alonso, d.g. 2017: aproximación al conocimiento del grupo opuntia mill. (s.l.) (cactaceae) en extremadura (españa). folia botanica extremadurensis, (11): 51-75. petrova, a.s., vladimirov, v. 2009: two alien species of bidens (asteraceae) new to the bulgarian flora. phytologia balcanica, 15 (3): 367-371. petrova, a., vladimírov v., georgiev, v. 2013: invasive alien species of vascular plants in bulgaria. institue of biodiversity and ecosystem research, bulgarian academy of sciences. 320 p. petrović, i., lakušić, d. 2017: refinding of the critically endangered species eranthis hyemalis (l.) salisb. in western and eastern serbia. botanica serbica, 41 (1): 79-82. doi: 10.5281/zenodo.454889 ranđelović, n. 1979-1980: šumska vegetacija planine seličevice. zbornik radova vi pmf priština. 123-136. ranđelović, n., stamenković, v. 1979: flora of the grdelica gorge. acta biologiae et medicinae experimentalis, 4: 3140. ranđelović, n., stamenković, v. 1984: flora i vegetacija rujan planine u jugoistočnoj jugoslaviji. leskovački zbornik, 24: 375-392. ranđelović, v. 1999: juncus capitatus weigel. in: stevanović, v. (ed.), crvena knjiga flore srbije, 1: 74-75, ministarstvo za životnu sredinu republike srbije, biološki fakultet univerziteta u beogradu, zavod za zaštitu prirode republike srbije, beograd. ranđelović, v., jušković, m., šarac, z. 2007: horološke i ekološke karakteristike stepskih elemenata flore na području istočne i jugoistočne srbije. proceedings 9 th symposium on the flora of southeastern serbia and neighbouring regions, niš: 83-100. ranđelović, v., zlatković, b., živojinović, lj. 2000: ugroženost flore suve planine. proceedings, 6 th symposium on flora of the southeastern serbia, sokobanja. 303-322. sarić, m., diklić, n., (eds.) 1986: flora sr srbije, 10. srpska akademija nauka i umetnosti, beograd. stamenković, v. 1985: flora donjeg toka reke vlasine i njene pritoke lužnice u jugoistočnoj srbiji sa biljnogeografskom analizom. leskovački zbornik, 25: 489-505. stevanović, v., (ed.) 1992: flora sr srbije, 1 (second edition). srpska akademija nauka i umetnosti, beograd. stevanović, v., (ed.) 1999: crvena knjiga flore srbije 1, iščezli i krajnje ugroženi taksoni. ministarstvo za životnu sredinu republike srbije, biološki fakultet univerziteta u beogradu, zavod za zaštitu prirode republike srbije, beograd. stevanović, v., (ed.) 2012: flora sr srbije, 2 (second edition). srpska akademija nauka i umetnosti, beograd. stevanović, v., niketić, m., lakušić, d. 1991: chorological additions to the flora of eastern yugoslavia. flora mediterranea, 1: 121-142. stevanović, v., niketić, m., lakušić, d. 1993: distribution of the vascular plants in yugoslavia (serbia, montenegro) and macedonia, 1. glasnik instituta za botaniku i botaničke bašte univerziteta u beogradu. nova serija, 24-25: 3354. stevanović, v., vladimirov, v., niketić, m., vukojičić, s., jakovljević, k., lubarda, b., tomović, g. 2014: plant species and subspecies discovered by dr. josif pančić 1distribution and floristic importance. botanica serbica, 38 (2): 251-268. tashev, a. 2012: characteristics of the opuntia humifusa (cactaceae) locality in the harmanli district, south bulgaria. phytologia balanica, 18 (1): 11-16. biologica nyssana 9 (2) ⚫ december 2018: 63-75 bogosavljević, zlatković ⚫ report on the new floristic data from serbia ii 75 tatić, b. 1999: aristolochia merxmuelleri greuter & e. mayer. in: stevanović, v. (ed.) crvena knjiga flore srbije, 1. iščezli i krajnje ugroženi taksoni. 367-368. ministarstvo za životnu sredinu republike srbije, biološki fakultet univerziteta u beogradu, zavod za zaštitu prirode republike srbije. beograd. tatić, b., žukowski, w. 1973: bidens vulgata greene in yugoslavia. glasnik instituta za botaniku i botaničke bašte univerziteta u beogradu, 8, nov. ser. 7(1-4): 125-128. tatić, b., krivošej, z., petković, b., atanacković, b., vasić, p. 1995: potreba za revizijom zastupljenosti vrste aristolochia pallida willd. u našoj flori. ii simpozijum o flori srbije (iv simpozijum o flori jugoistočne srbije). abstracts. vranje. 25-26. the plant list. 2013: version 1.1. published on the internet; http://www.theplantlist.org/ tomović, g., zlatković, b., niketić, m., perić, r., lazarević, p., duraki, š., stanković, m., lakušić, d., anačkov, g., knežević, j., szabados, k., krivošej, z., prodanović, d., vukojičić, s., stojanović, v., lazarević, m., stevanović, v. 2009: threat status revision of some taxa from “the red data book of flora of serbia 1”. botanica serbica, 33 (1): 33-43. trinajstić, i. 1990: aristolochia pallida willd. (aristolochiaceae) u flori hrvatske. acta botanica croatica, 49 (1): 143-146. tutin, t.g., heywood, v.h., burges, n.a., moore, d.m., valentine, d.h., walters, s.m., webb, d.a. (eds.) 1964: flora europaea, 1. lycopodiaceae to platanaceae. university press. cambridge. tutin, t.g., heywood, v.h., burges, n.a., moore, d.m., valentine, d.h., walters, s.m., webb, d.a. (eds.) 1968: flora europaea, 2. university press. cambridge. zlatković, b. 2011: flora i fitogeografska pripadnost doline reke pčinje u jugoistočnoj srbiji. doktorska disertacija. univerzitet u beogradu, biološki fakultet. beograd. zlatković, b., bogosavljević, s. 2014: report on the new floristic data from serbia. biologica nyssana, 5 (2): 123-129. zlatković, b., ranđelović, v. 1993-1994: ugroženost i zaštita flore sićevačke klisure. zaštita prirode, 46-47: 191-199. http://www.theplantlist.org/ anticancer compounds from medicinal plants biologica nyssana 5 (1)  september 2014: 31-35 matejić, j. et al.  antimicrobial potential of essential oil from... 31 original article received: 10 july 2014 revised: 21 august 2014 accepted: 10 september 2014 antimicrobial potential of essential oil from pastinaca sativa l. jelena s. matejić 1 , ana m. džamić 2 , tatjana mihajilov-krstev 3 , vladimir n. ranđelović 3 , zoran đ. krivošej 4 , petar d. marin 2 1 university of niš, faculty of medicine, 18000 niš, serbia 2 university of belgrade, faculty of biology, institute of botany and botanical garden “jevremovac”, 11000 belgrade, serbia 3 university of niš, faculty of science and mathematics, 18000 niš, serbia 4 university of priština, department of biology, faculty of natural sciences, 38220 kosovska mitrovica, serbia e-mail: jekamatejic@gmail.com abstract: matejić, j., džamić, a., mihajlov-krstev, t., ranđelović, v., krivošej, z., marin, p.: antimicrobial potential of essential oil from pastinaca sativa l. biologica nyssana, 5 (1), septmeber 2014: 31-35. the aim of the current investigation was to evaluate the antimicrobial effect of pastinaca sativa l. (apiaceae) essential oil. the aerial parts of plants were collected at kopaonik mountain (serbia) and the essential oil has been isolated by hydrodistillation from this plant material. essential oil was dominated by (z)-β-ocimene (10.8%), hexyl butanoate (10.4%), (e)-β-farnesene (6.1%) and lavandulyl acetate (5.2%). the antimicrobial activity of the essential oil was investigated using a micro-well dilution assay against the most common human gastrointestinal pathogenic microbial strains: escherichia coli, pseudomonas aeruginosa, salmonella enteritidis, bacillus cereus, listeria monocytogenes, staphylococcus aureus and yeast candida albicans. the results showed that minimal inhibitory concentrations (mic) and minimal fungicidal concentrations of essential oil ranged from 0.72 μg/ml (for the most sensitive b. cereus) to above 92.5 mg/ml for s. enteritidis and l. monocytogenes. this finding suggests that p. sativa may be considered as a natural source of antimicrobial agents. key words: antimicrobial potential, essential oil, pastinaca sativa l. apstract: matejić, j., džamić, a., mihajlov-krstev, t., ranđelović, v., krivošej, z., marin, p.: antimikrobni potencijal etarskog ulja vreste pastinaca sativa l. biologica nyssana, 5 (1), septmeber 2014: 31-35. cilj ovog rada bio je utvrđivanje antimikrobne aktivnosti etarskog ulja vrste pastinaca sativa l. (apiaceae). nadzemni deo biljke sakupljen je na kopaoniku (srbija). etarsko ulje nadzemnog dela izolovano je metodom hidrodestilacije. kao glavne komponente etarskog ulja bile su: (z)-β-ocimen (10.8%), heksil butanoat (10.4%), (e)-β-farnezen (6.1%) i lavandulil acetat (5.2%). antimikrobna aktivnost etarskog ulja ispitivana je korišćenjem mikrodilucione metode na patogene gastrointestinalnog trakta kao što su: escherichia coli, pseudomonas aeruginosa, salmonella enteritidis, bacillus cereus, listeria monocytogenes, staphylococcus aureus i candida albicans. rezultati su pokazali da su se minimalne inhibitorne koncentracije (mic) kao i minimalne fungicidne koncentracije kretale u opsegu od 0.72 μg/ml (za najosetljiviju bakterijsku vrstu b. 5 (1) • september 2014: 31-35 biologica nyssana 5 (1)  september 2014: 31-35 matejić, j. et al.  antimicrobial potential of essential oil from... 32 cereus) do 92.5 mg/ml za s. enteritidis i l. monocytogenes. na osnovu dobijenih rezultata slobodno se može reći da p. sativa može služiti kao prirodni izvor antimikrobnih agenasa. ključne reči: antimikrobni potencijal, etarsko ulje, pastinaca sativa l. introduction essential oils are complex mixtures of compounds, synthesized in plant tissues as secondary metabolites. the role of essential oil in plant is multiple: defense against pathogens and herbivores, attraction of pollinators, inhibition of other plant's seed germination, formation of protective layer which reduces transpiration and formation of specific microclimate. due to their lipophylic character, essential oils can interact with microbial membranes and achieve significant antimicrobial effect (s t o j a n o v i ć r a d i ć , 2012). in serbian flora, family apiaceae consists of 138 species. from the genus pastinaca, two species are found in serbia p. sativa l. and p. hirsuta panč. (n i k o l i ć , 1973). pastinaca sativa l. is biennial, more or less pubescent plant native to europe except the arctic. stem is hollow or solid, angled or terete. basal leaves are usually simply pinnate. fruit is broadly elliptical and vittae on the commissural face not reaching the ends of the fruit (tutin, 1968). the seed of p. sativa species contains essential oils. the dominant constituents in this oil are myristicin (64.20%), β-ocimene (29.10%) and βfarnesene (24.50%). for this reason p. sativa is used in food industry in serbia (j a n č i ć e t a l ., 1995). in the oil obtained from crushed seeds of p. sativa subsp. urens from turkey, 18 components were characterized representing 95% of the oil with octyl butyrate (79.5%) and octyl hexanoate (5.3%) as the major constituents (k u r k c u o g l u e t a l ., 2006). a whole plant is used for strengthening the appetite, better digestion, and as a diuretic. the fruit is bitter aromatics that increases milk yield (t u c a k o v , 1986). the root is rich in starch and sugar and is used as food (parsnip), animal fodder, and for wine making. the sap is liable to cause skin irritation by sensitizing skin to uv radiation (z e h u i & w a t s o n , 2005). the root is delicious baked, and it can be used in soups, added to cakes, pies and puddings (f a c c i o l a , 1990). leaves and young shoots can be usedcooked with other greens as a vegetable or added to soups (l a u n e r t , 1981; f a c c i o l a , 1990). the seed is used as a condiment (l a u n e r t , 1981). it is similar in taste to dill (f a c c i o l a , 1990). native parts of plants have been applied in feeding as a spice, pharmaceutical industry and folk medicine (n i k o l i ć , 1973). material and methods plant material and isolation of the essential oils aerial parts of p. sativa were collected in july 2003 from kopaonik mountain. a voucher specimen (psa 60316), has been deposited at the herbarium of the institute of botany and botanical garden “jevremovac”, faculty of biology, university of belgrade (beou). the oil from the dried aerial parts (210 g) was separately obtained by hydro distillation for 3 h using a clevenger-type apparatus. yield of the essential oil from the aerial parts was 0.1%, ρ=925.5 g/cm 3 . antimicrobial activity microbial strains the antimicrobial activity of all tested samples was evaluated using laboratory control strains obtained from the american type culture collection: gram (-) bacteria escherichia coli atcc 25922, pseudomonas aeruginosa atcc 9027, salmonella enteritidis atcc 13076; gram (+) bacteria: bacillus cereus atcc 10876, listeria monocytogenes atcc 15313, staphylococcus aureus atcc 25923 and yeast candida albicans atcc 10231. micro-well dilution assay the inocula of the microbial strains were prepared from the overnight broth cultures and suspensions were adjusted to 0.5 mcfarland standard turbidity (corresponding to 10 7 -10 8 cfu/ml, depending on genera consensus standard by the nccls (2003)). serial doubling dilutions of the tested samples (tested oil in 5% dmso) were prepared in a 96/well microtiter plate over the range of 0.05–100 μl/ml (for oil) in inoculated muellerhinton broth. the final volume was 100 μl and the final microbial concentration was 10 6 cfu/ml in each well. the plate was incubated for 24 h at 37 ° c. all experiments were performed in triplicate. two controls were included medium with 30% ethanol (negative control) and medium with streptomycin, chloramphenicol and nystatin (positive control). microbial growth was determined by adding 20 μl of 0.5% triphenyl tetrazolium chloride (ttc) aqueous solution (s a r t o r a t t o et al., 2004). biologica nyssana 5 (1)  september 2014: 31-35 matejić, j. et al.  antimicrobial potential of essential oil from... 33 minimal inhibitory concentration (mic) was defined as the lowest concentration of the samples inhibiting visible growth (red colored pellet on the bottom of the wells after the addition of ttc). to determine mbc/mfc, the broth was taken from each well without visible growth and inoculated in mueller-hinton agar (mha) for 24 h at 37 ° c. minimal bactericidal/fungicidal concentration (mbc/mfc) was defined as the lowest samples concentration killing 99.9% of bacterial/fungal cells. results and discussion chemical analysis of p. sativa essential oil was already tested and the main components were: (z)-β-ocimene (10.8%), hexyl butanoate (10.4%), (e)-β-farnesene (6.1%) and lavandulyl acetate (5.2%), and p. hirsuta was dominated by butyl butyrate (2.5%). in both species, hexyl butanoate is dominant (10.4 and 55.4%, resp.) (k a p e t a n o s e t a l ., 2008). yield of the essential oil for 13 components in this essential oil varied in the range of 5-1% (germacrene d, (+)-γ-terpinene, arcurcumene, α-zingiberene, β-sesquiterpene, (+)-(e)β-caryophyllene, geranyl acetone, myrcene, βbourbonene, nonanyl hexanoate, nonanal, (+)-βpinene, ethyl 2-phenylbutanoate (k a p e t a n o s e t a l . , 2008). in k u b e c z k a & s t a h l (1977) study, oils from the wild parsnip p. sativa were characterized by the presence of octyl acetate. the composition of essential oils in the roots of p. sativa changes during the course of plant development: at the seedling stage (0-18 days), the primary oil ducts between the pericycle cells contain mainly the sesquiterpene hydrocarbon trans-β-farnesene (34.9%) with smaller amounts of myristicin (14.4%) and terpinolene (3.1%); as the roots thicken during the secondary stage (23-30 days) the sesquiterpene content decreases, whereas the myristicin and terpinolene contents increase; and finally, at the adult stage (38-160 days) when secondary oil ducts develop, the percentage of sesquiterpene falls to 1.5%, while that of myristicin and perpinolene increase to 62.6 and 25.5%, respectively (stahlbiskup & wichtmann, 1991). in the oil of p. hirsuta, twenty-nine compounds were identified in root oil and thirteen compounds in fruit oil (92.8% and 90.8% of the total oils, respectively) using gc-fid and gc-ms. the principal components of the root oil were apiole (56.0%) and myristicin (21.0%), followed by βbisabolene (7.2%). the main compounds in the fruits oil were hexyl hexanoate (59.8%) and hexyl butanoate (21.4%) (p e t r o v i ć e t a l . , 2008). our results of antimicrobial activity obtained for the examined essential oil are presented in table 2. we were using the antibiotics streptomycin, chloramphenicol and nystatin for comparison. the essential oil of p. sativa showed the greatest activity against b. cereus strains (mic=mbc=0.72 mg/ml). bacterial strains s. enteritidis and l. monocytogenes showed the lowest susceptibility to the tested essential oil. terpenes, in general, possess a wide spectrum of biological activity through which they appear to play a role in plant defense mechanisms (f r a g a , 2001, 2003). in addition, it was possible that components present in lower amounts in the oil might be involved in some type of synergism with the other active compounds (m a r i n o et al., 2001). due to their bioactivity, the great number of terpenes has been evaluated for antibacterial or antifungal activity. components of essential oil such as p-cymene, α-pinene, β-pinene, limonene, α-terpinene, α–terpinolene, caryophyllene oxide, camphene, 1,8-cineole and linalool have been reported for their antimicrobial activity (v i l j o e n , 2003; k n o b l o c h , 1989; s ö k m e n , 2004). the main components of p. sativa essential oil were not up to now evaluated for their antimicrobial activity. b r k o v i ć e t a l . (2006) tested antimicrobial activity of water, ethanol and ethyl acetate extracts from p. sativa and 11 other plants from the family of apiaceae. comparison with the other species showed that p. sativa had the least antimicrobial activity. the root oil of p. hirsuta pančić was active against all tested microorganisms and exhibited the best activity against b. subtilis (mic 6.25 µg/ml), s. aureus, m. luteus and c. albicans atcc 10259 (mic 12.5 µg/ml). the oil from the fruits of p. hirsuta was active only against e. coli and c. albicans (the most active against c. albicans atcc 10259: mic 12.5 µg/ml) (p e t r o v i ć e t a l . , 2008). when comparing the results, we can conclude that the results of antimicrobial activity points to higher potential of p. hirsuta than the herein tested oil. the reason for this can be different chemical composition of the root and the fruits as well as different solvent in which the oil is dissolved during the antimicrobial testing. biologica nyssana 5 (1)  september 2014: 31-35 matejić, j. et al.  antimicrobial potential of essential oil from... 34 table 2. antimicrobial activity of pastinaca sativa essential oil against pathogenic microbial strains using micro-well dilution assay pathogenic microbial strains p. sativa essential oil (mic/mbc(mfc) in mg/ml) antibiotic (mic/mbc(mfc) in μg/ml) gram (-) bacteria streptomycin e. coli atcc 25922 23.1/92.5 16.0/16.0 p. aeruginosa atcc 9027 46.2/46.2 8.0/8.0 s. enteritidis atcc 13076 92.5/92.5 4.0/4.0 gram (+) bacteria chloramphenicol b. cereus atcc 10876 0.72/0.72 4.0/16.0 l. monocytogenes atcc 15313 92.5/92.5 8.0/16.0 s. aureus atcc 25923 46.2/46.2 1.0/8.0 yeast nystatin c. albicans atcc 10231 46.2/46.2 16.0/16.0 conclusion the essential oil from pastinaca sativa inhibited the growth of tested microorganisms. these microorganisms cause food spoilage and are recognized as human and animal pathogens. thus, the oil from this species deserves further investigation in search for natural food preservatives. acknowledgements. the authors are grateful to the ministry of education and science of the republic of serbia for financial support (grant no. 173029). references brković, l.d., čomić, lj., solujić-sukdolak, s. 2006: antibacterial activity of some plants from family apiaceae in relation to selected phytopathogenic bacteria. kragujevac journal of science, 28: 65-72. facciola, s. 1990: cornucopia a source book of edible plants. kampong publications, california, usa. fraga, b.m. 2001: natural sesquiterpenoids. natural product reports, 18:650–673. fraga, b.m. 2003: natural sesquiterpenoids. natural product reports, 20:392–413. jаnčić, r., stošić, d., mimicа-dukić, n., lаkušić, b. 1995: aromаtične biljke srbije. nip dečije novine, beogrаd-gornji milаnovаc. kapetanos, c., karioti, a., bojović, s., marin, p., veljić, m., skaltsa, h. 2008: chemical and principal-component analyses of the essential oils of apioideae taxa (apiaceae) from central balkan. chemistry and biodiversity, 5:101-119. knobloch, k., iberl, a.p.b., weigand, h., weis, n. 1989: antibacterial and antifungal properties of essential oil components. journal of essential oil research, 1:119–128. kubeczka, k. h., stahl e. 1977: essential oils from the apiaceae (umbelliferae). ii. the essential oils rom the above ground parts of pastinaca sativa. planta medica, 31:173-184. kurkcuoglu, m., baser, k.h.c., vural, m. 2006: composition of the essential oil of pastinaca sativa l. subsp. urens (req. ex godron) celak. chemistry of natural compounds, 42(1):114115. launert, e., 1981: edible and medicinal plants. hamlyn, london. marino m, bersani c, comi g 2001: impedance measurements to study the antimicrobial activity of essential oils from lamiaceace and compositae. international journal of food microbiology, 67: 187–195. nccls – national committee for clinical laboratory standards, performance standards for anti-microbial susceptibility testing: eleventh informational supplement, document m100-s11, 2003: national committee for clinical laboratory standard, wayne, pa, usa. biologica nyssana 5 (1)  september 2014: 31-35 matejić, j. et al.  antimicrobial potential of essential oil from... 35 nikolić v. 1973: fam. apiaceae. in: josifivić m. (ed.), flora of sr serbia 5, beograd, serbia: sanu, 183-348 p. petrović, s., pavlović, m., milenković, m., vučićević, d., couladis, m., tzakou, o., niketić, m. 2008: pastinaca hirsuta essential oils: composition and antimicrobial activity. planta medica, 74-pi7. sartoratto, a., machado, a.l.m., delarmelina, c., figueira, g.m., duarte, m.c.t., rehder, v.l.g. 2004: composition and antimicrobial activity of essential oils from aromatic plants used in brazil. brazilian journal of microbiology, 35:275–280. sökmen, a., gulluce, m., askin akpulat, h., daferera, d., tepe, b., polissiou, m., et al. 2004: the in vitro antimicrobial and antioxidant activities of the essential oils and methanol extracts of endemic thymus spathulifolius. food control, 15(8):627–634. stahl-biskup, e., wichtmann, e.m. 1991: composition of the essential oils from roots of some apiaceae in relation to the development of their oil duct systems. flavour and fragrance journal, 6:249-256. stojanović-radić, z. 2012: antimicrobial activity of the three commercial drug’s essential oils: chamomillae flos, calendulae flos and millefolii herba. biologica nyssana, 3 (2):69-76. tucakov, j. 1986: lečenje biljem, fitoterapija. izdavačka radna organizacija "rad", beograd. tutin, t.g. 1968: genus pastinaca. in: tutin, t.g., heywood, v.h., burges, n.a., moore, d.m., valentine, d.h., walters, s.m., webb, d.a. (eds.), flora europaea 2. cambridge university press, london, uk, p. 364. viljoen, a., van vuuren, s., ernst, e., klepser, m., demirci, b., baser, h., et al. 2003: osmitopsis asteriscoides (asteraceae)-the antimicrobial activity and essential oil composition of a capedutch remedy. journal of ethnopharmacology, 88(2–3):137–143. zehui, p., watson, m.f. 2005: 94. pastinaca linnaeus, sp. pl. 1: 262. 1753. flora of china, 14, 193. biologica nyssana 5 (1)  september 2014: 31-35 matejić, j. et al.  antimicrobial potential of essential oil from... 36 mirić 2022, biologica nyssana 13(1) 13 (1) september 2022: 27-31 doi: 10.5281/zenodo.7117534 population size of the bluethroat (cyanecula svecica) in the lower course of great bačka canal original article radislav mirić mir.radislav@gmail.com (corresponding author) received: september 01, 2021 revised: december 10, 2021 accepted: may 24, 2022 abstract: a census of the territorial males of the bluethroat (cyanecula svecica) was conducted in the year 2014 on the great bačka canal and surrounding wetlands. a total of 32 territorial males were recorded on the total transect length of 67.9 km. bluethroat population in the study area presents 12-20% of the total estimated population of this species in serbia. a significant short-term threat recorded was surface water regime maintenance since the removal of vegetation during this activity led to the destruction of habitat in several potential bluethroat territories. key words: breeding birds, habitat destruction, population density, wetland apstrakt: veličina populacije modrovoljke (cyanecula svecica) u donjem toku velikog bačkog kanala tokom 2014. godine sproveden je popis teritorijalnih mužjaka modrovoljke (cyanecula svecica) na velikom bačkom kanalu i okolnim vlažnim područjima. ukupno je zabeleženo 34 teritorijalna mužjaka na pređenih 67.9 km transekata. populacija modrovoljke zabeležena na ovom području predstavlja 11-19% celokupne procenjene populacije ove vrste u srbiji. kao značajan kratkoročni ugrožavajući činilac zabeleženo je održavanje kanalske mreže, pošto je uklanjanjem vegetacije tokom ove aktivnosti uništeno stanište na nekoliko mogućih teritorija modrovoljke. ključne reči: reproduktivno zrele ptice, uništavanje staništa, gustina populacije, vlažno introduction the bluethroat (cyanecula svecica) is a species of passerine bird belonging to the flycatcher family (muscicapidae). its breeding range covers a vast area from western europe across northern asia to the western coast of alaska (cramp, 1988). the species prefers ecotone habitats such as foresttundra, mountain steps, subalpine bush, coasts of lakes, ponds, and rivers with willows, reed beds, and other emergent vegetation (collar, 2019). it nests under the dense cover of mid-height vegetation with patches of bare ground (collar, 2019). there are 11 subspecies recognized in the world of which two are recorded in serbia. cyanecula svecica is a sporadic migrant, while c. cyanecula is a regular breeder in the northern part of the country. the population breeding in central europe prefers habitats covered with reeds natural and man-made ditches, canals, sewage farms, and fishponds (krüger, 1997; orłowski & sęk, 2005; petersen et al., 2005; van turnhout et al., 2010). the breeding period lasts from april to july, after which it remains in the vicinity until august and even mid-september (tomik, 2011). wintering range covers the mediterranean, sub-saharan africa, and south asia (kováts, 2009; collar, 2019). the breeding population in serbia is estimated at 170-280 breeding pairs with a stable population trend (puzović et al., 2015). although the current population is considered stable, historical data from the 19th century suggest a severe reduction of the breeding range (šćiban et al., 2015). in addition, we assume that bluethroat suffered a severe decline in serbia as a result of wetland drainage and vegetation succession as it happened in neighbouring countries (grüll, 2001). because threatening factors are still abundant, the species is considered near threatened in the red book of the fauna of serbia (mirić et al., 2018). the main aim of this paper is to quantify the population size and density of bluethroat in the lower course of the great bačka canal with surrounding wetlands and to evaluate it’s significance in comparison with a total country population. © 2022 mirić. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 27 materials and methods the study area is located within the vojvodina province (n 45.54°, e 19.90°) and includes a section of the great bačka canal that runs from srbobran to bačko gradište, lower courses of krivaja and beljanska bara rivers which are medium-sized rivers with the slow flow (in the further text slow-flowing rivers). adjacent 3-5 m wide and 1.5-3 m deep drainage ditches and surrounding ponds, that are in some cases completely overgrown with reed (ponds smaller than 10 ha) were part of the study area. listed wetlands are predominantly surrounded by arable lands and have suffered significant changes after reclamation activity that has been undertaken from 19th to the mid-20th century (tomić, 1974; stojšić, 1977). the water level is maintained artificially with little fluctuation throughout the year. the width of the floodplain is 50 m on average. the majority of the banks are covered with reeds which form a belt from 1 to 130 m in width. the water surface is at 78 m a.s.l., whilst surrounding land rises to 85 m a.s.l. (tomić, 1990). the dominant plant species is the common reed (phragmites australis) which creates many communities with the other species such as cattail (typha sp.), grey willow (salix cinerea), crack willow (salix fragilis), and white willow (salix alba) (lakušić et al., 2005; dobretić & stojšić, 2011). the census of bluethroats took place from the 10th to the 23rd of april 2014 on sunny days, without wind and rain, from 4 a.m. to 1 p.m. according to merilä & sorjonen (1994) the census should be ended before 10 a.m. because of a significant decrease in territorial male activity. in this study, since the level of bluethroat activity remained stable well after 8 a.m., census lasted until 1 p.m (fig. 1, fig. 2). a total transect length of 67.9 km was covered, of which: 19.8 km along great bačka canal, 16.3 km along beljanska bara river, 9.6 km along krivaja river, 13.3 km along ponds and 8.9 km along drainage ditches. sampling was undertaken based on the line transects with the stop points and the use of male call tape lure. along each transect, every 300 m, bluethroat song was played two times for 1 minute followed by 1 minute pause for listening after every song playback. distance between count points in this census was increased compared to 200 m that sorjonen & merilä (2000) and peris & mendes (2010) propose in order to reduce the chance of double counting. also, the terrain is deforested and flat, thus there are no obstacles for sound to spread. every male recorded singing or in display flight, which stands for possible and probable breeding in 28 biologica nyssana ● 13 (1) september 2022: 27-31 mirić ● population size of the bluethroat (cyanecula svecica) in the lower course of great bačka canal fig. 1. time of song/song flight recorded without song playback fig. 2. time of every song/song flight recorded european breeding bird atlas methodology (ebcc 2015), is considered territorial. area of suitable habitat, used to calculate bluethroat density, includes reed bed surface around surveyed water bodies, within a distance belt of 60 m from transects. the detection rate of bluethroats diminishes dramatically when they are more than 60 meters away from the observer (musseau & beslic, 2018), hence this distance is chosen. habitat surface was calculated using google earth pro and arcmap 10.8 software and in total it amounts to 171.88 ha, of which: 111.04 ha around slowflowing rivers, 44.34 ha on ponds, and 16.5 ha along drainage ditches. bluethroat density was estimated by dividing the number of territorial males by the area of suitable habitat. google earth pro was also used to calculate reed bed width and to digitalize the location of recorded birds. arcmap 10.8 was used for map production. results during the study period 32 territorial males were recorded (fig. 3). out of this number 18 males were counted around slow-flowing rivers, 9 were situated on ponds and 5 of them were in drainage ditches. the average bluethroat density is 1.86/10 ha. separately for each habitat type, bluethroat 29 densities are the following: 1.62/10 ha for slowflowing rivers, 2.03/10 ha for ponds and 3.03/10 ha for drainage ditches. discussion if we compare the number of territorial males registered in this study (n = 32) with puzović et al. (2015) population estimate for bluethroat in serbia, we can say it represents 11-19% of the total population of the country. number of males registered in study area is minimal, as it is known that bluethroat males significantly reduce their response to a tape lure after acquiring a mate (sorjonen & merilä, 2000) and since they have an evident habit of discretion if observer is too close to it (musseau & beslic, 2018). biologica nyssana ● 13 (1) september 2022: 27-31 mirić ● population size of the bluethroat (cyanecula svecica) in the lower course of great bačka canal however, the estimate given by puzović et al. (2015) was based on sporadic findings from large wetlands (e.g. kormanjoš, 1989; gergelj et al., 2000; žuljević & đapić, 2002; vig et al., 2009; radišić & tucakov, 2010; tucakov, 2011; vig et al., 2012) without any systematic research. this study indicates that small wetlands such as drainage ditches and ponds are significant for this species. this is also shown in the research performed in lower saxony (krüger, 2002), in which around 50% of territories are found on fig. 3. study area with potential bluethroat territories drainage ditches. when we consider that the province of vojvodina has over 20,000 km of drainage ditches (vranešević, 2015), and we multiply it by the 5 males found on 8.9 km of the same habitat in this study, we get an estimate of over 11,000 males just on drainage ditches. unfortunately, there is no available data from other parts of the country, so this estimate should be taken with reserve. a few conspicuous but under-researched habitatrelated occurrences were also noticed. on territories of 18 bluethroat males some amount of burnt reed was registered. some reed beds were reduced to only a few m2 which males used for perch and shelter. a similar case was recorded in poland where the fire occurred during the breeding season, but it did not chase birds away from their territories (orłowski & sęk, 2005). also, 24 bluethroat males were recorded in habitats with the one-year-old reed, which is not in line with the findings of vadász and csörgő (2009) who assumed that such habitat is deprived of species diversity and abundance. it is worth to mention that in the years following this study several cases of bluethroat habitat destruction were recorded in study area. on seven locations where bluethroats were recorded, habitats were destroyed, in some cases even during the breed30 ing season. habitat destruction is the consequence of surface water regime maintenance, such as drainage ditch maintenance and reed bed removal. this problem was recorded earlier in the vicinity of the study area, where two males lost their territories because of drainage ditch maintenance (šćiban, 2004). with this in mind, drainage ditches can be considered ecological traps for bluethroat. besides the aforementioned problem, additional threats for bluethroat in the entirety of its range in serbia and in central europe are e.g., long-term water level decrease, vegetation succession, reduction of wetlands, and possible predation pressure (grüll, 2001; vadász et al., 2011; čížková et al., 2013). these threats have the greatest impact on the birds that live in the habitats surveyed in this study, such as ponds, so developing national conservation plans for them could be important for bluethroat conservation in serbia. acknowledgements. i would like to thank draženko rajković and marko šćiban for advice given during paper writing,vukašin kartalović for helping with the graphic presentation, and milica krmar for english proofreading. references čížková, h., květ, j., comín, f.a., laiho, r., pokorný, j., pithart, d. 2013: actual state of european wetlands and their possible future in the context of global climate change. aquatic sciences, 75: 3-26. collar, n. 2019: bluethroat (cyanecula svecica). in: del hoyo, j., elliott, a., sargatal, j., christie, d.a., de juana (eds.), handbook of the birds of the world alive. lynx edicions, barcelona. cramp, s. 1988: the birds of the western palearctic, 5. oxford university press, oxford. dobretić, v., stojšić, v. 2011: park prirode “beljanska bara” predlog za stavljanje pod zaštitu zaštićeno područje iii kategorije. pokrajinski zavod za zaštitu prirode, novi sad. european bird census council (ebcc) (2015): ebba2 methodology. https://www.ebba2.info/wpcontent/uploads/2015/01/ebba2_methodology_ final.pdf (accessed on august 1st, 2021). gergelj, j., tot, l., frank, z. 2000: ptice potisja od kanjiže do novog bečeja. ciconia, 9: 121-154. grüll, a. 2001: investigations on the population of the white-spotted bluethroat (luscinia svecica cyanecula) in the area of lake neusiedl (burgenland, austria). egretta 44: 1-44. kováts, l. 2009: bluethroat luscinia svecica (linnaeus, 1758). in: csörgő, t., karcza, z., halmos, g., magyar, g., gyurácz, t., szép, t., bankovics, a., schmidt, s., schmidt, e. (eds.), magyar madárvonulási atlasz [hungarian bird migration atlas]. kossuth kiadó, budapest, hungary. 448-449 p. kormanjoš, r. 1989: modrovoljka luscinia svecica cyanecula (meisner, 1804) u okolini čoke. ciconia, 1: 52-53. krüger, t. 1997: das blaukehlchen luscinia svecica im oldenburger land. jahresbericht ornithologische arbeitsgemeinschaft oldenburg, 14: 46-69. krüger, t. 2002: verbreitung, bestand und habitatwahl des blaukehlchens (luscinia svecica cyanecula) in niedersachsen 2001: ergebnisse einer landesweiten erfassung. vogelkdl. ber. niedersachs. 34: 1-21. lakušić, d., blaženčić, j., ranđelović, v., butorac, b., vukojičić, s., zlatković, b., jovanović, s., šinžar-sekulić, j., žukovec, d., čalić, i., pavićević, d. 2005: staništa srbije – priručnik sa opisima i osnovnim podacima. in: lakušić, d. (ed), staništa srbije, rezultati projekta “harmonizacija nacionalne nomenklature u klasifikaciji staništa sa standardima međunarodne zajednice”, institut za botaniku i botanička bašta “jevremovac”, biološki fakultet, univerzitet u beogradu i ministarstvo za nauku i zaštitu životne sredine republike srbije. merilä, j., sorjonen, j. 1994: seasonal and diurnal patterns of singing and song-flight activity in bluethroats (luscinia svecica). the auk, 111(3): 556-562. mirić, r., balog, i., đapić, d. 2018: luscinia svecica. in: radišić, d., vasić, v., puzović, s., ružić, m., šćiban, m., grubač, b., vujić, a. (eds), crvena knjiga faune srbije iii – ptice. beograd: zavod za zaštitu prirode srbije, univerzitet u novom sadu, prirodno-matematički fakultet, departman za biologiju i ekologiju i društvo za zaštitu i proučavanje ptica srbije. 442-444 p. musseau, r., beslic, s. 2018: high densities of the french coastal endemic bluethroat (cyanecula svecica namentum) revealed in intertidal reed beds and conservation perspectives towards sea level rise. revue d’ecologie (terre et vie), 73(2): 115-121. orłowski, g., sęk, m. 2005: semi-natural reedbeds as breeding habitat of bluethroat (luscinia svecica l.) on a sewage farm inwrocław city (south-western poland). polish journal of ecology, 53:133-140. peris, j., mendes, s. 2010: comparación de dos métodos de censo para estimar la población reproductora del pechiazul luscinia svecica azuricollis. ardeola, 57 (especial):117-122. biologica nyssana ● 13 (1) september 2022: 27-31 mirić ● population size of the bluethroat (cyanecula svecica) in the lower course of great bačka canal 31 biologica nyssana ● 13 (1) september 2022: 27-31 mirić ● population size of the bluethroat (cyanecula svecica) in the lower course of great bačka canal petersen, b., krüger, t., zang, h. 2005: blaukehlchen – luscinia svecica (l., 1758). in: zang, h., heckenroth, h., südbeck, p. (eds): die vögel niedersachsens und des landes bremen – drosseln, grasmücken, fliegenschnäpper, naturschutz und landschaftspflege in niedersachsen, sonderreihe b heft 2.9. hannover. 50-62 p. puzović, s., radišić, d., ružić, m., rajković, d., radaković, m., pantović, u., janković, m., stojnić, n., šćiban, m., tucakov, m., gergelj, j., sekulić, g., agošton, a., raković, m. 2015: ptice srbije: procena veličina populacija i trendova gnezdarica 2008 – 2013. društvo za zaštitu i proučavanje ptica srbije i prirodno-matematički fakultet, departman za biologiju i ekologiju, univerzitet u novom sadu, novi sad. radišić, d., tucakov, m. 2010: sastav i status faune ptica ribnjaka kod bača u periodu 2000-2010. ciconia, 19: 33-47. sorjonen, j., merilä, j. 2000: response of male bluethroats luscinia svecica to song playback: evidence of territorial function of song and song flights. ornis fennica, 77: 43-47. stojšić, m. 1977: odvodnjavanje ravničarskog dela vojvodine. vodoprivreda, 45-46: 19-23. šćiban, m. 2000: podaci o nekim zanimljivim vrstama ptica uz veliki bački kanal od kule do čuruga. ciconia, 9:185-186. šćiban, m. 2004: bluethroath luscinia svecica. acrocephalus, 25: 234. šćiban, m., rajković, d., radišić, d., vasić, v., pantović, u. 2015: ptice srbije – kritički spisak vrsta. pokrajinski zavod za zaštitu prirode i društvo za zaštitu i proučavanje ptica srbije, novi sad. tomić, p. 1974: turija, fizičko – geografske odlike okoline. zemlja i ljudi, 24: 139-144. tomić, p. 1990: geografska monografija turije. prirodno-matematički fakultet, institut za geografiju novi sad, novi sad. tomik, a. 2011: inventarizacija gnijezdeće populacije modrovoljke erithacus svecicus i žutog voljića hippolais icterina, konačno izvješće. hrvatsko društvo za zaštitu ptica i prirode, osijek. 49 p. tucakov, m. 2011: potamišje – dinamično poplavno područje. iucn, beograd. vadász, c., csörgő, t. 2009: evaluation of regulations of the agro-environmental program focusing on reedbed management, based on their effects on the breeding passerine assemblage. természetvédelmi közlemények, 15: 235-245. vadász, c., mogyorósi, s., pellinger, a., aleksza, r., biró, c. 2011: results of the breeding passerine census carried out at the hungarian part of lake fertő in 2008. ornis hungarica, 19: 11-20. van turnhout, c.a.m., hagemeijer, e.j.m., foppen, r.p.b. 2010: long-term population developments in typical marshland birds in the netherlands. ardea, 98: 283-299. vig, l., balog, i., pete, g. 2009: ptice jegričke. društvo ljubitelja prirode “falco”, temerin. vig, l., đureković-tešić, o., marić, b., stojanović, t., puzović, s., stojnić, n., knežev, m., lazić, l., stojanović, v. 2012: slano kopovo. edicija “ramsarska područja vojvodine” knjiga iii. pokrajinski sekretarijat za urbanizam, graditeljstvo i zaštitu životne sredine, novi sad. vranešević, m. 2015: biotehničke mere kao mogućnost za povećanje efikasnosti sistema za odvodnjavanje. phd thesis. poljoprivredni fakultet, novi sad. žuljević, a., đapić, d. 2002: podaci o fauni ptica bare “jezero” kod stanišića u severozapadnoj bačkoj. ciconia, 11: 123-126. perišić, v., perišić, v., vukajlović, f., pešić, s., predojević, d., đekić, v., luković, k.: feeding preferences and progeny production of rhyzopertha dominica (fabricius 1792) (coleoptera: bostrichidae) in small grains. biologica nyssana, 9 (1). september, 2018: 55-61. biologica nyssana 9 (1) ⚫ september 2018: 55-61 perišić et al. ⚫ feeding preferences and progeny production of rhyzopertha… 55 original article received: 3 may 2018 revised: 3 june 2018 accepted: 6 july 2018 feeding preferences and progeny production of rhyzopertha dominica (fabricius 1792) (coleoptera: bostrichidae) in small grains vladimir perišić1, vesna perišić1, filip vukajlović2, snežana pešić2, dragana predojević2, vera đekić1, kristina luković1 1center for small grains, save kovačevića 31, 34000 kragujevac, serbia 2university of kragujevac, faculty of science, radoja domanovića 12, 34000 kragujevac, serbia * e-mail: vperisic@kg.ac.rs abstract: perišić, v., perišić, v., vukajlović, f., pešić, s., predojević, d., đekić, v., luković, k.: feeding preferences and progeny production of rhyzopertha dominica (fabricius 1792) (coleoptera: bostrichidae) in small grains. biologica nyssana, 9 (1). september, 2018: 55-61. the lesser grain borer, rhyzopertha dominica (fabricius, 1792) (coleoptera: bostrichidae) is a primary pest of stored wheat. many authors studied its development on this cereal. the wheat is commonly stored together with other grain, especially barley, rye, oats and triticale. the objectives of this study are to assess the feeding preferences for the small grains (wheat, barley, rye, oat and triticale) and to evaluate their susceptibility/resistance to progeny production of r. dominica. the mean survival rate of r. dominica adults, progeny emergence and amounts of insect-damaged grains and dockage in various small grain species were determined. also, influence of r. dominica feeding on chemical properties (moisture protein and ash contents in grains) was found out. the influence of the grain species on the development of r. dominica was significant. the highest mortality of parents and the lowest progeny counts in oats, while triticale is very suitable for development of r. dominica, much more then wheat, as a primary host. the biggest amount of damage kernals and dust were found in triticale. the contents of moisture, protein and ash in the grain has been changed due to feeding of r. dominica. key words: rhyzopertha dominica f., small grains, progeny, damaged kernels, dust apstrakt: perišić, v., perišić, v., vukajlović, f., pešić, s., predojević, d., đekić, v., luković, k.: preferencija u ishrani i pojava potomstva rhyzopertha dominica (fabricius 1792) (coleoptera: bostrichidae) u strnim žitima. biologica nyssana, 9 (1). septembar, 2018: 55-61. žitni kukuljičar (rizoperta) rhyzopertha dominica (fabricius, 1792) (coleoptera: bostrichidae) je primarna štetočina uskladištene pšenice. njen razvoj na ovoj vrsti strnih žita proučavali su brojni autori. pšenica se 9 (1) • september 2018: 55-61 doi: 10.5281/zenodo.1470852 biologica nyssana 9 (1) ⚫ september 2018: 55-61 perišić et al. ⚫ feeding preferences and progeny production of rhyzopertha… 56 obično skladišti sa drugim strnim žitima, posebno ječmom, raži, ovsom i tritikaleom. ovo istraživanje je sprovedeno u cilju ispitivanja preferencija u ishrani r. dominica u strnim žitima (pšenica, ječam, raž, ovas i tritikale) i ocene osetljvosti/otpornosti ovih žita na pojavu potomstva r. dominica. utvrđen je prosečan broj preživelih jedinki r. dominica, pojava potomstva i broj zrna oštećenih insektima, kao i količina prašine u različitim vrstama strnih žita. takođe, utvrđen je uticaj r. dominica na hemijske osobine strnih žita (sadržaj vlage, proteina i pepela u zrnu). uticaj vrste žitarica na razvoj r. dominica bio je značajan. najveća smrtnost roditelja i najmanja pojava potomstva utvrđena je kod ovsa, dok je tritikale veoma povoljan za razvoj r. dominica, mnogo više nego pšenica, kao primarni domaćin. najveća količina oštećenih zrna i prašine nađeni su u tritikaleu. ishrana r. dominica je dovela do promene sadržaja vlage, proteina i pepela u zrnu. ključne reči: rhyzopertha dominica, strna žita, potomstvo, oštećena zrna, prašina introduction the lesser grain borer, rhyzopertha dominica (fabricius, 1792) (coleoptera: bostrichidae) is a primary pest of stored grain, with the great economic importance in the republic of serbia and many regions of the world (kljajić, 2008). it feeds mostly on grains from families poeaceae (e.g. rice, wheat, sorghum, oats, pearl, millet, malt, barley) and fabaceae (e.g. chickpeas, peanuts, beans) (edde, 2012). a lot of research was conducted which dealt with the influence of the species and variety of the plant on the development of r. dominica and occurrence of the progeny (arthur et al., 2012, 2013; astuti et al., 2013; metwaly et al., 2015; pires, 2016). although it is one of the most important stored-grain insect pests in the world, most of research was conducted on wheat and rice (chanbang et al., 2008; mebarkia et.al, 2009; nawrot et al., 2010). wheat is commonly stored together with other grain, especially barley, rye, oats and triticale. however, there are no studies in which adults of r. dominica have been directly exposed to small grains and assessed the influence of these species on progeny production and feeding preferences. the larvae and adults of r. dominica spend most of their life inside the kernel, feeding on both the germ and endosperm, directly causing damages and changes in grain physicochemical properties (rees, 2004; edde, 2012). insect-damaged kernels are characterized by the presence of irregularly shaped holes (about 1 mm in diameter) of increasing depth that extends from the point of larval entry, around the edge of the kernel to the pupation chamber (rees, 2004). adult feeding activities produce large amount of dust, which often leads to the reduction of grain kernel to the pericarp. jood & kappor (1993), mebarkia et al. (2009) and nawrot et al. (2010) investigated the amount of grain consumed by r. dominica under experimental conditions, but the losses of nutrient value varied among the authors. small grains contain different quantities of main nutrition groups, which are very important for development and progeny production of r. dominica. the most commonly grown grain species in serbia are wheat, barley, rye, oats and triticale. according to serna-saldivar (2010a), this species differed in amount of proteins, starch and other nutrients. furthermore, above-mentioned species express differences in morphological properties, for example, presence/absence of a hull. nitrogen and phosphorus are particularly mismatched when herbivorous insects are compared with their host plants (huberty & dennno, 2006). the objectives of this study are to assess feeding preferences of r. dominica for small grains (wheat, barley, rye, oats and triticale) through determination of some grain properties, i.e. mass of infested-damaged kernels (idk) and dust, contents of moisture, proteins and ashes in grains infested with r. dominica and to evaluate their susceptibility/ resistance to progeny production of r. dominica. material and methods examination of the influence of different small grain species (wheat, barley, oats, rye and triticale) on the emergence of the progeny of r. dominica, on stored small grains, as well as effect of their presence on chemical properties of small grains were conducted in laboratories of center for small grains kragujevac, serbia during 2016. tested insects adults of r. dominica of both sexes and 2-4-weeks old were used during the experiment, reared in the center for small grains on the whole wheat kernels, in laboratory conditions with temperature (t) 26±1°c and relative humidity (rh) 60±5%. tested cereal species r. dominica was reared on five commercially available small grain varieties (wheat variety vizija, barley variety rekord, oat variety vranac, rye variety raša and triticale variety favorit), originating from the center for small grains kragujevac, serbia. different cereal species with the grain moisture 1112% were used during the testing. this value was biologica nyssana 9 (1) ⚫ september 2018: 55-61 perišić et al. ⚫ feeding preferences and progeny production of rhyzopertha… 57 chosen because it has been proposed level of the moisture for stored small grains (the official gazette of rs, 2016). the moisture content was determined with motomco moisture meter (motomco inc, 919, canada) according to aacc method 44-11 (aacc, 1995). examination of the influence of different small grain species (wheat, barley, oats, rye and triticale) on the emergence of the progeny of r. dominica, on stored small grains was based on the modified method according to arthur et al. (2013). after the harvest, grains were put in the refrigerator at 4 °c. before the incorporation of adults of r. dominica, grains were sieved on the 2 mm sieve and cleaned by hand. then, plastic vessels of 200 ml were filled with 50 g of grains and put in the incubator with controlled t 26±1 °c and rh 60±5% (incubator xo 1450 special, iskra, loka, slovenia). twenty-five adults of r. dominica were released into each vessel. vessels were then closed with cotton cloths fixed with a rubber band. for each small grain species, eight replications were conducted. hatching period is about seven days or less (crombie, 1940; cit.loc. edde, 2012), while we decided to determine the influence of commodity on feeding preferences and survival of r. dominica for a period of three weeks. parental adults were removed from the vessels after three weeks and classified as alive or dead. afterwards, damaged grains and dust (the feeding damage) were separated and their mass was measured on analytical balance (mettler 609-b6, zürich, switzerland) for each sample (50 g) of grain types, and then the contents were put back into the vessels. since egg-to-adult development of r. dominica could be shorter than five-six weeks on wheat (edde, 2012), the period of seven weeks was chosen for incubation, as duration of development was expected to be longer in non-preferred commodities. after seven weeks at the constant temperature and humidity, vessels with grains were opened and the progeny emergence was determined by counting insects separated from grains. damaged grains and dust were weighed again, after progeny counting. the whole procedure was repeated twice. determination of the influence of r. dominica feeding on chemical properties of small grains. infested and damaged kernel, as well as dust, were returned to the vessel after weighing. for this research, uninfested samples from the same year of production were used as a standard (control). all samples were milled and prepared for chemical analysis of moisture, total protein and ash contents. the moisture content was determined based on the loss of samples weight after drying on t 130-133 °c (icc standard no. 110/1: 1976). the total protein content of grains (n x 5.7 for wheat, rye and triticale, and n x 6.25 for oats and barley) was determined according to modified kjeldahl’s method (icc standard no. 105/2:194). ash content was estimated by burning a sample at 900 °c and measuring the residue (icc standard method no.104/1: 1990). results were expressed as percent (%) from the sample weight and represented as a dry matter. statistical analysis. results of examination were expressed in percent of mortality (%) with computed standard error (se). before analysis, results for the progeny number was transformed with formula log(x+1), while formula sqrt(x) was used for the amount of damaged grain and dust. recorded data were analyzed by one-way or two-way analysis of variance (anova) depending on the trial design. the significance of mean differences was determined according to duncan test (for p=0.05). pearson's coefficient at p=0.005 was used to determine linear correlation between the survival parents or the number of progeny and the number of damaged kernels, and correlation between mass of damaged kernels and the parameters of chemical analysis (moisture, proteins and ash contents). all data were processed on statsoft version 7.1 (statsoft inc., tulsa, oklahoma). results survival of r. dominica parents on different small grain species three weeks after the infestation ranged from 91.25% on oat to 99.25% on rye (tab. 1) and was significantly smaller in oat than in other examined cereals. the significantly smaller amount of insect-damaged grains and dust after two weeks was found in oats. the highest mass of damaged grains and dust was found in the triticale (3.87 g/1.96 g). mean r. dominica parent survival and feeding damage were compared using a linear correlation analysis with pearson’s coefficient and significant positive correlations were not detected for p<0.005, n=16 (wheat r=-0.123, p=0.650; barley r=0.287, p=0.281; rye r=-0.257, p=0.925; oats r=-0.564, p=0.836 and triticale r=0.232, p=0.386). based on the data given in tab. 2, our analyses show that the production of r. dominica progeny and feeding damage varied considerably in dependence of cereal type. the influence of the grain species on the development of r. dominica was significant. the highest progeny was recorded in triticale (430.35 in average), followed by rye (221.42 in average), wheat (150.2 in average), barley (91.1 in average) and the smallest in oats (35.15 in average). biologica nyssana 9 (1) ⚫ september 2018: 55-61 perišić et al. ⚫ feeding preferences and progeny production of rhyzopertha… 58 the amounts of insect-damaged grains and dust after the removal of the progeny on various grains is presented in tab. 3. the dust weight after progeny count was significantly lower in oats (0.45 g). the highest damage and dust were detected in the triticale, compared to the wheat, as primary host of r. dominica (9.60 g vs. 4.52 g). pearson’s correlation coefficient revealed a significant positive correlation between progeny production and damaged grain in wheat (r=0.948, p<0.005 ), barley (r=0.677, p<0.005 ), rye (r=0.807, p<0.005) and triticale (r=0.880, p<0.005), while no correlation was found between the two data in oats (r=-0.194, p=0.470). the infested samples, with an exception of oats, had significantly higher moisture content, compared to un-infested sample. moisture content and the weight of damaged grain (tab. 2) were compared using a linear correlation analysis with pearson’s coefficient and significant positive correlations were detected for wheat (r=0.564, p<0.005), triticale (r=0.847, p<0.005) and rye (r=0.867, p<0.005), the lowest for barley (r=0.135, p<0.005), while no correlation was found between the two data for oats (r=0.319, p=0.229). total content of proteins was significantly lower in infested sample in all types of grain, except in oats, where no difference was found (tab. 3). pearson’s correlation coefficient revealed a significant negative correlation between protein content and damaged grain in wheat (r=-0.738, p<0.005), barley (r=-0.786, p<0.005) rye (r=-0.909, p<0.005) and triticale (r=-0.483, p<0.005 ), while no correlation was found between the two data in oats (r=-0.189, p=0.317). significant differences in ash content between the tested variants were recorded only for rye and triticale where ash contents were significantly increased. pearson’s linear correlation coefficient between ash content and damaged grain revealed significant positive correlations in rye (r=0.647, p<0.005) and triticale (r=0.847, p<0.005), while no correlation was recorded for wheat (r=0.198, p=0.038), barley (r=0.119, p=0.201) and oats (r=0.194, p=0.104). table 1. mean survival (% ± se) of rhyzopertha dominica adults and amounts of insect-damaged grains and dockage (g ± se) after three weeks of infestation of various cereal grains species. grain type adult survival (% ± se) damaged kernels (g ± se) dust (g ± se) wheat 98.25±0.11 b* 1.84±0.03d 0.43±0.05c barley 96.75±1.28 b 0.97±0.12b 0.18±0.06b rye 99.25±0.08 b 1.58±0.18c 0.89±0.09d oat 91.25±0.15 a 0.09±0.09a 0.02±0.01a triticale 98.25±0.89 b 3.87±0.23e 1.96±0.11e f 11.163 59.56 74.22 p < 0.05 < 0.05 < 0.05 * within each column, means followed by the same letter are not significantly different, in all cases df=4.75; duncan test at p>0.05. table 2. progeny emergence ( x ± se) and amounts of insect-damaged grains and dockage (g ± se) after the removal of the progeny from various cereal grains species grain type number of progeny ( x ± se) damaged kernels (g ± se) dust (g ± se) wheat 150.2±0.67c* 5.42±0.05c 1.69±0.06c barley 91.1±0.40b 2.61±0.09b 0.95±0.04b rye 221.42±0.79d 5.17±0.10c 3.33±0.09d oats 35.15±0.19a 0.45±0.03a 0.21±0.02a triticale 430.35±0.42g 9.60±0.11d 4.52±0.08e f 72.08 43.42 50.12 p < 0.05 < 0.05 < 0.05 * within each column, means followed by the same letter are not significantly different, in all cases df=4.75; duncan test at p>0.05. biologica nyssana 9 (1) ⚫ september 2018: 55-61 perišić et al. ⚫ feeding preferences and progeny production of rhyzopertha… 59 discussion mechanisms of tolerance of cereal grains are complex and depend on many factors, such as the physicochemical and biochemical grain properties as well as the insects’ capacity for biochemical and physical adaptation to post-harvest conditions (nawrot et al., 2006). it should be noted that physical and chemical properties of cereal grain have a fundamental influence on the developmental rate of r. dominica. the physical properties significantly affect the percentage of grain infestation of r. dominica (kavallieratos et al., 2010). keskin & ozkaya (2013) stated that grain hardiness represents one of the main reasons for stronger or weaker infestation of r. dominica. in our study, the highest progeny was found in triticale, which grain is classified as the softest (perišić et al., 2009a) and rye, which is significantly softer than wheat, while significantly smaller number of progeny was found in the barley, whose grain is harder than wheat (serna-saldivar, 2010b). also, triticale has the biggest 1000-kernel mass (am ≈ 45 g), compared to the oats and rye (am ≈ 29.35 and 36 g, respectively) (perišić et al., 2009b). according to kavallieratos et al. (2010), smaller size and kernel shape of grain could discourage the lesser grain borer from laying eggs in clusters on the kernels. edde (2012) claims that the texture of larval food can have significant influence on the rate of brood development. our data confirmed results reported by nawrot et al. (2010), who established high resistance of some varieties of wheat to sitophilus granarius (linnaeus, 1758) infestation. this resistance was associated with vitreosity-grain hardness and lower 1000-kernel mass and kernel diameter. it must be emphasized that kernel of barley and oats have a hull as an outer layer, namely hull stayed intact on the grain of this species after the harvest. hull of barley is completely concrescence with pericarp of kernel, while oats’ hull could be mechanically removed from the grain. mebarkia et al. (2009) noted that barley grain covered with its cellulose envelopes is unfavorable for s. granarius, but it is not disadvantage for r. dominica. the development of progeny in our study on barley can therefore be explained only by the greater hardness and with some chemical properties (content of proteins). table 3. moisture, protein and ash content (% ± se) in various cereal grains species after rhyzopertha dominica progeny count. sample moisture, protein and ash contents (% ± se) in various grain types wheat barley rye oats triticale moisture content infested sample 14.4±±0.05b* 12.3±0.05b 13.4±0.05b 11.3a 14.2±0.05b un-infested sample (control) 12.1 ±0.05a 11.7±0.05a 11.7±0.05a 10.8±0.05a 11.8±0.05a f 56.49 68.04 15.23 1.8 72.91 p < 0.05 < 0.05 < 0.05 0.204 < 0.05 protein content infested sample 12.5±0.05 b 11.73±0.05b 15.37±0.05b 13.26±0.05a 10.90±0.05b un-infested sample (control) 13.21±0.05a 12.04±0.05a 15.78±0.05a 13.30±0.05a 12.34±0.05a f 95.3 42.6 33.36 2 195.94 p < 0.05 < 0.05 < 0.05 0.149 < 0.05 ash content infested sample 1.83a 2.45b 2.08b 3.41a 2.81b un-infested sample (control) 1.71a 2.36a 1.91a 3.38a 2.25a f 1.21 3.745 22.05 0.42 98.91 p 0.280 0.07 < 0.05 0.52 < 0.05 * for each parameter separately, means within columns followed by the same letter are not significantly different, in all cases df=1.30; duncan test at p>0.05. un-infested sample (control) dockage and insect free sample biologica nyssana 9 (1) ⚫ september 2018: 55-61 perišić et al. ⚫ feeding preferences and progeny production of rhyzopertha… 60 in our study, the highest mortality of parents and the low progeny count was determined in oats. among all studied species, only oats have a hull which is not grown up with grain and which is covered with hair. the hull is unpalatable, dry, and brittle outer layer which accounts for approximately 25–36% of the total dry weight of the oat (welch, 1995). according to nawrot et al. (2010), the hull in oats is composed mainly of silica, which protects the kernel against insects and can slow down the infestation by these pests. these authors also claim that an additional factor which can be taken into consideration is the compact structure of the lemma and palea. reproduction insect ecology postulate is that the oviposition preference of a female should be correlated with the host’s suitability for the development of its offspring. hull on oats covers its grain as well as the clusters on the grain where r. dominica lays eggs which significantly reduces this phase of development. the amounts of damaged kernel and dust were in correlation with the emergence of progeny. since the highest number of progeny was determined in triticale, it was quite expected that aforementioned parameters were greatest in this species. mebarkia et al. (2009) examined the influence of grain species (soft and hard wheat, barley, rice, maize) on the development and the way of feeding of r. dominica. their results were similar to our study, with established differences between examined species for progeny number, length of development and the loss of dry matter generated after feeding of this pest. furthermore, loss of a dry matter as a consequence of the development of r. dominica progeny was different in examined species of small grains (wheat, barley, rice, and maize). all samples showed a significant change in moisture content, except in oats. as more damage was found in those grains, the increase in moisture was expected. pearson’s correlation coefficient also confirmed the association of these two parameters. as r. dominica mostly feed on seed endosperm and germ, it is able to reduce the grain merely to its pericarp (edde, 2012) and increased infestation and higher grain damage are expected to reduce total protein contents in untreated grains. jood & kapor (1993) also reported reduced protein contents in wheat seeds but only under intensive r. dominica infestation of 75%, while ozkaya et al. (2009) determined significant increase of protein, moisture and ash contents. the explanation for these opposites between different researches is based on the presence of impurities and uric acid, while the real content of protein was decreased as a consequence of r. dominica feeding. a significant difference in ash content was detected in triticale and rye. changes in ash content were caused by r. dominica presence and feeding, which was confirmed by pearson’s correlation coefficient. our data are in agreement with those reported by jood et al. (1992), who found that r. dominica influence the content of mineral matter in grain by increasing it under strong infestation. keskin & ozkaya (2013) confirmed the same effect of s. granarius as this insect also feeds mostly on seed endosperm and germ. these experiments were conducted only on wheat, with no results on the other small grain. small grains wheat, barley, rye and triticale can be considered as ''nutritionally suitable'' for r. dominica development. triticale is the most susceptible to attacks and suitable for development of r. dominica. it is clearly demonstrated that oats is less infested and is not a suitable host for the strain of r. dominica used in the present study. the presence of the hull in oats proved to be important morphological characteristic of a kernel which has an effect on development and feeding of the examined pest. the role of the hull can be objective of future research, i.e. is it represents a physical barrier only or possesses some chemical substances which act as a repellent against r. dominica. acknowledgements. this study was carried out within a project supported by the ministry of education, science and technological development of the republic of serbia, grant no oi 173038. references arthur, f.h., ondier, g.o., siebenmorgen, t.j. 2012: impact of rhyzopertha dominica (f.) on quality parameters of milled rice. journal of stored products research, 48: 137-142. arthur, f.h., laura starkus, l., smith, m.c., phillips, t.w. 2013: methodology for determining susceptibility of rough rice to rhyzopertha dominica and sitotroga cerealella. journal of pest science, 86: 499–505. astuti, l.p., mudjiono, g., rasminah, ch.s., rahardjo, b.t. 2013: susceptibility of milled rice varieties to the lesser grain borer (rhyzopertha dominca f.). journal of agricultural science, 5: 145-149. chanbang, y., arthur, f.h., wilde, g.e., throne, j.e. 2008: hull characteristics as related to susceptibility of different varieties of rough rice to rhyzopertha dominica (f.) (coleoptera: bostrichidae). journal of stored product research, 44: 205–212. biologica nyssana 9 (1) ⚫ september 2018: 55-61 perišić et al. ⚫ feeding preferences and progeny production of rhyzopertha… 61 edde, p., 2012: a review of the biology and control of rhyzopertha dominica (f.). the lesser grain borer. journal of stored products research, 48: 1-18. huberty, a.f., denno, r.f. 2006: plant water stress and its consequences for herbivorous insects: a new synthesis. ecology, 85: 83-98. icc standard methods, international association for cereal science and technology (1976): determination of the moisture content of cereals and cereal products (practical method). icc standard method no. 110/1. icc, vienna, austria. icc standard methods, international association for cereal science and technology (1990): determination of ash in cereals and cereal products. icc standard method no. 104/1. icc, vienna, austria. icc standard methods, international association for cereal science and technology (1994): determination of crude protein in cereals and cereal products for food and for feed. icc standard method no. 105/2. icc, vienna, austria. jood, s., kapor, a., singh, r., 1992: mineral content of cereal grains as affected by storage and insect infestation. journal of stored products research, 28 (3): 147-151. jood, s., kapor, a.c., 1993: protein and uric acid contents of cereal grains as affected by insect infestation. food chemistry, 46: 143-146. kavallieratos, n.g., athanassiou, c.g, vayias, b.j., kotzamanidis, s., synodis, s. 2010: efficacy and adherence ratio of diatomaceous earth and spinosad in three wheat varieties against three stored-product insect pests. journal of stored products research, 46: 73-80. keskin, s., ozkaya, h. 2013: effect of storage and insect infestation on the mineral and vitamin contents of wheat grain and flour. journal of economic entomology, 106 (2): 1058-1063. kljajić, p. 2008: suzbijanje štetnih insekata uskladištenog žita. in. kljajić, p. (ed.), zaštita uskladištenih biljnih proizvoda od štetnih organizama. 67-100 p. institut za pesticide i zaštitu životne sredine, beograd. mebarkia, a., guechi, a., mekhalif, s., mekhalif, m. 2009: biochemical composition effect of the some cereal species on the behavior of sitophilus granarius l. and rhyzoperta dominica f. species in semi-arid zone of setif, algeria. journal of agronomy, 8 (2): 60-66. metwaly, m.r., abou-ghadir, m. f., abdu-allah, g. m., abdel-nasser, m. k. 2015: susceptibility of certain wheat varieties to the infestation by rhyzopertha dominica (f.) and tribolium confusum (du val). journal of phytopathology and pest management, 2 (3): 1-8. nawrot, j., warchalewski, j.r., piaseckakwiatkowska, d., niewiada, a., gawlak, m., grundas, s.t., fornal, j. 2006: the effect of some biochemical and technological properties of wheat grain on granary weevil (sitophilus granarius l.) (coleoptera: curculionidae) development. 9th international working conference on stored product protection, 400407 p. nawrot, j., gawlak, m., szafranek,j., szafranek, b., synak, e., warchalewski, j.r., piaseckakwiatkowska, d., b1aszczak, w., jelinski, t., fornal, j. 2010: the effect of wheat grain composition, cuticular lipids and kernel surface microstructure on feeding, egg-laying, and the development of the granary weevil, sitophilus granarius (l.). journal of stored products research, 46: 133-141. ozkaya, h., ozkaya, b., colakoglu, a. 2009: technological properties of a variety of soft and hard bread wheat infested by rhyzopertha dominica (f.) and tribolium confusum du val. journal of food, agriculture and environment, 7 (3-4): 166-172. pires, m., nogueira, r., pina, d., manica, c., faroni, r., moreira, p. 2016: walking stability of rhyzopertha dominica (fabricius, 1792) (coleoptera: bostrichidae). brazilian journal of biology, 76 (3): 568-576. perišić, v., milovanović, m., staletić, m., nikolić, o. 2009a: delija new spring triticale cultivar. the iv congress of the serbian genetic society, tara, 285-289 p. perišić, v., milovanović, m., ćulaković, b., janković, s., staletić, m. 2009b: produktivnost kragujevačkih sorata ozime pšenice, ječma i jarog ovsa. poljoprivredne aktuelnosti, 3-4: 5-14. rees, d.p. (ed.). 2004: insects of stored products. csiro publishing, australia. 181 p. serna-saldivar, o. 2010a: chemical composition of cereal grains. in: serna-saldivar, o. (ed.), cereal grains: properties, processing and nutritional attributes, 3: 81-108, crc press, taylor & francis group, new york. serna-saldivar, o. 2010b: physical properties, grading, and specialty grains. in: sernasaldivar, o. (ed.), cereal grains: properties, processing and nutritional attributes, 2: 43-80, crc press, taylor & francis group, new york. the official gazette of the republic of serbia. 2016: pravilnik o kvalitetu žita, mlinskih i pekarskih proizvoda, 68/16. welch, r. 1995: the chemical composition of oats. in: welch, r. (ed.), the oat crop, 279–320. springer, the netherlands. http://www.scielo.br/scielo.php?script=sci_serial&pid=1519-6984&lng=en&nrm=iso http://www.scielo.br/scielo.php?script=sci_serial&pid=1519-6984&lng=en&nrm=iso nikolić-milojević et al 2020, biologica nyssana 11(2) 11 (2) december 2020: 121-128 doi: 10.5281/zenodo.4393967 influence of the time of maceration on phenolic composition of wines produced from the indigenous variety prokupac original article natalija lj. nikolić-milojević status doo, 65a vase albanca st, svrljig, serbia lunaelumen84@gmail.com ivana s. mošić aromatika doo, jadranska st, niš, serbia ivanamosic@gmail.com ivana t. karabegović faculty of technology, university of niš, 128 bulevar oslobođenja st, leskovac, serbia ivana.karabegovic@gmail.com miodrag l. lazić faculty of technology, university of niš, 128 bulevar oslobođenja st, leskovac, serbia lmiodrag@yahoo.com nada č. nikolić faculty of technology, university of niš, 128 bulevar oslobođenja st, leskovac, serbia nadanikolic64@yahoo.com sanja r. perić academy of vocational studies south serbia, department of agricultural and food studies, 1 ćirila i metodija st, 18400 prokuplje, serbia sanjaperics@yahoo.com slađana s. golubović academy of vocational studies south serbia, department of agricultural and food studies, 1 ćirila i metodija st, 18400 prokuplje, serbia golubovicsladjana1@gmail.com dejan n. davidović academy of vocational studies south serbia, department of agricultural and food studies, 1 ćirila i metodija st, 18400 prokuplje, serbia dejandavidovic7@gmail.com dragan t. veličković academy of vocational studies south serbia, department of agricultural and food studies, 1 ćirila i metodija st, 18400 prokuplje, serbia dvelickovic7@ptt.rs (corresponding author) received: january 20, 2020 revised: september 15, 2020 accepted: september 20, 2020 abstract: the influence of the time of maceration on phenolic composition of wine (white, rosé and red wine) produced from the indigenous variety prokupac was studied in this paper. white wine was produced from free run, without maceration. in the production of rosé wine, cool-temperature extraction prior to fermentation in 2-hour period was used, while for the production of red wine, maceration was carried out at 15-20 °c for 2 weeks. with the increase of the time of maceration, the concentration of total phenolic compounds also increases, whereby, the lowest content was detected in white wine (565.4 mg/l gae). the phenolic content in red wine was almost 2.5 times higher when the time of maceration was extended to 14 days (1433.8 mg/l gae). a significant increase in total flavonoids content was detected: 153.4 mg/l ce (white wine), 179.3 mg/l ce (rosé wine), and 1205.0 mg/l ce (red wine). the total flavonoid share in total phenolic content of the wine increases with the increase of the time of maceration and its value is as follows: 27.1% (white wine), 34.3% (rosé wine) and 84.0% (red wine). key words: maceration, parameters of quality, prokupac, wine apstract: uticaj vremena maceracije na fenolni sastav vina proizvedenih od autohtone sorte grožđa prokupac u ovom radu je proučavan uticaj vremena maceracije na fenolni sastav vina (belo, roze i crveno vino) proizvedeno od autohtone sorte prokupac. belo vino je proizvedeno bez maceracije. u proizvodnji roze vina vršena je hladna maceracija pre fermentacije u dvosatnom periodu, dok je za proizvodnju crvenog vina maceracija vršena na 15-20 °c tokom 2 nedelje. sa povećanjem vremena maceracije povećava se i koncentracija ukupnih fenolnih jedinjenja, pri čemu je najmanji sadržaj otkriven u belom vinu (565,4 mg/l gae). sadržaj fenola u crvenom vinu bio je skoro 2,5 puta veći kada je vreme maceracije produženo na 14 dana (1433,8 mg/l gae). otkriven je značajan porast ukupnog sadržaja flavonoida: 153,4 mg/l ce (belo vino), 179,3 mg/l ce (vino roze), i 1205,0 mg/l ce (crveno vino). ukupan udeo flavonoida u ukupnom fenolskom sadržaju vina povećava se sa povećanjem vremena maceracije i njegova vrednost je sledeća: 27,1% (belo vino), 34,3% (vino roze) i 84,0% (crveno vino). ključne reči: maceracija, parametri kvaliteta, prokupac, vino introduction grape varieties, climate factors, soil and vineyard practices are crucial for the composition of the grapes, but the main wine attributes are highly influenced by winemaking procedures. in general, the application of different winemaking practices does not have a great effect on basic wine parameters (ethanol content, ph-value, total and volatile acidity), but significantly enhances the color, polyphenolic and volatile profile of wines (gonzález-neves et al., 2013). these characteristics are of great importance © 2020 nikolić-milojević et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work noncommercially under the same license as the original. 121 for the red wines as they are crucial for the sensorial properties, flavor, mouthfeel, aging ability of wines and value in the marketplace. also, it is well known that pre-fermentative practices, such as cold maceration or macerating enzymes application, considerably affects the extraction of compounds responsible for the phenolic composition and sensory characteristics of red wines (gonzález-neves et al., 2013; casassa et al., 2019). besides that, red wines with a higher concentration of phenolic compounds or antioxidant activity (kocabey et al., 2016; alencar et al., 2018), with better sensory properties (şener, 2018) can be produced by prolonged maceration stage which occurs simultaneously with alcoholic fermentation. according to some authors, antioxidant activity of different brazilian (alencar et al., 2018), portuguese (jordão et al., 2012) and australian (yoo et al., 2011) red wines increased with maceration time prolongation, but after reaching its maximum value, it remained stable till the end of the process. many studies showed that the basic quality parameters for teran (plavša et al., 2012), castelão (spranger et al., 2004), bombino nero (suriano et al., 2015), plavacmali (budić-leto et al., 2008) red wines do not deviate substantially with maceration time prolongation. on the other hand, the duration of maceration significantly affected the content of total anthocyanins, low molecular weight proanthocyanidins (budić-leto et al., 2008; suriano et al., 2015) and tannins (şener, 2018) in wine. furthermore, the higher antioxidant capacity of karaoglan (kocabey et al., 2016), merlot and vranec (kostadinović et al., 2012) wine was observed in samples produced with longer maceration time. sensory analysis of cabernet sauvignon (şener and yildirim, 2013), monastrell, syrah (busse-valverde et al., 2012), teran (plavša et al., 2012), karaoğlan (yilmaztekin et al., 2015), cabernet franc (cadot et al., 2012) wines showed that maceration time significantly affected sensory characteristics, but some researchers (barbará et al., 2020; plavša et al., 2012; yilmaztekin et al., 2015) highlighted that the wine produced with ten days maceration treatment gives the most harmonious wine, with the best aroma and flavor attributes. it was also shown that wine astringency and color intensity increased with longer maceration time (cadot et al., 2012; kocabey et al., 2016; şener, 2018). all these results indicate the importance of maceration treatment in winemaking, as well as the fact that desirable quality of wine can be produced by optimizing maceration duration. there are many indigenous grape varieties in serbia, but prokupac receives most of the attention in the local and international markets. thus, the objective of this study was to examine the effect of maceration duration on basic wine parameters and to verify the effects on the total content of anthocyanins, phenolic compounds and flavonoids in order to obtain premium quality wine from these grape varieties. material and methods wines three wines were used for the experiment (white, rosé and red wine) produced from the prokupac grape variety (vitis vinifera, 2016 vintage), producer from aleksandrovac (republic of serbia). the wines were made from the same grapes, where the key difference was duration of maceration. white wine was produced from wine dripping without maceration, and alcohol fermentation is carried out in the stum, i.e. in unfermented grape juice. for the production of rosé wine, short-term cold maceration was carried out for 2 hours in the press, while for the production of red wine the maceration lasted during the whole fermentation period of 2 weeks at temperature of 15°c to 20°c. relative density pycnometric analysis was used. the relative density is calculated by the formula: where: relative density of wine, mwine mass of pycnometer with wine, mwater mass of pycnometer with water, m0 mass of empty pycnometer. alcohol content pycnometric analysis with distillation was used. from the calculated relative density in the formula below, the value expressing the alcohol content of the wine is read through the tables: where: relative density of distillate, mdist mass of pycnometer with distillate, mwater mass of pycnometer with water, m0 mass of empty pycnometer. total extract after the wine has been distilled to determine the alcohol content, a residue used to determine the extract content of the wine is left in the distillation balloon. the relative density is determined, where the rinsing, quantitative transfer, refilling, thermostating and measuring are performed as in the previous cases, and the contents of the extract in g/l are read from the tables (blesić, 2016). 122 biologica nyssana ● 11 (2) december 2020: 121-128 nikolić-milojević et al. ● influence of the time of maceration on phenolic composition of wines produced from the indigenous variety prokupac 123 total acids into an erlenmeyer flask 10 ml of wine and 20 ml of distilled water are poured. a few drops of the bromo-thymol blue indicator are added. titration with a solution of naoh 0.1n is carried out. total acids (g/l, calculated on tartaric acid) are calculated as: total acids = vnaoh . 0.75 volatile acids about 300 ml of distilled water is poured into a larger normal vessel and 50 ml of wine into a smaller one. the normal water vessel is heated, and the created water vapor is introduced into the wine. when about 100 ml has been distilled, the wine is heated and at least 200 ml of wine is collected. 3-4 drops of phenolphthalein are added and titrated with a solution of naoh (0.1n). volatile acids (g/l, calculated on acetic acid) are calculated as: volatile acids = vnaoh . 0.12 . 16.67 sugar free extract sugar free extract is the difference between total dry extract and total sugars. ash into the previously measured porcelain cup 10 ml of wine is poured. annealing is carried out at the temperature which rises from 200 °c to 500 °c, and when the content is completely charred, 5 ml of distilled water is added and evaporation is carried out in a water bath, as well as re-annealing and cooling in a desiccator. for determination of the ash content (g/l), measuring was performed on an analytical scale, while the mass of the empty cup was subtracted from the obtained mass, and the value obtained was multiplied by 100. reducing sugars a fast french method based on the oxidoreduction processes of fehling’s solution and sugar was used, whereby divalent copper (cuso4) from fehling’s solution was reduced to monovalent (cu2o) and the sugar was oxidized to the corresponding acids. in an erlenmeyer flask 5 ml of fehling i and fehling ii was poured and 20 ml of distilled water were added. the content of the erlenmeyer flask is heated to boiling and the filtrate is added from the burette. during the titration, the content in the erlenmeyer flask is kept around the boiling point. the filtrate is added until the last traces of blue colour disappear, indicating that the reduction of fehling’s solution has been completed. a red precipitate is formed at the bottom of the erlenmeyer flask and the liquid above the precipitate is colorless. the content of wine sugar (g/l) was calculated as: free and total sulphur dioxide the aspiration method was used. into a flat bottom balloon 10 ml of h2o2 is added together with a few drops of methyl red and methylene blue indicator mixture. the colour should be grey, dirty blue, indigo blue or greenish. if a purple color is obtained, it should be neutralized with a solution of naoh (0.01n). as soon as the purple color disappears, adding of naoh solution is stopped. usually, only a few drops are needed for neutralization. into a round bottom balloon 10 ml of ortho-phosphoric acid and 20 ml of wine are poured. then cooling by the condenser and aspiration pumps is activated. after 15 minutes, the whole free so2 goes into the flat bottom balloon, where the colour changes to purple. titration with a solution of naoh 0.01n is performed until the colour changes to green. the consumed volume of naoh solution can be read on the burette and free so2 content (mg/l) is calculated as: free so2 = vnaoh . 16 the same sample of wine, heated for 10 minutes, is used to determine bound so2, while h2o2 and the mixture of indicators are prepared once again. after 10 minutes, titration with naoh solution is carried out until the color changes to green. the amount of bound so2 is calculated in the same way. total sulphur is the sum of bound and free sulphur. ph-value the ph-value was measured with a ph-meter, threepoint calibration. total phenols a spectrophotometric method using folin-ciocalteu reagent was used. the absorbance is measured at 765 nm. into a normal 10 ml vessel 0.1 ml of wine is poured. red wines require necessary dilution, usually at a ratio of 1:5. then 7.9 ml of distilled water is added, 0.5 ml of folin-ciocalteu reagent and 1.5 ml of na2co3 sodium-carbonate solution. the same mixture is prepared for the blank assay, only distilled water is used instead of wine. the absorbance was measured with a spectrophotometer and the results were read from a calibration curve. index of total phenols determination of total phenol index is a spectrophotometric method. the method is simple, but the results obtained do not provide the information on what phenolic compounds are present in wine and at what concentration. red wine biologica nyssana ● 11 (2) december 2020: 121-128 nikolić-milojević et al. ● influence of the time of maceration on phenolic composition of wines produced from the indigenous variety prokupac sugars = 50a 124 requires necessary dilution of 1:100, rosé wine from 1:10 to 1:50, and white wine 1:10. the absorbance was measured at 280 nm, in quartz cuvettes with an optical path of 10 mm, in relation to distilled water as blank assay. the total phenolic index of tpi is obtained by multiplying the absorbance by the dilution factor: tpi = a280 . f total flavonoids spectrophotometric method with aluminum chloride (alcl3) was used. into a normal vessel with 4 ml of distilled water, 0.1 ml of wine is added. in case of red wines, a dilution of 1:5 is usually used. at zero moment, 0.3 ml of 5% nano2 solution is added to the normal vessel, 0.3 ml of 10% alcl3 solution is added after 5 minutes, and 2 ml of naoh 1 mol/ dm3 solution is added in the sixth minute, and the vessel is refilled with the distilled water to the index mark. the absorbance was measured at 510 nm, in comparison to the distilled water as blank assay. on the basis of the measured values from the calibration curve, the concentration of total flavonoids (mg/l) was determined. total anthocyanins the ribereau-gayon method was used. one ml of wine was mixed with 1 ml of 0.1% hcl (in 96% ethanol) and 20 ml of 2% hcl solution. the obtained mixture was stirred and 10 ml of the mixture were separated into two test tubes. into the first tube 4 ml of distilled water is added and into the second tube 20% of a solution of sodium bisulfite na2s2o3. after 20 minutes, the optical density at 520 nm was measured using a spectrophotometer. from the difference in optical density between the two tubes, the anthocyanin content (mg/l) was calculated based on a working curve for which malvidin-3 glucoside was used as a standard. results and discussion the results of testing the basic physicochemical parameters are shown in tab. 1. the alcohol content ranged from 11.43 vol% to 11.85 vol%, meaning that the maceration conditions had no effect on the alcohol content in the wine. the content of reducing sugars ranged from 1.1 g/l to 1.4 g/l, indicating that the degree of sugar overfermentation was complete, regardless of the maceration conditions. with the extension of the maceration time in wines, an increase in the content of the sugar-free extract and the ash content were determined. in this respect, the extraction of various compounds from the solid parts of the grapes (skin, seeds) into stum is prolonged, whereby the content of the sugar-free extract and the ash content in wine are increased. the lowest content of sugar-free extract was found in white wine (18.6 g/l), with virtually no maceration. with rosé wine, where the maceration lasted for two hours, the content of sugar-free extract was just slightly higher, and was 18.9 g/l. the highest content of sugar-free extract was found in red wine at 21.3 g/l. the lowest ash content was also found in white wine (1.86 g/l), while the highest content was in red wine (2.34 g/l). total acid content ranged from 4.34 g/l to 5.36 g/l and volatile acid content from 4.5 meq/l to 6.5 meq/l. duration of maceration caused decrease in the content of total acids, so the lowest content was in red wine of 4.34 g/l. the reduction of total acids during maceration is explained by the release of minerals that pass into the wine and react with tartaric acid during maceration period, resulting in table 1. basic physicochemical parameters of quality of white, rosé and red wine parameters white wine rosé wine red wine relative density, 20 °c 0.9922 0.9918 0.9927 alcohol, % v/v 11.43 11.76 11.85 total extract, g/l 19.0 19.0 21.6 reducing sugars, g/l 1.4 1.1 1.3 sugar free extract, g/l 18.6 18.9 21.3 total acids, g/l 5.36 5.16 4.34 volatile acids, meq/l 6.5 4.5 5.7 ph-value 3.24 3.26 3.75 free so2, mg/l 26.4 28.8 12.8 total so2, mg/l 148.0 110.4 28.8 ash, g/l 1.86 1.99 2.34 biologica nyssana ● 11 (2) december 2020: 121-128 nikolić-milojević et al. ● influence of the time of maceration on phenolic composition of wines produced from the indigenous variety prokupac the formation of salts. with reduction of the total acid content, the ph value of the wine increases, so that a maximum value of 3.75 was measured in red wine, while with white wine it was 3.24. according to the available results (budić-leto et al., 2008) the maceration time had no effect on the relative density, alcohol content, total extract of reducing sugars, acidity and ash in red wine. within the experimental part of this paper, the intensities and shades of wine colors were analyzed. tab. 2 shows the chromatic characteristics of the tested wines. the results are presented as the mean value out of three repetitions, with standard deviation. the color intensity of the wines (i) is the lowest in white wine; when rosé wine is concerned, short maceration led to a significant increase in colour intensity, while with red wine there is a significant rise in measured value. the shade of colour (n), which is mostly used as a parameter of aging wine, is highest in white and lowest in red wine. the colour of rosé wine is non-specific. according to glories (1984), the optimum ratio of the colour components to the type of rosé wine is 35:55:10, and in the case of the tested rosé wine is 64:33:3. it is characterized by an extremely light colour with a predominant yellow-orange tone. maceration duration changed some chemical constituents and color of red wines (kocabey et al., 2016). extended maceration duration resulted in increase in the concentration of phenolic compounds, reflecting the antioxidant activities of wine. the highest concentrations of total and individual phenolic compounds as well as antioxidant activities were found in wines macerated for 15 days. the maceration process plays an important role in the composition of the colour and sensory properties of red wine (şener, 2013, 2018). macerations carried out at low temperature ranges (10 °c to 15 °c) resulted in red wines with the highest levels of total phenolic content, anthocyanin and colour intensity, and richer fruity, flowery and spicy aroma. prolonged maceration leads to a stable red colour, too. chromatic characteristics of red wines were observed four times during the year (babincev et al., 2016). intensity, hue and brilliance of color of these wines were determined. the highest value of color intensity was observed in young wines (merlot 2.26, vranac 2.24) and the lowest value was recorded for prokupac, aged 12 months (1.18). with wine ageing the color intensity slightly decreased. the maximum value for hue in a very young wine was found in merlot (0.86) and in 12-month-old wines cabernet (0.99). brilliance of wine also decreased with wine ageing and it was most pronounced in young vranac (47.1) and the lowest value was found in 12-month old prokupac (27.7). test results for total phenols, total phenol index (tpi), total flavonoids, and total anthocyanins are shown in tab. 3 as the mean value out of three repetitions with standard deviation. 125 table 2. chromatic characteristics of white, rosé and red wine parameters white wine rosé wine red wine a420 0.098 ± 0.005 0.187 ± 0.002 1.455 ± 0.030 a520 0.030 ± 0.003 0.096 ± 0.002 1.882 ± 0.020 a620 0.001 ± 0.001 0.008 ± 0.002 0.483 ± 0.019 i 0.129± 0.006 0.291± 0.006 3.820±0.013 n 3.337± 0.388 1.955± 0.046 0.773± 0.021 a420% 76.44± 2.70 64.33± 0.88 38.09± 0.66 a520% 23.05± 1.90 32.91± 0.53 49.26± 0.61 a620% 0.51± 0.87 2.75± 0.90 12.65± 0.52 da% / / 48.49± 1.24 table 3. total flavonoids and anthocyanins in white, rosé and red wine parameters white wine rosé wine red wine total phenols, mg/l gae 565.4 ± 17.0 523.4 ± 3.5 1433.8 ± 90.9 tpi (a280) 12.29 ± 0.49 13.83 ± 0.41 31.77 ± 4.19 total flavonoids, mg/l ce 153.4±1.8 179.3±2.1 761.7±15.9 total anthocyanins, mg/l / 2.8 154.0±2.9 biologica nyssana ● 11 (2) december 2020: 121-128 nikolić-milojević et al. ● influence of the time of maceration on phenolic composition of wines produced from the indigenous variety prokupac 126 total phenols, in contrast from expected, are higher in white wine than in rosé wine. most are found in red wine and their value is 1433.8 mg/l. on the other hand, the index of total phenols in rosé wine (13.83) is slightly higher than in white wine (12.29), while it is the highest in red wine (31.77). total flavonoids are the lowest in white wine (153.4 mg/l) and the highest in red wine (761.7 mg/l). anthocyanins are below the lower detection limit in white wine, 2.8 mg/l, and in red wines they are 154 mg/l. here the influence of maceration becomes obvious. according to the results of alencar et al. (2018), the content of phenolic compounds increased up to 15th day during the process of maceration, and anthocyanins up to 20 days. extending the maceration time thus also improved the antioxidant activity of the wine. chemometric analysis of wine samples is shown in fig. 1 and 2, which were made in the program statistica v.8.0 (statsoft inc., tulsa, ok, usa). conclusion maceration has a very large influence on the physical and chemical parameters of wine quality. during the maceration process, many substances are extracted, and transferred from the solid parts into the wine. as a result, red wines have a much more complex chemical composition than white and rosé wines. in our samples, the duration of maceration had no significant effect on the relative density and alcohol content. the effect is significant on the content of the sugar-free extract and the ash content due to the higher extraction of nitrogen compounds, minerals and numerous other substances. chromatic analysis showed that the extraction of coloured substances was much higher with red wine, where the maceration lasted for two weeks at temperature ranging from 15 °c to 20 °c, compared to rosé wine, which only used short-term cold maceration. total phenols in red wine are almost three times higher than in white and rosé wines, and total flavonoids by slightly more than four times. the difference is greatest when it comes to anthocyanins extraction, which is almost nonexistent with white wine, and with rosé wine 55 times less concentrated than in red wine. the tannin concentration is also highest in red wine and lowest in white wine. the parallel examination of wine produced from the same, autochthonous prokupac variety, indicates fig. 1. dendrogram obtained by cluster analysis using means of basic physicochemical parameters of white, rosé and red wine quality single linkage euclidean distances 30 40 50 60 70 80 90 linkage distance red wine rose wine white wine fig. 2. dendrogram obtained by cluster analysis using means of chromatic characteristics and total flavonoids and anthocyanins of white, rosé and red wine biologica nyssana ● 11 (2) december 2020: 121-128 nikolić-milojević et al. ● influence of the time of maceration on phenolic composition of wines produced from the indigenous variety prokupac 127 the potential of this variety and the opportunities it offers to producers for the application of different technologies. acknowledgement. this work was supported by the ministry of education, science and technological development of the republic of serbia under the program of financing scientific re search work, number 451-032842/2019-14/172047. the authors are grateful to prof. nebojša deletić, phd, university of priština, faculty of agriculture, kosovska mitrovica lešak, for his help with the statistical processing of the results. references alencar, n.m.m., cazarin, c.b.b., corrêa, l.c., maróstica junior m.r., biasoto, a.c.t., behrens, j.h. 2018: influence of maceration time on phenolic compounds and antioxidant activity of the syrah must and wine. journal of food biochemistry, 42 (2): e12471. babincev m. lj., gurešić, m.d., simonović m.r. 2016: spectrophotometric characterization of red wine color from the vineyard region of metohia. journal of agricultural sciences, 61(3): 281-290. barbará, j.a., nicolli, k.p., souza-silva, e.a., biasoto, a.c.t., welke, j.e., zini, c.a. 2020: volatile profile and aroma potential of tropical syrah wines elaborated in different maturation and maceration times using comprehensive twodimensional gas chromatography and olfactometry. food chemistry, 308: 125552. blesić, m. 2016: tehnologija vina. praktikum, univerzitet u sarajevu, poljoprivredno-prehrambeni fakultet. budić-leto, i., gracin, l., lovrić, t., vrhovsek, u. 2008: effects of maceration conditions on the polyphenolic composition of red wine ‘plavac mali’. vitis, 47 (4): 245-250. busse-valverde, n., bautista-ortín a.b., gómezplazae., fernández-fernández j.i., gil-muñoz, r. 2012: influence of skin maceration time on the proanthocyanidin content of red wines. european food research and technology, 235: 1117-1123. cadot, y., caillé, s., samson, a., barbeau, g., cheynier, v. 2012: sensory representation of typicality of cabernet franc wines related to phenolic composition: impact of ripening stage and maceration time. analytica chimica acta, 732: 9199. casassa, l.f., huff, r., steele, n.b. 2019: chemical consequences of extended maceration and postfermentation additions of grape pomace in pinot noir and zinfandel wines from the central coast of california (usa). food chemistry, 300: 125-147. glories, y. 1984: la couleur des vins rouges. lre partie : les équilibres des anthocyanes et des tanins. journal international des sciences de la vigne et du vin, 18 (3): 195-217. gonzález-neves, g., gil, g., favre, g., baldi, c., hernández, n., traverso, s. 2013: influence of winemaking procedure and grape variety on the colour and composition of young red wines. south african journal of enology & viticulture, 34 (1): 138-146. jordão, a.m., simões, s., correia, a.c., gonçalves, f.j. 2012: antioxidant activity evolution during portuguese red wine vinification and their relation with the proanthocyanidin and anthocyanin composition. journal of food processing and preservation, 36: 298-309. kocabey, n., yilmaztekin, m., hayaloglu, a.a. 2016: effect of maceration duration on physicochemical characteristics, organic acid, phenolic compounds and antioxidant activity of red wine from vitis vinifera l. karaoglan. journal of food science and technology, 53(9): 3557-3565. kostadinović, s., wilkens, a., stefova, m., ivanova, v., vojnoski, b., mirhosseini, h., winterhalter p. 2012: stilbene levels and antioxidant activity of vranec and merlot wines from macedonia: effect of variety and enological practices. food chemistry, 135(4): 3003-3009. plavša, t., jurinjak, n., antunović d., peršurić, đ., kovačević ganić, k. 2012: the influence of skin maceration time on the phenolic composition and antioxidant activity of red wine teran (vitis vinifera l.). food technology and biotechnology, 50(2): 152-158. şener, h. 2018: effect of temperature and duration of maceration on colour and sensory properties of red wine: a review. south african journal of enology & viticulture, 39(2): 227-234. şener, h., yildirim, h.k. 2013: influence of different maceration time and temperatures on total phenols, colour and sensory properties of cabernet sauvignon wines. food science and technology international, 19(6): 523-33. spranger, m.i., clı́maco, m.c., sun, b., eiriz, n., fortunato, c., nunes, a., leandro, m.c., avelar, m.l., belchior, a.p. 2004: differentiation of red wine making technologies by phenolic and volatile composition. analytica chimica acta, 513(1): 151161. suriano, s., basile, t., tarricone, l. gennaro, biologica nyssana ● 11 (2) december 2020: 121-128 nikolić-milojević et al. ● influence of the time of maceration on phenolic composition of wines produced from the indigenous variety prokupac d.d., tamborra, p. 2015: effects of skin maceration time on the phenolic and sensory characteristics of bombino nero rosé wines. italian journal of agronomy, 10(1): 21-29. yilmaztekin, m., kocabey, n., hayaloglu, a.a. 2015: effect of maceration time on free and bound volatiles of red wines from cv. karaoğlan (vitis vinifera l.) grapes grown in arapgir, turkey. journal of food science, 80(3): c556-63. yoo, y.j., prenzler, p.d., saliba, a.j., ryan, d. 2011: assessment of some australian red wines for price, phenolic content, antioxidant activity, and vintage in relation to functional food prospects. journal of food science, 76: 1355-1364. 128 biologica nyssana ● 11 (2) december 2020: 121-128 nikolić-milojević et al. ● influence of the time of maceration on phenolic composition of wines produced from the indigenous variety prokupac grigorov & pavlovic 2021, biologica nyssana 12(2) 12 (2) december 2021: 123-129 doi: 10.5281/zenodo.5759850 quality control, phenolic content and in vitro antioxidant potential of orange mullein flower and leaf original article maja grigorov department of pharmacy, faculty of medicine, university of niš, bulevar dr zorana ðinđića 81, 18000 niš, serbia maja.grigorov@gmail.com (corresponding author) dragana pavlović department of pharmacy, faculty of medicine, university of niš, bulevar dr zorana ðinđića 81, 18000 niš, serbia received: august 23, 2021 revised: november 24, 2021 accepted: december 02, 2021 abstract: verbascum species are traditionally used in the treatment of respiratory disorders. orange mullein (verbascum phlomoides l.) is one of three species that are the official source of drug mullein flower (verbasci flos). our study aimed to determine the overall quality of the v. phlomoides flos and v. phlomoides folium, the contents of polyphenols, tannins, flavonoids, and phenylpropanoid derivatives alongside the antioxidant capacity of their ethanolic extracts. quality control tests were conducted according to monographs of ph. eur. 10.0, secondary plant metabolites were quantified spectrophotometrically while the antioxidant potential was estimated by two complementary methods (dpph free radical scavenging and β-carotene bleaching assay). the higher contents of examined compounds were determined in flower extract although the leaf extract showed a stronger antioxidant capacity. we can conclude that verbascum phlomoides is a rich source of biologically valuable phenolics, it possesses considerable antioxidant capacity and investigated herbal drugs fulfill pharmacopeia’s quality requirements. key words: verbascum phlomoides, overall quality, polyphenols, flavonoids, antioxidant activity apstrakt: ispitivanje opšteg kvaliteta, sadržaj fenola i in vitro antioksidativni potencijal cveta i lista krupnocvetne divizma vrste roda verbascum se tradicionalno koriste u lečenju respiratornih poremećaja. krupnocvetna divizma (verbascum phlomoides l.) je jedna od tri vrste koje se koriste za dobijanje biljne droge cvet divizme (verbasci flos). cilj naše studije bio je da se utvrdi opšti kvalitet droge v. phlomoides flos i v. phlomoides folium, kao i sadržaj polifenola, tanina, flavonoida i derivata fenilpropanoida i ispita antioksidativna aktivnost etanolnih ekstrakata. opšti kvalitet droge ispitan je u skladu sa ph. eur. 10.0, sekundarni biljni metaboliti su određeni spektrofotometrijski, dok je antioksidativna aktivnost ispitivana pomoću dve metode (dpph metoda i test inhibicije obezbojavanja β-karotena). veći sadržaji ispitivanih jedinjenja utvrđeni su u ekstraktu cveta, iako je ekstrakt lista pokazao jači antioksidativni kapacitet. možemo zaključiti da je verbascum phlomoides bogat izvor biološki vrednih fenola, da poseduje značajan antioksidativni kapacitet i da ispitani biljni lekovi ispunjavaju zahteve farmakopeje u pogledu kvaliteta. ključne reči: verbascum phlomoides, ispitivanje opšteg kvaliteta, polifenoli, flavonoidi, antioksidativna aktivnost introduction orange mullein (verbascum phlomoides l.) belongs to the genus verbascum from the family scrophulariaceae (georgiev et al., 2011). this genus comprises about 360 species around the world. they are mostly represented in europe (95 species, 23 of which are also found in serbia), asia, and north america (georgiev et al., 2011; mihailović et al., 2016). plant species of this genus are known as mullein (georgiev et al., 2011). mulleins are flowering biennial plants (rarely perennial or annual), which form a rosette of leaves in the first year and flowering stems in the second year (georgiev et al., 2011; marian et al., 2018). they reach a height of 0.3-2 m (georgiev et al., 2011). yellow flowers are grouped under bracts. they bloom from june to august, sometimes in september (marian et al., 2018). traditionally, verbascum species are used in © 2021 grigorov & pavlović. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 123 the treatment of respiratory disorders, such as dry cough, bronchitis, tuberculosis, asthma (georgiev et al., 2011; selseleh et al., 2019). yet, these species show beneficial effects in hemorrhoids, wounds, rheumatism, diarrhea, eczema, and other types of inflammatory conditions (luca et al., 2019), burns and, according to some literature data, are also effective against headache (mihailović et al., 2016). a mild sedative, as well as mild diuretic and soothing effects on the ureter, have been noticed. verbascum species are sometimes used to reduce urinary inflammation (süntar et al., 2010; selseleh et al., 2019), and as a tea to reduce abdominal pain (süntar et al., 2010). mulleins have been used since ancient times and in recent years their popularity is growing (georgiev et al., 2011). nowadays, herbal products for tea preparation from flowers of v. thapsus, v. densiflorum and v. phlomoides can be found in american and european health stores (mihailović et al., 2016). according to data from 2010, verbascum spp. is used in six european union countries as traditional herbal supplements or herbal medicinal products (mihailović et al., 2016). verbascum phlomoides is one of three species of the genus verbascum (the other two are: v. densiflorum bartol, and, rarely used due to some reported deficiencies, v. thapsus l. (georgiev et al., 2011)) that are the source of the drug mullein flower (verbasci flos) (ema, 2007). it is a biennial plant, known as a very rich phytochemical source (paun et al., 2016). the most common compounds of the drug verbasci flos are iridoid glycosides (aucubin, catalpol, 6-xylosilaukubin, and 6-xylosylcatalpol), flavonoids (tamarixetine 7-rutinoside, tamarixetin 7-glucoside, apigenin, drimolphein and luteolin), phenylethanoid glycosides (verbascoside), triterpene saponins (verbascosaponin), polyphenols, polysaccharides, phytosterols, oleic acid (ema, 2007). due to such complex composition, verbascum species show many beneficial effects on human health. numerous studies have confirmed that the fractions of saponins, iridoids, and phenylethanoids are responsible for their anti-inflammatory effect (mihailović et al., 2016). in numerous developing countries, traditional medicines are preferred over conventional ones. the interest in herbal products is in constant increase in developed countries too. consequently, the quality, safety and efficacy of herbal products must be at the highest level (he et al., 2015; van wyk & prinsloo, 2020). that is why a comprehensive specification must be developed for each herbal substance even if the starting material for the manufacture of the finished product is a herbal preparation. the specification should be established based on recent scientific data and the monographs of the european pharmacopoeia (sarfaraz, 2007) and other pharmacopeias (like german pharmacopoeia, swiss pharmacopoeia, british herbal pharmacopoeia). considering the mentioned pharmacological potential of verbascum species, our study aimed to determine the overall quality of the verbasci flos and verbasci folium as well as the content of polyphenols, tannins, flavonoids, and phenylpropanoid derivatives in their ethanolic extracts. estimation of antioxidant activity of these extracts was added into the study since this activity could be linked to many beneficial health outcomes. materials and methods plant material and extracts the plant material (flowers and leaves) was collected in the vicinity of bosilegrad during august 2019. botanical identification was performed by prof. bojan zlatković (faculty of science and mathematics, university of niš). the voucher specimens are stored in the herbarium of the faculty of science and mathematics, university of niš (herbarium code: 14506). after drying at room temperature, in a dark place with a constant flow of air, the plant material was ground using a laboratory mill and stored in glass bottles until the examination. extracts were prepared by percolation with 50% ethanol (vpf – v. phlomoides flower extract and vpl – v. phlomoides leaf extract) according to european pharmacopeia 10.0 (2019) (european pharmacopeia, 2019), followed by total removal of the solvent in a rotary vacuum evaporator at 40 °c. yields after evaporation are expressed in % of dry plant material used and as der (drug:extract ratio specifies the initial amount of drug used for the preparation of a certain amount of extract). the dry extracts were preserved for one month in the refrigerator until examinations. the determinations were performed in extracts dissolved in 50% alcohol. chemical reagent and instrumentation all reagents used in this study were of analytical grade. rutin and gallic acid were purchased from sigma-aldrich co. (sigma-aldrich co., st louis, mo), and catechin from extrasynthese (lyon, france). spectrophotometric measurements were performed on an evolution 60 thermo scientific spectrophotometer (fisher scientific, loughborough, u.k.) and multiskan ascent no354 (thermo labsystems, finland) elisa microplate reader. for incubation incuterm raypa® trade (catalonia, spain) was used. 124 biologica nyssana ● 12 (2) december 2021: 123-129 grigorov & pavlović ● quality control, phenolic content and in vitro antioxidant potential of orange mullein flower and leaf 125 quality control the overall quality examinations of herbal drugs included a swelling index, loss on drying, total ash, ash insoluble in hydrochloric acid following the regulations of ph. eur. 10.0 (2019) and by the monograph of the examined herbal drug verbasci flos (european pharmacopeia, 2019). these parameters are listed in the part tests of the monograph. total polyphenols and total tannins the content of total polyphenols was determined by folin–ciocalteu colorimetric method (makkar et al., 2000). the absorbances of the samples and standards were measured at 725 nm, and the results were expressed as gallic acid equivalents per g of extract. the total tannin content was determined by the same folin-ciocalteu procedure (makkar et al., 2000) after removal of tannins using insoluble binder (polyvinylpolypyrrolidone, pvpp). the test was performed with a clear supernatant, and the results were also expressed as gallic acid equivalents per g of extract. phenylpropanoid derivates the content of total phenylpropanoid derivatives was determined spectrophotometrically, according to the arnow method (arnow, 1937). this method is based on the reaction of derivatives of o-dihydroxycinnamic acid with sodium molybdate. absorbance was measured at 525 nm, and the phenylpropanoid content was expressed as the content of the corresponding hydroxycinnamic acid derivative. total flavonoids the total flavonoid content was determined spectrophotometrically by the alcl3 method at 430 nm (lamaison & carnat, 1990). results were expressed as a microgram of rutin per milligram of the sample (mg ru/mg). antioxidant activity radical scavenging activity was determined using stable free radical 1,1-diphenyl-2-picrylhydrazyl (dpph). the determination was done according to the method described by chang et al (2010) (chang et al., 2010) with certain modifications. dpph free radical inhibition in percent was calculated according to: % dpph = (ab as / ab) x 100, where ab presents the absorbance of the control reaction (containing ethanol instead of the test solution) and as is the absorbance of the sample. a regression curve was constructed, and the obtained results were presented as the ic50 value, which indicates the sample concentration required to remove 50% of dpph free radicals. rutin was used as a reference compound. concentrations are expressed in µg/ml. β-carotene bleaching test (bcbt) was done according to the method described by koleva et al. (2002) with slight modifications. the absorbance was measured using an elisa reader at a wavelength biologica nyssana ● 12 (2) december 2021: 123-129 grigorov & pavlović ● quality control, phenolic content and in vitro antioxidant potential of orange mullein flower and leaf ta bl e 1. r es ul ts o f t he o ve ra ll qu al ity te st in g of o ra ng e m ul le in fl ow er a nd le af sw el lin g in de x l os s on d ry in g (% ) t ot al a sh (% ) a sh in so lu bl e in hy dr oc hl or ic a ci d (% ) % o f d ry pl an t m at er ia l us ed d e r * ph . e ur .1 0 ph . e ur .1 0 ph . e ur .1 0 ph . e ur .1 0 v er ba sc i flo s 9. 20 ±1 .3 2 >9 9. 06 ±0 .4 7 <1 2 3. 5± 0. 92 <6 0. 97 ±0 .1 2 <2 40 .8 2% 2. 45 :1 v er ba sc i fo liu m 14 .7 5± 1. 86 >9 7. 28 ±0 .3 1 <1 2 6. 05 ±0 .0 6 <6 1. 76 ±0 .2 3 <2 12 .9 3% 7. 73 :1 * d ru g/ ex tra ct ra tio 126 of 450 nm after shaking the plate on a microplate shaker. the initial absorbance was measured, the plate was placed in an incubator at 47 °c for 120 minutes, afterward, the absorbance was measured again. the ability to inhibit β-carotene decolorization is expressed as a percentage of inhibition according to the formula: % inhibition = (a120 / a0) x 100, where a120 is the absorbance measured at t=120 min and a0 is the absorbance measured at t=0 min. after the regression curve was constructed, the ic50 value (concentration required to inhibit β-carotene bleaching to 50%) was calculated and expresses as the average value of three independent experiments in µg/ml. statistical analysis all tests and experiments were performed in triplicates, and the experimental results were presented as the mean of three consecutive measurements ± standard deviation. results results of the overall quality testing of dry flowers and leaves alongside pharmacopoeial demands are presented in tab. 1. tab. 2 shows the contents of total polyphenols, total tannins, phenylpropanoid derivatives, and total flavonoids and the results of in vitro antioxidant activities of ethanolic extracts vpf and vpl. discussion the obtained results of the overall quality testing of the herbal drug are in accordance with the recommendations of the ph. eur. 10.0 (european pharmacopeia, 2019). the extract yields were 40.82% and 12.93% for flower and leaf extract, respectively. many herbal drugs are of specific therapeutic or pharmaceutical utility because of their swelling properties (e.g., gums and those containing an appreciable amount of mucilage, pectin or hemicellulose) (who, 1998). the swelling index of v. phlomoides flower and leaf define mucilage content and present a valuable parameter of drug quality. in our study, verbasci folium showed a higher swelling index than verbasci flos, although both parameters are in accordance with pharmacopoeial demand (>9). polyphenols show numerous biological effects, such as anti-inflammatory, antioxidant, immunomodulatory, anticancer, cardioprotective effects (yahfoufi et al., 2018). their content was determined by the colorimetric folin–ciocalte method, which is based on the reducing ability of the hydroxyl groups of polyphenolic compounds. the polyphenolic compounds are oxidized, and the resulting phenoxide anion reduces the folinciocalteu reagent to a blue-colored ion. based on the results, shown in tab. 2, the plant species v. biologica nyssana ● 12 (2) december 2021: 123-129 grigorov & pavlović ● quality control, phenolic content and in vitro antioxidant potential of orange mullein flower and leaf phlomoides is rich in polyphenols and their higher content was determined in the vpf compared to the vpl. these results are close to the results of the determination of polyphenols obtained by mihailović et al. (2016) in the aqueous extract (51.3±0.3 mg gallic acid equivalents (gae)/g extract), while content in the methanol extract in the mentioned study was slightly higher (88.2±1.6 mg gae/g extract) (mihailović et al., 2016). the total polyphenol content expressed as a percentage of gallic acid is 4.91, a bit higher than the 4.18%, which is the content of polyphenols given by ema (ema, 2007). our results are also similar to the results of determining polyphenols in the butanol extract of v. phlomoides (grigore et al., 2013). numerous epidemiological data indicate beneficial effects of tannins in the treatment of inflammation and skin injuries, as well as in preventing the development of chronic diseases (badal mccreath & delgoda, 2017). flavonoids are a group of polyphenols, with a lot of beneficial effects on human health. their antioxidant effect is most often discussed, but their anti-inflammatory, table 2. contents of total polyphenols, total tannins, phenylpropanoid derivatives and total flavonoids in ethanolic extracts of orange mullein flower and leaf and ic50 values of dpph test and bcbt total polyphenols (mg ga1/g extract) total tannins (mg ga1/g extract) phenylpropanoid derivates (ha2/g extract) total flavonoids (mg r3/g extract) ic50 in dpph test (µg/ml) ic50 in β-carotene bleaching test (µg/ml) vpf 49.15±2.70 25.19±4.17 6.32±0.19 16.71±0.91 257.61±2.62 164.24±7.11 vpl 39.70±4.12 16.56±1.82 7.10±0.01 12.81±0.49 157.04±3.86 132.59±5.08 1ga – gallic acid, 2ha – hlorogenic acid, 3r – rutin 127 biologica nyssana ● 12 (2) december 2021: 123-129 grigorov & pavlović ● quality control, phenolic content and in vitro antioxidant potential of orange mullein flower and leaf anticancer, antimutagenic effects are equally important (panche et al., 2016). the results of the determination of tannins and flavonoids show that the vpf is richer in these compounds than the vpl. this could be one of the explanations why is the mullein flowers rather than leaves used in folk medicine. our results of total flavonoids determination are close to the results obtained by mihailović et al. (2016) for the aqueous extract but lower than their results for the methanol extract (mihailović et al., 2016). armatu et al. (2011) determined the chemical compositions of two extracts of v. phlomoides collected in the southeastern part of romania. their results for the total flavonoid content are close to ours, with the butanol extract being richer in flavonoids, compared to the ethanol (armatu et al., 2011). paun et al. (2016) in their work determined the content of polyphenols and flavonoids in aqueous and ethanol extract of v. phlomoides and reported a higher content of polyphenols and flavonoids compared to ours (paun et al., 2016). the content of polyphenols and flavonoids, as well as other compounds in plant species, is influenced by numerous factors, such as the region in which the species grows, the conditions in which it grows, the time in which plant material is collected, storage methods and many others (elmastaş et al., 2017). additionally, the determination of phenylpropanoids was included in this study since this class of secondary metabolites besides anti-inflammatory, antimicrobial, cytoprotective and wound healing effects could be beneficial for skin regeneration (pavlović et al., 2013). the total phenylpropanoid derivatives content in vpl was slightly higher than in vpf (tab. 2). there are several different methods for assessing the antioxidant capacity of natural products. however, given the range of different types of free radicals that could be generated under in vivo conditions and the different reaction mechanisms involved in the antioxidant capacity of antioxidants, there is still no universal method to determine antioxidant capacity accurately and qualitatively (mihailović et al., 2016). in our work, we used the dpph and the bcbt tests. the obtained results of the dpph test show that the vpf has a lower antioxidant capacity (ic50=257.61±2.62 µg/ml) compared to the vpl (ic50=157.04±3.86 µg/ml). the antioxidant activity tested in the bcbt test was also higher for vpl extracts compared to vpf extracts, although the difference in ic50 values is not so pronounced (132.59±5.08 µg/ml and 164.24±7.11 for vpl and vpf, respectively). it is important to underline that the antioxidant capacity was dosedependent (higher doses of extract demonstrated higher effects which was the base for the construction of regression curves). since plant extracts are a mixture of different compounds, more different antioxidant tests are needed for a full understanding of the properties of one particular extract (pavlović et al., 2013). as expected, antioxidant activities of rutin, used as reference substance (positive control), were much lower (ic50 values in dpph and bcbt: 2.68±0.25 µg/ml and 33.29±4.06 µg/ml, respectively) compared with antioxidant activities of tested extracts. conclusions based on the presented results, we can conclude that the plant species verbascum phlomoides contains significant amounts of polyphenols, tannins, and flavonoids, and due to the well-known positive effects of these compounds on human health, this species deserves to remain in the focus of our study. also, the contents of these compounds were higher in flowers compared to leaves. this could be one of the explanations why is the mullein flowers rather than leaves used in folk medicine for topical applications. yet, the leaf extract showed a stronger antioxidant capacity in applied tests. further investigations and detailed chemical profiling are needed to assess the relevance of ethnopharmacological application and define the potential health benefits of orange mullein. references armatu, a., bodirlau, r., nechita, c.b., niculaua, m., teaca, c.a., ichim, m., spiridon, i. 2011: characterization of biological active compounds from verbascum phlomoides by chromatography techniques. i. gas chromatography. romanian biotechnological letters, 16(4): 6297-6304. arnow, e. 1937: colorimetric determination of the components of 3,4-dihydroxyphenylalaninetyrosine mixtures. journal of biological chemistry, 118: 531-537. badal mccreath, s., delgoda, r. 2017: pharmacognosy: fundamentals, applications and strategies. elsevier science. 2246 p. chang, l.c., zhang, j.l., chen, y.r., kuo, y.l.m., huang, p.j., huang, c.h., lee, h.k., wu, c.y., kuo, h.y. 2010: antioxidant and antiinflammatory phenylpropanoid derivatives from calamus quiquesetinervius. journal of natural products, 73: 1482–1488. council of europe 2019: european pharmacopeia. 10th edition. council of europe: european directorate for the quality of medicines and healthcare, strasbourg. dimitrova, p., alipieva, k., grozdanova, t., simova, s., bankova, v., georgiev, m.i., popova, m.p. 2018: new iridoids from verbascum nobile and their effect on lectin-induced t cell activation and proliferation. food and chemical toxicology, 111: 605–615. elmastaş, m., demir, a., genç, n., dölek, ü., güneş, m. 2017: changes in flavonoid and phenolic acid contents in some rosa species during ripening. food chemistry, 235: 154-159. ema 2018: assessment report on verbascum thapsus l., v. densiflorum bertol. (v. thapsiforme schrad) and v. phlomoides l., flos. london: european medicinal agency. georgiev, m.i., ali, k., alipieva, k., verpoorte, r., choi, h.y. 2011: metabolic differentiations and classification of verbascum species by nmr-based metabolomics. phytochemistry, 72: 2045–2051. grigore, a., colceru-mihul, s., litescu, s., panteli, m., rasit, i. 2013: correlation between polyphenol content and anti-inflammatory activity of verbascum phlomoides (mullein). pharmaceutical biology, 51(7): 925–929. he, t.t., oi lam ung, c., hu, h., wang, y.t. 2015: good manufacturing practice (gmp) regulation of herbal medicine incomparative research: china gmp, cgmp, whogmp, pic/s andeu-gmp. european journal of integrative medicine, 7(1): 55-66. koleva, i.i., van beek, t.a., linssen, j.p.h., de groot, a., evstatieva, l.n. 2002: screening of plant extracts for antioxidant activity: a comparative study on three testing methods. phytochemical analysis, 13: 8–17. lamaison, l.j., carnat, a. 1990: teneurs en principaux flavonoids des fleurs de crataegeus monogyna jacq et de crataegeus laevigata (poiret d.c.) en fonction de la vegetation. pharmaceutica acta helvetiae, 65: 315-320. luca, s.v., czerwińska, m.e., miron, a., aprotosoaie, a.c., marcourt, l., wolfender, j., granica, s., skalicka-woźniak, k. 2019: high-performance countercurrent chromatographic isolation of acylated iridoid diglycosides from verbascum ovalifolium donn ex sims and evaluation of their inhibitory potential on il-8 and tnf-α production. journal of pharmaceutical and biomedical analysis, 166: 295–303. makkar, h.p.s., hagerman, a., mueller-harvey, i. 2000: quantification on tannins in tree foliage-a laboratory manual. fao/iaea, vienna. 26 p. 128 biologica nyssana ● 12 (2) december 2021: 123-129 grigorov & pavlović ● quality control, phenolic content and in vitro antioxidant potential of orange mullein flower and leaf marian, e., vicas, l.g., jurca, t., muresan, m., pallag, a., stan, r.l., sevastre, b., diaconeasa, z., ionescu, m.l.c., hangan, a.c. 2018: salivia officinalis l. and verbascum phlomoides l. chemical, antimicrobial, antioxidant and antitumor investigations. revista de chimie, 69(2): 365-370. mihailović, v., kreft, s., tavčar benković, e., ivanović, n., stanković, m.s. 2016: chemical profile, antioxidant activity and stability in stimulated gastrointestinal tract model system of three verbascum species. industrial crops and products, 89: 141–151. panche, n.a., diwan, d.a., chandra, r.s. 2016: flavonoids: an overview. journal of nutritional science, 5(47): 1-15. paun, g., neagu, e., albu, k., radu, g.l. 2016: verbascum phlomoides and solidago virgaureae herbs as natural source for preventing neurodegenerative diseases. journal of herbal medicine, 6(4): 180-186. pavlović, d.r., tasić-kostov, m., marčetić, m., lakušić, b., kitić, d., savić, s., kovačević, n. 2013: evaluation of in vivo effects on surfactantirritated human skin, antioxidant properties and phenolic composition of five ericaceae species extracts. rivista italiana delle sostanze grasse, 90(4): 255-264. sarfaraz, n.k. 2007: handbook of preformulation: chemical, biological and botanical drugs. informa healthcare usa, inc. new york. 446 p. selseleh, m., hadian, j., ebrahimi, s.n., sonboli, a., georgiev, m.i., mirjalili, m.h. 2019: metabolic diversity and genetic association between wild populations of verbascum songaricum (scrophulariaceae). industrial crops & products, 137: 112–125. süntar, i., tatlı, i.i., küpeli akkol, e., keleş, h., kahraman, ç., akdemir, z. 2010: an ethnopharmacological study on verbascum species: from conventional wound healing use to scientific verification. journal of ethnopharmacology, 132: 408–413. van wyk, a.s., prinsloo, g. 2020: health, safety and quality concerns of plant-based traditional medicines and herbal remedies. south african journal of botany, 133: 54-62. who 1998: quality control methods for herbal materials. updated edition of quality control methods for medicinal plant materials. who, geneva. 187 p. 129 biologica nyssana ● 12 (2) december 2021: 123-129 grigorov & pavlović ● quality control, phenolic content and in vitro antioxidant potential of orange mullein flower and leaf yahfoufi, n., alsadi, n., jambi, m., matar, c. 2018: the immunomodulatory and antiinflammatory role of polyphenols. nutrients, 10(11): 1618. cvetković et al 2020, biologica nyssana 11(2) 11 (2) december 2020: 129-138 doi: 10.5281/zenodo.4393969 effects of different sucrose concentrations on some parameters of the life cycle in two wild drosophila species original article vladimir j. cvetković university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia biovlada@yahoo.com (corresponding autor) saša s. stanković university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia sasasta@gmail.com vladimir žikić university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia zikicvladimir@gmail.com nikola jovanović university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia nikolajov90@gmail.com jovana dimitrijević university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia jovana.dimitrijevic1@pmf.edu.rs aleksandra cvetanović university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia aleksandra.cvetanovic1@pmf.edu.rs tatjana mitrović university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia tatjanamitrovic3@gmail.com received: may 06, 2020 revised: november 19, 2020 accepted: november 30, 2020 abstract: the fruit flies collected from the wild can be easily cultured in laboratory for research purposes. on that occasion many similar recipes for cornmealbased feeding media can be used for drosophila cultivation. however, in these recipes concentrations of ingredients may differ what might imply a need to choose the recipe which is the most appropriate for culturing of wild drosophila species. the aim of this study was to check how two wild species, drosophila melanogaster and drosophila suzukii respond to different sucrose concentration in feeding media by monitoring of life cycle key parameters. the total number of oviposited eggs, formed pupae and eclosed adults as well as dynamics of pupae formation and adult eclosion were recorded. the results showed that the sucrose concentrations of 160 gl-1 caused significantly lower number of formed pupae and eclosed adults in d. suzukii than at concentrations of 40 gl-1 and 80 gl-1. prolonged and stretched dynamics of pupation and eclosion were recorded in d. melanogaster only at concentration of 160 gl-1. all tested drosophila showed no differences in life cycle parameters between sucrose concentration of 40 gl-1 and 80 gl-1. presented results can be helpful in deciding which cornmeal-based feeding media recipe to choose for cultivation of mentioned wild drosophila species. key words: fruit fly, drosophila melanogaster, drosophila suzukii, feeding media, life cycle, sucrose concentration apstract: efekat različitih koncentracija šećera na neke parametre životnog ciklusa kod dve divlje vrste drosophila voćne mušice, prikupljene u prirodi, mogu se jednostavno gajiti u laboratoriji za potrebe različitih istraživanja. za tu svrhu se može upotrebiti jedan od mnogih recepata sličnih sastojaka za pravljenje hranljive podloge, bazirane na kukuruznom grizu za gajenje drozofila. međutim, u tim receptima koncentracije sastojaka mogu da variraju što može da podrazumeva potrebu da se odabere recept koji najviše odgovara za gajenje divljih vrsta drozofila. cilj ovog istraživanja je da se praćenjem ključnih parametara životnog ciklusa proveri kako dve divlje vrste drosophila melanogaster i d. suzukii reaguju na različite koncentracije šećera u hranljivoj podlozi. praćeni su ukupan broj položenih jaja, ulutkanih i eklodiranih imaga kao i dinamike ulutkavanja i eklodiranja. rezultati su pokazali da koncentracija šećera od 160 gl-1 izaziva značajno manje ulutkavanja i eklodiranja kod d. suzukii nego što je to pri koncentracijama od 40 gl-1 i 80 gl-1. produžena i rastegnuta dinamika ulutkavanja i izleganja su zabeležene kod d. melanogaster samo pri koncentraciji od 160 gl-1. kod svih testiranih drozofila nisu detektovane značajne razlike u praćenim parametrima životnog ciklusa između tretmana sa koncentracijama šećera od 40 gl-1 i 80 gl-1. prikazani rezultati mogu biti od koristi prilikom odabira hranljivog medijuma baziranog na kukuruznom grizu za gajenje pomenutih divljih vrsta drozofila. ključne reči: voćna mušica, drosophila melanogaster, drosophila suzukii, hranljiva podloga, životni ciklus, koncentracija šećera © 2020 cvetković et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 129 introduction the common fruit fly, drosophila melanogaster meigen is a famous model organism that has very wide implementation in biological sciences, primarily in genetic researches. there are many stock centers in the world that provide an array of different drosophila stocks intended for experimental purposes. nevertheless, for some research purposes, there is a need for drosophila species that are obtained from the wild. on that occasion, the flies are caught and transferred into a laboratory ambient for further experiments. for instance, species from the genus drosophila caught in the wild are very often processed in this way, for example d. melanogaster (anagnostou et al., 2010; lin et al., 2014; gao et al., 2018). spotted wing drosophila, drosophila suzukii (matsumura) is a very serious pest that has been extensively explored in laboratory conditions (lin et al., 2014; tochen et al., 2014; silva-soares et al., 2017; fellous & xuéreb, 2017; schlesener et al., 2017). since this species is introduced worldwide, it has been already recognized as a real threat on production of certain soft fruits that are of economic importance (de ros et al., 2013; benito et al., 2016; mazzi et al., 2017). also, the presence of this fruit pest fly is confirmed for the first time in serbia in 2014 by toševski et al. (2014). thus, good laboratory practice is to provide optimal conditions including appropriate feeding media for such species to be transferred from the wild to a laboratory to obtain correct results from conducted experiments. apart from laboratory drosophila stocks, it is well-known that wild drosophila species can be successfully cultured on cornmeal-based feeding media, even d. suzukii (schlesener et al. 2017). there are many available recipes that are relevant for drosophila culturing, for example at the site of bloomington drosophila stock center (https://bdsc. indiana.edu) or in papers (lewis, 1960; schlesener et al. 2017; jovanović et al., 2018; cvetković et al., 2020). however, different recipes for cornmealbased feeding media imply different amounts of some ingredients such as carbohydrates and yeast. in addition, numerous articles are dealing with the influence of dietary yeast on d. melanogaster (anagnostou et al., 2010) and in d. suzukii on life-history traits (bellutti et al., 2018) or protein and carbohydrate ratio on larvae in d. suzukii (silva-soares et al., 2017) and dietary protein and carbohydrates balance in d. melanogaster (lee, 2015). these studies suggested that carbohydrates and proteins play very important role in drosophila lifespan so the special attention should be paid on the concentration of these two ingredients in the feeding media. according to lushchak et al., (2014) the influence of dietary carbohydrate type and dosage is still poorly understood. in addition, we believe that among different recipes, the most appropriate recipe should be selected for wild drosophila species that are transferred from natural to laboratory conditions because they cannot be adapted to artificial feeding media to the same extent as drosophila laboratory stocks. here, we ran a study to rapidly screen the degree of tolerance of the two wild species on three different sucrose concentrations in cornmeal-based feeding media at standard laboratory conditions by monitoring of the life cycle parameters. we believe that this will help to depict which cornmeal-based feeding media, in relation to sucrose concentration, is the most suitable for laboratory cultivation of wild d. melanogaster and d. suzukii collected in southeastern serbia. also, the study should contribute to overall knowledge about influence of dietary sucrose on the life cycle of two drosophila species. material and methods culturing conditions and types of feeding media the fruit flies were cultured at standard laboratory conditions which implies 60% of relative humidity, 12h/12h of the day/night light regime and constant ambient temperature at about 25±1 °c. standard feeding media based on cornmeal was used for culturing of all flies from this experiment. particularly, agar (6 gl-1), cornmeal (96 gl-1), sucrose (80 gl-1) and yeast (20 gl-1) prepared by cooking in dh2o with subsequent addition of fungicide as explained in our previous studies (cvetković et al., 2015; jovanović et al., 2016). for purposes of the experiment three different feeding media were prepared: all previously mentioned components were used in the same concentrations, except for the sucrose concentration that varied. medium with sucrose concentration of 40 gl-1 – labeled as half, medium with sucrose concentration of 80 gl-1 – labeled as normal and medium with sucrose concentration of 160 gl-1 – labeled as double, were used. fruit flies the wild d. melanogaster specimens were caught in the city of niš, near the faculty of sciences and mathematics (43°18’32.5”n; 21°55’22.9”e) in traps using grape as bait, during the september of 2018., leg. v. j. cvetković. the adult flies were immediately transferred on the standard feeding media for culturing. for the starter generation, d. suzukii adults were incubated from larvae from the infested raspberries collected in private raspberry orchard near lebane town (southeastern serbia), 130 biologica nyssana ● 11 (2) december 2020: 129-138 cvetković et al. ● effects of different sucrose concentrations on some parameters of the life cycle in two wild drosophila species 131 42°55’56.1”n; 21°45’21.6”e, during the august of 2018., leg. s. s. stanković. immediately after eclosion they were transferred onto fresh standard media for culturing (fig. 1). captured fruit fly species were determined by v. žikić and s. s. stanković. in order to establish stock, the fruit flies from the wild were cultured on standard (normal) feeding media (see culturing conditions and types of feeding media) for a dozen generations during 4 months. the referent laboratory strain of wild-type d. melanogaster oregon-r-c strain (bloomington drosophila stock center at indiana university, usa), was used in this study as referent line because this strain is adapted on artificial cornmeal-based feeding media that was used in this experiment. experimental protocol the whole experiment was performed at standard laboratory conditions (as explained in culturing conditions and types of feeding media). for the d. melanogaster – referent line and two wild species flies, three different types of vials were prepared, ten of each, that contained half, normal and double concentration of sucrose in experimental feeding media. from referent laboratory stock culture (d. melanogaster) and wild flies established stocks (d. melanogaster and d. suzukii), pairs of young flies at the same age (5-7 days old) were chosen for further analyses, similarly as it was done in our previous research (jovanović et al., 2018). in total, ten pairs were transferred into ten vials in each type of experimental feeding media to mate and lay eggs for two days. afterwards, the adult flies were discarded and the oviposited eggs were counted immediately. each day, number of formed pupae and subsequently eclosed adults was recorded. statistical analysis the statistical analysis was performed within each of the three drosophila group. the comparisons were performed by the following parameters: 1) the total numbers of oviposited eggs, 2) the total numbers of formed pupae and 3) the total numbers of eclosed adults, for each of the three sucrose concentrations. the percentage of pupation (percentage of formed pupae from hatched and survived larvae) and eclosion (percentage of eclosed adults from formed pupae) were calculated as it was described by kinjo et al. (2014). the obtained values for the same parameter (percentage of pupation or eclosion) in feeding media with different sucrose concentration were compared within each of the drosophila group. statistically significant differences were determined with the kruskall-wallis anova test for p<0.05. further, post hoc mann-whitney test was used to determine significant differences between any of two compared values. statistical analysis was conducted by using software package spss (spss inc., chicago, il, usa). results statistical analysis of the life cycle parameters i.e. the total number of oviposited eggs, formed pupae and eclosed adults are presented in tab. 1. the presented mean values and standard deviations were obtained based on 10 replicates for each strain, d. fig. 1. drosophila suzukii the starter generation. a – larvae (showed with blue arrow) from infested raspberry. b – eclosed adults, incubated from infested raspberries, transferred on standard fresh feeding media for culturing. c – pupae (showed with yellow arrows) within the feeding media, the first laboratory generation of captured d. suzukii. photos by v. j. cvetković biologica nyssana ● 11 (2) december 2020: 129-138 cvetković et al. ● effects of different sucrose concentrations on some parameters of the life cycle in two wild drosophila species 132 melanogaster-referent line, wild d. melanogaster and d. suzukii. all calculations were done for each of three feeding media types. the only significant difference in percentage of pupation between normal and double concentration were recorded in d. suzukii (fig. 2). the only significant differences in percentage of eclosion were observed between normal and double group within wild d. melanogaster and normal compared to half and double within d. melanogaster – referent line (fig. 3). in d. melanogaster – referent line, the peak of larval pupation was recorded on the fourth day, fifth and the seventh day in half, normal and double sucrose concentration, respectively (fig. 4). the peak of adult eclosion was recorded at the fifth day in half and normal and at the eighth day in double sucrose concentration (fig. 4). in addition, the peaks were higher and the pupation, as well as eclosion, lasted shorter in groups with half and normal sucrose concentration than in the group with double of the sucrose concentration. in the wild d. melanogaster, the peak of larval pupation was recorded on the fourth day in normal and double and the eighth day in half concentration of sucrose (fig. 5). the peak of adult eclosion was recorded also on the fourth day in half and normal and on the eighth day in a double concentration of sucrose (fig. 5). in addition, the peaks were higher and the pupation, as well as eclosion, species sucrose concentration oviposited eggs formed pupae eclosed adults d . m el an og as te r half 57.63 30.13 29.63 ±19.04 ±11.42 ±11.61 normal 71.5 41.1 40.7 ±21.19 ±14.49 ±14.24 double 65.87 37.88 36.38 ±29.43 ±11.51 ±11.36 d . m el an og as te r re fe re nt li ne half 71.22 62.22 59.11 ±24.54 ±20.28 ±17.88 normal 75.38 * 70.38 * 69.63 * ±20.70 ±16.49 ±16.55 double 55.4* 50.1 * 48.00 * ±19.98 ±18.54 ±17.51 d . s uz uk ii half 11.5 8.6† 8.1† ±8.22 ±5.68 ±5.5 normal 12.2 8.8‡ 8.5‡ ±6.05 ±2.3 ±3.2 double 7.1 2.9 †‡ 2.7 †‡ ±5.09 ±3.6 ±3.3 table 1. life cycle parameters in d. melanogaster-referent line, wild d. melanogaster and d. suzukii cultured on feeding media with different sucrose concentrations. mean values are presented with ± standard deviation. fig. 2. percentage of pupation in d. melanogaster, d. melanogaster-referent line and d. suzukii cultured on feeding media with half, normal and double concentration of sucrose. the lower case letter “a” marked the two values that are significantly different. differences were determined using kruskall-wallis anova and post hoc mann-whitney test. statistical significance was considered for p<0.05 -values marked with the same symbol * are significantly different within d. melanogaster – referent line -values marked with the same symbol † and/or ‡ are significantly different within d. suzukii group. -significant differences were considered for p<0.05 biologica nyssana ● 11 (2) december 2020: 129-138 cvetković et al. ● effects of different sucrose concentrations on some parameters of the life cycle in two wild drosophila species lasted shorter in groups with half and normal sucrose concentration than in double of the sucrose concentration. in d. suzukii line, the peak of larval pupation was recorded on the third, fourth and the seventh day in half, normal and double sucrose concentration, respectively (fig. 6). the peak of adult eclosion was recorded on the fourth, fifth and the eighth day in half, normal and double concentration of sucrose, respectively (fig. 6). additionaly, the peaks were higher in groups with half and normal sucrose concentration than in double of the sucrose concentration but the duration of the pupation and eclosion was mostly the same in all three groups. discussion all three parameters of the life cycle – the total number of oviposited eggs, formed pupae and 133 fig. 3. percentage of eclosion in d. melanogaster, d. melanogaster-referent line and d. suzukii cultured on medium with half, normal and double concentration of sucrose. the same lower cases letter (a, b or c) marked the two values that are significantly different. differences were determined using kruskall-wallis anova and post hoc mann-whitney test. statistical significance was considered for p<0.05. fig. 4. dynamics of pupation and eclosion in d. melanogaster – referent line. a – dynamics of larval pupation and b – dynamics of adult eclosion. half, normal and double refers to sucrose concentration in feeding media. biologica nyssana ● 11 (2) december 2020: 129-138 cvetković et al. ● effects of different sucrose concentrations on some parameters of the life cycle in two wild drosophila species 134 eclosed adults were not significantly different in the wild d. melanogaster cultivated on feeding media with different sucrose concentrations. according to these results, the wild d. melanogaster strain used in this experiment were tolerant to variation of sucrose concentration in feeding media at standard ambient conditions. in d. suzukii, at double sucrose concentration, the total number of formed pupae and eclosed adults were significantly lower (p<0.05) than in half and normal sucrose concentrations. however, the total number of oviposited eggs was not significantly different between groups with different sucrose concentrations. in some way, the higher sucrose concentrations disrupted incubation of the eggs and/or development of the hatched larvae until the pupation stage. also, in the d. melanogaster-referent line significantly lower (p<0.05) numbers of oviposited eggs, formed pupae and eclosed adults were observed in double than in normal sucrose concentration. a possible explanation is that the higher sucrose concentration in feeding media probably stimulates the increase of the products of fermentation. the higher levels of the fermentation products (ethanol, carbon dioxide etc.) may have a negative impact on larval hatching, development as well as pupation. also, it might have a negative effect on the number of oviposited eggs that was significantly lower in double concentration in d. melanogaster – referent line. this hypothesis is waiting to be confirmed or rejected in further experiments because this time we did not measure the levels of fermentation products in our experiment. a lower percentage of pupation were recorded in all three types of feeding media in the wild d. fig. 5. dynamics of pupation and eclosion in wild d. melanogaster. a – dynamics of larval pupation and b – dynamics of adult eclosion. half, normal and double refers to sucrose concentration in feeding media. biologica nyssana ● 11 (2) december 2020: 129-138 cvetković et al. ● effects of different sucrose concentrations on some parameters of the life cycle in two wild drosophila species 135 melanogaster while in the d. melanogaster – referent line was higher. this means that the laboratory drosophila strain is well adapted on applied cornmeal feeding media, as it was expected. the percentage of pupation values in d. suzukii were about 90% in average. as we were not able to count the hatched larvae, the much lower percentage of pupation could mean that many of the eggs were not fertilized or/and that many larvae have died before pupation in wild d. melanogaster. thus, these results indicate that standard laboratory conditions and applied feeding media (for medium details see section: culturing conditions and types of feeding media) influenced pupation in wild d. melanogaster. besides previous observations, in wild d. melanogaster percentage of pupations were not significantly different in all three sucrose concentrations, as in the d. melanogaster – referent line, while in d. suzukii percentage of pupation was significantly lower in double concentration compare to normal sucrose concentration. we propose one possible explanation for such a result. during pupation, larvae of d. melanogaster mostly went out of the feeding media and formed pupae on the walls of the vials where they were cultivated. in d. suzukii, pupae are formed mostly within the feeding media. thus, the explained effects of high sucrose concentrations in feeding media and the consequent presence of higher levels of fermentation products like ethanol and carbon dioxide might affect the pupation process in this case by affecting the larvae hatching or/and eggs incubation or/and larvae survival. as we did not count the number of hatched larvae, so we cannot state at which state the detrimental effect occurred, fig. 6. dynamics of pupation and eclosion in wild d. suzukii. a – dynamics of larval pupation and b – dynamics of adult eclosion. half, normal and double refers to sucrose concentration in feeding media. biologica nyssana ● 11 (2) december 2020: 129-138 cvetković et al. ● effects of different sucrose concentrations on some parameters of the life cycle in two wild drosophila species this observation remains to be tested in forthcoming analysis. gao et al. (2018) showed that both adults and larvae of d. melanogaster were more tolerant of environmental ethanol than d. suzukii (gao et al., 2018). this is consistent with our observation about the influence of possible higher levels of ethanol in double sucrose concentrated media. in addition, it has long been known that d. melanogaster was more tolerant on alcohol compared to other drosophila species (mckenzie & parsons, 1972). on the other hand, a minor difference in the percentage of eclosion in both wild drosophila species as in the d. melanogaster – referent line in any type of the tested feeding media. this could mean that the process of incubation from pupal to the adult stage was not affected in the same percentage as the pupation process by different sucrose concentrations in any experimental group since pupal mortality was very low in all drosophila groups. only significant differences showed that the percentage of eclosion was significantly affected by double sucrose concentration comparing with normal within the wild d. melanogaster group. also, within d. melanogaster – referent line, in double and half concentration percentage of eclosion was significantly affected compared to normal concentration (the percentages of eclosion were above 95%). however, these differences were minimal in both d. melanogaster groups. interestingly there were no significant differences between the percentage of eclosed flies in different feeding media within d. suzukii group, so the different sucrose concentrations did not affect d. suzukii at the pupal stage. it was already stated that in the wild d. melanogaster there were no significant differences for the total number of formed pupae and eclosed adults at different sucrose concentrations. but the differences were observed at dynamics of pupation and eclosion. the peaks for the dynamic of these parameters in half and standard concentration was on the fourth day while in double was about the eighth day. the pupation and eclosion were very prolonged and stretched at double concentration compared with the half and standard within the wild d. melanogaster. analyzing these parameters in the d. melanogaster – referent line group and d. suzukii group, a similar pattern could be observed in the double sucrose concentration. nevertheless, in d. suzukii the other two groups, half and normal, also express prolonged pupation and eclosion without pronounced peaks unlike both d. melanogaster groups. thus, due to its detrimental effect in all groups the double sucrose concentration in feeding media caused prolonged and extended pupation and eclosion. the reason might be the same, explained in previous paragraph, the detrimental effect of the higher level of fermentation products. on the other hand, half concentration did not cause any visible effect on dynamics of pupation and eclosion in any drosophila group. overall, our findings is in accordance with the conclusion of fellous & xuéreb (2017) that high sugar concentrations are detrimental for development of the d. suzukii. also, lushchak et al., (2014) stated that sucrose, commonly used for drosophila laboratory food, shorten lifespan and contribute to lower egg-laying capability in d. melanogaster. according to the results and previous observations, the detrimental effects, especially of the highest applied sucrose concentration are obvious in drosophila. even more, d. suzukii was more sensitive to higher sucrose concentration than d. melanogaster. there are many available cornmeal-based feeding media recipes that can be applied for laboratory culturing of drosophila species, but the concentrations of ingredients may vary in different recipes. despite differences in ingredients, many of them can be used for drosophila culturing. for example, some fly food recipes are available at the site of bloomington drosophila stock center from indiana university in usa (https://bdsc.indiana.edu). schlesener et al. (2017) reported modified cornmealyeast-glucose-agar medium that are suitable for d. suzukii rearing. the composition of standard feeding media that we use in this experiment were not the same but it is quite similar as in schlesener et al. (2017). we found that one of the differences between the feeding media that we used from the mentioned one is in yeast concentration. particularly, we used almost twice smaller concentration of yeast, as a protein source, what in addition to the highest applied sucrose concentration also might influenced development of the d. suzukii in our experiment. however, we did not vary yeast concentration in this experiment because it was not the subject of this research and in all treatment media was added in same amount according to one of the recipe (see section: culturing conditions and types of feeding media) that we use for laboratory drosophila stocks culturing. also, fellous & xuéreb (2017) reported that intermediate protein concentration is optimal for d. suzukii development. these observations indicate that protein concentration in feeding media should be taken into consideration when choosing appropriate feeding media for wild d. suzukii and we believe also for d. melanogaster because it is known that dietary balance of proteins and carbohydrates concentrations is important because its influence on drosophila lifespan (lushchak et al., 2014; lee, 2015). 136 biologica nyssana ● 11 (2) december 2020: 129-138 cvetković et al. ● effects of different sucrose concentrations on some parameters of the life cycle in two wild drosophila species conclusion according to our findings the sucrose concentration of 160 gl-1 in the feeding media caused significantly lower number of formed puape and eclosed adults in d. suzukii than at concentrations of 40 gl-1 and 80 gl-1. in all tested drosophila there were no significant differences in analyzed life cycle parameters between sucrose concentrations of 40 gl-1 and 80 gl-1. moreover, there were no significant differences between any of three tested sucrose concentrations for numbers of laid eggs, pupation and eclosion in wild d. melanogaster. the dynamics of pupation and eclosion were prolonged and stretched in wild d. melanogaster at 160 gl-1 sucrose concentration while in d. suzukii was mostly the same as in 40 gl-1 and 80 gl-1. we believe that the presented results might be helpful in deciding which cornmeal-based feeding media, in relation to the sucrose concentration, should be selected for the cultivation of mentioned drosophila species that originating from the wild. also, the obtained results represent contribution to the overall knowledge of the influence of dietary sucrose on lifecycle of two drosophila species. acknowledgement. this study was supported by the ministry of education, science and technological development of the republic of serbia (contract number: 451-03-68/2020-14/200124). references anagnostou, c., dorsch, m., rohlfs, m. 2010: influence of dietary yeasts on drosophila melanogaster life‐history traits. entomologia experimentalis et applicata, 136(1): 1-11. bellutti, n., gallmetzer, a., innerebner, g., schmidt, s., zelger, r., koschier, e.h. 2018: dietary yeast affects preference and performance in drosophila suzukii. journal of pest science, 91: 651-660. benito, n.p., lopes-da-silva, m., santos, r.s. 2016: potential spread and economic impact of invasive drosophila suzukii in brazil. pesquisa agropecuária brasileira, 51(5):571-578. cvetković, v.j., jovanović, b., lazarević, m., jovanović, n., savić-zdravković, d., mitrović, t., žikić, v. 2020: changes in the wing shape and size in drosophila melanogaster treated with food grade titanium dioxide nanoparticles (e171) – a multigenerational study. chemosphere. dec 1;261: 127787. cvetković, v. j., mitrović, t. l., jovanović, b., stamenković, s. s., todorović, m., đorđević, m., radulović, n. 2015: toxicity of dimethyl sulfoxide against drosophila melanogaster. biologica nyssana, 6(2): 91-95. de ros, g., anfora, g., grassi, a., ioriatti, c. 2013: the potential economic impact of drosophila suzukii on small fruits production in trentino (italy). iobc-wprs bulletin, 91:317-321. fellous, s. & xuéreb, a. 2017: a geometric analysis of the macronutrient needs of drosophila suzukii larvae. drosophila information service, 100: 158-167. gao, h.h., zhai, y.f., chen, h., wang, y.m., liu, q., hu, q.l., ren, f.s., yu, y. 2018: ecological niche difference associated with varied ethanol tolerance between drosophila suzukii and drosophila melanogaster (diptera: drosophilidae). florida entomologist, 101(3): 498-504. jovanović, b., cvetković, v.j., mitrović, t.l. 2016: effects of human food grade titanium dioxide nanoparticle dietary exposure on drosophila melanogaster survival, fecundity, pupation and expression of antioxidant genes. chemosphere, 144: 43-49. jovanović, b., jovanović, n., cvetković, v.j., matić, s., stanić, s., whitley, e.m., mitrović, t.l. 2018: the effects of a human food additive, titanium dioxide nanoparticles e171, on drosophila melanogaster-a 20 generation dietary exposure experiment. scientific reports, 8: 17922. kinjo, h., kunimi, y., nakai, m. 2014: effects of temperature on the reproduction and development of drosophila suzukii (diptera: drosophilidae). applied entomology and zoology, 49: 297-304. lee, k.p. 2015: dietary protein: carbohydrate balance is a critical modulator of lifespan and reproduction in drosophila melanogaster: a test using a chemically defined diet. journal of insect physiology, 75:12-19. lewis, e.b. 1960: a new standard food medium. drosophila information service, 34: 117– 118. lin, q.c., zhai, y.f., zhang, a.s., men, x.y., zhang, x.y., zalom, f.g., zhou, c.g., yu, y. 2014: comparative developmental times and laboratory life tables for drosophlia suzukii and drosophila melanogaster (diptera: drosophilidae). florida entomologist, 97(4): 1434-1442. lushchak, o.v., gospodaryov, d.v., rovenko, b.m., yurkevych, i.s., perkhulyn, n.v., lushchak, v.i. 2014: specific dietary carbohydrates differentially influence the life span and fecundity of drosophila melanogaster. journals of gerontology 137 biologica nyssana ● 11 (2) december 2020: 129-138 cvetković et al. ● effects of different sucrose concentrations on some parameters of the life cycle in two wild drosophila species series a: biomedical sciences and medical sciences, 69(1):3-12. mazzi, d., bravin, e., meraner, m., finger, r., kuske, s. 2017: economic impact of the introduction and establishment of drosophila suzukii on sweet cherry production in switzerland. insects, 8(1):18. mckenzie, j.a., parsons, p.a. 1972: alcohol tolerance: an ecological parameter in the relative success of drosophila melanogaster and drosophila simulans. oecologia, 10: 373–388. schlesener, d.c., wollmann, j., krüger, a.p., martins, l.n., geisler, f.c., garcia, f.r. 2017: rearing method for drosophila suzukii and zaprionus indianus (diptera: drosophilidae) on artificial culture media. drosophila information service, 100: 185-189. silva-soares, n.f., nogueira-alves, a., beldade, p., mirth, c.k. 2017: adaptation to new nutritional environments: larval performance, foraging decisions, and adult oviposition choices in drosophila suzukii. bmc ecology, 17: 21. tochen, s., dalton, d.t., wiman, n., hamm, c., shearer, p.w., walton, v.m. 2014: temperaturerelated development and population parameters for drosophila suzukii (diptera: drosophilidae) on cherry and blueberry. environmental entomology, 43(2): 501-510. toševski, i., milenković, s., krstić, o., kosovac, a., jakovljević, m., mitrović, m., cvrković, t., jović, j. 2014: drosophila suzukii (matsumura, 1931)(diptera: drosophilidae): a new invasive pest in serbia. zaštita bilja, 65(3): 99-104. https://bdsc.indiana.edu 138 biologica nyssana ● 11 (2) december 2020: 129-138 cvetković et al. ● effects of different sucrose concentrations on some parameters of the life cycle in two wild drosophila species stojanović et al., 2019, biologica nyssana 10(2) 10 (2) december 2019: 189-197 doi: 10.5281/zenodo.3600205 evaluation of the reference genes in human adipose tissue and lipoma samples original article sanja stojanović university of niš, faculty of medicine, department of biology and human genetics and department for cell and tissue engineering, 18000 niš, serbia s.sanja88@gmail.com (corresponding author) stevo najman university of niš, faculty of medicine, department of biology and human genetics and department for cell and tissue engineering, 18000 niš, serbia stevo.najman@medfak.ni.ac.rs vladimir cvetković university of niš, faculty of sciences and mathematics, department of biology and ecology, 18000 niš, serbia biovlada@yahoo.com aleksandra korać university of belgrade, faculty of biology, 11000 belgrade, serbia aleksandra.korac@bio.bg.ac.rs received: november 1, 2019 revised: december 12, 2019 accepted: december 21, 2019 abstract: housekeeping genes, by definition and function, should be constitutively and stably expressed in all cells of the organism, regardless of the cell type and function they accomplish. however, it was observed that expression of housekeeping genes, used as reference genes in gene expression analysis, varies greatly in different cells and tissue types and is also dependent on the experimental conditions and treatments. the aim of our study was to examine the expression patterns of the most frequently used reference genes (gapdh, actb and rrn18s) in the samples of human subcutaneous adipose tissue (scwat) and benign adipose tissue tumors (lipomas). the obtained results have shown that the expression of all three examined housekeeping genes is slightly lower in lipoma samples compared to scwat samples and that gapdh is the most stable housekeeping gene in examined samples so it can be recommended as an optimal reference gene for gene expression analysis in human scwat and lipoma samples. key words: reference genes, gene expression, adipose tissue, lipoma, scwat apstract: evaluacija referentnih gena u uzorcima humanog masnog tkiva i lipoma housekeeping geni bi, po definiciji i funkciji, trebalo da se konstitutivno i stabilno eksprimiraju u svim ćelijama organizma, nezavisno od tipa ćelija i funkcije koju one obavljaju. primećeno je međutim da ekspresija housekeeping gena, koji se koriste kao referentni geni u analizi ekspresije gena, varira u velikoj meri zavisno od tipa ćelija i tkiva i da zavisi takođe od eksperimentalnih uslova i tretmana. cilj našeg istraživanja je bio ispitati obrazac ekspresije najčešće korišćenih referentnih gena (gapdh, actb i rrn18s) u uzorcima humanog potkožnog masnog tkiva (scwat) i benignih tumora masnog tkiva (lipoma). dobijeni rezultati su pokazali da je ekspresija sva tri ispitivana gena nešto niža u uzorcima lipoma u odnosu na uzorke scwat, da je gapdh najstabilniji housekeeping gen u ispitivanim uzorcima i da se može preporučiti kao optimalan referentni gen za analizu ekspresije gena u uzorcima humanog scwat i lipoma. ključne reči: referentni geni, genska ekspresija, masno tkivo, lipomi, scwat introduction housekeeping genes, by definition and function, should be constitutively and stably expressed in all cells of the organism, regardless of the cell type function and they accomplish, and are considered to be the minimal set of genes necessary for the life of an organism (eisenberg and levanon, 2013). since they are essential for maintaining the basic cellular structures and functions, they are expressed at all stages of the development and growth of an organism, as well as in physiological and pathophysiological states (eisenberg and levanon, 2013). in biomedical research, housekeeping genes are widely used as reference genes, i.e. internal controls. the largest application in this field is for the purposes of gene expression analysis using qrt-pcr (quantitative reverse-transcription polymerase chain reaction) method, where housekeeping genes play a role of endogenous control for normalizing the expres© 2019 stojanović et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 189 sion of a gene of interest (goi) (bustin and nolan, 2004; eisenberg and levanon, 2013; caracausi et al., 2017). many studies have shown that the results obtained using this method are largely influenced by various factors, such as experimental conditions, sample size and rna isolation techniques, but also rna integrity, and the efficiency of reverse transcription reaction (rna reversely transcribed into cdna) and the quality of the cdna obtained (bustin, 2002; bustin and nolan, 2004). the influence of these factors is due to the multistage manipulations of the samples prior to the qpcr reaction itself (rna isolation, reverse transcription etc.), as well as the use of different reagents as obligatory components of the kits that are necessary during all the above steps. all of these can significantly affect the efficacy and specificity of the qpcr reaction itself, so certain internal controls need to be included in the analysis to increase efficiency and to make the final result as realistic and highly specific as possible for the studied goi. housekeeping (reference) genes are used to correct, i.e. to normalize variations in the expression of the goi from sample to sample, thereby avoiding errors and increasing the efficiency of the rt-pcr method when quantifying the expression of the goi. in order for a housekeeping gene to be considered as the most suitable reference gene used for normalization in gene expression analysis it should meet the following criteria: to be expressed at a sufficient level so it can be easily detected, to have stable/constant expression in different cell types in an examined organism and within the same cell type undergoing different treatments or experimental conditions and to be ubiquitously expressed (caracausi et al., 2017). thus, the proper selection of the reference gene for studying gene expression by qrt-pcr is extremely important and can be very challenging (andersen et al., 2004; derveaux et al., 2010; zhang et al., 2016). although many different reference genes have been used in the research to date, no “ideal” reference gene has been found yet (radonić et al., 2004; andersen et al., 2004). the stability of the commonly used reference genes has been shown to be relative, since it was observed that their expression varies greatly in different cell and tissue types and also depending on the experimental conditions and different treatments (thellin et al., 1999; schmittgen and zakrajsek, 2000; suzuki et al., 2000; warrington et al., 2000; tricarico et al., 2002; andersen et al., 2004; dheda et al., 2004). in the literature, among the reference genes used in the studies of gene expression in biomedical research, various genes can be found (eisenberg and levanon, 2013; taube et al., 2015; zhang et al., 2016; almeida-oliveira et al., 2017), however among the most frequently used, regardless of the sample type and organism, are genes for: glyceraldehyde-3-phosphate dehydrogenase (gapdh), beta actin (actb) and 18s ribosomal rna (rrn18s) (gabrielsson et al., 2005; almeida-oliveira et al., 2017), while in the studies that analyzed gene expression in different adipose tissue samples, in addition to these three the most common, genes for ldl receptor related protein 10 (lrp10), beta-2-microglobulin (b2m) and beta-glucuronidase (gusb) were also used. lipomas represent one of the most common soft tissue neoplasms of mesenchymal origin (mohammed et al., 2014; omonte et al., 2016) with still unclear etiology and pathogenesis. although lipomas are very common soft tissue tumors, there is insufficient information on their molecular characteristics and the potential mechanisms of their formation. to understand the mechanisms of lipoma formation and to find potential ways for their adequate treatment, detailed characterization of these tumors at molecular level needs to be performed in addition to already performed studies on the implications of mesenchymal stem cells in the pathogenesis of lipoma (stojanović et al., 2018; stojanović and najman, 2019). gene expression analysis gives us insight into the changes that have occurred in adipose tissue that may have led to its dysfunction and tumor development, so it is necessary to have reliable reference genes to perceive the real state of transcriptome and to find target places for eventual therapy. considering the very different data on reference genes used in gene expression analysis and the extent to which their expression varies, both in different tissue types and in different samples of the same tissue type, the aim of this study was to examine the expression patterns of the most frequently used reference genes (gapdh, actb and rrn18s) in the samples of human subcutaneous adipose tissue (scwat) and benign adipose tissue tumors (lipomas). materials and methods tissue samples lipoma tissue samples were obtained after surgical removal of the lipomas, previously clinically diagnosed as benign adipose tissue tumors, while scwat samples were taken during other non-cancerous surgeries patients underwent. all samples were obtained at clinical center niš, serbia, and all patients included in the study gave their informed written consent. the study was approved by the local ethics committee of the faculty of medicine, university of niš, serbia (approvals 01-6481-15 and 12-6316-2/4). a total of 37 tissue samples were analyzed, of which 24 lipoma and 13 scwat samples, patients of both sexes aged 21 to 70 years. tissues 190 biologica nyssana ● 10 (2) december 2019: 189-197 stojanović et al. ● evaluation of the reference genes in human adipose tissue and lipoma samples resis gel. the expression pattern of the examined housekeeping (reference) genes, as well as the variation of their expression among the samples of both study groups, was analyzed based on the obtained ct (cycle threshold) values. statistical analysis results in the form of obtained ct values were statistically processed. as indicators of central tendency mean and median values were calculated, while standard deviation (sd) and coefficient of variation (cv) were calculated as indicators of the variability. the results are presented as boxplots, made in graphpad prism 5, with median values and the range of minimum and maximum values. statistically significant differences in the expression of the examined reference genes between lipoma and scwat samples were analyzed by anova and mann – whitney u tests. values of p < 0.05 were considered as statistically significant. results the specificity of qpcr reactions was confirmed by melting curve analysis and the results are presented in fig. 1. based on the obtained melting curves, it can be seen that in all qpcr reactions the specific primer binding products were obtained. only one peak for rrn18s (a), actb (b) and gapdh (c) for each qpcr sample reaction that can be observed in the graphs indicates the presence of only one specific reaction product per sample. the results of the gene expression pattern analysis of gapdh, actb and rrn18s housekeeping genes are presented as ct values in fig. 2. it can be noticed that rrn18s is highly expressed in all samples, as shown by low ct values (fig. 2a). the expression of rrn18s is slightly lower in lipoma than in scwat samples, although the resulting difference is not statistically significant (p = 0.86). the expression of rrn18s is highly variable in both examined groups of samples, which can be noticed based on the distribution of ct values (fig. 2a). the expression of actb was statistically significantly lower in lipoma compared to scwat samples (p < 0.05) (fig 2b). broad range and uneven distribution of the values can be noticed in both lipoma and scwat group, indicating a relatively large variability in actb expression in different samples within both study groups (fig. 2b). the results of gapdh expression (fig. 2c) indicate statistically significantly lower level of expression of this gene in lipoma compared to scwat samples (p < 0.05), which can be seen from the higher ct values in lipoma samples. on the other hand, narrower range and more uniform distribution of ct values can be observed in the case of gapdh expression compared to the expression were sampled from several body localizations such as: head, oral cavity, neck, arm, leg, abdomen, back, hip, knee and groin. all patients had a body mass index less than 30, which excluded pathologically obese patients. gene expression analysis rna isolation and reverse transcription immediately after sampling, parts of the lipoma and scwat were stored in rnalater® at -80 °c until rna isolation. total rna was isolated using rneasy lipid tissue mini kit (qiagen, venlo, netherlands) according to the manufacturer’s instructions, after tissue homogenization in trizol reagent (qiagen), using tissue homogenizer (th220, omni international, sad). on column removal of residual genomic dna was performed during rna isolation using dnase i rnase-free kit (qiagen). rna concentration was determined immediately after isolation on the qubit® fluorimeter (thermo scientific, waltham, ma, usa) using qubit™ rna br assay kit (thermo scientific), and then isolated rna was stored at -80 °c until further use. total rna was subsequently transcribed into a single-stranded cdna using high-capacity cdna reverse transcription kit (applied biosystems, foster city, ca, usa), according to the manufacturer’s instructions. the reverse transcription reaction was carried out in surecycler8800 (agilent technologies, santa clara, ca, usa), with 400 ng of total rna per reaction per sample. the reverse transcription reaction was performed according to the following program: 10 min at 25 °c, 120 min at 37 °c, 5 min at 85 °c and cooling at 4 °c. the synthesized cdna was stored at -80 °c and used later to determine relative gene expression. real-time pcr analysis of the relative gene expression in tissue samples, by sybr green based real-time qpcr method, was performed in mic qpcr cycler (bio molecular systems, australia). the reactions were prepared using luna® universal qpcr master mix (new england biolabs, ipswich, ma, usa) according to the manufacturer’s instructions. universal rox dye was used as a reference dye. predesigned primer sets (quantitect primer assay kits, qiagen) were used for the following genes: gapdh (qt00079247), rrn18s (qt00199367) and actb (qt00095431). the protocol conditions were as follows: (1) enzyme activation: 60 s at 95 oc (1 cycle); (2) denaturation: 15 s at 95 oc and annealing/extension (with data acquisition): 30 s at 60 oc (40 cycles). the specific binding of primers was confirmed by melting curve analysis for each reaction and by visualizing specific length products on electropho191 biologica nyssana ● 10 (2) december 2019: 189-197 stojanović et al. ● evaluation of the reference genes in human adipose tissue and lipoma samples 192 fig. 1. melting curves for all qpcr products; reference genes: rrn18s (a), actb (b) and gapdh (c) fig. 2. expression pattern of rrn18s (a), actb (b) and gapdh (c) housekeeping genes in lipoma and scwat samples; the results are presented as ct values; boxplot charts with median and the range of minimum and maximum values biologica nyssana ● 10 (2) december 2019: 189-197 stojanović et al. ● evaluation of the reference genes in human adipose tissue and lipoma samples of previously analyzed rrn18s and actb genes. the cv, as a measure of deviation from the mean, is the lowest in the case of gapdh gene in both lipoma and scwat group of samples (tab. 1) which confirms the findings that gapdh is the most stable reference gene in our study in lipoma and scwat samples. for all tested genes, mean and median ct values were higher in lipoma compared to scwat group of samples, indicating that the expression of all three examined reference genes was slightly lower in lipoma samples compared to scwat samples. statistically significant difference (p < 0.05) in gene expression between lipoma and scwat samples was noticed for actb and gapdh, but not for rrn18s. similar values of the mean and median can be observed for all tested samples in the case of all examined genes, indicating a relatively symmetric distribution of values in both groups. discussion to adequately analyze the expression of goi in examined samples and to increase the efficiency of qpcr reactions, it is very important to choose the optimal housekeeping gene. gapdh, actb and rrn18s are the most commonly used reference genes that can be found in published studies that dealt with gene expression analyses wherein gapdh was recommended as the reference gene with the most stable expression. however, numerous literature data indicates that there is a great variability in the expression of these genes both in samples of different tissues and samples of the same tissue and cell type as well as under different experimental conditions, treatments and metabolic states. there are various computer programs, statistical methods and software tools such as normfinder, genorm, bestkeeper and global pattern recognition for identification and selection of an optimal reference gene (de spiegelaere et al., 2015; perez et al., 2017) that can predict the expression pattern of housekeeping genes in the desired samples, but they cannot confidently predict the experimental conditions and how it will affect the expression of reference genes. when adipose tissue is analyzed good prediction of this type cannot be done since adipose tissue is an endocrine and paracrine organ that is greatly influenced by various factors such as nutrition, hormonal status, metabolic status, physical activity, temperature, etc. and also its characteristics vary from organism to organism and within different anatomical localizations of the same organism. therefore, the aim of this study was to examine the expression pattern of three the most commonly used housekeeping genes in the scwat and lipoma samples, and to determine which housekeeping gene is the most optimal to be used as a reference gene in gene expression analysis by real-time pcr. due to interand intraindividual differences in housekeeping gene expression in human tissue samples, some authors consider that, in the case of comparison, more than one housekeeping gene should be used to normalize the level of the rna of the target gene (tricarico et al., 2002). in the study that analyzed the expression patterns of housekeeping genes in colon and bladder cancer samples by rt-pcr, housekeeping genes ubc, gapdh and tpt1 were shown to be suitable for normalization in colon cancer samples, while hspcb, tegt and atp5b were suitable for normalization in bladder cancer samples (andersen et al., 2004). in the study in which the expression of several housekeeping genes was analyzed in 16 different human tissue samples by real-time pcr, the rpii gene has been shown to be suitable as a reference gene for a large number of different tissues (radonić et al., 2004), but authors have noticed that the expression level of examined genes was signifi193 table 1. statistical parameters of reference genes’ expression analysis in lipoma and scwat samples rrn18s actb gapdh lipomas scwat lipomas scwat lipomas scwat mean 6.89 6.82 16.13 15.58 17.03 16.61 median 6.94 6.79 16.11 15.33 17.05 16.65 minimum 6.05 5.33 15.06 14.83 15.86 15.63 maximum 7.63 7.67 17.52 17.30 18.05 17.32 standard deviation 0.53 0.68 0.64 0.79 0.58 0.44 coefficient of variation (%) 7.69 9.97 3.97 5.10 3.41 2.65 statistical significance p = 0.86 p < 0.05 p < 0.05 biologica nyssana ● 10 (2) december 2019: 189-197 stojanović et al. ● evaluation of the reference genes in human adipose tissue and lipoma samples 194 cantly different in different tissue types. in the same study, in performed in vitro experiments on cell cultures, it has been shown that different treatments have the least effect on rpii expression (radonić et al., 2004). when expression level of 10 commonly used reference genes was analyzed (gadph, ppia, actb, ywhaz, rrn18s, b2m, ubc, tbp, rplp and hprt) in human heart tissue samples, ppia, rplp and gadph genes have been shown to be the most stable as estimated by genorm and normfinder and were recommended for the analysis of this tissue type (pérez et al., 2007). no statistically significant difference was found between the rats on the lipid-rich diet and the control group when the expression levels of the reference genes rrn18s, actb, gapdh and 36b4 were examined in three different types of rat adipose tissue of both groups of rats, but it was noticed that rrn18s was the most expressed gene in all samples (zhang et al., 2016) which correlates with the results obtained in our study. the stability of reference genes’ expression was examined by genorm, normfinder and bestkeeper softwares and the results showed that gapdh was the most stable reference gene in ewat (epididymal white adipose tissue) samples, but in ibeat (inguinal beige adipose tissue) and bat (brown adipose tissue) samples its expression was the most variable (zhang et al., 2016). the results obtained with genorm and normfinder differed from those obtained by other analyzes, so the authors proposed 36b4 and gapdh as the best combination of reference genes for ewat samples, while for ibeat and bat samples they suggested 36b4 and actb (zhang et al., 2016). changes in the expression of housekeeping genes were monitored during adipogenic differentiation of primary preadipocytes as well as under various experimental conditions and treatments of human primary mature adipocytes and its has been shown that the expression of gapdh and tfr housekeeping genes was the most stable in different experimental conditions (gorzelniak et al., 2001). the effect of adipogenic differentiation, cryopreservation and medium supplementation on the stability of 11 reference genes was examined in adscs in vitro and estimated by genorm software (dessels and pepper, 2019). it has been shown in this study that different experimental conditions significantly influence the expression stability of reference genes while the most stable expression in all experimental conditions was observed for ywhaz, hprt, tbp and actb (dessels and pepper, 2019). expression of 12 commonly used reference genes was analyzed in different murine adipose tissue samples of animals subjected to different treatments and the results showed that the tbp gene has stable expression in examined adipose tissue samples of the control group and is proved to be suitable for the analysis of gene expression in bat of the control and obese mice (almeida-oliveira et al., 2017). the atpf1 gene was stably expressed in the subcutaneous and perigonadal adipose tissue samples of control and obese mice and was proved to be the best for normalizing gene expression in adipose tissue samples of streptozotocin-treated animals (almeida-oliveira et al., 2017). authors of this study conclude that there is no ideally stable reference gene under all experimental conditions, but on the basis of the results obtained, they propose tbp and atpf1 as suitable reference genes for gene expression normalization in mouse adipose tissue samples in various metabolic states and disorders (almeida-oliveira et al., 2017). when several commonly used reference genes as well as some new candidates were analyzed by qpcr and microchip analysis in perirenal bat and subcutaneous adipose tissue, among commonly used, rplp0, lrp10, ywhaz and gapdh showed the lowest variability in examined samples in this study as evaluated by normfinder and genorm software (taube et al., 2015). the authors of this study conclude that all frequently used reference genes, except rrn18s, show acceptably low variability (taube et al., 2015). also, authors identified potentially new reference genes such as: gnb1, gnb2 and psmb2, which proved to be even more stable than the commonly used reference genes (taube et al., 2015). our results have shown that the expression of gapdh was the most stable in both groups of examined samples (lipoma and scwat), and varied the least between different patients within examined groups. expression analysis of a large number of reference genes in subcutaneous and omental adipose tissue samples has shown that rplp0 is the most suitable reference gene for adipose tissue. however, in one study it was shown that the expression of rplp0, when analyzed in relation to the lrp10, was significantly influenced by the diet-induced weight loss (gabrielsson et al., 2005). in examined adipose tissue samples in that study, it was shown that gapdh expression varied in greater extent in all examined groups compared to rrn18s and actb, which is contrary to what was observed in cultures of primary human adipocytes in vitro for the same genes, where expression of gapdh was stable in all samples and experimentally conditions (gorzelniak et al., 2001). this finding in tissue samples is contrary to what we obtained in our study. gabrielsson et al. (2005) propose lrp10, as the most suitable reference gene for gene expression analysis in human adipose tissue samples. expression of ipo8 and fbxl10, as potential reference genes, was examined by real-time pcr in omental and subcutaneous adipose tissue samples as biologica nyssana ● 10 (2) december 2019: 189-197 stojanović et al. ● evaluation of the reference genes in human adipose tissue and lipoma samples 195 well as in cultured primary preadipocytes and it has been shown that ipo8 expression was more stable in tissue samples of both adipose tissue depots as well as in preadipocytes isolated from subcutaneous adipose tissue, compared to fbxl10 expression that was more stable in preadipocytes from omental adipose tissue (hurtado del pozo et al., 2010). as the expression level of these two genes did not change during adipogenesis, the authors of this study concluded that both ipo8 and fbxl10 are good candidates for reference genes for gene expression analysis in human omental and subcutaneous adipose tissue samples as well as in differentiation of preadipocytes isolated from these tissues (hurtado del pozo et al., 2010). perez et al. (2017) analyzed the expression stability of a large number of reference genes by δct method in genorm, normfinder and bestkeeper software, using different adipocyte and muscle cell cultures in vitro, under different treatments and conditions, in animal muscle and adipose tissue samples of different metabolic states as well as in human muscle and adipose tissue samples. different results were obtained for different tissue types of different origin and under different conditions but general observation was that rrn18s was highly expressed in human samples and that gene for β-actin, although often used alone as a reference gene in rt-qpcr, is one of the most unstable genes in all muscle and adipose tissue samples, and is not recommended for this type of research (perez et al., 2017). the selection of an optimal reference gene for gene expression analysis is of great importance, because its stability in different samples and conditions will determine the final results obtained for the expression of goi and their reliability. zhang et al. (2016) demonstrated how the reference genes influence the expression of lep and ucp-1 gene in different adipose tissue samples of rats on a high-fat diet and control normal-fed rats. the expression of both examined goi was normalized by the expression of the most stable and the least stable reference genes. different gene expression patterns were obtained for lep and ucp-1 gene in different adipose tissue samples and different groups of animals when expression was normalized with the most and the least stable reference genes previously determined for each type of examined adipose tissue (zhang et al., 2016). based on the literature data, we can conclude that it is difficult to find an “ideal” reference gene that will have stable expression in different tissue types and samples of the same tissue type but under different experimental conditions and treatments. it is a common occurrence that one reference gene is suitable for one tissue type or treatment but not for the others, so it is difficult to compare the expression of goi between different samples. in our study, gapdh was shown to be the least variable and the most stable examined housekeeping gene in both lipoma and scwat group which is of great importance for the gene expression analysis and comparison between lipoma and scwat samples that could reveal the molecular mechanisms involved in the formation of lipoma. it can be noted that the expression of all examined housekeeping genes is slightly lower in lipoma samples compared to scwat, which obviously represents a characteristic of lipomas and reflects their basal metabolic status. conclusion based on the obtained results we can conclude that the expression of all three examined housekeeping genes (rrn18s, actb and gapdh) is slightly lower in lipoma samples compared to scwat samples and that expression of gapdh is the least variable and the most uniform in both examined groups of samples. we can consider gapdh as the most stable housekeeping gene in our study and recommend it as an optimal reference gene for gene expression analysis in human scwat and lipoma samples and its use for normalization of gene expression in realtime pcr method. acknowledgments. this study was supported by the ministry of education, science and technological development, of the republic of serbia (grant no. iii 41017). references almeida-oliveira, f., leandro, j.g.b., ausina, p., sola-penna, m., majerowicz, d. 2017: reference genes for quantitative pcr in the adipose tissue of mice with metabolic disease. biomedicine & pharmacotherapy, 88: 948-955. andersen, c.l., jensen, j.l., ørntoft, t.f. 2004: normalization of real-time quantitative reverse transcription-pcr data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. cancer research, 64(15): 5245-50. bustin, s.a. 2002: quantification of mrna using real-time reverse transcription pcr (rtpcr): trends and problems. journal of molecular endocrinology, 29(1): 23-39. bustin, s.a., nolan, t. 2004: pitfalls of quantitative real-time reverse-transcription polymerase chain reaction. journal of biomolecular techniques, 15(3): 155-66. caracausi, m., piovesan, a., antonaros, f., strippoli, p., vitale, l., pelleri, m.c. 2017: biologica nyssana ● 10 (2) december 2019: 189-197 stojanović et al. ● evaluation of the reference genes in human adipose tissue and lipoma samples 196 systematic identification of human housekeeping genes possibly useful as references in gene expression studies. molecular medicine reports, 16(3): 2397–2410. derveaux, s., vandesompele, j., hellemans, j. 2010: how to do successful gene expression analysis using real-time pcr. methods, 50(4): 227-30. de spiegelaere, w., dern-wieloch, j., weigel, r., schumacher, v., schorle, h., nettersheim, d., bergmann, m., brehm, r., kliesch, s., vandekerckhove, l., fink, c. 2015: reference gene validation for rt-qpcr, a note on different available software packages. plos one, 10(3): e0122515. dessels, c., pepper, m.s. 2019: reference gene expression in adipose-derived stromal cells undergoing adipogenic differentiation. tissue engineering. part c, methods, 25(6): 353-366. dheda, k., huggett, j.f., bustin, s.a., johnson, m.a., rook, g., zumla, a. 2004: validation of housekeeping genes for normalizing rna expression in real-time pcr. biotechniques, 37(1): 112-4, 116, 118-9. eisenberg, e., levanon, e.y. 2013: human housekeeping genes, revisited. trends in genetics, 29(10): 569-74. gabrielsson, b.g., olofsson, l.e., sjögren, a., jernås, m., elander, a., lönn, m., rudemo, m., carlsson, l.m. 2005: evaluation of reference genes for studies of gene expression in human adipose tissue. obesity research, 13(4): 649-52. gorzelniak, k., janke, j., engeli, s., sharma, a.m. 2001: validation of endogenous controls for gene expression studies in human adipocytes and preadipocytes. hormone and metabolic research, 33(10): 625-7. hurtado del pozo, c., calvo, r.m., vesperinasgarcía, g., gómez-ambrosi, j., frühbeck, g., corripio-sánchez, r., rubio, m.a., obregon, m.j. 2010: ipo8 and fbxl10: new reference genes for gene expression studies in human adipose tissue. obesity, 18(5): 897-903. mohammed, u., samaila, m.o., abubakar, m. 2014: pattern of adipose tissue tumors in ahmadu bello university teaching hospital, zaria, nigeria. annals of nigerian medicine, 8(1): 8–10. omonte, s.v., de andrade, b.a., leal, r.m., capistrano, h.m., souza, p.e., horta, m.c. 2016: osteolipoma: a rare tumor in the oral cavity. oral surgery, oral medicine, oral pathology and oral radiology, 122(1): e8–e13. perez, l.j., rios, l., trivedi, p., d’souza, k., cowie, a., nzirorera, c., webster, d., brunt, k., legare, j.f., hassan, a., kienesberger, p.c., pulinilkunnil, t. 2017: validation of optimal reference genes for quantitative real time pcr in muscle and adipose tissue for obesity and diabetes research. scientific reports, 7(1): 3612. pérez, s., royo, l.j., astudillo, a., escudero, d., alvarez, f., rodríguez, a., gómez, e., otero, j. 2007: identifying the most suitable endogenous control for determining gene expression in hearts from organ donors. bmc molecular biology, 8: 114. radonić, a., thulke, s., mackay, i.m., landt, o., siegert, w., nitsche, a. 2004: guideline to reference gene selection for quantitative realtime pcr. biochemical and biophysical research communications, 313(4): 856-62. schmittgen, t.d., zakrajsek, b.a. 2000: effect of experimental treatment on housekeeping gene expression: validation by real-time, quantitative rt-pcr. journal of biochemical and biophysical methods, 46: 69-81. stojanović, s., najman, s. 2019: the effect of conditioned media of stem cells derived from lipoma and adipose tissue on macrophages’ response and wound healing in indirect co-culture system in vitro. international journal of molecular sciences, 20(7): 1671. stojanović, s., najman, s., korać, a. 2018: stem cells derived from lipoma and adipose tissue— similar mesenchymal phenotype but different differentiation capacity governed by distinct molecular signature. cells, 7(12): 260. suzuki, t., higgins, p.j., crawford, d.r. 2000: control selection for rna quantitation. biotechniques, 29: 332-7. taube, m., andersson-assarsson, j.c., lindberg, k., pereira, m.j., gäbel, m., svensson, m.k., eriksson, j.w., svensson, p.a. 2015: evaluation of reference genes for gene expression studies in human brown adipose tissue. adipocyte, 4(4): 280-5. thellin, o., zorzi, w., lakaye, b., de borman, b., coumans, b., hennen, g., grisar, t., igout, a., heinen, e. 1999: housekeeping genes as internal standards: use and limits. journal of biotechnology, 75(2-3): 291-5. tricarico, c., pinzani, p., bianchi, s., paglierani, m., distante, v., pazzagli, m., bustin, s.a., orlando, c. 2002: quantitative real-time reverse transcription polymerase chain reaction: normalization to rrna or single housekeeping genes is inappropriate for human tissue biopsies. biologica nyssana ● 10 (2) december 2019: 189-197 stojanović et al. ● evaluation of the reference genes in human adipose tissue and lipoma samples 197 analytical biochemistry, 309(2): 293-300. warrington, j.a., nair, a., mahadevappa, m., tsyganskaya, m. 2000: comparison of human adult and fetal expression and identification of 535 housekeeping/maintenance genes. physiological genomics, 2: 143-7. zhang, w.x., fan, j., ma, j., rao, y.s., zhang, l., yan, y.e. 2016: selection of suitable reference genes for quantitative real-time pcr normalization in three types of rat adipose tissue. international journal of molecular sciences, 17(6): e968. biologica nyssana ● 10 (2) december 2019: 189-197 stojanović et al. ● evaluation of the reference genes in human adipose tissue and lipoma samples anticancer compounds from medicinal plants biologica nyssana 5 (1)  september 2014: 1-10 stojković, m. et al.  antioxidant potential of tanacetum vulgare l. … 71 short communication received: 14 august 2014 revised: 26 august 2014 accepted: 10 september 2014 several records of tachinidae (diptera) reared from their hosts in serbia and montenegro saša s. stanković1, vladimir žikić1, boženka hric2, hans-peter tschorsnig3 1department of biology and ecology, faculty of science and mathematics, university of niš, 33 višegradska street, 18 000 niš, serbia 2jovana popovića 72, 22300 stara pazova, serbia. 3staatliches museum für naturkunde, rosenstein 1, 70191 stuttgart, germany. * e-mail: sasasta@gmail.com abstract: stanković, s.s., žikić, v., hric, b., tschorsnig, h.p.: several records of tachinidae (diptera) reared from their hosts in serbia and montenegro. biologica nyssana, 5 (1), septmeber 2014: 71-73. during the year 2013 over fifty tachinid flies were reared. nine tachinid species form two subfamilies were reported for the territory of serbia and montenegro. species erynniopsis antennata and phryxe hirta are recorded for the first time in serbia. key words: tachinidae, parasitoids, serbia, montenegro. sažetak: stanković, s.s., žikić, v., hric, b., tschorsnig, h.p.: nekoliko zapisa iz familije tachinidae (diptera) odgajene sa domaćina na teritoriji srbije i crne gore. biologica nyssana, 5 (1), septmeber 2014: 71-73. tokom 2013. godine sakupljeno je i odgajeno preko pedeset jedinki tahinida. devet vrsta tahinida iz dve potfamilije su zabeležene na teritoriji srbije i crne gore. vrste erynniopsis antennata i phryxe hirta su po prvi put registrovane u srbiji. ključne reči: tachinidae, parazitoidi, srbija, crna gora. introduction the family tachinidae is one of the most diverse group among diptera order, so far over 10 000 species have been described worldwide (i r w i n e t a l ., 2003). all members of the family are endoparasitoids of other insects, but few genera attack centipedes, scorpions and spiders (w i l l i a m s e t a l ., 1990; v i n c e n t , 1985). many species parasitize economically important insects such are many species of lepidoptera, coleoptera and symphyta (e g g l e t o n & b e l s h a w , 1993) therefore, they have been often used as biocontrol agents against phytophagous pest insects (g r e n i e r , 1988). most tachinid species parasitize larval stage of their hosts. they deposit eggs on or near the host since they lack an ovipositor, unlike parasitic hymenoptera to which they share similar life history. the family tachinidae comprises four subfamilies: exoristinae, 5 (1) • september 2014: 71-73 biologica nyssana 5 (1)  september 2014: 71-73 stanković, s. et al.  several records of tachinidae (diptera) … 72 dexiinae, phasiinae and tachininae (h e r t i n g & d e l y d r a s k o v i t s 1993) according to fauna europaea, internet database 877 species have been recorded so far. from that number, 242 species are recorded for the territory of ex serbia and montenegro state. during the year 2013 several tachinids were reared in serbia and montenegro from a number of lepidopterous, hymenopterous, and coleopterous hosts. the material was bred by b. hric, z. kojičić, a. petrović, s. stanković and v. žikić. the annotated results are listed below. the remarks are, as far as they are not referenced in detail, based on a catalogue on palaearctic tachinid-host records which is currently compiled by the last author (tschorsnig). two new species are newly reported for the investigated territory, and they are marked by an asterisk (*). the arrangement and nomenclature of the tachinids follow herting & dely-draskovits (1993). the data mean the date of collection of the host material. the material is stored at the department of biology and ecology, faculty of science and mathematics, university of niš. species list subfamily dexiinae voria ruralis (fallén, 1810) 3 ♂, 1 ♀, serbia, stara pazova, 4.vii 2013, ex autographa gamma linnaeus [lep. noctuidae], leg. b. hric. – remark: voria ruralis is a common parasitoid of noctuidae-plusiinae, with a. gamma as a very often reared host. subfamily exoristinae ceromasia rubrifrons (macquart, 1834) 1 ♂, serbia, tara, mitrovac, 27.vi 2013, ex p. aporia crataegi linnaeus [lep. pieridae], leg. v. žikić. – remark: ceromasia rubrifrons typically develops in caterpillars of zygaena spp. [lep. zygaenidae]. other lepidopterous host families are only rarely parasitized by this species. among the pieridae, a. crataegi was not yet known as host, but pieris brassicae linnaeus was. compsilura concinnata (meigen, 1824) 12 ♂, 14 ♀, montenegro, visitor, 8–10.vii 2013, ex l. nymphalis antiopa linnaeus [lep. nymphalidae] on salix caprea, leg. a. petrović. – remark: compsilura concinnata is the tachinid species with the largest number of hosts; many records are known from n. antiopa. drino inconspicua (meigen, 1830) 2 ♂, 3 ♀, serbia, jelašnička gorge, 14.v 2013, ex neodiprion sertifer geoffroy [hym. diprionidae] on pinus sp., leg. v. žikić. – 1 ♂, montenegro, visitor, 10.vii 2013, ex nymphalis antiopa linnaeus [lep. nymphalidae] on salix caprea, leg. a. petrović. – 4 ♂, 2 ♀, montenegro, plužine, 8.vii 2013, ex nymphalis polychloros linnaeus on salix caprea, leg. v. žikić. – remark: neodiprion sertifer is, among other diprionidae, a usual host of drino inconspicua. this tachinid is also known from many lepidopterous hosts, but the two often reared nymphalidae, n. antiopa and n. polychloros are untypical. only a single rearing from another nymphalid host, vanessa cardui linnaeus, was known before (richter, 1996). *erynniopsis antennata (rondani, 1861) 3 ♂, 2 ♀, serbia, sićevačka gorge, 28.v 2013, ex xanthogaleruca luteola müller [col. chrysomelidae], leg. z. kojičić. – remark: erynniopsis antennata is an important parasitoid of the elm leaf beetle (see silvestri, 1910: under the erroneous name erynnia nitida). this tachinid fly was not yet known from serbia (compare hubenov, 2008). *phryxe hirta (bigot, 1880) 2 ♀, serbia, vlasina lake, 15.vi 2013, ex p. heterogynis sondereggeri de freina [lep. heterogynidae] on chamaecytisus heuffelii, leg. v. žikić. – remark: phryxe hirta is well-known as a tachinid specialized on the genus heterogynis (see de freina & tschorsnig, 2005), but h. sondereggeri as a new described moth for the world fauna has recorded only once (žikić et al., unpublished). p. hirta is herewith also recorded for the first time from serbia. senometopia separata (rondani, 1859) 1 ♀, serbia, bovansko lake, 9.vi 2013, ex lymantria dispar linnaeus [lep. lymantriidae] on prunus domestica, leg. v. žikić. – remark: s. separata is a common parasitoid of l. dispar. records from this host were also already known from serbia (sisojević, 1959). sturmia bella (meigen, 1824) 1 ♀, serbia, stara pazova, 5.vii 2013, ex vanessa atalanta linnaeus [lep. nymphalidae], leg. b. hric. – 1 ♀, serbia, stara pazova, 2.vii 2013, ex vanessa cardui linnaeus [lep. nymphalidae], leg. b. hric. – remark: sturmia bella is a common parasitoid of nymphalidae. abundant records exist for the hosts v. atalanta and biologica nyssana 5 (1)  september 2014: 71-73 stojković, m. et al.  antioxidant potential of tanacetum vulgare l. … 73 v. cardui, for the latter also from serbia (lehrer & dobrivojević, 1967). winthemia quadripustulata (fabricius, 1794) 1 ♀, serbia, vlasina lake, 21.vii 2013, ex l. cucullia lanceolata villers [lep. noctuidae] on verbascum thaspus, leg. s. stanković. – 1 ♂, serbia, jerma canyon, 19.vii 2013, ex cucullia sp., leg. v. žikić. – remark: cucullia spp. belong to the preferred hosts of w. quadripustulata, but the species c. lanceolata becomes known for the first time. here we report nine tachinid species form two subfamilies for the territory of serbia and montenegro. species erynniopsis antennata and phryxe hirta are recorded for the first time in serbia. also, for two species of tachinidae we report new hosts. cucullia lanceolata is a new host for winthemia quadripustulata, while aporia crataegi is new host for ceromasia rubrifrons. acknowledgements. thanks are due to daniel bartsch (stuttgart) who identified the host pupa of aporia crataegi. this study is supported by the ministry of education and science of the republic of serbia (iii43001). references de freina, j. j., tschorsnig, h. p. 2005: raupenfliegen (diptera: tachinidae) aus heterogynis spp. (lepidoptera: heterogynidae). nachrichtenblatt der bayerischen entomologen, 54: 95-100. eggleton, p., belshaw, r. 1993; comparisons of dipteran, hymenopteran and coleopteran parasitoids: provisional phylogenetic explanations. biological journal of the linnean society, 48: 213-226. grenier, s. 1988: applied biological control with tachinid flies (diptera, tachinidae): a review. anzeiger für schädlingskunde, pflanzenschutz, umweltschutz, 61 (3): 49-56. herting, b., dely-draskovits, a. 1993. family tachinidae. in: soós, a., papp, l. (ed.), catalogue of palaearctic diptera 13: 118-624, hungarian natural history museum, budapest. hubenov, z. 2008. composition and zoogeographical characteristics of the family tachinidae (diptera: insecta) in serbia and bulgaria. in: makarov, s. e., dimitrijević, r. n. (ed.), advances in arachnology and developmental biology pp. 375-394, vienna, belgrade and sofia. irwin, m. e., schlinger, e. i., thompson, f. c. 2003. diptera, trueflies. in: goodman, s. m., benstead, j. p. (ed.), the natural history of madagascar, pp. 692-702.: univ. chicago press. chicago/london 1728pp. lehrer, a. z., dobrivojević, k. 1967: fichier bioécologique et morphologique de diptères entomophages obtenus d’élevage, vii–xiii. bulletin et annales de la société royale d’entomologie de belgique, 103: 53-62. richter, v. a. 1996: on the fauna of tachinids (diptera, tachinidae) of the crimea. entomologiceskoe obozrenie 75: 908-929 [in russian; english translation in entomological review 76 (7): 900-918]. silvestri, f. 1910: contribuzioni alla conoscenza degli insetti et dei loro simbionti. i. galerucella dell’olmo (galerucella luteola f. müll.). bollettino del laboratorio di zoologia generale e agraria della facoltà agraria in portici, 4: 246-288. sisojević, p. 1959: ecological studies of tachinid parasites of the gipsy moth. preliminary communication (annual report for 1958). zaštita bilja, 10: 165-166 (in serbo-croatian with english summary). vincent, l. s. 1985: the first record of a tachinid fly as an internal parasitoid of a spider (diptera: tachinidae; araneae: antrodiaetidae). panpacific entomologist, 61: 224-235. williams, s. c., arnaud, p. h., lowe, g. 1990: parasitism of anuroctonus phaiodactylus (wood) and vaejovis spinigerus (wood) (scorpiones: vaejovidae) by spilochaetosoma californicum smith (diptera: tachinidae), and a review of parasitism in scorpions. myia, 5: 11-27. žikić, v., stanković, s. s, hric, b., mitroiu, m. d., schwartz, m., tschorsnig, h. p.: first records of parasitoids (hymenoptera: ichneumonidae and pteromalidae; diptera: tachinidae) of heterogynis sondereggeri de freina (lepidoptera: heterogynidae), (unpublished). anticancer compounds from medicinal plants biologica nyssana 2 (2)  december 2011: 00-00 nahirnić a.  supplements of butterfly fauna… 77 original article phytogeographical analysis of the flora of miljkovačka gorge in eastern serbia milica miljković, novica ranđelović, vladimir ranđelović university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia * e-mail: milica.zeka@yahoo.com abstract: miljković, m. ranđelović, n., ranđelović, v.: phytogeographical analysis of the flora of miljovačka gorge in eastern serbia. biologica nyssana, 3 (2), december 2012: 77-90. as a result of two-year investigation of the miljkovacka gorge flora, carried out during the 2010. and 2011., 331 plant taxa belonging to 245 genera and 70 families were recorded and sorted into 112 floristic elements, 15 area groups and 9 area types. phytogeographical analysis showed that the species of eurasian area type are the most abundant ones. key words: area group, area type, floristic element, miljkovačka gorge introduction one of the major biodiversity hotspots is balkan peninsula (h o r v a t et al., 1974), and the miljkovacka gorge is situated in eastern serbia, se of niš, bordered by mt kalafat (837 m), mt kamenički vis (813 m) from south and hillsides of mt devica (1187 m) from north and northeast, which belong to carpathian-balkan group of mountains (fig. 1). bela river that flows beneath mt devica and kopaljkosarska river that flows beneath mt kalafat are flowing into each other near popsica village making popsica river. during its passage through the village kravlje, it changes name to kravljanska river. on its way to south morava, it changes its name twice more: miljkovačka river and toponička river. as toponička river, it finally flows into south morava. the geological structure of the miljkovačka gorge is represented by mesozoic limestone. the pedological substrate is predominantly consisted of cambisoil and red soil. the climate of this area is humid continental with two extremely wet and cold periods (from january to may and from september to december) and one arid period during the summer. miljkovačka gorge is not well explored area. the first data concerning this area were collected by spas sotirov in 1977, who found the new locality of endemic-relict plant species, ramonda serbica in the valley of kravljanska river. m a r t i n o v i ć et al. (1985) published paper which was the first phytogeographical investigation of miljkovačka gorge. the more significant and widely extended breakthrough in the studies of the flora and vegetation of the miljkovačka gorge was made during 2010. and 2011. (m i l j k o v i ć , 2011; m i l j k o v i ć et al., 2012). materials and methods the study is based on literature data (s o t i r o v , s t a n o j e v i ć , 2012), herbarium collections (hmd), and continuous field observations carried out during the 2010 and 2011. herbarium specimens are deposited at the herbarium moesiacum (hmn). identification of the collected plants was performed according to flora 3 (2) • december 2012: 77-90 mailto:%20branko_jotic@live.com biologica nyssana 3 (2)  december 2012: 77-90 miljković m. et al.  phytogeographical analysis of the flora… 78 europaea (t u t i n et al.., 1964-1980) and the regional floras relevant for the investigated area (j o r d a n o v , 1963-1979, v e l c h e v 1982-1995, j o s i f o v i ć , 1970-1977, m i c e v s k i , 1985-1995, s a r i ć , 1986, 1992). the nomenclature follows med-checklist (g r e u t e r et al., 1984-1989), flora europaea (t u t i n et al., 1964-1980), and iopi – international organization for plant information (http://plantnet.rbgsyd.nsw.gov.au/iopi/iopihome.ht m). for the area types classification m e u s s e l et al. (1965, 1978), m e u s s e l and j ä g e r (1992) and s t e v a n o v i ć (1992) were used. results as a result of floristic investigations of the miljkovačka gorge in eastern serbia, region in the central part of the balkan peninsula, 331 plant taxa belonging to 245 genera and 70 families were recorded (tab. 1). figure 1. geographical position of investigated area table 1. list of vascular flora with area types, area groups and floristic elements taxa area type area group flostic element equisetopsida equisetaceae equisetum arvense l. hol circumcholarct. -(arct-merid) equisetum maximum lam. hol europ.-north amer.-(bor-merid) aspleniaceae polypodiopsida asplenium ceterach ea asplenium ruta-muraria l. hol asplenium trichomanes l. cosm cosm.-amfiatl. origin magnolyopsida acanthaceae acanthus balcanicus heywood & i. b. k. richardson med-smed balk http://plantnet.rbgsyd.nsw.gov.au/iopi/iopihome.htm http://plantnet.rbgsyd.nsw.gov.au/iopi/iopihome.htm biologica nyssana 3 (2)  december 2012: 77-90 miljković m. et al.  phytogeographical analysis of the flora… 79 taxa area type area group flostic element aceraceae acer monspessulanum l. med-smed acer tataricum l. ea ce-m-p-or adoxaceae adoxa moschatelina l. hol circumcholarkct.-(temp-submer) anacardiaceae cotinus coggygria scop. msm apiaceae aegopodium podagraria l. ea eurasian-(bor-merid) berula erecta (hudson) coville hol circumcholarkct.-(bor-merid) carum carvi l. ea eurasian-(temp-submer) danaa cornubiensis (l.) burnat med-smed daucus carota l. phol-ptrop central europ.-medit.-pont.orient.-turan.-east african eryngium campestre l. ea falcaria vulgaris bernh. ea heracleum sphondylium l. ce central european pastinaca sativa l. ea eurasian-(bor-merid) pimpinella saxifraga l. ea eurasian-(temp-mer) seseli rigidum waldst. & kit. med-smed skarp-balk seseli varium trev. ea ce-med torilis arvensis (hudson) link ea ce-med araliaceae hedera helix l. ea ea(w) aristolochiaceae aristolochia clematitis l. med-smed asarum europaeum l. ea eurasian asclepiadaceae cynanchum vincetoxicum (l.) pers. ea ea(w) europ.-medit.-west-asian asteraceae achillea crithmifolia waldst. & kit. msm med-pont panon.-skard.-pind.-mac.-trak.moes.-danub. achillea millefolium l. ea eurasian-(bor-submer) achillea millefolium l. forma rubra ea eurasian-(bor-submer) arctium lappa l. ea eurasian-(temp-submer) artemisia absinthium l. ea eurasian-(subbor-mer) artemisia alba l. med-smed artemisia annua l. ea artemisia scoparia waldst. & kit. ea artemisia vulgaris l. hol circumcholarkct.-(subbor-mer) bellis perennis l. ea ce-med central european-medit.submedit. bidens tripartita l. ea eurasian-(bor-temp) bombyciealena erecta (l) smolj. ea ce-med-pont carduus acanthoides l. ce central european carduus nutans l. ea carthamus lanatus l. ea ce-med-pont biologica nyssana 3 (2)  december 2012: 77-90 miljković m. et al.  phytogeographical analysis of the flora… 80 taxa area type area group flostic element centaurea solsticialis l. msm medit.-submedit.-pont.-south sibir-orijent.-turan. centaurea stoebe l. ea ea(w) chrysanthemum parthenium l. ea ea(w) cirsium palustre (l.) scop. bor eur.-west asian-(bor-temp) crepis pulchra l. ea ce-m-p-or crepis setosa haller fil. med-smed medit.-submedit. crupina vulgaris cass. msm med-pont echinops sphaerocephalus l. ea erigeron canadensis l. adv north american eupatorium cannabium l. ea ea(w) europ.-medit.-west-asian galinsoga parviflora cav. adv south american hieracium pilosella l. ea ea(w) europ.-medit.-west-asian inula conysa dc. ea ce-m-p-or sarm.-medit.-smed.-pont.-orijent. inula germanica l. ea ce-m-p-or inula helenium l. ea ce-m-p-or inula oculus-christi l. ea ce-med-pont lactuca perennis l. ea ce-med-pont matricaria trichophylla (boiss.) boiss. ea ce-med herc.-panon.-east submedit. mycelis muralis (l.) dumort. ea ea(w) europ.-medit.-west-asian petasites hybridus (l.) p. gaertner, b. meyer & scherb. ea eurasian-(bor-submer) ptilestemon afer (jacqu.) greut. med-smed south carp.-moes-mac.-trac.skard. pind. senecio jacobaea l. ea ce-med-pont senecio vernalis waldst. & kit. ea ea(w) europ.-medit.-west-asian stenactis annua (l.) less. ea ce-med-pont taraxacum officinale weber ea eurasian-(bor-temp) tussilago farfara l. ea central european-medit.submedit.-pont.-south sibir.central asian xeranthemum annuum l. med-smed submedit. xeranthemum cylindraceum szb. et sm. msm med-pont berberidaceae berberis vulgaris l. ea ea(w) boraginaceae anchusa officinalis l. ea ce-med-pont central european-east submedit.-west pont. echium italicum l. med-smed echium vulgare l. ea eurasian-(bor-merid) symphytum officinale l. ea ea(w) atlant-subatlant.-medit.submedit.-pont.-south-sibir. symphytum tuberosum l. msm med-pont medit.-submedit.-pont. brassicaceae alliaria officinalis andrz. ex bieb. ea ea(w) europ.-medit.-west-asian alyssum alyssoides l. ea ea(w) alyssum murale waldst. & kit. msm med-pont alyssum saxatile l. ea ce-med-pont arabis recta will. ea ce-med-pont biologica nyssana 3 (2)  december 2012: 77-90 miljković m. et al.  phytogeographical analysis of the flora… 81 taxa area type area group flostic element conringia orientalis (l.) dumort. ea ea(w) draba aizoides l. ea ce-med erysimum comatum pancic med-smed balk moesian-skard. pind. erysimum diffusum ehrh. ea ce-m-p-or east medit.-pont.-panon.-south sibir.-turan. hesperis matronalis l. ea ea(w) campanulaceae campanula bononiensis l. ea campanula lingulata waldst. & kit. ea ce-med karp.-panon.-balk. campanula rapunculoides l. ea ea(w) europ.-medit.-west-asian campanula trachelium l. ea eurasian-(bor-merid) cannabaceae humulus lupulus l. ea caprifoliaceae sambucus ebulus l. ea central european-medit.submedit.-pont.-south sibirorijent.-turan. caryophyllaceae cerastium brachypetalum pers. ea ce-med cerastium pumilum curtis ea ce-med cucubalus baccifer l. ea dianthus armeria l. ea ce-med dianthus petraeus waldst. & kit. ea ce-med ckarp-balk dianthus pontederae a. kerner ea ce-med herniaria hirsuta l. ea ce-m-p-or central european-submedit.-west asian-(temp-merid) holosteum umbellatum l. ea lychnis coronaria (l.) desr. ea ce-m-p-or central european-submedit.-kavk.turan. moehringia muscosa l. eam csem central-south-europ.-mont. petrorhagia illyrica (l.) p. w. ball & heywood med-smed petrorhagia saxifraga (l.) link ea ea(w) europ.-medit.-west-asian saponaria officinalis l. ea eurasian-(temp-submer) silene alba (miller) w. greuter & burdet ea eurasian-(bor-merid) silene flavescens waldst. & kit. ea ce-med silene viridiflora l. ea ce-m-p-or central european-central medit.east submedit.-orijent.-turan. silene vulgaris (moench) garcke ea eurasian-(bor-merid) stellaria holostea l. ea ea(w) europ.-medit.-west-asian stellaria media (l.) vill. cosm cosm-eurasian origin celastraceae euonymus europaeus l. ea ce-med europ.-submedit. chenopodiaceae polycnemum arvense l. ea cistaceae helianthemum nummularium (l.) miller ea ea(w) europ.-medit.-west-asian convolvulaceae calystegia sepium (l.) r. br. cosm cosm.-europ.-northamer. origin biologica nyssana 3 (2)  december 2012: 77-90 miljković m. et al.  phytogeographical analysis of the flora… 82 taxa area type area group flostic element convolvulus arvensis l. cosm cosm.mediter. origin convolvulus cantabricus l. ea ce-med cornaceae cornus mas l. ea ce-med cornus sanguinea l. ce corylaceae carpinus orientalis miller ea ce-med crassulaceae ce-med sedum acre l. ce ce-med atlant.-central europeansubmedit. sedum dasyphyllum l. med-smed sedum hispanicum l. med-smed e smed submedit. sedum ochroleucum chaix med-smed e smed sedum telephium l. ssp. maximum (l.) krocker med-smed e smed sempervivum marmoreum griseb. ea med-pont cucurbitaceae bryonia alba l. ea ce-m-p-or cuscutaceae cuscuta epithymum l. ea eur.-med.-smed.-pont.-orijent.turan.-himal. dipsacaceae cephalaria flava (sibth. & sm.) szabó med-smed balk moesian-skard. pind. dipsacus laciniatus l. ea ea(w) knautia drymeia heuffel eam csem alp.-balk. scabiosa ochroleuca l. ea ea(w) europ.-medit.-west-asian euphorbiaceae euphorbia amygdaloides l. ea ea(w) europ.-medit.-west-asian euphorbia cyparissias l. ea eurosib.-(bor-merid) euphorbia polychroma a. kerner med-smed euphorbia salicifolia host ea ce-med-pont euphorbia taurinensis all. med-smed mercurialis perennis l. ea ea(w) europ.-medit.-west-asian fabaceae astragalus cicer l. ea ce-med galega officinalis l. ea med-pont pontsko-istočnosubmedit. genista tinctoria l. ea ea(w) europ.-medit.-west-asian lathyrus venetus (miller) wohlf. ea ce-med-pont lathyrus vernus (l.) bernh. ea ce-med europ.-medit.-west-asian medicago arabica (l.) hudson ea ce-med-pont medicago falcata l. ea medicago prostrata jacq. ea ce-med-pont melilotus officinalis (l.) pallas ea eurasian ononis spinosa l. ea ce-med atlant.-central europeansubmedit. trifolium alpestre l. ea ea(w) europ.-medit.-west-asian trifolium arvense l. ea ea(w) europ.-medit.-southsib. trifolium campestre schreber ea ce-m-p-or central european-medit.submedit.-pont.-orijent.-turan. biologica nyssana 3 (2)  december 2012: 77-90 miljković m. et al.  phytogeographical analysis of the flora… 83 taxa area type area group flostic element trifolium dalmaticum vis. med-smed e smed trifolium pratense l. ea ea(w) europ.-submedit.-pont.-southsibir. trifolium repens l. hol circumcholarkct.-(arct-submer) vicia lathyroides l. ea ea(w) vicia villosa roth ea ea(w) europ.-medit.-west-asian fagaceae fagus moesiaca k.maly ce moesian quercus cerris l. med-smed quercus frainetto ten. ea ce-med quercus pubescens willd. ea ce-med gentianaceae ce-med centaurium erythraea rafin. ea ea(w) europ.-medit.-west-asian geraniaceae geranium lucidum l. ea geranium molle l. ea geranium robertianum l. cosm sec. cosm. -eurasian. origin gesneriaceae ramonda serbica panč. med-smed balk moesian-skard. pind. hypericaceae hypericum boissieri petrovic med-smed balk hypericum perforatum l. ea europ.-medit.-west-asian hypericum rumeliacum boiss. med-smed skarp-balk lamiaceae acinos arvensis (lam.) dandy ea ce-med-pont central european-submedit.pont.-south sibir. acinos hungaricus (simonkai) [ilic ea ce-med-pont herc.-panon.-south sarmat.medit.-submedit.-pont. ajuga reptans l. ea ce-med central european-submedit. calamintha sylvatica bromf. ea ce-m-p-or sublant.-central europeansubmedit.-pont.-orijantal.-turan. clinopodium vulgare l. ea ce-med-pont eurasian-(subbor-submer) glechoma hederacea l. ea eurasian-(subbor-submer) glechoma hirsuta waldst. & kit. ea ce-med-pont subatl.-ce-medit.-pont. hyssopus officinalis l. med-smed lamium amplexicaule l. hol phol-ptrop eurasian-(temp-smer)-east-afric. lamium galeobdolon l. ce central european lamium garganicum l. ea(m) csem lamium maculatum l. ea ce-med-pont central european-submedit.pont. leonurus cardiaca l. ea ce-m-p-or herc.-sarmat.-submedit.-pont.south sibir.-orijental.-turan. lycopus europaeus l. ea ea(w) europ.-medit.-west-asian marrubium peregrinum l. ea ce-med-pont melissa officinalis l. msm medit.-east submed.-orijent.turan. melittis melissophyllum l. ce mentha aquatica l. hol phol-ptrop europ.-(subbor-mer)-east.-afric.(borsubtro)-afric-(austr) biologica nyssana 3 (2)  december 2012: 77-90 miljković m. et al.  phytogeographical analysis of the flora… 84 taxa area type area group flostic element mentha longifolia (l.) hudson hol phol-ptrop europ.-west-asian-(temp-mer)east-afric.-(borstro)-afric-(austr) mentha pulegium l. ea micromeria cristata (hampe) griseb. med-smed balk moesian origanum vulgare l. ea eurasian-(temp-mer) prunella laciniata l. ea ea(w) atlant.-central-europ.-medit.orijental prunella vulgaris l. ea ea(w) europ.-medit.-west-asian salvia nemorosa l. ea ce-m-p-or central european-submedit.pont.-orijent.-turan. salvia sclarea l. ea ce-m-p-or satureja kitaibelii wierzb. med-smed scutellaria columnae all. med-smed sideritis montana l. ea ce-med stachys germanica l. msm med-pont medit.-submedit.-pont. stachys palustris l. hol circumcholarkct.-(bor-submer) stachys recta l. ea ea(w) stachys sylvatica l. ea ea(w) europ.-medit.-west-asian teucrium chamaedrys l. ea ea(w) europ.-medit.-west-asian teucrium montanum l. ea(m) csem teucrium polium l. med-smed thymus marschalianus willd. ea ce-med thymus pulegioides l. ce central european lythraceae lythrum salicaria l. cosm eurasian disjunct-(subbor-mer)southeastern austr. (austr.) malvaceae alcea rosea l. med-smed althaea officinalis l. ea oenotheraceae circaea lutetiana l. hol circumcholarkct.-(bor-merid) epilobium hirsutum l. hol phol-ptrop euras.-(subbor-mer)-afric(borsubtro-austrosubtro) epilobium lanceolatum sebastiani & mauri ea ea(w) epilobium montanum l. ea eurasian oenothera biennis l. adv oleaceae fraxinus ornus l. ea ce-med ligustrum vulgare l. ea ce-med-pont syringa vulgaris l. ea ce-med-pont paeoniaceae paeonia decora g. anderson msm med-pont papaveraceae chelidonium majus l. ea ea(w) amfiatlant.-central-europ.medit.-west-asian-(bor-merid) biologica nyssana 3 (2)  december 2012: 77-90 miljković m. et al.  phytogeographical analysis of the flora… 85 taxa area type area group flostic element corydalis bulbosa (l.) dc ea ea(w) european-west asian papaver rhoeas l. ea european-medit.-submedit.-pont.south sibir.-orijental. plantaginaceae plantago lanceolata l. ea eurasian-(subbor-temp) plantago major l. cosm cosm.-europ.-northamer. origin plantago media l. ea eurasian-(temp-submer) polygonaceae bilderdykia convolvulus (l.) dumort. ea eurasian-(bor-merid) polygonum hydropiper l. ea eurasian-(temp) portulacaceae portulaca oleracea l. adv primulaceae anagallis arvensis l. cosm cosm.mediter. origin lysimachia nummularia l. ce central european lysimachia punctata l. ea ce-pont lysimachia vulgaris l. ea eurasian-(temp-submer) ranunculaceae anemone ranunculoides l. ce central european clematis vitalba l. ea ea(w) europ.-medit.-west-asian helleborus odorus waldst. & kit. ce central european (temp-submer) hepatica nobilis schreber ce isopyrum thalictroides l. ea ce-med-pont herc.-sarmat.-pan.-medit.submedit.-pont.-southsib. nigella arvensis l. ea ce-med ranunculus millefoliatus vahl med-smed ranunculus repens l. ea eurasian-(bor-merid) ranunculus serbicus vis. eam sem apen.-balk. ranunculus stevenii andrz. ex besser msm med-pont pont.-submedit. thalictrum lucidum l. ce central european rosaceae agrimonia eupatoria ledeb. ea evropsko-mediteransko-pontskoorijentalno-južnosibirskoturanski aremonia agrimonioides (l.) dc. eam ea(w)m sem-south-west-asian-mont. filipendula hexapetala gilib. ea fragaria vesca l. cosm sekundarni kosmop. evroaz. porekla geum urbanum l. hol europ.-west asian-north amer.(bor-temp) potentilla argentea l. ce central european potentilla cinerea chaix ex vill. ea ce-med-pont potentilla detommasii ten. eam csem apen.-balk.-anatol. potentilla recta var. sulfuraea lam. ea ea(w) potentilla reptans l. ea prunus spinosa l. ea ea(w) rubus caesius l. ea eurasian-(temp-submer) sanguisorba minor scop. ea ea(w) europ.-medit.-west-asian waldsteinia geoides willd. ea ce-med-pont biologica nyssana 3 (2)  december 2012: 77-90 miljković m. et al.  phytogeographical analysis of the flora… 86 taxa area type area group flostic element rubiaceae asperula longiflora waldst. & kit. med-smed alp-balk asperula purpurea (l.) ehrend. med-smed asperula taurina l. ea ce-med south central europ.-submedit. galium album miller ce subatlant.-central european galium aparine l. ea eurasian-(bor-merid) sherardia arvensis l. cosm rutaceae dictamnus albus l. ea salicaceae salix fragilis l. ea ea(w) europ.-west-asian-(tempsubmer) salix purpurea l. ea eurasian-(bor-merid) santalaceae comandra elegans roch. med-smed southkarp.-balk. saxifragaceae saxifraga rotundifolia l. eam ea(w)m sem-anatol.-kavk. scrophulariaceae digitalis lanata ehrh. ce panon.-balk. lathraea squamaria l. ea linaria concolor gris. med-smed balk moesian-macedon.-trac. linaria genistifolia (l.) miller ea ce-med-pont central european-cmed.-pont.south-sibir. linaria vulgaris miller ea ea(w) europ.-medit.-west-asian melampyrum arvense l. ea(w) odontites rubra besser ea ce-med central european-medit.submedit. verbascum phlomoides l. ea ce-med central european-medit.submedit. veronica beccabunga l. ea ea(w) europ.-medit.-west-asian veronica hederifolia l. ea ce-med-pont central european-submedit.pont. veronica polita fries ea ea(w) veronica serpyllifolia l. cosm cosm-eurasian origin solanaceae solanum dulcamara l. ea eur.-medit.-pont.-orijent.-turan. urticaceae parietaria officinalis l. ea ce-med central european-medit.submedit. parietaria serbica pancic med-smed balk moesian urtica dioica l. hol circumcholarkct.-(bor-temp) valerianaceae valeriana officinalis l. ea eurasian-(subbor-temp) valerianella carinata loisel. ea ce-m-p-or valerianella turgida (steven) betcke med-smed verbenaceae verbena officinalis l. cosm cosm-eurasian origin biologica nyssana 3 (2)  december 2012: 77-90 miljković m. et al.  phytogeographical analysis of the flora… 87 taxa area type area group flostic element violaceae viola canina l. hol circumcholarkct.-(bor-submer) viola kitaibeliana schultes ea ce-med viola silvestris lam. p.p. ea liliopsida alysmataceae alisma plantago-aquatica l. hol circumcholarkct.-(bor-submer) araceae arum maculatum l. ea ce-med-pont ce-submedit.-west pont. cyperaceae carex bueckii wimmer ea ea(w) scirpus sylvaticus l. adv eurasian-(bor-temp) dioscoreaceae tamus communis l. ea ea(w) atlant.-central-europ..-medit.submedit.-orijental iridaceae iris pseudoacorus l. ea ea(w) lemnaceae lemna minor l. cosm cosm. liliaceae allium flavum l. msm med-pont medit.-submedit.-pont. allium rotundum l. ea ce-m-p-or sublant.-central europeansubmedit.-orijantal. allium saxatile bieb. msm med-pont asparagus tenuifolius lam. msm med-pont colchicum autumnale l. ea ce-med atlant.-central european-medit. erythronium dens-canis l. ea eurasian-(bor-merid) lilium martagon l. ea subatlant.-central europeansubmedit.-central sibir. polygonatum odoratum (miller) druce ea eurasian-(bor-submer) orchidaceae cephalanthera damasonium (miller) druce ea himantoglossum hircinum (l.) sprengel ea ce-med poaceae aegilops triaristata willd. nom. illegit. med-smed agrostis alba l. hol agrostis capillaris l. hol circumcholarkct.-(bor-merid) alopecurus pratensis l. ea eurasian-disjunct.-(bor-submer) anthoxanthum odoratum l. ea eurasian-disjunct.-(bor-submer) brachypodium sylvaticum (hudson) beauv. ea chrysopogon gryllus (l.) trin. msm med-pont dasypirum villosum (l.) cand. med-smed dichanthium ischaemum (l.) roberty cosm cosm.mediter. origin echinochloa crus-galli (l.) beauv. cosm hordeum leporinum (linc) arcangeli) hol biologica nyssana 3 (2)  december 2012: 77-90 miljković m. et al.  phytogeographical analysis of the flora… 88 taxa area type area group flostic element koeleria macrantha (ledeb.) schultes hol circumcholarkct.-(bor-submer) lolium perenne l. ea eurasian-(bor-merid) melica ciliata l. ea ea(w) europ.-medit.-west-asian orysopsis virescens (trin.) g. beck msm med-pont-or poa bulbosa l. ea eur.-medit.-submedit.-pont.south sibir.-orijent.-turan.central asian poa nemoralis l. hol circumcholarkct.-(temp) poa pratensis l. cosm sesleria rigida heuffel ex reichenb. ea(m) csem setaria viridis (l.) beauv. ea eurasian-(temp-mer) stipa capillata l. ea eurasian-(temp-mer) sparganiaceae sparganium erectum l. ea ea(w) europ.-medit.-west-asian typhaceae typha latifolia l. cosm cosm.circumcholarct. origin typha laxmanii lepechin ea ea(w) table 2. chorological spectrum of the mijkovačka gorge flora areal type (group) species % boreal (bor) 2 0.60 eurasian mountane (eam) 0.00 central south european mountane (eam/csem) 6 1.81 west eurasian mountainous (eam/ea(w)m 2 0.60 south european mountainous (eam/sem) 1 0.30 central european (ce) 16 4.83 meditteranenan-submeditteranean (med-smed)) 26 7.85 alpine-balkan (med-smed/alp-balk) 1 0.30 balkan (med-smed/balk) 8 2.42 european-submeditteranean (med-smed/e smed) 4 1.21 meridional-submeridional (msm) 0.00 mediterranean-pontic (msm/med-pont) 12 3.63 meditteranean-pontic-oriental (msm/med-pont-or) 1 0.30 meditteranean-pontic-oriental-carpathian (msm/med-pont-or-ca) 3 0.91 eurasian (ea) 72 21.75 european-west asian (ea/ea (w)) 53 16.01 central european-mediterranean (ea/ce-med) 33 9.97 mediterranean-pontic (ea/med-pont) 2 0.60 central european-mediterranean-pontic (ea/ce-med-pont) 27 8.16 central european-mediterranean-pontian-oriental-turanian (ea/ce-m-p-or) 17 5.14 central european-pontian (ea/ce-pont) 1 0.30 holarctic (hol) 18 5.44 paleoholarctic-paleotropic (hol/phol-ptrop) 5 1.51 adventive (adv) 4 1.21 cosmopolitan (cosm) 17 5.14 total 331 100.00 biologica nyssana 3 (2)  december 2012: 77-90 miljković m. et al.  phytogeographical analysis of the flora… 89 figure 2. area spectrum of the flora of the miljkovacka gorge disscusion for the phytogeographical analysis, all plant taxa were classified into 112 floristic elements, sorted in 15 area groups and 9 areal types (tab.2). as shown in fig.2, domination of eurasian area type (ea) with 205 taxa (62%) indicates the accessibility of miljkovačka gorge for colder influences coming from the northwest through the valley of the south morava river. from the other side, svrljiške mountain massif (kalafat and kamenički vis) prevent the penetration of warmer effects coming from the mediterranean by the valleys of nišava and south morava rivers. european-west asian areal group were represented with 53 taxa (16.01%), where 27 taxa belongs to european-meditteranean-west-asian floristic elements: stellaria holostea, veronica beccabunga, prunella vilgaris, cynanchum vincetoxicum, campanula rapunculoides, etc. the high presence of plants which belong to meditteranenan-submeditteranean area type (medsmed (12%), or 39 plant taxa) and meridionalsubmeridional area type (msm (5%), or 16 taxa), can be explained by the ground degradation, presence of limestone that heats easily and more easily colds, and small amount of soil covering. almost all endemic taxa, parietaria serbica, hypericum boissieri, linaria concolor, erysimum comatum, ramonda serbica, acanthus balcanicus, micromeria cristata, cephalaria flava belong to med-smed area type, while parietaria serbica and micromeria cristata represent edificatory species of ass. micromerio-pariterietum serbicae miljković, m. 2012., and ramonda serbica represents edificatory taxa of ass. ceterachi-ramondietum serbicae jovanović, r. 1953 on limestones. holarctic area type includes 23 taxa (7%) with 4 borealmeridional and 4 borealsubmeridional floristic elements, while others have disjunctive distribution area. percentage of 5% of species which belong to central european area type is product of the presence of mesophilous beach forest communities (fagetum moesiacae montanum blečić et lakušić 1970. above 700 m,, carpinio-querco-fagetum mixtum, prov., located near the stream that flows next to the monastery st. archangel gabriel in popsica) (đorđević, v. et al. 2006). low percentage of eurasian-mountain (3%) and boreal (0.6%) chorological types point to relatively low altitude of this gorge as well as to weak phytogeographical links with the high mountain region of the balkan peninsula. the significant presence in the composition of the miljkovačka gorge flora has cosmopolitan (cosm) area type with many ruderal species: plantago major, stellaria media, convolvulus arvensis, verbena officinalis, etc. small number of adventive area type taxa (4 (1%)) can be explained by the distance of the miljkovačka gorge valley and the significant migratory routes of adventive flora, which coincides with the nearby road traffic. bor 1% eam 3% med-smed 12% msm 5% eurasian (ea) 22% ea(w) 16% ce 5% ce-med 10% ce-med-pont 8% ce-m-p-or 5% hol 7% adv 1% cosm 5% biologica nyssana 3 (2)  december 2012: 77-90 miljković m. et al.  phytogeographical analysis of the flora… 90 references đorđević, v., ranđelović, n., avramović, d., lilić, a., 2006: prilog vegetaciji leskovika. ekološka istina 2006. sokobanja. greuter, w., burdet, h.m., long, g., eds., 19841989: med-checklist, 1, 3, 4. gèneve. horvat, i., glavač, v., ellemberg, h., 1974: vegetation sudosteuropa. gustav fischer verlag. stuttgart. jordanov, d. (ed.). 1963–1979. flora republ. popularis bulgaricae. vols. 1-7. in aedibus acad. sci. bulgaricae, serdicae (in bulgarian). josifović, m. (ed)., 1970-1977: flora sr srbije iix. sanu. beograd. martinović, ž., sotirov., s., ranđelović, n., 1985: biljno-geografske karakteristike miljkovačke klisure. simpozijum stogodišnjica flore okoline niša. niš, 51-56. meussel, h., jäger, e., weinert, e., 1965: vergleinchende chorologie der zentraleuropaischen flora. veb. gustav fischer verlag, 1. jena. meussel, h., jäger, e., raischert, s., weinert, e.,1978: vergleinchende chorologie der zentraleuropaischen flora. veb. gustav fischer verlag, 2. jena. meussel, h., jäger, e., 1992: vergleinchende chorologie der zentraleuropaischen flora. veb.gustav fischer verlag, 3. jena. micevski, k. 1985: flora na republika makedonija. manu, 1(1). skopje. micevski, k., 1993: flora na republika makedonija. manu, 1(2). skopje. micevski, k., 1995: flora na republika makedonija. manu, 1(3). skopje. miljković, m., 2011: flora i vegetacija doline miljkovačke reke. diplomski rad. miljković, m., ranđelović, v. ranđelović, n., 2012: the boulder vegetation of miljkovacka gorge valley, eco-ist '12, 84-91. sotirov, s., stanojević, v., 2012: treći alen kamen, cerjanska pećina i topilo, bajka na domaku niša. niš. sarić, m (ed)., 1986: flora sr srbije x. sanu. beograd. sarić, m. (ed)., 1992: flora srbije i (2.izd.) sanu. beograd. stevanović, v., 1992: floristička podela teritorije srbije sa pregledom viših horiona i odgovarajućih flornih elemenata. in sarić, m. (ed.): flora srbije, i. (drugo izdanje). sanu. beograd. 49 – 70. the euro+med plantbase the information recourse for euro-meditteranean plant diversity (http://ww2.bgbm.org/europlusmed/). the international organisation for plant information (iopi ( http://plantnet.rbgsyd.nsw.gov.au/iopi/iopihome. htm)). tutin, t.g., heywood, v.h., burges, n.a., moore, d.m., valentine, d.h., walters, s.m., webb, d.a., eds., 1964-1980: flora europaea, i-v. cambridge, university press. london. velchev, v. (ed.). 1982–1989. flora republ. popularis bulgaricae, vols 8-9. in aedibus acad. sci. bulgaricae, serdicae (in bulgarian). http://ww2.bgbm.org/europlusmed/ http://plantnet.rbgsyd.nsw.gov.au/iopi/iopihome.htm) http://plantnet.rbgsyd.nsw.gov.au/iopi/iopihome.htm) urosevic et al. 2021, biologica nyssana 12(2) 12 (2) december 2021: 151-157 doi: 10.5281/zenodo.5763846 new findings of kotschy’s gecko, mediodactylus kotschyi (steindachner, 1870) in serbia, with a particular focus on recently recorded populations in niš and sremska mitrovica original article aleksandar urošević institute for biological research “siniša stanković”, national institute of republic of serbia, university of belgrade, bulevar despota stefana 142, belgrade, serbia aurosevic@ibiss.bg.ac.rs (corresponding author) marko maričić institute of zoology, faculty of biology, university of belgrade, studentski trg 16, belgrade, serbia tijana vučić institute of zoology, faculty of biology, university of belgrade, studentski trg 16, belgrade, serbia vladimir žikić faculty of sciences and mathematics, university of niš, višegradska 33, 18000 niš, serbia saša s. stanković faculty of sciences and mathematics, university of niš, višegradska 33, 18000 niš, serbia mirko šević institute of zoology, faculty of biology, university of belgrade, studentski trg 16, belgrade, serbia marko anđelković institute for biological research “siniša stanković”, national institute of republic of serbia, university of belgrade, bulevar despota stefana 142, belgrade, serbia received: october 26, 2021 revised: november 24, 2021 accepted: december 03, 2021 abstract: kotschy’s gecko (mediodactylus kotschyi) is native to the eastern mediterranean basin but well known as a successful colonizer. the most probable way of its spreading is cargo and passenger traffic. in serbia, m. kotschyi is assumed native only in prizren. anthropogenic introductions were confirmed in belgrade, novi sad, pančevo and smederevo. this study aimed to summarize data of m. kotschyi findings with reference to recently confirmed population in niš and newly recorded population in sremska mitrovica. these two populations are well-established with multiple pieces of evidence of reproduction. pholidosis data were collected for several individuals to confirm their specific status. new and previously recorded introduced populations of kotchy’s gecko in serbia belong to m. kotschyi bibroni based on morphology. the introduction was most likely related to railways and further spread in urban zones is expected. it seems that introduced populations do not threaten native ecosystems as these populations are appeared to be highly localized in urban habitats. key words: anthropogenic introduction, colonizing species, distribution, m. kotschyi bibroni apstrakt: novi nalazi kočijevog gekona, mediodactylus kotschyi (steindachner, 1870) u srbiji, sa posebnim fokusom na nedavno zabeležene populacije u nišu i sremskoj mitrovici kočijev gekon (mediodactylus kotschyi) se autohtono javlja u basenu istočnog sredozemlja ali je dobro poznat i kao uspešan kolonizator. najverovatniji načini njegovog širenja su teretni i putnički saobraćaj. u srbiji, kočijev gekon se smatra autohtonim samo na području prizrena. antropogene introdukcije potvrđene su u beogradu, novom sadu, pančevu i smederevu. cilj ove studije je da sumira podatke o nalazima m. kotschyi sa posebnom pažnjom na nedavno potvrđeno prisustvo populacije u nišu i novozabeleženu populaciju u sremskoj mitrovici. ove dve populacije su dobro uspostavljene, sa višestrukim dokazima reprodukcije. podaci o folidozi prikupljeni su za veći broj jedinki da bi se potvrdio njihov status vrste. nove i prethodno zabeležene populacije kočijevog gekona u srbiji prema morfologiji pripadaju m. kotschyi bibroni podvrsti. introdukcije su najverovatnije vezane za železnički saobraćaj i očekivano je dalje širenje ove vrste u urbanim zonama. pretpostavlja se da introdukovane populacije ne ugrožavaju autohtone ekosisteme jer su po svoj prilici veoma lokalizovane na urbana staništa. ključne reči: antropogena introdukcija, kolonizujuća vrsta, distribucija, m. kotschyi bibroni introduction lizards, and particularly geckos, are well known as adept colonizers that significantly spread their range via human activity (flower, 1933; davis, 1974; conant & collins, 1998; jesus et al., 2002). it was even assumed that many populations of the hemidactylus (gekkonidae, squamata) and tarentola (phyllodactylidae, squamata) geckos in the mediterranean basin are of anthropogenic origin (harris et al., 2004; kasapidis et al., 2005). kotschy’s gecko (mediodactylus kotschyi (steindachner, 1870), gekkonidae, squamata) is a small, crepuscular, or nocturnal gecko species © 2021 urošević et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 151 native to the eastern mediterranean basin (böhme et al., 2009). recently, based on nuclear and mitochondrial dna analysis, it was discovered that it is a species complex consisting of five species – m. kotschyi (mainland of balkan peninsula, most of aegean islands, italy), m. orientalis (levant, cyprus, southern anatolia, south-eastern aegean islands), m. danilewskii (black sea region, southwestern anatolia and gavdos island, greece), m. bartoni (crete and surrounding islets) and m. oertzeni (southern dodecanese islands) (kotsakiozi et al., 2018). there are known cases of individuals of m. kotschyi species complex introduction outside the native range in hungary (farkas et al., 1999), serbia (ajtić, 2009; tomović et al., 2014; balej & jablonski, 2015; urošević, 2016; urošević et al., 2016, 2019), romania, bulgaria (koynova et al., 2020) and italy (mares & novarni, 2020). in serbia, it is assumed native only in prizren, metohija (ajtić & tomović, 2001), according to the distribution of mediterranean fauna in serbia and its neighbouring countries (haxhiu, 1998; ajtić & тomović, 2001; ajtić, 2004). other populations that were discovered outside of metohija are presumed to be introduced during the historical times or, more likely, recently (ajtić, 2009; tomović et al., 2014; balej & jablonski, 2015; urošević, 2016; urošević et al., 2016, 2019). the introductions have been confirmed in novi sad (ajtić, 2009; balej & jablonski, 2015;), belgrade [vračar (balej & jablonski, 2015), stari grad – kneginje zorke st., zemun (urošević et al., 2016)], smederevo [smederevo fortress (urošević, 2016; urošević et al., 2016)] and pančevo (urošević et al., 2019). finding from niš has been mentioned in the literature (ajtić, 2009; urošević et al., 2016), but it was based only on one reported sighting in a local coffee warehouse, and it has lacked an expert confirmation. with the recent taxonomic revision of the m. kotschyi group (kotsakiozi et al., 2018), the need to correctly assess the specific status of the introduced populations emerged. although the molecular analyses are not congruent with most of the described subspecies, the newly described species are very close to the main groups defined by beutler (1981). also, ajtić (2014) showed that discrimination between the main groups (for instance, the mainland kotschyi and danilewskii) is feasible via morphological characters. our main assumption was that the introductions occurred by land, from the continental parts of the balkan peninsula. since the geckos could have originated from the black sea coast region and therefore belong to the m. danilewskii group (d. jablonski, personal communication) we wanted to assess meristic characters through which we could identify the approximate origin of the colonized geckos, before we conduct the dna analyses. the main aim of this study is to provide a comprehensive list of the published and unpublished known findings of the kotschy’s gecko in serbia, and morphologically assess specific/subspecific status of the populations in niš (confirmed for the first time) and sremska mitrovica (newly discovered), since they are well-established, numerous and with multiple proofs of successful reproduction observed by the authors. materials and methods the presence of kotschy’s geckos at the studied sites was detected by visual observation or by listening to 152 biologica nyssana ● 12 (2) december 2021: 151-157 urošević et al. ● new findings of kotschy’s gecko, mediodactylus kotschyi (steindachner, 1870) in serbia, with a particular focus on recently recorded populations in niš and sremska mitrovica fig. 1. meristic data. a) rdt – number of rows of dorsal tubercles; b) supl. – number of supralabial tubercles; infl. – number of infralabial tubercles; otic tubercles – arrangement, c) pap – number of preanal pores; d) lct – number of lateral caudal tubercles 153 the characteristic “vocalization”, usually during the late afternoon or in the evenings, after sunset, during the summer and early autumn of 2020 and 2021. for confirmation, voucher photos of the animals were taken with a digital camera, and the precise locations were georeferenced (via application gps measurer – area, perimeter, distance, poi; v. 1.6.6 for android, edsa). when possible, the animals were collected by hand to examine pholidosis and take a tail tip as a sample for the subsequent dna analyses. the detailed pholidosis data was collected for several individuals in niš and sremska mitrovica. activities such as mating and feeding, as well as presence of juvenile animals or gravid females were also noted if observed. the pholidosis was compared to the diagnostic characters of the m. kotschyi former subspecies (biserkov et al., 2007; ajtić, 2009 and references there in) with an emphasis on the former subspecies native to the continental part of the balkans – m. k. bibroni (beutler & gruber, 1977), m. k. skopjensis biologica nyssana ● 12 (2) december 2021: 151-157 urošević et al. ● new findings of kotschy’s gecko, mediodactylus kotschyi (steindachner, 1870) in serbia, with a particular focus on recently recorded populations in niš and sremska mitrovica (karaman, 1965), m. k. rumelicus (müller, 1940) and m. k. danilewskii (strauch, 1887), since we assumed that the multiple introductions were most likely via the continental transportation routes. specific traits that we examined were: 1) layout of the tubercles around the ear opening, 2) shape of the dorsal tubercles, 3) number of rows of the dorsal tubercles (rdt), 4) number of supralabials (supl.) and infralabials (infl.), 5) number of the preanal pores (pap), and 6) number of lateral caudal tubercles (lct) (fig. 1). results the intensive research re-confirmed the presence of kotschy’s geckos at most of the known locations from the literature (fig. 2, supplementary table 1). the new findings were reported from the town of sremska mitrovica and a few localities in belgrade, as well as the city of niš, where the expert confirmation of the introduction was provided for the first time. in novi sad, belgrade, smederevo and niš, the findings were often localized to specific walls, buildings, or streets. in those places, the species seems to prefer the walls that are oriented towards the south and/or west, and that receive a lot of insolation during the day. in sremska mitrovica, the geckos seem to be frequent throughout the old town centre, probably due to the abundance of old, mutually connected buildings which provide suitable habitats. the pholidosis characters of the observed individuals in niš and sremska mitrovica are given in tab. 1. all animals had between 11 and 13 rows of dorsal tubercles (most had 12), between 8 and 10 supralabial scales (most having 9), 6 or 7 infralabial scales (most having 7) and 2 or 3 lateral caudal tubercles. all males in the sample had 4 preanal pores. all animals had large, keeled dorsal tubercles and almost all individuals had otic tubercles above and in front of ear opening (only one had above, in front and behind). in niš, mating was observed and one gravid female with two visible eggs was caught (fig. 3). in sremska mitrovica, three caught animals were subadults. we also observed feeding on arachnids in niš. many animals in both populations had regenerated tails, which is a strong indication that they are subject to predation (by wall lizards, feral cats, etc.) or aggressive intraspecific behaviour. discussion mediterranean geckos are known as successful colonizers and their spread to the north is constrained only by their association with anthropogenic habitats fig. 2. map of m. kotschyi distribution in serbia. empty blue dots represent literature reports, blue dots with black centre represent literature records reconfirmed in the field during the last survey, and red dots denote new distribution records. population in prizren, that is considered as native, is presented as a large blue dot 154 biologica nyssana ● 12 (2) december 2021: 151-157 urošević et al. ● new findings of kotschy’s gecko, mediodactylus kotschyi (steindachner, 1870) in serbia, with a particular focus on recently recorded populations in niš and sremska mitrovica and climate (meshaka et al., 2005). in serbia, the establishment of kotschy’s geckos in the urban areas is well known and documented (ajtić, 2009; tomović et al., 2014; balej & jablonski, 2015; urošević, 2016; urošević et al., 2016, 2019). by now, only the population in prizren is considered to be autochthonous (ajtić & tomović, 2001). due to its isolation from the rest of the native range of m. kotschyi, it is hypothesized that the prizren population could also be a result of the historical introduction (ajtić & tomović, 2001; ajtić, 2004, 2009). however, the isolation could be explained by the lack of distribution data from the north albania and metohija regions (haxhiu, 1998), since these areas generally lack systematic faunistic research (ajtić & tomović, 2001; ajtić, 2009; tomović et al., 2014). the rest of the populations in serbia are considered to be anthropogenic introductions (ajtić, 2009; urošević et al., 2016). since the discovered population in novi sad appeared stable, it was supposed that the introduction took place long ago (ajtić, 2009). the other populations that were discovered afterwards also seemed well established, albeit localized, and could be detected throughout consecutive years, with most being reconfirmed during the last field surveys. hypotheses of historical introduction could not explain how well-established populations of partially diurnal geckos were not noticed earlier. the deliberate introductions in hungary showed that these geckos can establish breeding colonies quickly (farkas et al., 1999). urošević et al. (2016) proposed a scenario of table 1. meristic data for the collected individuals. abbreviations: ind. – individual; rdt – number of rows of dorsal tubercles; supl. – number of supralabial tubercles; infl. – number of infralabial tubercles; pap – number of preanal pores; lct – number of lateral caudal tubercles; sdt – shape of dorsal tubercles; subad. – subadult. locality ind. sex rdt supl. infl. pap lct sdt otic tubercules note niš ni001 m 12 9 7 4 3 large, keeled above and in front niš ni002 f 12 10 6 / 2 large, keeled above and in front gravid niš ni003 m 12 9 7 4 / large, keeled above and in front niš ni004 f 11 9 7 / 3 large, keeled above and in front sremska mitrovica sm001 f 13 9 7 / 3 large, keeled above and in front subad. sremska mitrovica sm002 f 11 8 6 / 3 large, keeled above and in front sremska mitrovica sm003 m 13 9 7 4 3 large, keeled above, in front and behind sremska mitrovica sm004 f 12 10 7 / 3 large, keeled above and in front sremska mitrovica sm005 f 13 9 7 / 2 large, keeled above and in front sremska mitrovica sm006 f 11 8 7 / 2 large, keeled above and in front subad. sremska mitrovica sm007 f 13 9 7 / 2 large, keeled above and in front subad. sremska mitrovica sm008 f 12 8 6 / 2 large, keeled above and in front sremska mitrovica sm009 m 12 9 7 4 2 large, keeled above and in front 155 biologica nyssana ● 12 (2) december 2021: 151-157 urošević et al. ● new findings of kotschy’s gecko, mediodactylus kotschyi (steindachner, 1870) in serbia, with a particular focus on recently recorded populations in niš and sremska mitrovica fig. 3. evidence of m. kotschyi reproduction in the niš population. a) mating pair; b) gravid female with visible eggs relatively recent (15–20 years ago), simultaneous introduction into the large urban centres. ajtić (2009) assumed that geckos were introduced accidentally throughout the known localities in serbia, via cargo shipments from south or east. through the centuries, balkan peninsula harboured an important network of trading routes. for instance, via militaris (later known as “tsarigrad road”) had been traced in the 1st century ad. some of the places where gecko populations were confirmed (prizren, belgrade, sremska mitrovica, pančevo, smederevo, and niš) were the important trading hubs and caravan stations during the roman and ottoman empires (simonović, 2003; ajtić, 2009; urošević, 2016; urošević et al., 2016). today, these places are well connected by road and railroad networks. railroads are suggested as an especially probable means of introductions, since most of the known introductions in serbia are situated up to 1.5 km far from the railroad stations and junctions, and railroad beds can also provide good migratory corridors for reptiles (gherghel et al., 2009; urošević et al., 2019). this could also have facilitated the spread of geckos via cargo or passenger traffic (urošević, 2016). according to the meristic characters interpretable as diagnostic for the subspecies (biserkov et al., 2007; ajtić, 2009), geckos from the populations in niš and sremska mitrovica clearly belonged to the former m. kotschyi bibroni subspecies, characteristic for the central and southern balkans – males had four preanal pores, all examined individuals had two or three lateral caudal tubercles, most animals had otic tubercles only above and in front of the ear opening (which rules out m. kotschyi rumelicus) and dorsal tubercles were large and strongly keeled. animals observed in belgrade, novi sad and smederevo that were photographed also had strongly keeled dorsal tubercles and otic tubercles only above and in front of the ear opening. the number of rows of the dorsal tubercles ruled out the geographically closest subspecies, m. kotschyi skopjensis, since it usually has only 10 rows of tubercles, while the animals we observed had between 11 and 13 (most 12) rows, which corresponds with m. kotschyi bibroni group (ajtić, 2009). according to the molecular data, the group m. kotschyi bibroni, which inhabits the mainland balkans, belongs to the m. kotschyi species (kotsakiozi et al., 2018). although kotschy’s gecko seems to be spreading in serbia, there are no known negative effects which it can have on native ecosystems (urošević et al., 2016). all known introduced populations are localized in the urban zones, sometimes only on specific buildings. in the north of its native range, the species is also almost exclusively associated with human dwellings, in urban or suburban zones (ajtić & tomović, 2001; arnold & ovenden, 2002; ajtić, 2009). we expect further spread of kotschy’s gecko predominantly into urban areas, towns and cities that, due to the “heat island effect“, provide 156 biologica nyssana ● 12 (2) december 2021: 151-157 urošević et al. ● new findings of kotschy’s gecko, mediodactylus kotschyi (steindachner, 1870) in serbia, with a particular focus on recently recorded populations in niš and sremska mitrovica adequate thermal conditions for the predominantly mediterranean species. acknowledgements. we would like to thank ljiljana tomović and ilias strachinis for their constructive reviews that greatly improved our manuscript, ana miletić, jelena pavlović and sara krunić for the help in the field, and dunja čolak and ksenia prądzyńska for sharing their field data. the study was partially financed by the serbian ministry of the education, science and technological development, grant nos. 451-03-9/2021-14/ 200007 and 451-03-9/2021-14/ 200178. animals and dna samples were collected with permissions from the serbian ministry of environmental protection, permit nos. 353-012876/2019-04 and 353-01-2716/2020-04. references ajtić, r., tomović, l. 2001: first record of kotschy’s gecko cyrtodactylus kotschyi (steindachner, 1870) (gekkonidae, lacertilia) in fr yugoslavia. archives of biological sciences, 53: 23–24. ajtić, r. 2004: morphological and ecological characteristics of population of kotchy’s gecko (cyrtodactylus kotchyi, steindachner, 1870) (gekkonidae, lacertilia) from part of the balkan peninsula. protection of nature, 56: 69–78. ajtić, r. 2009: morfološke, biogeografske i ekološke odlike kočijevog gekona (cyrtodactylus kotschyi steindachner, 1870) sa kopnenog dela areala. master thesis. odsek za biologiju i ekologiju, prirodno-matematički fakultet, univerzitet u nišu. niš. ajtić, r. 2014: morphological, biogeographical and ecological characteristics of kotschy’s gecko (cyrtodactylus kotschyi steindachner, 1870 gekkonidae) from the mainland portion of its distribution range. fauna balkana, 3: 1–70. arnold, n., ovenden, d. 2002: a field guide to the reptiles and amphibians of britain and europe. harper collins publishers, london. balej, p., jablonski, d. (eds.) 2015: balcanica.info. http:// www.balcanica.cz/ (accessed 26.03.2015) beutler, a. 1981: cyrtodactylus kotschyi (steindachner 1870) agaischer bogenfingergecko. in: boehme, w. (ed.), handbook der reptilien und amphibien europas. akademische verlagsgesellschaft, wiesbaden. biserkov, v. (ed.), naumov, b., tsankov, n., stoyanov, a., petrov, b., dobrev, d., stoev, p. 2007: opredelitel na zemnovodnite i vlechugite v balgariya. zeleni balkani, sofiya. böhme, w., lymberakis, p., ajtić, r., disi, a. m. m., werner, y., tok, v., ugurtas, i. h., sevinç, m., hraoui-bloquet, s., sadek, r., crochet, p. a., haxhiu, i., corti, c., sindaco, r., kaska, y., kumlutaş, y., avci, a., üzüm, n., yeniyurt, c., akarsu, f., crnobrnja-isailović, j. 2009: mediodactylus kotschyi. in: iucn 2013. iucn red list of threatened species. version 2013.2. www. iucnredlist.org conant, r., collins, j. t. 1998: a field guide to reptiles and amphibians of eastern and central north america. houghton mifflin co., new york. 640p. davis, w. k. 1974: the mediterranean gecko, hemidactylus turcicus in texas. journal of herpetology, 8: 77–80. farkas, b., újvári, b., kőszegi, g. 1999: cyrtopodion kotschyi (kotschy’s gecko). herpetological review, 30: 173–174. flower, s. 1933: notes on the recent reptiles and amphibians of egypt, with a list of the species recorded from that kingdom. proceedings of the zoological society of london, 1933: 735–851. gherghel, i., strugariu, a., sahlean, t. c., zamfirescu, o. 2009: anthropogenic impact or anthropogenic accommodation? distribution range expansion of the common wall lizard (podarcis muralis) by means of artificial habitats in the north-eastern limits of its distribution range. acta herpetologica, 4: 183–189. harris, d. j., batista, v., lymberakis, p. carretero, m. a. 2004: complex estimates of evolutionary relationships in tarentola mauritanica (reptilia: gekkonidae) derived from mitochondrial dna sequences. molecular phylogenetics and evolution, 30: 855–859. haxhiu, i. 1998: the reptilia of albania: species composition, distribution, habitats. bonner zoologische beiträge, 48: 35–57. jesus, j., freitas, a. i., brehm, a., harris, d. j. 2002: an introduced population of hemidactylus mabouia (moreau de jonnes, 1818) on madeira island. herpetozoa, 15: 179–180. kasapidis, p., magoulas, a., mylonas, m., zouros, e. 2005: the phylogeography of the gecko cyrtopodion kotschyi (reptilia: gekkonidae) in the aegean archipelago. molecular phylogenetics and evolution, 35: 612–623. kotsakiozi, p., jablonski, d., ilgaz, ç., kumlutaş, y., avcı, a., meiri, s., itescu, y., kukushkin, o., gvoždík, v., scillitani, g., roussos, s.a., jandzik, 157 biologica nyssana ● 12 (2) december 2021: 151-157 urošević et al. ● new findings of kotschy’s gecko, mediodactylus kotschyi (steindachner, 1870) in serbia, with a particular focus on recently recorded populations in niš and sremska mitrovica d., kasapidis, p., lymberakis, p., poulakakis, n. 2018: multilocus phylogeny and coalescent species delimitation in kotschy’s gecko, mediodactylus kotschyi: hidden diversity and cryptic species. molecular phylogenetics and evolution, 125: 177– 187. koynova, t., doichev, d., natchev, n. 2020: new data on the distribution of the bulgarian bent-toed gecko (mediodactylus danilewskii strauch, 1887) in shumen town (ne bulgaria). biharean biologist, 14: 122–124. mares, g., novarini, n. 2020: a likely population of the alien gecko mediodactylus kotschyi (steindachner, 1870) in the province of belluno (northeastern italian alps). bollettino del museo di storia naturale di venezia, 71: 83–88. meshaka, w. e. jr., smith, h. t., engeman, r. m., dean, c. l., moore, j. a., o’brien, w. e. 2005: the geographically contiguous and expanding coastal range of the northern curlytail lizard (leiocephalus carinatus armouri) in florida. southeastern naturalist, 4: 521–526. simonović, z. 2003: roads, caravan traffic and security on the medieval serbian roads. peščanik, istorijski arhiv niš, 1: 25-34. tomović, l., ajtić, r., ljubisavljević, k., urošević, a., jović, d., krizmanić, i., labus, n., đorđević, s., kalezić, m. l.,vukov, t., džukić, g. 2014: reptiles in serbia – distribution and diversity patterns. bulletin of the natural history museum, 7: 129–158. urošević, a. 2016: introdukovana populacija kočijevog gekona (mediodactylus kotschyi, steindachner 1870) u smederevu. smederevski zbornik, 5: 11–25. urošević, a., tomović, l., ajtić, r., simović, a., džukić, g. 2016: alterations in the reptilian fauna of serbia: introduction of exotic and anthropogenic range expansion of native species. herpetozoa, 28(3/4): 115–132. urošević, a., maričić, m., mrkić, d., anđelković, m. 2019: mediterranean expats – populations of kotschyi’s gecko thriving at the edge of the pannonian plain in serbia. tibe 2019: biodiversity, ecology and evolution in mediterranean ecosystems, 04 06 december, cibio, vairão, portugal. savić-zdravković et al., 2020, biologica nyssana 11(1) 11 (1) september 2020: 1-7 doi: 10.5281/zenodo.4060278 affordable chironomid housing: proposed modifications of standard oecd substrate for testing of chemicals on aquatic midges original article dimitrija savić-zdravković department of biology and ecology, faculty of sciences and mathematics, university of niš, serbia dimitrija.savic@pmf.edu.rs (corresponding author) aca đurđević department of biology and ecology, faculty of sciences and mathematics, university of niš, serbia djukiamphibia@gmail.com zorana lazarević department of biology and ecology, faculty of sciences and mathematics, university of niš, serbia zorana.lazarevic92@gmail.com nastasja manić department of biology and ecology, faculty of sciences and mathematics, university of niš, serbia nastasja.mitic@gmail.com djuradj milošević department of biology and ecology, faculty of sciences and mathematics, university of niš, serbia djuradj@pmf.ni.ac.rs received: february 19, 2020 revised: march 23, 2020 accepted: march 26, 2020 abstract: this study was conducted in order to formulate a modification of standard substrate for laboratory bioassays (oecd protocol number 218: ”sediment water chironomid toxicity test using spiked sediment“), with as few constituents as possible, yet enabling the highest larval survival of model organism: chironomus tentans (chironomidae, diptera). laboratory experiment consisted of two bioassays: first with substrate mixture of two and more ingredients tested: standard oecd substrate, standard substrate with medical clay, peat + clay, peat + sand, clay + sand; second with every ingredient individually tested: coarse sand, fine sand, peat, clay and no substrate. highest larval survival was observed in sand + peat (≈85%) and coarse sand substrate (≈82%), whilst clay + peat and peat substrate caused the highest larval mortality (≈65% and 46% respectively). no larvae survived in treatment without any substrata, indicating the absolute necessity and importance of substrate presence for larval survival. key words: chironomus tentans, bioassay, life traits, oecd guidelines, sediments, substrate, toxicity tests apstract: pristupačno stanovanje za hironomide: predlog izmene standardne podloge oecd-a za ispitivanje hemijskih agenasa na vodenim komarcima ovo istraživanje sprovedeno je radi formulisanja modifikovane standardne podloge namenjene laboratorijskim biološkim testovima (protokol oecd-a broj 218: “test toksičnosti na hironomidama u sistemu sediment-voda, sa supstancom u sedimentu”), sa što manje sastojaka koja omogućava visoku stopu preživljavanja larvi model organizama: chironomus tentans (chironomidae, diptera). laboratorijski eksperiment sastojao se od dva biološka testa: u prvom je testirana mešavina dva ili više sastojaka: standardna oecd podloga, standardna podloga sa medicinskom glinom, treset + glina, treset + pesak i glina + pesak; u drugom je testiran svaki sastojak podloge individualno: krupan pesak, sitan pesak, treset, glina i tretman bez podloge. najveće preživljavanje uočeno je u podlozi sa sastavom pesak + treset (≈85%) i podlozi sa krupnim peskom (≈82%), dok su podloge sastavljene iz mešavine gline i treseta ili samo treseta izazvale najveći mortalitet larvi (≈65% i 46% respektivno). ni jedna larva nije preživela u tretmanu bez ikakve podloge, ukazujući na potpunu neophodnost i značaj prisustva podloge za preživljavanje larvi. ključne reči: chironomus tentans, biološki test, životni parametri, oecd smernice, sedimenti, podloga, testovi toksičnosti introduction in aquatic ecosystems, sediments represent a vital and specific habitat for many organisms providing feeding, spawning, hiding, and rearing areas. on the other hand, sediments in aquatic habitats can accumulate anthropogenic chemicals and waste materials, which can be directly toxic to sediment-dwelling organisms or bioaccumulate in the food chain contributing to a variety of environmental problems (den besten and munawar, 2016; savić-zdravković et al. 2020). considering this enormous environmental problem, ecotoxicological testing of water and sediment based on information from chemical analyses, but also on effect measurements, i.e. bioassays or toxicity tests, has been developed over the years (den besten and munawar, 2016). these tests have been standardized and numerous regulations and guidelines have been developed, providing direct, quantifiable evidence of biological con© 2020 savić-zdravković et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work noncommercially under the same license as the original. 1 sequences of sediment contamination (taylor and scroggins, 2013). great efforts has been invested to harmonize testing and to facilitate the mutual recognition of data by american public health association (apha), american society for testing and materials international (astm), environment canada (ec), european committee for standardization or comite´ europe´en de normalisation (cen), international organization for standardization (iso), organisation for economic co-operation and development (oecd) and united states environmental protection agency (us epa) (taylor and scroggins, 2013). in europe, toxicity tests are conducted by the guidelines developed by oecd (oecd, 2020). the results from oecd test guidelines are intended to be accepted by all eu member states by the mad principle (oecd agreement on mutual acceptance of data in the assessment of chemicals, http://www. oecd.org/env/ehs/mutualacceptanceofdatamad.htm). a wide range of toxicity tests and test organisms exist for both freshwater and marine organisms, but there is a need for adjusting the existing protocols in a sense of new chemical substances and new possible tested endpoints (hund-rinke et al. 2016) and there is allowance for case-specific final modifications of the testing methodology. very important part of the macrozoobenthos in the freshwater ecosystems is the group of chironomids, known as non-biting midges (dickman and rygiel, 1996). chironomidae are a family of the order diptera, representing world-wide distributed midge flies with exceptional species richness and abundance (ferrington, 2008). the family is divided into 11 subfamilies and 22 nominal tribes (ferrington, 2008). chironomids could be found in all sorts of habitats, from aquatic to terrestrial ones, but a total of 339 genera and 4,147 species are strictly aquatic in the larval stage (ferrington, 2008). occupying almost every type and conditions of aquatic habitats and having extraordinary ecological range and sensitivity makes them very good environmental indicators (dickman and rygiel, 1996). chironomids usually exhibit specific phenotypes as a response to the presence of different stressors (wiederholm, 1984). in polluted areas midges with deformed phenotypes were found, which lead to conclusion that aquatic pollutants cause deformities in chironomids, thus making them widely used as indicators of environmental stress in studies assessing the ecological impact of human activities (lenat, 1993). hamilton and saether (1971) were the first to propose the usage of chironomid deformities as biological indicators of freshwater quality, and a lot of research has been done ever since. in fact recent meta-analysis found 41 studies on chemical effects on chironomid deformities in laboratory experiments, especially larval mouthpart deformities (gagliardi et al. 2016). this study, by gagliardi et al. 2016, also emphasizes the inconsistency in the published data due to several factors, amongst which are mortality effects and background stressors (referring to all stressors apart from the one that is tested in the particular study, including inbreeding, parental effects, inadequate substrates and unknown stressors in substrates and food), that influence the “reliability” of conducted studies. the design of the appropriate bioassay depends on the intended application of the test. when examining chemicals’ effects on structures in chironomid larvae, especially mouthparts, it is of great importance to have minimal wear of the structures and minimize additional deformities caused by sediment they are reared in. in few papers, substrate consisting only on quartz sand has been proposed as suitable for this kind of studies (bird, 1997; langer-jaesrich et al. 2010), but no detailed studies have been conducted. in this study, modifications of the standard oecd protocol number 218, entitled ”sediment water chironomid toxicity test using spiked sediment“, designed to assess the effects of prolonged exposure of chemicals to the sediment-dwelling larvae, have been made. this was done in order to formulate a substrate with as few constituents as possible for bioassays where morphological changes on the larvae are examined as an endpoint. the aim of this study was to compare the effect of different standard substrate modifications on the survival on c. tentans larvae and to propose a simplified substrate formulation (lowering the possibility of damage or deformation of the larval mouth apparatus) that will enable favorable larval survival. the proposed modifications of sediment standards are based upon the laboratory experiments on chironomus tentans species. material and methods experimental organisms specimens of chironomus tentans, fabricius, 1805, (diptera, chironomidae), used in the experiments were from the stock cultures reared in the laboratory of the faculty of science and mathematics, university of niš (niš, serbia). stock cultures were reared under the oecd guidelines (oecd; 2004). specimens were reared in glass tanks with water temperature 21.0±0.5 °c, a light-dark cycle of 16:8h artificial daylight and under constant aeration. the artificial sediment used for rearing the laboratory cultures was shredded paper sediment. larvae were fed with tetramin® fish food flake mixture. about a week before the start of the experiment, fresh egg cases have been isolated from the stock aquaria into 2 biologica nyssana ● 11 (1) september 2020: 1-7 savić-zdravković et al. ● affordable chrionomid housing: proposed modifications of standard oecd substrate for testing of chemicals on aquatic midges petri dishes filled with water from the stock culture aquaria for hatching. experimental design sediment bioassays were performed following oecd guidelines for the testing of chemicals, section 2, specifically the test number 218: sedimentwater chironomid toxicity using spiked sediment (oecd, 2004), further referred to as oecd test 218. larvae were exposed to sediment in the test vessels (glass 600 ml beakers measuring 8 cm in diameter) which were composed of 2 cm sediment layer surrounded by a mixture of deionized and tap water (ph 6.5±0.4; hardness 7.3 °dh; electrical conductivity 325 μs/cm3). twenty freshly hatched 1st instar larvae were placed in each test vessel (larvae were 2 days old), at 21 ± 1 °c, with gentle aeration and 16:8 light-dark cycle. three replicas were made for each treatment, i.e. tested substrate type. on termination of the experiment (each bioassay was terminated after 12 days when larvae reached 4th stadium), the number of larvae recovered was recorded from each substrate. survival (s) was calculated by the formula: s=ne/na, where ne is the sum of midge larvae survived at the end of the experiment per vessel and na is number of larvae introduced per vessel. in order for test to be valid, the average survival of c. tentans must be greater than or equal to 70% in the control at the end of the test, according to the protocol. modification of the formulated substrate the following formulated substrate, based on the artificial soil used in oecd test 218 (oecd, 2004), is recommended: 1. 4-5% (dry weight) peat: as close to ph 5.5 to 6.0 as possible; it is important to use peat in powder form, finely ground (particle size less than 1 mm) and only air dried. 2. 20% (dry weight) kaolin clay (kaolinite content preferably above 30%). 3. 75-76% (dry weight) quartz sand (fine sand should predominate with more than 50 percent of the particles between 50 and 200 μm). deionised water is added to obtain a moisture content of the final mixture in a range of 30 to 50 % and calcium carbonate of chemically pure quality (caco3) is added to adjust the ph of the final mixture of the sediment to 7.0 ± 0.5. organic carbon content of the final mixture should be 2% (±0.5%) and is to be adjusted by the use of appropriate amounts of peat and sand, according to the proposed proportions. the experiment consisted of the two identically structured bioassays with different modifications of thhe recommended standard oecd substrate. 3 the modifications consisted of simplification, i.e. excluding one or more of the requested constituents. in the first bioassay we tested the mixture of two and more ingredients, whilst in the second we tested every ingredient individually (for sterilization each ingredient was thoroughly rinsed with water, than incubated 3 h at 500 °c, except peat, which was air dried as suggested by the protocol). each tested substrate (treatment) had three replicas and in each replica the ratio of the depth of the substrate layer to the depth of the overlying water was 1:4 (overlaying water consisted of mixture of dechlorinated tap and deionized water in 1:1 ratio). first bioassay was conducted with following five treatments, i.e. substrate mixtures: 1. standard oecd substrate (sand + peat + clay) (further referred to as standard) 2. standard substrate with medicinal clay (sand + peat + medicinal clay) (further referred to as s+p+mc) 3. standard substrate without sand (peat + clay) (further referred to as p+c) 4. standard substrate without clay (peat + sand) (further referred to as p+s) 5. standard substrate without peat (clay + sand) (further referred to as c+s) second bioassay was conducted with following five treatments, i.e. individual ingredient substrates: 1. coarse quartz sand (canary grain sand which is available from pet stores with >50% of the particles were in the range of 400–1000 μm) (further referred to as cs) 2. fine sand (commercially available sandbox sand with >50% of the particles were in the range of 50–200 μm) (further referred to as fs) 3. peat (further referred to as p) 4. clay (further referred to as c) 5. without substrate (vessels with only water) (further referred to as 0) data analysis the datasets from both bioassays were of normal distribution (kolmogorov-smirnov with lilliefors significance correction and shapiro-wilk test of normality, significance <0.05), therefore the data was analyzed with parametric tests (one way anova). in order to test the significant differences in mortality rates between all tested combinations of substrates, the datasets from both experiments were analyzed separately, than were pooled together for the analysis. for post hoc comparison of larval mortality between groups, i.e. tested substrates, tukey’s honestly significant difference (tukey hsd) test was used, with significance set at p<0.05. all statistical analyses were conducted in ibm spss statistics software version 19 © spss inc. 1989, 2010. biologica nyssana ● 11 (1) september 2020: 1-7 savić-zdravković et al. ● affordable chrionomid housing: proposed modifications of standard oecd substrate for testing of chemicals on aquatic midges 4 results bioassay with ingredient mixture substrate types the 1st bioassay was terminated after 12 days. according to protocol propositions, the average survival of c. tentans larve must be greater than or equal to 70% in the control at the end of the test, which was the case for only two tested substrates: standard and p+s substrate. one way anova showed statistically significant differences in mean survival values among the groups, i.e. tested substrates (p=0.049, f=3.518). post hoc test showed that survival of chironomid larvae in substrate p+c was the lowest and significantly different (p<0.05, mean difference = – 0.316) than in the substrate p+s, where the highest survival was recorded (tab. 1). bioassay with individual ingredient substrate types the 2nd bioassay was terminated after 12 days. according to protocol propositions, the average survival of c. tentans larve must be greater than or equal to 70% in the control at the end of the test, which was the case for only two tested substrates: cs substrate and fs substrate. one way anova showed statistically significant differences among the groups, i.e. tested substrates (p=0.001, f=11.019). post hoc test showed statistically significant differences between the following groups (tukey hsd p<0.05): none of the larvae survived in the substrate 0, which was significantly lower survival than in all other substrates, accept in substrate p. survival of chironomid larvae in substrate p was the second lowest and significantly different than in substrate cs, where the highest survival was recorded (tab. 2). all substrate types when combining the results from both bioassays (fig. 1), and excluding the treatment 0 (in which no larvae survived), one way anova showed statistically significant differences among the groups, i.e. tested substrates (p=0.025, f=2.2998). larval survival in the substrate cs and p+s was the highest among all substrates and significantly higher only than survival in substrate p, which was the lowest (tukey hsd p<0.05). discussion when conducting eco-toxicological tests on chironomids it is essential to propose a simple and effective bioassay design that will enable detecting the influence of examined stressor with desired specific endpoints. if we consider sublethal effects of the tested stressor as specific endpoints we desire to investigate (i.e. larval deformities, stress enzyme activity, dna damage etc.), the bioassay that ensures survival of at least 70% of the population in the control, with proper substrate formulation that reduces the morphological alteration, variability of important population parameters and other background stressors, is essential. to propose optimal simplified substrate formulation for laboratory bioassays designed to assess the effects of prolonged exposure of chemicals to the sediment-dwelling freshwater chironomid biologica nyssana ● 11 (1) september 2020: 1-7 savić-zdravković et al. ● affordable chrionomid housing: proposed modifications of standard oecd substrate for testing of chemicals on aquatic midges table 1. survival mean values ± sd in % of c. tentans larvae in different ingredient mixture substrate types. calculated as the mean values of three replicas for each tested substrate. code substrate type mean survival ± sd% standard standard oecd substrate (sand + peat + clay) 76.7 ± 7.6 s+p+mc standard substrate with medicinal clay (sand + peat + medicinal clay) 61.7 ± 15.3 p+c standard substrate without sand (peat + clay) 53.3 ± 12.6 b p+s standard substrate without clay (peat + sand) 85 ± 15. a c+s standard substrate without peat (clay + sand) 63.3 ± 2.9 treatment a is statistically significantly different from treatment b (tukey hsd p<0.05) table 2. survival and mortality mean values ± sd in % of c. tentans larvae in different individual ingredient substrate types. calculated as the mean values of three replicas for each tested substrate. code substrate type mean survival ± sd % cs coarse sand 81.67 ± 17.55 a fs fine sand 73.3 ± 12.5 p peat 35 ± 31.2 b c clay 61.6 ± 7.6 0 without substrate 0.0 ± 0.0 c the treatment a is statistically significantly different from treatments b and c, whilst treatment c is statistically significantly different from all other treatments accept b (tukey hsd p<0.05) 5 larvae, we compared the effect of different standard oecd substrate modifications on the survival on c. tentans larvae. a total of 10 substrate types, i.e. treatments, were tested: 9 combinations of substrate constituents and one treatment with no substrate at all. acceptable survival (70% and above) was recorded in only following four substrates (listed by average survival in descending order): p+s > cs > standard > fs. in all other substrates the recorded survival was lower than 70%, whilst the survival in treatment with no substrate was 0%. most of the existing internationally harmonized ecotoxicological bioassays were developed through research and development, validation and peer review before they were officially published as documents with standard conditions, procedures, and guidance (davis, 1977). typically, several years of development precedes establishment of bioassay protocols and new additions to existing rules and regulations are regularly made in line with contemporary trends and market demand. therefore, these protocols can be modified with respect to their function depending on the specific requirements of the tested species and the tested hypothesis (iso, 2003). sediment dwelling larvae of chironomids represent ideal test organisms for sediment ecotoxicity tests, especially since they usually exhibit specific phenotypes as a response to the presence of different stressors (wiederholm, 1984) and are most diverse and abundant group out of all macroinvertebrates (milošević, 2013). the standard oecd test 218, recommends using formulated substrate because of several advantages: reduced experimental variability, reduced costs in comparison to using field sediments as substrate, replicability and comparison of results between studies (davis, 1977; oecd, 2004). however, modifications of the recommended substrate are approved, as long as clean and uncontaminated substrate which provides satisfactory environment for larval development and 70% or higher survival (oecd, 2004). providing more economical and simpler design which leads to same results in a more cost-effective way is always encouraged. this is why in this study we investigated the possibility of creating a simplified substrate that provides sufficient survival rate, with as few ingredients as possible to reduce possible influence on larval mouth apparatus morphology, reduce contamination, cost and time of substrate preparation and therefore increase the efficiency of laboratory bioassays. to simplify the existing oecd formulated substrate, we firstly excluded only one of the standard three recommended substrate constituents, formulating the substrate out of two mixed ingredients. thereunto we tested the standard oecd substrate and standard substrate with medicinal clay (commercially available and less expensive clay than proposed kaolin clay). we recorded the highest survival in the s + p substrate (≈85%) and in the standard (≈77%), whilst the lowest survival was in c + p sediment (≈53%) (tab. 1). we then excluded two out of three oecd substrate constituents, formulating the substrate out of only one ingredient. thereunto we tested the larval survival in vessels containing only water and no substrate at all. we recorded the highest survival in cs substrate (≈82%), following by the high survival in the fs substrate (≈73%). no larvae survived the end of the experiment in treatment 0, the jars without any substrata (tab. 2), indicating the absolute necessity fig. 1. larval survival in all tested combinations of substrate types, i.e. treatments, presented together. treatments from the 1st bioassay are colored blue and treatments form the 2nd bioassay are colored green. a – boxplots of survival rates with sd values; b – average survival rates sorted from highest to lowest. treatments a are statistically significantly different from treatments b (tukey hsd p<0.05). biologica nyssana ● 11 (1) september 2020: 1-7 savić-zdravković et al. ● affordable chrionomid housing: proposed modifications of standard oecd substrate for testing of chemicals on aquatic midges 6 of substrate presence for larval survival. just few previous studies conducted the usage of different substrate formulations in the experiments with chironomids as model organisms: the study of bird, (1997), where c. tentans larvae were cultured on four different substrates (shredded paper, coarse sand, fine sand and silty clay), showed the highest survival in paper sediment. this study of bird suggests that substrates with sand are less suitable for chironomids because some of the food is lost between the sand grains and is not accessible to the larvae. however, this study encourages the use of coarse sand if the bioassay is designed to examine larval mentum deformities, since larvae reared in coarse sand had less worn (p<0.05) teeth than those reared in the other substrates. chironomus tentans larvae belong to gatherers/collectors functional feeding group (ffg), meaning that they collect small food particles (<1mm) that get lodged between rocks or in deep pools (brabec et al., 2017). given the size of the particles they can eat, it is possible for the larvae to ingest the substrate particles and mistake them for food, if they are less than 1 mm in size, therefore lowering the amount of food eaten and increasing mortality rates. the substrate composed of coarse sand (with particles around 1 mm) is too large to be ingested by the larvae and therefore does not cause higher mortality, deformation and damage of the oral apparatus. in our study, the survival recorded on both coarse and fine sand substrate was within the allowed survival rate, and in the coarse sand it was even greater than in a standard substrate. when combining the results from both bioassays and comparing all possible substrate combinations (fig. 1), the descending order of suitable substrates (from highest to the lowest larval survival recorded) is the following: c+s > standard > f+s > c+s > c > s+p+mc > p+c > p > 0. significant variation in larval survival on different substrates was observed, but the only significant difference was between p substrate (where the lowest survival of ≈35% is recorded) and cs and p+s substrate (where the highest survival is recorded: ≈82% and ≈85% respectively). the average larval survival in the standard substrate was among the highest of all treatments (≈77%), but when excluding clay form the standard mixture or excluding clay and peat (leaving only coarse sand sediment) the larval survival increased, justifying our aim to simplify the substrate formulation. considering that acceptable survival of above 70% was recorded in only four substrates: c+s, cs, standard and fs, only these combinations of ingredients could be recommended for further usage as substrata in further laboratory bioassays. those four tested substrates were not mutually statistically significantly different in terms of larval survival in the present experiment. using the substrate composed of peat and sand sediment has shown to be the most convenient in the terms of survival of the larvae, but sediments that consist of only peat, or peat mixed with other ingredients, such as clay, have shown to cause higher mortality. high survival is evident in the sediments containing sand, which is in accordance with research of langer-jaesrich et al. (2010) which suggested the use of quartz sand substrate in bioassays (particle size 0.1–0.3 mm) as free of possible organic contaminations, and proposed by several other authors (bird, 1997, meregalli and ollevier, 2001; meregalli et al., 2001). there has never been a systematic proposal for modification of oecd test 218 (oecd, 2004; oecd, 2005). this could be due to the fact that research and development of a test method is typically the most time-consuming aspect of the standardization process, as it requires one to become familiar with all aspects of the test organism and experimental design. the importance of uniformity and consistency in ecotoxicological studies is indisputable, but in order to have solid comparative framework there are still a lot of problems that need to be overcome in the future. on the basis of this study, conducted under controlled laboratory conditions, we could propose further usage of substrate consisting only of quartz sand (especially coarse canary grain sand which is available from pet stores with >50% of the particles in the range of 400–1000 μm) as substrate in laboratory bioassays on c. tentans species. the substrate consisting only of one ingredient could be lowering possible negative effects and “background noise” in bioassays and increasing the reproducibility and ease of performing experiments: sand could be obtained by cheaper prices in the pet stores, could be easily sterilized (thoroughly rinsed with water, than burned 3 h at 500 °c) and chironomid larvae could be better visible and more easily separated from the sediment at the end of the experiment. further study on the influence of substrate to more specific, sublethal endpoints, such as mentum and mandible ware, deformity rates, body weight, larval shape and size has to be conducted on all four proposed suitable substrates in order to confirm the preposition of their usage in bioassays where sublethal effects on the larvae are examined. acknowledgements. this work was financed by the ministry of education, science and technological development, republic of serbia, grant no. iii43002. references brabec, k., janecek, b.f.u., rossaro, b., spies m., bitusik, p., syrovatka, v., schmidt-kloiber, a. 2017: chironomidae indicator database. biologica nyssana ● 11 (1) september 2020: 1-7 savić-zdravković et al. ● affordable chrionomid housing: proposed modifications of standard oecd substrate for testing of chemicals on aquatic midges 7 euro-limpacs project (contract no. gocect-2003-505540), dataset “chironomidae”, workpackage, 7. https://www.freshwaterecology. info/ bird, g. a. 1997: deformities in cultured chironomus tentans larvae and the influence of substrate on growth, survival and mentum wear. environmental monitoring and assessment, 45(3): 273-283. den besten, p.j., & munawar, m. 2016: ecotoxicological testing of marine and freshwater ecosystems: emerging techniques, trends and strategies. crc press, boca raton. 297 p. davis, j.c. 1977: standardization and protocols of bioassaystheir role and significance for monitoring, research and regulatory usage. in proceedings of the 3 rd aquatic toxicity workshop, halifax, nova scotia nov. 2-3, 1976, environment canada, tech. report. dickman, m., & rygiel, g. 1996: chironomid larval deformity frequencies, mortality, and diversity in heavy-metal contaminated sediments of a canadian riverine wetland. environment international, 22(6): 693-703. ferrington, l. c. 2008: global diversity of nonbiting midges (chironomidae; insecta-diptera) in freshwater. hydrobiologia, 595(1): 447. gagliardi, b. s., pettigrove, v. j., long, s. m., & hoffmann, a. a. 2016: a meta-analysis evaluating the relationship between aquatic contaminants and chironomid larval deformities in laboratory studies. environmental science & technology, 50(23): 12903-12911. hamilton, a.l. & saether, o.a. 1971: the occurrence of characteristic deformities in the chironomid larvae of several canadian lakes. the canadian entomologist, 103(3): 363-368. hund-rinke, k., baun, a., cupi, d., fernandes, t.f., handy, r., kinross, j.h., navas, j.m., peijnenburg, w., schlich, k., shaw, b.j., scottfordsmand, j.j. 2016: regulatory ecotoxicity testing of nanomaterials–proposed modifications of oecd test guidelines based on laboratory experience with silver and titanium dioxide nanoparticles. nanotoxicology, 10(10): 1442-1447. international organization for standardization, 2003: soil quality-guidance on the ecotoxicological characterization of soils and soil materials. iso. https://www.iso.org/obp/ui/#iso:std:iso:15799:ed2:v1:en langer-jaesrich, m., köhler, h. r., & gerhardt, a. 2010: can mouth part deformities of chironomus riparius serve as indicators for water and sediment pollution? a laboratory approach. journal of soils and sediments, 10(3): 414-422. lenat, d. r. 1993: using mentum deformities of chironomus larvae to evaluate the effects of toxicity and organic loading in streams. journal of the north american benthological society, 12(3): 265-269. meregalli, g., & ollevier, f. 2001: exposure of chironomus riparius larvae to 17α-ethynylestradiol: effects on survival and mouthpart deformities. science of the total environment, 269(1): 157-161. milošević, d., simić, v., stojković, m., čerba, d., mančev, d., petrović, a., paunović, m. 2013: spatio-temporal pattern of the chironomidae community: toward the use of non-biting midges in bioassessment programs. aquatic ecology, 47(1): 37-55. oecd 2020: oecd guidelines for the testing of chemicals, section 2. effects on biotic systems. https://doi.org/10.1787/20745761 oecd 2005: guidance document on the validation and international acceptance of new or updated test methods for hazard assessment, (env/jm/ mono(2005)14). http://www.oecd.org/officialdocuments/publicdisplaydocumentpdf/?doclanguage= en&cote=env/jm/mono(2005)14 oecd 2004: test no. 218: sediment-water chironomid toxicity using spiked sediment, oecd guidelines for the testing of chemicals, section 2. oecd publishing, paris, https://doi. org/10.1787/9789264070264-en. savić‐zdravković, d., milošević, d., uluer, e., duran, h., matić, s., stanić, s., vidmar, j., ščančar, j., dikic, d., jovanović, b. 2020: a multiparametric approach to cerium oxide nanoparticle toxicity assessment in non‐biting midges. environmental toxicology and chemistry, 39(1):131-140. taylor, l.n., & scroggins, r.p. 2013: standardization of ecotoxicological tests: the process. in: férard, j.f., blaise, c. (ed): encyclopedia of aquatic ecotoxicology. springer, dordrecht, 1221 p. wiederholm, t. 1984: incidence of deformed chironomid larvae (diptera: chironomidae) in swedish lakes. hydrobiologia, 109(3): 243-249. biologica nyssana ● 11 (1) september 2020: 1-7 savić-zdravković et al. ● affordable chrionomid housing: proposed modifications of standard oecd substrate for testing of chemicals on aquatic midges mitra et al., 2020, biologica nyssana 11(1) 11 (1) september 2020: 35-44 doi: 10.5281/zenodo.4060292 isolation and impacts of rhizobacteria from saussurea obvallata (dc.) edgew. (brahma kamal) original article debasis mitra department of biotechnology, graphic era (deemed to be university), dehradun, uttarakhand, india debasismitra3@gmail.com (corresponding author) navendra uniyal department of biotechnology, graphic era (deemed to be university), dehradun, uttarakhand, india navendraunniyal121@gmail.com komal sharma department of biotechnology, graphic era (deemed to be university), dehradun, uttarakhand, india komal.sharma638@gmail.com anju rani department of life sciences, graphic era (deemed to be university), dehradun, uttarakhand, india teotia_anju29@rediffmail.com lok man singh palni vice chancellor, graphic era (deemed to be university), dehradun, uttarakhand, india lmspalni@rediffmail.com akansha chauhan department of biotechnology, graphic era (deemed to be university), dehradun, uttarakhand, india akansha0911chauhan@gmail.com prabhakar semwal uttarakhand state council for science and technology (ucost), vigyan dhaam, dehradun, uttarakhand, india. semwal.prabhakar@gmail.com poonam arya department of biotechnology, graphic era (deemed to be university), dehradun, uttarakhand, india aryapoonam73@gmail.com received: november 06, 2019 revised: january 21, 2020 accepted: january 26, 2020 abstract: the rhizospheric association of bacteria in terrestrial plants across the hilly region has tremendous potential for plant growth-promotion (pgp) and protection. this is the first study which aims to isolate some rhizo-bacteria from the different rhizospheric soil samples of s. obvallata in the himalayan region. genotypic, biochemical and pgp traits shows genetically similar bacteria species to have diverse pgp potential as shown by iaa production of 87.567 μg ml-1 by bacillus sp. d5 and phosphate solubilization potential 30 mm halo zones are shown by bacillus sp. d9. growth of isolates was in the ranges of temperature (0-60 °c), salt (0-20%), which shows their tendencies towards surviving harsh environmental conditions. the isolates were further evaluated on amaranthus cruentus, which shows a significant improvement in growth compared to control. the novel study finds the bacterial community at high altitudes to survive extreme climatic variability through association with plants by developing a commensalistic relationship with host. key words: s. obvallata, indigenous rhizobacteria, plant growth promotion, amaranth apstract: izolacija i uticaj rizobakterija iz saussurea obvallata (dc.) edgew (brahma kamal) bakterijska rizosferna asocijacija na terestričnim biljkama širom brdskog regiona ima ogroman potencijal za stimulaciju biljnog rasta (pgp) i zaštitu. ovo je prva studija koja ima za cilj izolovanje nekih rizobakterija iz različitih uzoraka zemljišta rizosfere vrste saussurea obvallata u regionu himalaja. genotipska, biohemijska i pgp svojstva pokazuju da genetski slične vrste bakterija imaju raznovrstan pgp potencijal, kao što pokazuje iaa produkcija od 87.567 µg/ml od strane bacillus sp. d5 i potencijali za solubilizaciju fosfata od 30 mm halo zone od strane bacillus sp. d9. rast izolata je bio u rasponu temperature (0-60°c) i soli (0-20%), što pokazuje njihove tendencije prema preživljavanju surovih uslova životne sredine. izolati su dalje procenjeni na vrsti amaranthus cruentus, što je pokazalo značajno poboljšanje rasta u odnosu na kontrolu. nova studija otkriva da bakterijska zajednica na velikim nadmorskim visinama preživljava ekstremne klimatske varijacije povezivanjem sa biljkama i razvijanjem komensalističke veze sa domaćinom. ključne reči: s. obvallata, autohtone rizobakterije, stimulacija biljnog rasta, amaranthus introduction saussurea obvallata (dc.) edgew. (brahma kamal), the state flower of uttarakhand, india is an endemic herb of the himalayan region (encompassing the indian himalayan region, northern burma and southwest china). the plant is distributed at an altitudinal range of 3000–4800 m (semwal and pant, 2013). brahma kamal is used for the preparation of traditional medicines by the local peoples in tibet and other places including garhwal himalayas. it is well known that indigenous rhizobacteria exert beneficial effects on plants productivity and sustenance of soil health through their capacity for phosphate solubilization (sarikhani et al., 2019), indole acetic acid (iaa) (selvakumar et al., 2008), ammonia, hcn and cell wall degrading enzyme production (tsegaye et al., 2019) etc. pgpr are heterogeneous groups of microbes associated with the plant’s root in diverse ways (nath et al., 2015). in the rhizobac© 2020 mitra et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 35 terial population, 2-5% of total the beneficial rhizobacteria are involved in plant growth promotion (bakker and schippers, 1987; antoun and kloepper 2001; ahemad and kibret, 2014). over 95% of the rhizobacteria existing in the roots and plants obtain many nutrients through the soil bacteria (ji et al., 2014). different pgpr, including associative and symbiotic bacteria such as pseudomonas sp., azospirillum sp., azotobacter sp., rhizobium sp., klebsiella sp., enterobacter sp., alcaligenes sp., arthrobacter sp., burkholderia sp., bacillus sp., and serratia sp. groups have been used for their beneficial effects on plant growth and improvement (kloepper and beauchamp, 1992; höflich et al., 1994). rhizospheric microbial population mainly promote plant health, they stimulate plant growth directly by producing or changing the concentration of plant growth regulators (kloepper et al., 1980; ramette et al., 2003; sparzak-stefanowska et al., 2019) like gibberellic acid, iaa, cytokinins (glick, 1995; vessey, 2003), asymbiotic n2 fixation (ardakani et al., 2010; bhattacharyya and jha, 2012). these populations also achieve antagonism against phytopathogenic microorganisms by producing antibiotics, fluorescent pigment, enzyme, cyanide, phenolics, signal compounds (niranjan et al., 2005), as well as solubilization of mineral phosphates and other nutrients (johri et al., 1999; sharma and johri 2003). inoculums have been used to increase plant yields in several countries, which is reported in research articles (dursun et al., 2019) and commercial products are currently available (backer et al., 2018). we are addressing for the first time isolation of high altitude rhizobacteria from s. obvallata (brahma kamal) rhizospheric soil in the himalayan region and their beneficial effects, harsh environment survival strategy and plant growth promotion in amaranthus cruentus. we proposed that these isolates may also have the ability to express activity towards promoting sustainability in harsh environments. material and methods the four rhizospheric soil samples of s. obvallata were collected from different place of kedarnath valley, uttarakhand, india and samples collection sites were described by semwal et al. (2018) and mitra et al. (2019). the soil samples were serially diluted and 100 μl of dilution sample was plated onto nutrient agar medium (nam) (himedia, mv002, india) for the isolation of potential pgp. all isolated strains were stored at -20 °c in nutrient broth containing 15% (v/v) glycerol. morphological and biochemical characterization tests viz. imvic test, citrate utilization, starch hydrolysis, phenylalanine deaminase, urease test, casein hydrolysis, h2s production, peroxidase test, nitrate utilization, and growth in different media macconkey’s agar, esculine hydrolysis, mannitol salt agar tests were carried out followed by macfaddin, 1985; harley and prescott, 2002; anija, 2003; dubey and maheshwari, 2007; cappuccino and sherman, 2008; brown, 2009 respectively. to identify the morphological characteristic of all isolates, gram staining, negative staining, shape, margin, elevation, form, motility and pigment were recorded. a loopful of bacteria was inoculated and incubated into pre-sterilized peptone broth containing 1% of tryptone for 48 h at 28 °c. after 48 h, 1 ml of kovac’s reagent (himedia, india) was added to all tubes including control, gently shaked for few second and left to react for 5-10 minutes. the appearance of red/ pink ring at the top is the clear indication of iaa production (loper and schroth, 1987; dubey and maheshwari, 2007). as described in gordon and weber (1951), the standard graph of iaa was prepared for the quantitative estimate of iaa production. different iaa concentrations were prepared as aqueous solution of iaa ranging from 5-150 μg ml-1. to each 1 ml of the standard, 2 ml of 2% fecl3 (0.5 m) in 35% perchloric acid i.e. salkowaski reagent was added and readings were taken after 30 minutes at 535 nm using uv-visible spectrophotometer (thermo scientific™ genesys-10s-uv). the standard graph was finally prepared by plotting concentration of iaa in ppm vs optical density at 535 nm. bacterial cultures were grown for 48 h in nutrient broth. fully grown cultures were centrifuged at 4000 rpm for 20 minutes at 4 °c. the supernatant (1 ml) was mixed with 4 ml of salkowaski reagent. samples were left at 28 °c for 30 minutes. development of pink color indicates the production of iaa. optical density was measured at 535 nm with a spectrophotometer. the concentration of iaa produced by bacterial cultures was measured with the help of standard graph of iaa (loper and schroth, 1987). phosphate solubilization ability of the isolates was evaluated in pikovskaya’s (pkv) agar medium (himedia, india) incorporated with tri-calcium phosphate (tcp) [ca3 (po4)5] as insoluble phosphate. pkv media plate was prepared and point inoculation was transferred in each plate at 28 °c for 5 days. after incubation, positive phosphate solubilizing bacteria which give clear zone around the colony were measured (fiske and subbarow, 1925). isolated strains were grown overnight in 10% trypone soy agar supplemented with glycine (4.4 gly l-1) (himedia, india) (bakker et al., 2003). a whatman no. 1 filter paper soaked in 2% sodium carbonate and 0.4% picric acid solution was placed to the underside of the petri dish lids (macfaddin, 1980; results explained by cook, 1993) and kept at 28 °c for 4 days. for the ammonia production, all isolated 36 biologica nyssana ● 11 (1) september 2020: 35-44 mitra et al. ● isolation and impacts of rhizobacteria from saussurea obvallata (dc.) edgew. (brahma kamal) bacterial strain was grown in 4% peptone broth for 48 h at 28 °c. detection of ammonia production was done by adding dropwise of 1.5 ml nessler’s reagent (himedia, india) (bakker et al., 2003). cellulase enzyme production was determined from clear zone test in cmc (carboxyl methyl cellulose) agar (cmc 100.00 g, peptone 5.0, agar: 15.00 g, distilled water 1000 ml, ph: 7.2) and czapekmineral salt medium (nano3 2.2 g, k2hpo4 1.0 g, mgso4.7h2o 0.7 g, kcl 0.5 g, cmc 5.0 g, peptone 2.5 g, agar 18.0 g, distilled water 1000 ml, ph 6.0) (bakthavatchalu et al., 2012). two days after incubation, 2 ml of congo red solution (0.1% congo red in 1 m nacl) was added in each plate. all the plates were then gently shaken. a positive result was indicated when a clear zone and light develops (qadri et al., 1988). protease activity (casein degradation) was determined from the clear zone in skim milk agar (skim milk powder 100.0 g, peptone 5.0 g, agar 15.0 g, distilled water 1000 ml and ph 7.2) (vijayaraghavan and vincent, 2013). the biofilm experiment was performed in 10 ml glass bottles. three milliliters of nutrient broth (without nacl) was dispensed and inoculated with an overnight experiment culture. the bottles were incubated under the static condition for 5 days, the wells were washed thrice with distilled water and the biofilm was stained with 1% crystal violet dye solution for 15 min, and again the wells were rinsed thrice with distilled water to wash off the unbound dye. quantification of the biofilm biomass was done by absolute alcohol and recording the absorbance at 600 nm according to srinandan et al. (2010). antibiotic resistance testing was determined by disc diffusion method (penicillin: sd028-1vl, erythromycin: sd083-1vl, streptomycin: sd091-1vl, chloramphenicol: sd153-1vl and tetracycline: sd1331vl, himedia, india) using nutrient agar plates. the bacterial suspension was inoculated on the nutrient agar plate by swabbing to give a smooth lawn and antibiotic discs were properly placed on the plate for overnight. the plates were observed and recorded the zone of inhibition around the antibiotic disc. all isolates antibiotic profile diversity was analyzed by ntsys 2.02e software, where 0 denotes susceptible and 1 denotes resistance to a particular antibiotic source. the data were analyzed using the similarity for qualitative data subroutine of ntsys-pc2 package. the isolates were grouped as per jaccard’s similarity index by the unweighted paired group method using arithmetic means (upgma) and depicted as dendrogram (ansari et al., 2014). antibiotics test result were graphically determined by the r-heatmap plot analysis where 0 denotes susceptible and 1 denotes resistance to a particular antibiotic source. 37 to find out the optimal conditions for the growth of bacterial isolate, the effect of different temperature levels on the development of isolates was examined. 0 °c, 4 °c, 28 °c, 37 °c and 60 °c were mainly used and bacterial suspension (100 μl) was placed in a tube containing 10 ml nutrient broth. the tube was incubated at the predetermined temperatures for 48 hours. the effect of different salt concentration on bacterial growth was studied with nutrient broth containing salt concentrations ranging from 0-20%. the effect on different salt concentration (0% and 8% nacl) for indole-3-acetic acid production in tryptone broth. this was investigated by using tryptone broth containing 0% and 8% nacl and following 48 h of incubation, the effects on growth rate were measured by spectrophotometric measurements at 660 nm. the pot experiment was conducted (two times) in the net-house, graphic era (deemed to be university), dehradun (30°19’n, 78°04’e, 650 m) with two times sterile soil. amaranthus cruentus seeds were surface sterilized, the seeds were dried for 2 hours in sterile conditions and subsequently planted into the pots prepared for treatments. there were five replicates per treatment with 15 seeds planted per pot. two bacterial isolates viz. bacillus sp. d5 and bacillus sp. d9 were selected based on pgp activity. soil treatments consisted of 0.5 kg lignite (carrier material) and 100 ml of inoculum (d5 and d9; separately) solution and sterile distilled water was the control. after 30 days, the length and mass of shoots and roots were taken to determine the effect of treatments on the growth of a. cruentus plants. diversity and statistical analysis were done by using ntsys 2.02e software. experimental data were analyzed using standard analysis of variance (anova) and hau, hisar online statistical analysis software results ten pgpr isolates (pseudomonas sp. d1, bacillus sp. d2, bacillus sp. d3, bacillus sp. d4, bacillus sp. d5, bacillus sp. d6, bacillus sp. d7, bacillus sp. d8, bacillus sp. d9 and bacillus sp. d10) were chosen from a total of eighty-five isolates using the serial dilution technique of rhizospheric soil samples of s. obvallata. all isolates were screened and selected on the basis of growth rate, morphological (fig. 1) and biochemical characteristics. this is the first research report that the d1, d2 and d3 isolated from hathi parwat (hp), d4 and d5 from maha panth (mp), d6 and d7 from madhu ganga (mg) and d8, d9 and d10 from cheer ganga (cg) of kedarnath valley. similarly, mitra et al. (2019) reported that some dominant fungi viz. phanerochaete chrysosporium, aspergillus fumigatus and trichoderma longibrachiatum was found and identified through 16s rdna sequencing (its4 and its5) from the same biologica nyssana ● 11 (1) september 2020: 35-44 mitra et al. ● isolation and impacts of rhizobacteria from saussurea obvallata (dc.) edgew. (brahma kamal) 38 rhizospheric soil samples of s. obvallata. biological communities in the indian himalayas include one of the biggest altitudinal encompass in the world and a portion of the more extravagant gatherings of wild and medicinal plants are found in this area (chauhan et al., 2015). presentation and exploitation of pgpr in agro-biological systems improve plant-microbes interactions that may influence biological communities supportability, farming efficiency, and ecological quality (zahid et al., 2015). the rhizosphere is a special specialty for different kinds of microorganisms in the soil (agrawal and johri, 2014). semwal et al. (2018) reported that s. obvallata rhizospheric and non-rhizospheric soils macro micro nutrients analysis and range of different nutrients availability viz. nitrogen, phosphorus, potassium, zinc, copper, iron, manganese in normal and suggested that they are helpful for development and growth for this endangered plant. so, in our present study, ten pgpr bacterial isolates were isolated and screened for pgp abilities (kloepper et al., 1988). this study was conducted to isolate the native low temperature growing potential pgpr from the himalayan region rhizospheric soil of s. obvallata plant and showed that some common pgprs like bacillus sp. and pseudomonas sp. were present in this rhizospheric area. similarly, majeed et al. (2015) reported that they are isolated some pgpr for the root endosphere and rhizosphere of wheat from the himalayan region and isolates showed pgp ability like iaa production, n2 fixation and p solubilization. agrawal and johri (2014) reported that fiftyfive rhizobacteria were isolated, characterized and screened pgp attribute from the central himalayan region, forest sites; altitude 3500, 12000 and 3100 ft. biochemical test results of all bacterial isolates were analyzed by heatmap plot and represented the results where black colour denotes negative and gray colour denotes positive activity (fig. 2). heatmap plot colour analysis showed that most (approx. <50%) isolates has positive biochemical activity. iaa production is a natural property of rhizobacteria that enhance and facilitate plant growth and development. quantitative assay of iaa production of rhizobacteria was done by the spectrophotometfig 1. the figure shows the percentage abundances of bacteria as determined under microscope fig 2. biochemical test results represented by heatmap plot (mr and vp: mr/vp test, c: citrate utilization, sh: starch hydrolysis, pd: phenylalanine deaminase, ut: urease test, ch: casein hydrolysis, h2s: h2s production, pt: peroxidase test, nr: nitrate utilization, ma: macconkey’s agar, eh: esculine hydrolysis and msa: mannitol salt agar) biologica nyssana ● 11 (1) september 2020: 35-44 mitra et al. ● isolation and impacts of rhizobacteria from saussurea obvallata (dc.) edgew. (brahma kamal) 39 ric analysis. the concentration of iaa produced by bacterial cultures was measured with the help of the standard graph of iaa. in this study, six qualitative screened positive bacterial isolates were taken for the quantitative assay and d5 isolate iaa production is 87.567 μg ml-1. phosphate solubilizing bacteria used as inoculants simultaneously increases phosphate uptake by the plant and crop yield (rodríguez and fraga, 1999). phosphate solubilization activity was determined by the development of the clear zone around the bacterial colony. isolate d9 is a high phosphate solubilizer bacterial strain which is used for the treatments. d9 showed the 30 mm zone (diameter) on pkv agar media (fig. 3a) after 5 days of incubation. four phosphate solubilizer bacteria strains were grown in pkv medium (ph 6.35) for 3 and 5 days at 28 °c. after the given time, ph of the culture medium was checked by ph meter and decreased ph rate can be observed in fig. 3b. in the similar study, panday et al. (2002) reported and isolated p. corrugata from the low-temperature location of sikkim and reported that all isolates could grow and solubilize phosphate in the range of 4 to 35 °c. all isolates were grown in 10% tryptone soy agar with glycine (4.4 gly l-1) on the plates sealed with parafilm®. the production of hcn was determined by the change in colour of filter paper from yellow to red-brown. after the incubation of all bacterial culture in medium, the nessler’s reagent was added. ammonia produced by bacterial isolates formed the yellowish-brown colour in the medium. cell wall degrading enzyme production was determined by congo red method and protease activity was determined by clear zone formation on the test plate. by this, the production of the most important plant growth hormones was recorded (fig. 4). fig 3. a bacillus sp. d9 isolate phosphate solubilization zone on pkv media after 5 days; b ph level in pkv broth fig 4. isolates plant growth-promoting test results are evaluated graphically by heatmap plot where gray color: (+) pgpr and black colour: (-) pgpr. (iaa: iaa production, ps: phosphate solubilization, hcn: hcn production, ap: ammonia production, c: cellulase test and p: protease test) fig 5. biofilm formation by isolates biologica nyssana ● 11 (1) september 2020: 35-44 mitra et al. ● isolation and impacts of rhizobacteria from saussurea obvallata (dc.) edgew. (brahma kamal) 40 quantification of the biofilm biomass results by the bacterial isolates are presented in fig. 5. isolates d2, d5, d7 and d9 showed high biofilm formation after 5 days of incubation. the antibiotic sensitivity was determined by disc diffusion method, where five antibiotics were used and the results of resistance and susceptibility of the isolates are presented in fig. 6a. antibiotic diversity of the isolates was analyzed by ntsys and dendrogram, where d2 and d5 showed sensitivity to all tested antibiotics (fig. 6b). heatmap plot showed that d6, d7, d8, d9 and d10 are resistant to penicillin and d1 showed resistance to erythromycin and tetracycline (fig. 6c). bacterial growth under different temperature and nacl concentration were determined by measuring the optical density (od) at 660 nm by spectrophotometer (fig. 7a, b). in this study d2, d5 and d9 bacterial isolates demonstrated the highest growth rate at 0 c, while 28 and 37 were optimal growth temperatures for most other isolated strains. all isolates showed low growth rate at 60 °c. our study suggested that the most suitable salt concentrations differed among the strains. the result showed that optimal growth and survival were at 0% for d8, 0.5% for most of the strains (d1, d2, d3, d7, d9 and d10), while three strains (d4 d5 d6) had the best growth at 5% nacl concentration (fig. 7b). iaa is a common product of l-tryptophan metabolism by numerous microorganisms but in the medium salt concentrations affects the growth rate of microorganism. all isolates were grown in tryptone broth with different salt concentration. d6 and d7 growth rate were higher at 8% nacl and d9 growth rate was constant at 0% and 8% nacl (fig. 8). the results of the soil treatments with potential pgpr isolates viz. d5 and d9 on the growth of a. cruentus showed significant effect in enhancing the plant’s shoot root height and weight of inoculated plants as compared to uninoculated control (fig. 9). in experiment 1, d5 and d9 showed a positive result in the development of shoot but in experiment 2, d5 showed high effect in enhancing shoots and roots as compared to control. discussion pgpr are originally free-living soil bacteria that colonize the rhizosphere or the tissues of living plants. once the bacteria are associated with the plant by colonizing the rhizosphere around the roots, the rhizoplane (root surface) or the root itself (rodriguez-navarro et al., 2007), the bacteria can increase the development of the plant through indirect or direct mechanisms. in our study, pgpr isolates viz. d4, d5, d6 and d9 showed positive for phosphate solubilization and ph ranged from 4.00 – 5.00. d9 bacterial isolates showed the highest zone in pkv plate. highest biofilm formation showed by rhizobacterial isolates d1, d2, d5, d7 and d9 was very evident at 600 nm readings. from the compatibility results, d1, d5 and d9 isolates were selected for the pot experiment to assess its performance in terms of growth, nutrient uptake and stress conditions survival ability. pot experiment results showed fig 6. a effect of antibiotics to different isolates; b antibiotics diversity of isolates analyzed by ntsys; c antibiotics test results analyzed by heatmap plot where gray color: resistance and black color: susceptibility (p10: penicillin, e10: erythromycin, s25: streptomycin, c25: chloramphenicol, t10: tetracycline biologica nyssana ● 11 (1) september 2020: 35-44 mitra et al. ● isolation and impacts of rhizobacteria from saussurea obvallata (dc.) edgew. (brahma kamal) 41 fig 7. a isolates growth under the different temperature; b isolates growth under the different salt concentration fig 8. growth rate in tryptone broth with different salt concentration biologica nyssana ● 11 (1) september 2020: 35-44 mitra et al. ● isolation and impacts of rhizobacteria from saussurea obvallata (dc.) edgew. (brahma kamal) that soil treatments with these isolates significantly enhanced the plant growth of inoculated amaranth plants as compared to uninoculated control. conclusion in our study, we used bacillus sp. d5 and bacillus sp. d9 as a bio-inoculum and phosphate solubilizer from the rhizospheric soil samples of s. obvallata (brahma kamal) in the himalayan region. the present study has clearly demonstrated the pgp trails and application on amaranths with bacillus sp. d5 and bacillus sp. d9 having a positive influence in growth parameter in comparison with the control. the study suggested that low-temperature bacterial community growth has multifunctional characteristics in growth promotion and develops a green biofertilizer for hill farming. acknowledgements. we are grateful to the founder and president, prof. (dr.) kamal ghanshala, graphic era (deemed to be university), dehradun and giving student research grants for this research work. we are also grateful to prof. ashish thapliyal, professor and head, department of biotechnology, graphic era (deemed to be university), dehradun. first author thanks to devvrat, ph.d. scholar, graphic era (deemed to be university), dehradun, india and ansuman senapati, srf, icarnrri, cuttack, india for his help in data analysis references agrawal, p.k., johri, b.n. 2014: characterization of plant growth promoting rhizobacteria from rhizospheric soil of himalayan region. octa journal of biosciences, 2(2): 69-75. ahemad, m., kibret, m. 2014: mechanisms and applications of plant growth promoting rhizobacteria: current perspective, journal of king saud university – science, 26(1): 1-20. biologica nyssana ● 11 (1) september 2020: 35-44 mitra et al. ● isolation and impacts of rhizobacteria from saussurea obvallata (dc.) edgew. (brahma kamal) anija, k.r. 2003: experiments in microbiology. plant pathology and biotechnology, isbn: 81-2241494-x. ansari, p.g., rao, d.l.n., pal, k.k. 2014: diversity and phylogeny of soybean rhizobia in central india. annals of microbiology, 64(4): 1553-1565 antoun, h, kloepper, j.w. 2001: plant growthpromoting rhizobacteria. in: brenner s, miller jh, (eds). encyclopedia of genetics: 1477–1480, academic, new york. ardakani, s.s., heydari, a., tayebi, l., mohammedi, m. 2010: promotion of cotton seedlings growth characteristics by development and use of new bio formulations. international journal of botany, 6(2): 95–100. backer, r., rokem, j.s., ilangumaran, g., lamont, j., praslickova, d., ricci, e., subramanian, s, smith, d.l. 2018: plant growth-promoting rhizobacteria: context, mechanisms of action, and roadmap to commercialization of biostimulants for sustainable agriculture. frontiers in plant science: 9: 1473. bakker, a.w., schippers, b. 1987: microbial cyanide production in the rhizosphere in relation to potato yield reduction and pseudomonas sp. mediated plant growth stimulation. soil biology & biochemistry, 19: 451–457. bakker, p.a.h.m., ran, l.x., pieterse, c.m.j., van loon, l.c. 2003: understanding the involvement of induced systemic resistance in rhizobacteria-mediated biocontrol of plant diseases. canadian journal of plant pathology, 25: 5–9. bakthavatchalu, s., shivakumar, s., sullia, b.s. 42 fig 9. biomass and physiological parameter of the treated plant after 30 days biologica nyssana ● 11 (1) septembeer 2020: 35-44 mitra et al. ● isolation and impacts of rhizobacteria from saussurea obvallata (dc.) edgew. (brahma kamal) 2012: identification of multi-trait pgpr isolates and evaluation of their potential as biocontrol agents. acta biologica indica, 1(1): 61-67. bhattacharyya, p.n., jha, d.k. 2012: plant growthpromoting rhizobacteria (pgpr): emergence in agriculture. world journal of microbiology and biotechnology, 28(4): 1327-1350. brown, a.e. 2009: benson’s microbiological applications: laboratory manual in general microbiology. 11th ed. mcgraw-hill companies, new york, ny, usa. cappuccino, j.g., sherman, n. 2008: microbiology: a laboratory manual, 8th ed. pearson benjamin cummings. san francisco, ca, usa. chauhan, a., shirkot, c.k., kaushal, r., rao, d.l.n. 2015: plant growth-promoting rhizobacteria of medicinal plants in now himalayas: current status and future prospects. in: egamberdieva, d., shrivastava, s., varma, a. (eds. plant-growth-promoting rhizobacteria (pgpr) and medicinal plants: 381412, springer, cham, cook, r.j. 1993: making greater use of introduced microorganisms for biological control of plant pathogens. annual review of phytopathology, 31: 53–80. dubey, r.c., maheshwari, d.k. 2007: prac. microbiol. isbn: 81-219-2153-8 dursun, a., yildirim, e., turan, m., ekinci, m., kul, r., parlakova karagoz, f. 2019: determination of the effects of bacterial fertilizer on yield and growth parameters of tomato. journal of agricultural science and technology, 21(5): 1227-1234. fiske, c.h., subbarow, y. 1925: the colorimetric determination of phosphorus. journal of biological chemistry, 66: 375–400. glick, b.r. 1995: the enhancement of plant growth by free-living bacteria. canadian journal of microbiology, 41(2):109–117. gordon, s.a., weber, r.p. 1951: colorimetric estimation of indole acetic acid, plant physiology, 26:192–195. harley, j.p., prescott, l.m. 2002: laboratory exercises in microbiology, 5th ed. mcgraw-hill higher education, new york, ny, usa. höflich, g., wiehe, w., ku¨hn, g. 1994: plant growth stimulation with symbiotic and associative rhizosphere microorganisms. experientia, 50: 897– 905. ji, h.s., gururanib, a.m., chun, s. 2014: isolation and characterization of plant growth promoting endophytic diazotrophic bacteria from korean rice cultivars. microbiological research, 169: 83– 98. johri, j.k., surange, s., nautiyal, c.s. 1999: occurrence of salt, ph and temperature tolerant phosphate solubilizing bacteria in alkaline soils. current microbiology, 39: 89–93. kloepper, j.w., beauchamp, c.j. 1992: a review of issues related to measuring of plant roots by bacteria. canadian journal of microbiology, 38: 1219– 1232. kloepper, j.w., hume, d.j., scher, f.m., singleton, c. 1988: plant growth promoting rhizobacteria on canola (rape seed). plant disease, 72: 42-46. kloepper, j.w., leong, j., teintze, m., scroth, m.n. 1980: enhanced plant growth by siderophores produced by plant growth-promoting rhizobacteria. nature, 286: 885–886. loper, j.e., schroth, m.n. 1987: influence of bacterial sources of indole-3-acetic acid on root elongation of sugar beet. phytopathology, 76: 386–389. macfaddin, j. 1985: media for isolation-cultivation-identification-maintenance of medical bacteria. williams and wilkins, baltimore md. vol. 1. macfaddin, j.f. 1980: biochemical tests for identification of medical bacteria.williams and wilkins, baltimore majeed, a., abbasi, m.k., hameed, s., imran, a., rahim, n. 2015: isolation and characterization of plant growth-promoting rhizobacteria from wheat rhizosphere and their effect on plant growth promotion. frontiers in microbiology, 6:198. mitra, d., rani, a., palni, l. m. s., sharma, k., uniyal, n., chauhan, a., semwal, p., arya, p. 2019. isolation and characterization of dominant fungi from rhizospheric soil of saussurea obvallata (dc.) edgew. (brahma kamal) of the indian himalayan region. journal of pure and applied microbiology, 13(3):1509-1515. nath, r., sharma, g.d., barooah, m. 2015: plant growth promoting endophytic fungi isolated from tea shrubs of assam, india. applied ecology and environmental research, 13(3): 877-891. niranjan, r., shethy, s.h., reddy, s.m. 2005: plant growth promoting rhizobacteria: potential green alternative for plant productivity. in: z.a. siddiqui (ed.), pgpr: biocontrol and biofertilization: 197–216. springer, dordrecht, the netherlands. pandey, a., palni, l.m.s., mulkalwar, p., nadeem, m. 2002: effect of temperature on solubilization of tricalcium phosphate by pseudomonas corrugate, journal of scientific and industrial research, 43 61(6): 457-460. qadri, f., hossain, sa., ciznár, i., haider, k., ljungh, a., wadstrom, t., sack, d.a. 1988: congo red binding and salt aggregation as indicators of virulence in shigella species, journal of clinical microbiology, 26(7):1343–1348. ramette, a., frapolli, m., défago, g., moënneloccoz, y. 2003: phylogeny of hcn synthaseencoding hcnbc genes in biocontrol fluorescent pseudomonads and its relationship with host plant species and hcn synthesis ability, molecular plantmicrobe interactions, 16(6): 525–535. rodríguez, h., fraga, r. 1999: phosphate solubilizing bacteria and their role in plant growth promotion. biotechnology advances, 17: 319–339. rodriguez-navarro, d.n., dardanelli, m.s., ruiz-sainz, j.e. 2007: attachment of bacteria to the roots of higher plants. fems microbiology letters, 272: 127-136. sarikhani, m. r., khoshru, b., greiner, r. 2019: isolation and identification of temperature tolerant phosphate solubilizing bacteria as a potential microbial fertilizer. world journal of microbiology & biotechnology, 35(8): 126. selvakumar, g., mohan, m., kundu, s., gupta, a. d., joshi, p., nazim, s., gupta, h. s. 2008: cold tolerance and plant growth promotion potential of serratia marcescens strain srm (mtcc 8708) isolated from flowers of summer squash (cucurbita pepo). letters in applied microbiology, 46(2): 171175. semwal, p., pant, m. 2013: brahma kamal – the spiritually revered, scientifically ignored medicinal plant. current science, 104(6): 685-686. semwal, p., palni, l.m.s., verma, s., sharma, p., thapliyal, a. 2018: nutrient analysis of rhizospheric and non-rhizospheric soil of saussu44 biologica nyssana ● 11 (1) september 2020: 35-44 mitra et al. ● isolation and impacts of rhizobacteria from saussurea obvallata (dc.) edgew. (brahma kamal) rea obvallata (dc.) edgew. (brahma kamal) from kedarnath, uttarakhand, india. journal of graphic era university, 6(1):1-6. sharma, a., johri, b.n. 2003: growth promoting influence of siderophore–producing pseudomonas strains grp3a and prs9 in maize (zea mays l.) under iron depriving conditions. microbiological research, 158: 243– 248. sparzak-stefanowska, b., krauze-baranowska, m., hałasa, r. 2019: influence of plant growth regulators on the shoot culture of phyllanthus glaucus and accumulation of indolizidine alkaloids with evaluation of antimicrobial activity. acta physiologiae plantarum, 41(1), 6. srinandan, c.s., jadav, v., cecilia, d., nerurkar, a.s. 2010: nutrients determine the spatial architecture of paracoccus sp. biofilm. biofouling, 26: 449–459. tsegaye, z., gizaw, b., tefera, g., feleke, a., chaniyalew, s., alemu, t., assefa, f. 2019: isolation and biochemical characterization of plant growth promoting (pgp) bacteria colonizing the rhizosphere of tef crop during the seedling stage. journal of plant science and phytopathology, 3: 013-027. vessey, j.k. 2003: plant growth promoting rhizobacteria as biofertilizers. plant and soil, 255: 571– 586. vijayaraghavan, p., vincent, p.g.s. 2013: a simple method for the detection of protease activity on agar plates using bromocresolgreen dye. journal of biochemical technology, 4(3): 628-630. zahid, m., abbasi, m.k., hameed, s., rahim, n. 2015: isolation and identification of indigenous plant growth promoting rhizobacteria from himalayan region of kashmir and their effect on improving growth and nutrient contents of maize (zea mays l.). frontiers in microbiology, 6: 207. ostojić et al., 2020, biologica nyssana 11(1) 11 (1) september 2020: 9-22 doi: 10.5281/zenodo.4060283 natural values and concept of protection of the nature park “šargan-mokra gora” original article dragana ostojić institute for nature conservation of serbia, belgrade, serbia dragana.ostojic@zzps.rs (corresponding author) aleksandar dragišić institute for nature conservation of serbia, belgrade, serbia aleksandar.dragisic@zzps.rs danko jović institute for nature conservation of serbia, niš, serbia danko.jovic@zzps.rs verica stojanović institute for nature conservation of serbia, belgrade, serbia verica.stojanovic@zzps.rs bojan zlatković faculty of sciences and mathematics, university of niš, niš, serbia bojanzlat@yahoo.com vladimir nikolić ministry of agriculture, forestry and water management forest administration, belgrade, serbia vladimir.nikolic@zzps.rs miloš radaković institute for nature conservation of serbia, belgrade, serbia milos.radakovic@zzps.rs nenad sekulić institute for nature conservation of serbia, belgrade, serbia nеnad.sekulic@zzps.rs vladan bjedov institute for nature conservation of serbia, belgrade, serbia vladan.bjedov@zzps.rs mila ristić institute for nature conservation of serbia, belgrade, serbia mila.ristic@zzps.rs ana petković institute for nature conservation of serbia, belgrade, serbia ana.petkovic@ zzps.rs received: october 08, 2019 revised: april 30, 2020 accepted: may 08, 2020 abstract: this paper presents the results of the research and valorization of natural values of the šargan and mokra gora area, which were conducted by the expert team of the institute for nature conservation of serbia in 2005, 2008, 2015, 2016, and 2017. the area was protected in 2005 as outstanding natural landscape “šargan mokra gora”. in contrast, in 2008, the northwestern borders of the protected area were extended to include the sites “ljuto polje” and “dobro polje”, and the type of protected area has also been changed from the outstanding natural landscape to nature park. the paper further elaborates the results of the revision of natural values of the protected area, which has been done in 2015 for the purpose of harmonizing legal acts with the defined protection regimes according to the cadastre, and which has proposed a total protected area of 11,379.78 ha. increasing the area under protection was aimed for the conservation of natural values, sustainable development, and proper management of the protected area. key words: natural values, nature park šargan-mokra gora, protection concept, protection regimes i, ii and iii. apstract: prirodne vrednosti i koncept zaštite parka prirode „šargan-mokra gora“ u radu su prikazani rezultati istraživanja i valorizacije prirodnih vrednosti područja šargana i mokre gore, koje je uradio stručni tim zavoda za zaštitu prirode srbije tokom 2005., 2008., 2015., 2016. i 2017. godine. područje je stavljeno pod zaštitu 2005. godine kao predeo izuzetnih odlika „šargan mokra gora“, a 2008. godine proširena je granica zaštićenog područja na severozapadnoj strani lokalitetima „ljuto polje“ i „dobro polje“ i promenjena vrsta zaštićenog dobra iz predela izuzetnih odlika u park prirode. prikazani su i rezultati revizije prirodnih vrednosti zaštićenog područja urađene 2015. godine za potrebe usaglašavanja zakonskih akata sa definisanim režimima zaštite po katastru, kojom je predloženo da ukupna površina pod zaštitom bude 11.379,78 ha. povećanje površine pod zaštitom ima za cilj očuvanje prirodnih vrednosti, održivi razvoj i efikasno upravljanje zaštićenim područjem. ključne reči: prirodne vrednosti, park prirode „šargan mokra gora“, koncept zaštite, režim i, ii i iii stepena zaštite introduction the šargan mokra gora nature park is located on the territory of užice municipality, in southwestern serbia. it is situated between the massifs of tara (1,544 m) and zlatibor (1,359 m), including the mokra gora structural basin and part of the kremna structural basin, as well as the rivers kamišna, bratešina, beli rzav, and crni rzav. it is bordered by the tara national park (site zaovine) and the © 2020 ostojić et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 9 13th symposium on the flora of southeastern serbia and neighboring regions zlatibor nature park. šargan and mokra gora represent one of the phytogeographically most interesting areas of southwestern serbia and possess special natural values. based on these values, in 2005 the area was designated as an outstanding natural landscape measuring 3,678.99 ha. the revision of the natural values in 2008 resulted in the increase of the protected area to 10,813.73 ha, and the type of protected natural area was changed to the nature park, with a protection regime iii established in the expanded part of the protected area. according to the mid-term and annual program for the protection of natural resources and the spatial plan of the special purpose area of the tara national park (official gazette of the rs, no. 100/10), which also included the area of šargan and mokra gora, a legal obligation was introduced to evaluate the expanded area in detail. to revise natural values, a detailed multidisciplinary research was conducted in 2015, which slightly modified the outer boundary of the nature park. in addition, revaluation of existing sites was carried out along with the establishment of new sites with protection regimes i and ii, in comparison with those defined by the decree of 2005 and 2008. in the newly revised 2015 conservation proposal, the total area of this natural property was 11,379.78 ha (ostojić et al., 2015). the paper is based on the protection study of the outstanding natural landscape “šargan-mokra gora”, of 2005, and the protection study of the nature park “šargan-mokra gora” (ostojić et al., 2015). material and methods for valorization of natural values, field surveys were conducted to collect data and evaluate the data obtained. it is based on the national and international criteria for valorizing natural values. geological and hydrological characteristics have been selected according to general geological surveys of the area (mojsilović et al., 1977, 1978; olujić and karović, 1986a, 1986b; dimitrijevic, 1995 and others). in order to obtain as complete a picture as possible of the natural values for each area studied, existing field and literature data were used and mapped in the field. pursuant to the rulebook on the proclamation and protection of strictly protected and protected wild species of plants, animals and fungi (“official gazette of the rs”, nos. 05/10, 47/11, 32/16 and 98/16) (hereinafter: rulebook ) and the decree on the control of use and trade in wild flora and fauna (official gazette of the rs, no. 31/05, 45/05, 22/07, 38/08, 9/10 and 69/11) “strictly protected” and “protected wild species”, as well as species that are collected and traded therein have been determined. endangered taxa have been determined on the basis of field research and analysis of the threat factors and effects on populations in the wild, as well as according to the categorization of internationally accepted iucn criteria 1. the findings of the endangered taxa in the territory of serbia were also taken into account. species that are on the preliminary red list of flora of serbia have also been selected (stevanović (ed.), 2002). for the determination of forest phytocoenoses, the modern nomenclature was used with the latest principles of the syntaxonomy of forest vegetation of serbia, which were given in the editions: vegetation of serbia 2 (1) (jovanović et al., 1997) and vegetation of serbia 2 (2) (dinić et al., 2006). samplings of ichthyofauna were realized by the method of electrofishing to determine data on fish populations such as species composition, age structure, quantitative analyzes and other parameters. in the study of amphibians and reptiles fauna, sites were carefully selected for analysis, which were adapted to the weather conditions conducive to the daily activities of the herpetofauna. ornithofauna studies were based on the analysis and presence of endangered species closely related to particular habitats. they were derived according to the international standard classification of landscape types (corine, 1990), which were adapted to yugoslav conditions (stevanović, v., vasić, v., 1995). the evaluation of the investigated area as a natural resource was carried out in accordance with the rulebook on criteria for evaluation and procedure of categorization of protected areas (“official gazette of the rs”, no. 97/15), and based on based on the traits such as autochthony, rarity, representability, originality, etc. these criteria reflect the state of the natural values and their spatial distribution in the field. results and discussion the state and valorization of natural values geological characteristics the šargan-mokra gora nature park is situated in the area of the inland dinarides tectonic unit, and it is built of magmatic, metamorphic and sedimentary rocks that have been created over a long geological history. the oldest rock masses in the structure of the nature park area are sediments, mainly carbonates of the middle and upper triassic and upper cretaceous, and the most widespread are the jurassic ultramafics. 10 biologica nyssana ● 11 (1) september 2020: 9-22 ostojić et al. ● natural values and concept of protection of the nature park “šargan-mokra gora” in the long geological evolution of the carbonate mass of the karst plateau of tara and the peripheral parts of the peridotite massif of the nearby plateau of zlatibor, the tectonic movements caused the appearance of large blocks, which subsequently changed the original appearance to a large extent due to the erosion forces, and led to the formation of very dynamic relief (cvijić, j., 1924/1991). general epeirogenic ascent of the dinarides caused the appearance of deep-cut gorges and canyons in the area of šargan and mokra gora and the split in the relief of tusto brdo, putno brdo, kobilovac, vao brdo, njivičke kose, tabačke kose, skakavac, đoga, kozja stena and viogor. above these morphological units rise the peaks of kršanjska glava (1424 m) (fig. 1), carevića vis (1426 m), runjeva glava (1438 m), gavran (1453 m), iver (1478 m) and zborište (1544 m), which is the highest peak of the tara mountain. hydrological characteristics the hydrographic potential of the šargan and mokra gora areas consists of groundwater, springs and karst springs, larger and smaller river flows and thermal mineral waters, many of which are captivated for the needs of water supply. the waters of the mokra gora structural basin flow into the drina basin, whereas the waters of kremna structural basin flow into the basins of zapadna and velika morava. the watershed between the rivers kamišna and đetinja, that is, the drina and morava, is the šargan mountain that separates these two structural basins. springs and karst springs occur in the altitudinal zone of the peripheral area of šargan, tara and viogor, as well as in river valleys at the intersection of the valley side and the valley floor. the river network of the area consists of the kamišna, beli and crni rzav and their tributaries, the source-rivers of đetinja (bratešina and konjska river) the užice stream and a range of rather small rivers and streams, with numerous gorge valleys with rapids and waterfalls (veliki and mali skakavac on kamišna river), which represent interesting landscape units. the characteristics of flora the flora of this area was, for the first time, described by the famous serbian botanist josif pančić in 1860. pančić summarized his data in the capital 11 work of the serbian botany “flora of the principality of serbia” (pančić, 1874) and in the “addendum to the flora of the principality of serbia” (pančić, 1884), in which he says that mokra gora has an environment rich in rare plants. in his works, pančić described several species hitherto unknown to science. of the seven species described by pančić himself, or together with the italian professor of botany roberto visiani (18011878), as new to the science from this area (visiani et pančić 1862, 1866, 1870), five species still have the status of “good” or “undisputed” species. pančić points out that the flora and vegetation of mokra gora are distinguished by the presence of an impressive number of serpentinophytes, which is a term referring to specialized plant species of the characteristic and striking features, unique, very striking appearance, the occurrence of which is conditioned by a specific serpentinitic bedrock. the results of previous studies have shown that floristic richness and the diversity of the plant world of the mokra gora and šargan area are reflected in the presence of 722 taxa from the group of higherranked plants, excluding moss species (bryophyta) classified into 355 genera, or 92 families. it is assumed that the finite number of species is slightly higher. of the total flora species in this area, 6.2% are endemic and subendemic taxa, indicating that it is an area characterized by a diverse flora of distinct autochthonous character and originality. the largest number of endemics found in this area belongs to the group of balkan endemics. among the endemics, the most prominent are edraianthus jugoslavicus lakušić, micromeria croatica (pers.) schott, halacsya sendtneri (boiss.) dorfler, reichardia macrophylla (dc.) freyn, stachys anisochila vis. & pančić, alyssum markgrafii o. e. biologica nyssana ● 11 (1) september 2020: 9-22 ostojić et al. ● natural values and concept of protection of the nature park “šargan-mokra gora” fig 1. kršanjska glava (1424 m), photo: taken by camera drone 12 schulz ex markgraf, scrophularia tristis (k. maly) šilić, linaria rubioides vis. & pančić, helleborus serbicus adamović, euphorbia pancicii g. beck, euphorbia glabriflora vis., lathyrus binatus pančić and others. in the mokra gora area, 190 species are considered internationally and nationally important. according to the rulebook on the proclamation and protection of strictly protected and protected wild species of plants, animals and fungi, 102 species of the mokra gora area are being protected, 14 of them as strictly protected and 88 as protected species. in the mokra gora area 17 species are protected according to the cites convention on the international trade in endangered species of wild flora and fauna, and 35 plant species are protected according to the decree on the control of the use and trade in wild flora and fauna. the preliminary red list of endangered taxa of the flora of serbia (stevanović, ed. 1997) contains 49 species recorded during the research. also, the first volume of the red book of flora of serbia i lists rhaponticoides alpina (l.) m.v.agab. & greuter) (fig. 2), which grows only in ograđenica above mokra gora. this species, in accordance with the rulebook of the international union for the conservation of nature (iucn), has been categorized as the critically endangered taxon (cr) of the flora of serbia. the characteristics of vegetation in terms of vegetation, the mokra gora and šargan area belongs to the mesophilic western balkan forests of sessile oak and hornbeam (querco-carpinetum illyricum horv. et al. 1974) and beech forests of the fagenion illyricum horvat (1938) 1950 alliance. in the extended and cultivated parts of mokra gora, forests of alder and willow grows of the salicion albae soo (1930) 1940 alliance. in the serpentinitic gorges surrounding mokra gora, in addition to streams and rivers, there is the community of salicetum eleagni moor 1958 em. oberdorfer 1962, which is built by the gray willow. on thermophillic slopes, however, with a deeper soil layer, the forests of oak (quercetum cerris e. vukićević 1966) are developed, while in the humid valleys of the lower region and on the slopes rising above the streams the mesophillic forests of sessile oak and hornbeam belonging to the association querco petraea – carpinetum betuli rudski (1940) 1949 of the alliance carpinion betuli illiyrico-moesiacum ht. 1956 occur. the tree layer is composed of sessile oak (quercus petraea (mattuschka) liebl.) and hornbeam (carpinus betulus l.), while he herbaceous plant layer is mostly composed of: pulmonaria officinalis l., aposeris foetida (l.) less., corydalis solida (l.) clairv., lunaria rediviva l., epimedium alpinum l. and other forest species. the presence of valley lily (maianthemum bifolium (l.) f. w. schmidt), a species that is extremely rare in serbia and is regionally characterized as an endangered taxon (en) is of particular importance. on the mountains surrounding the mokra gora structural basin at altitudes of 700-800 m, sessile oak forests have been developed, building the quercetum montanum serpentinicum b. jovanović 1972 association. these are mostly degraded communities, developed on serpentinitic bedrock. the tree layer is dominated by sessile oak and in the ground layer semi-shrubs of winter heath (erica carnea l.) and daphne (daphne blagayana freyer) occur. steep slopes of ograđenica and tusto brdo (hill), occuring on shallow and rocky limestone, are overgrown with the ostryo carpinifoliae-fraxinetum orni aichinger 1933 community built by south european flowering ash and the european hop-hornbeam. a characteristic of this floristically rich community is the presence of peony (paeonia officinalis l.). it is a community of thermophilic, sub-mediterranean character, which in addition to edifying species is also built by an abundance of shrub species such as: acer monspessulanum l., evonimus verucosa scop., crataegus monogyna jacq., rhamnus saxatilis jacq, rosa canina l. and others. the rocky forest glades overgrown with smoke bush, especially on the ograđenica plateau, are very important since biologica nyssana ● 11 (1) september 2020: 9-22 ostojić et al. ● natural values and concept of protection of the nature park “šargan-mokra gora” fig 2. rhaponticoides alpina, photo by: m. niketić 13 they include endangered or rare species: frangula rupestris (scop.) schur, paеonia officinalis l., phyteuma orbiculare l., rhaponticoides alpina (l.) m.v.agab. & greuter, lathyrus binatus pančić, etc. the forests of european black pine develop on serpentinite, and in the mokra gora area two associations, erico-pinetum nigrae serpetinicum (z. pavlovic 1951) b. jovanović 1972 and ostryo-pinetum nigrae čolić 1965, occur. pine forests with winter heath (erico-pinetum nigrae serpetinicum) distribute all over šargan and viogor, as well as in the gorges and slopes descending towards mokra gora structural basin. these are light forests, where the ground layer is composed of winter heath (erica carnea l.) and of european blueberries (vaccinium myrtillus l.) at higher altitudes. the frequently occurring herbaceous plants in these forests are asplenium cuneifolium viv., potentilla malyana maly, stachys scardica (griseb.) hayek, veronica chamaedrys l., centaurea triumfetii all. and others. in more thermophillic and rocky sites grows spurge (euphorbia glabriflora vis.), while the steep slopes are almost 100% grown with sesleria serbica (adamovic) ujhelyi, protecting them from erosion. on the slopes of the entrance part of the beli rzav gorge, tusto brdo and vao peak, there is an another association of european black pine forests (ostryo-pinetum nigrae čolić 1965) (fig. 3). this association is composed of european black pine (pinus nigrae arn.) and hop hornbeam (ostrya carpinifolia scop.), in addition to which fraxinus ornus l., rhamnus fallax boiss., juniperus communis l. and others also occur in the community. this community is distinctively xerothermophilic, developed on limestone bedrock and on poor and shallow soil. beech communities consisting of fagetum moesiacae montanum bleč. et lakušić 1970 and fagetum submontanum moesiacum (rudski 1940) b. jovanović 1967 are developed in several places, whereby the largest areas of their distribution are the limestone soils of tusto brdo at the altitudinal range of 600 to 1100 m. the vegetation of rocky terrains and serpentinitic gorges is built by the communities of the alliances centaureo – bromion fibrosi blečić et all.1960 and potentillion visiani h. ritter-studnička 1970 of the order halacsyetalia sendtneri h. ritter-studnička 1970, which is a unique type of vegetation of serpentinitic ground. steep sites of serpentinitic gorges and steep serpentinitic rocky terrains of the mokra gora area are overgrown with the community festuco duriusculae – euphorbietum glabriflorae s. jov. et r. jov 1992., which has also been described in zlatibor (jovanović et al., 1992). the endemic, decorative euphorbia glabriflora vis. is dominant in this community, and this community consists of about one hundred plant species, the most frequent of which are festuca duriuscula l., galium purpureum l., teucrium montanum l., thymus jankae celak. and others. the particular importance of the community is reflected in the presence of endemic taxa such as halacsya sendtneri (boiss.) dorfler, stipa novakii martinovsky, genista friwaldskyi boiss, iris reichenbachii heuffel and other species. in gorges and canyons, the presence of numerous hazmophytic communities that develop in the crevices and recesses of the rocks is recorded. these areas are dominated by communities of the order asplenietea trichomanis br.-bl. 1934 corr. oberd. 1977 that are particularly well developed in serpentinitic gorges. on the other hand, the main characteristic of the vegetation of serpentinitic rocks are endemic serpentinophytes halacsya sendtneri (boiss.) dorfler and drymocallis malacophylla (borbás) kurtto. the two species are basic builders of the characteristic association halacsyo sendtneripotentilletum mollis pavlović 1955, which is particularly well developed in the kamišna river gorge and on the cliffs of kozja stena. beli rzav canyon is one of the characteristic sites where the rock vegetation is well developed (fig. 4). the vegetation of talus slopes (scree) occurs in the forest belt of oak and oak-hornbeam on limestone or serpentinitic bedrock. this vegetation consists of herbaceous plants, the roots of talus slopes grasses, or plants that look biologica nyssana ● 11 (1) september 2020: 9-22 ostojić et al. ● natural values and concept of protection of the nature park “šargan-mokra gora” fig. 3: the association of european black pine forests (ostryo-pinetum nigrae) at the šišatovac site, photo: v. nikolić 14 like low shrubs with very long semi-woody, creeping shoots that bind loose scree. it is a common occurrence that the vegetation of rocks and talus slopes is found in the same site, as is the case on ograđenica (fig. 5). the vegetation of limestone talus slopes is represented by the community of corydaletum euochroleucae b.tatić et b. atanacković 1972 and the community of saturejo montanae-achnatheretum calamagrostis r. jovanović et s. jovanović 1986. of particular importance is the occurrence of the species reichardia macrophylla (dc.) freyn, an endemic species of the southwestern part of the balkan peninsula, whereas the special characteristic of this vegetation is the occurrence of endemorelic species halacsya sendtneri (boiss.) dorfler on the limestone. the vegetation of serpentinitic talus slopes consists of roots of talus slope grasses, primarily the roots of achnatherum calamagrostis (l.) beauv. and medium-high endemic serpentinophytes: scrophularia tristis (k. maly) šilić, alyssum markgrafii o. e. schulz ex markgraf and linaria rubioides vis. & pančić. in these habitats often develop the brushwood of cotinus coggygria scop., which is formed by the suppression of forests. the characteristics of fauna within the boundaries of the protected area, a total of 6 species of fish from 3 families were recorded, namely: brown trout (salmo trutta linnaeus, 1758) from the trout family (fam. salmonidae), bleak (alburnoides bipunctatus bloch, 1782), danube barbel (barbus balcanicus kotlík, tsigenopoulos, ráb & berrebi, 2002), eurasian minnow (phoxinus phoxinus linnaeus, 1758), european chub (squalius cephalus linnaeus, 1758) from the carp family (fam. cyprinidae) and european bullhead (cottus gobio linnaeus, 1758) from the family cottidae (fig. 6). in terms of fish diversity in watercourses, the most species were recorded in beli rzav (5 species brown trout, bleak, danube barbel, european chub and bullhead), followed by kamišna river (3 species bleak, danube barbel and european chub), bratešina river (2 species – danube barbel and eurasian minnow) and dubošac (1 species danube barbel). of the total of 6 species of fish recorded in the waters of this area, only european bullhead has the status of strictly protected species according to the rulebook on the proclamation and protection of strictly protected and protected wild species of plants, animals and fungi, while 4 species have the status of protected wild species, namely: brown trout, bleak, danube barbel and european chub. the only source of data related to earlier research of batrachofauna and herpetofauna of šargan and mokra gora were data collected by radovanović (1951), as well as data collected during surveys conducted in the surrounding areas (tara national park) in 2002 and 2003. a total of 17 amphibian and reptile species (eight species from the amphibia biologica nyssana ● 11 (1) september 2020: 9-22 ostojić et al. ● natural values and concept of protection of the nature park “šargan-mokra gora” fig. 4: vegetation of rocks in beli rzav canyon, photo: taken by camera drone fig. 5: vegetation of the talus slopes and rocks on ograđenica, photo: photo documentation of the institute for nature conservation of serbia 15 class and nine species from the reptilia class) have been recorded in the šargan and mokra gora. in the lower bed of the kamišna river canyon horned viper vipera ammodytes linnaeus, 1758 has been recorded, whereas in the upper course of the river the yellow-bellied toad (bombina variegatа linnaeus, 1758) has been recorded in several sites. the twelve recorded species are strictly protected, while two species have the status of protected wild species pursuant to the rulebook on the proclamation and protection of strictly protected and protected wild species of plants, animals and fungi. of the 12 strictly protected species, 7 species are from the genus amphibia amphibians, of which two species belong to the tailed amphibians: fire salamander (salamandra salamandra linnaeus, 1758) and smooth newt (lissotriton vulgaris linnaeus, 1758), the other five species belong to the order of tailless amphibians (frogs): yellow-bellied toad (bombina variegata linnaeus, 1758), common toad (bufo bufo linnaeus, 1758), green toad (pseudepidalea viridis laurenti, 1768), greek stream frog (rana graeca boulenger, 1891), and agile frog (rana dalmatina fitzinger, 1838). the remaining 5 strictly protected species are from the genus reptilia reptiles and all species belonging to the snakes group (serpentens): aesculapian snake (zamenis longissimus laurenti, 1768), smooth snake (coronella austriaca laurenti, 1768), grass snake (natrix natrix linnaeus, 1758), dice snake (natrix tessellata laurenti, 1768) and common european viper (vipera berus linnaeus, 1758). considering the threat to these species, all species are on the list of the red book of fauna of serbia i amphibians (kalezić et al. 2015) and the red book of fauna of serbia ii reptiles (tomović et al. 2015). the initial ornithological survey of the wider area of šargan and mokra gora was carried out by the ornithologist othmar reiser during july 1899. based on the surveys conducted so far in the nature park “šargan mokra gora”, 120 bird species have been recorded, which, if compared with the bird diversity in the whole of serbia including 352 species (šćiban et al., 2015), represents 34% of the total bird diversity in serbia. most species of birds in šargan and mokra gora (101 species) are strictly protected according to the rulebook on the proclamation and protection of strictly protected and protected wild species of plants, animals and fungi, which includes the prohibition of killing, collecting eggs, disturbing nesting sites and other activities that may endanger the survival of these species. a total of 18 bird species are protected, which is regulated, in addition to the aforementioned rulebook, also by the rulebook on the closed season for protected game animal species (official gazette of rs, no. 75/2010). this group also includes game animal species such as quail, partridge, pheasant, mallard, wood pigeon and turtle dove. the importance of the area is reflected in the presence of nationally and internationally important species, as well as in the presence of vulnerable and endangered species closely related to particular habitats, which primarily refers to species such as: golden eagle (aquila chrysaetos linnaeus, 1758), the short-toed snake eagle (circaetus gallicus gmelin, 1788), peregrine falcon (falco peregrinus tunstall, 1771), capercaillie (tetrao urogallus linnaeus, 1758), corn crake (crex crex linnaeus, 1758), the collared flycatcher (ficedula albicollis temminck, 1815), the red-breasted flycatcher (ficedula parva bechstein, 1794), willow tit (parus montanus conrad von baldenstein, 1827) and the wallcreeper (tichodroma muraria linnaeus, 1758). at the international level, the birds of šargan and mokra gora are protected by various international conventions and directives. in this area, 23 species are listed in appendix i of the european birds directive (209/147/ec), according to which eu member states are required to designate special protected areas (spas) for the purpose of these birds protection. available professional and scientific papers (petrov, 1992; savić et al., 1995; paunović and milenković, 1996; kryštufek, et all., 1997; savić et all., 1997; marinović, 1997; paunović et al., 2011), as well as the results of field surveys of the institute for nature conservation of serbia in the area of šargan and mokra gora, indicate that this area is biologica nyssana ● 11 (1) september 2020: 9-22 ostojić et al. ● natural values and concept of protection of the nature park “šargan-mokra gora” fig. 6: european bullhead (cottus gobio), photo: n. sekulić permanently or occasionally inhabited by at least 49 mammal species, which makes up about half of the number of species registered so far in serbia. it is considered that this number is not definitive, given the still poor exploration of the whole space and individual groups. the largest group consists of bats (chiroptera) and rodents (rodentia), with 13 species of each order. all species of bats in serbia have the status of strictly protected species according to the rulebook on the proclamation and protection of strictly protected and protected wild species. as far as rodents are concerned, there are three strictly protected species in this area: lesser mole-rat (spalax leucodon nordmann, 1840), forest dormouse (dryomys nitedula pallas, 1778) and hazel dormouse (muscardinus avellanarius linnaeus, 1758), as well as two protected species: squirrel (sciurus vulgaris linnaeus, 1758) and edible dormouse (glis glis linnaeus, 1766). they are followed by carnivores (carnivora) with 11 species and species of the order eulipotyphla 8 of them. all these species fall into the category of “protected species”, with the carnivores such as otter (lutra lutra linnaeus, 1758) and the brown bear (ursus arctos linnaeus, 1758), and “shrew-form” (neomys fodiens pennant, 1771), which are classified as “strictly protected species” according to the same rulebook. the fauna of carnivores in the mokra gora and šargan currently includes the following species: wolf (canis lupus linnaeus, 1758), golden jackal (canis aureus linnaeus, 1758), red fox (vulpes vulpes linnaeus, 1758), brown bear (ursus arctos linnaeus, 1758), least weasel (mustela nivalis linnaeus, 1766), european polecat (mustela putorius linnaeus, 1758), european pine marten (martes martes linnaeus, 1758), beech marten (martes foina erxleben, 1777), badger (meles meles linnaeus, 1758), otter (lutra lutra linnaeus, 1758) and wildcat (felis silvestris schreber, 1775). to the smallest group belong the ungulates (artiodactyla) with 3 species: wild boar (sus scrofa linnaeus, 1758), chamois (rupicapra rupicapra linnaeus, 1758) and roe deer (capreolus capreolus linnaeus, 1758). lagomorphs (lagomorpha) are represented with 1 protected species european hare (lеpus еurоpаеus pallas, 1778). the characteristics of “šargan mokra gora” nature park the results of research and valorization of natural values of the area of šargan and mokra gora have shown that it is characterized by the following core values that account for its status of a nature park and its protection regimes: biologica nyssana ● 11 (1) september 2020: 9-22 ostojić et al. ● natural values and concept of protection of the nature park “šargan-mokra gora” • originality (autochthonous character) – the area is relatively well conserved and without significant and visible negative human intervention. a special characteristic of this area is the geological bedrock in the form of old masses of magmatic, sedimentary and metamorphic rocks, which were formed as a result of long geological history, and on which the especially rare and specific natural values of biodiversity were formed. the combination of fragments of high plains on watersheds with pastures and high pine trees of steep valley sides and deep-cut river valleys overgrown with dense centuries-old forests and here and there narrowed settlements give this area a distinct feature and specific beauty with a high degree of attractiveness. the pronounced presence of scots pine and black pine forests, in the form of pure and mixed stands, is a distinguishing feature of the autochthonous habitat, and thus more rapid and qualitative forest restoration. an example of a particularly important conserved old forest complex is the site “jelovac šišatovac”, in which a rare and endangered western capercaillie and other characteristic species of old black pine forests have been recorded. • representativeness – is reflected in the presence of large complexes of coniferous and mixed forests, certain representatives of flora, fauna and vegetation. in addition, this area is best represented by the species of plants that pančić first described here for science. on limestone rocky grounds and rocks, pančić discovered stachys anisochila vis. & pančić), and on serpentinitic rocky grounds he discovered haplophyllum boisserianum vis. & pančić, linaria rubioides vis. & pančić, euphorbia glabriflora vis. and potentilla visianii pančić. strictly protected species of ichthyofauna – european bullhead is an important resident of the waters of beli rzav. representatives of the insectivore fauna are the eurasian water shrew and mountain shrew, while the representatives of rodent fauna are: lesser mole-rat, forest dormouse and hazel dormouse. notable representatives of the carnivorous fauna are the brown bear and otter, as strictly protected species. the main brown bear habitats are in the beli rzav canyon, the sites tusto brdo and vao limestone hill. otter is a resident of mountainous, conserved, permanent watercourses of beli rzav, crni rzav and kamišna. • rareness – is expressed in highly conserved and pronounced biodiversity in a relatively small protected area. the criterion is evident in the distribution of natural black pine habitats on serpentinite, which are singled out as rare habitats in serbia. 16 biologica nyssana ● 11 (1) september 2020: 9-22 ostojić et al. ● natural values and concept of protection of the nature park “šargan-mokra gora” on the limestone massif of the site “ograđenica” the only habitat of rhaponticoides alpina (l.) m.v.agab. & greuter in serbia has been identified, which is a natural rarity in the category of critically endangered taxa (cr), the population of which is estimated at only a few specimens. in the mesophillic oak and hornbeam forests a species that is extremely rare in serbia has been recorded, namely maianthemum bifolium (l.) f. w. schmidt. rare representatives of the animal world are mountain eurasian water shrew and mountain shrew, wildcat and chamois. concerning the presence of rodents, there is forest dormouse, a rare species that inhabits thermophillic deciduous forests with a rich bush layer and rocky grounds within this layer, and with one typical habitat on the site “ograđenica”. • diversity is pronounced in landscape features deep-cut gorges, canyons, and dissected plateaus. the richness of species and ecosystem diversity is reflected by the presence of 722 species of vascular flora, classified into 355 genera or 92 families, 10 most represented and most important communities of forest ecosystems, 7 representative communities of the vegetation of rocks, talus slopes and rocky terrains. the importance of floristic diversity is further enhanced if it is known that a small area contains 22% of the total number of serbian flora, of which 6.2% of the area’s flora consists of endemic and subendemic taxa that represent a rare, specific from the point of biodiversity conservation view, and an extremely valuable group of obligate serpentinophytes. particularly valuable in terms of floristic features are the extremely rare serpentinophyte communities occurring on the rocky terrains with festuco duruusculae-euphorbetum glabriflorae and in the gorges with potentillo-halascyetum sentdneri, as well as the hazmophytic community of endemic edraiathion alliance on limestone, in which the endemic taxa form unique phytocoenological combinations. the fauna of this area is characterized by six (6) species of fish, 17 species of amphibians and reptiles, 120 species of birds and 49 species of mammals. most registered species and builders of determined communities have legal protection status, since they represent endemic and relict taxa and communities, as well as rare and endangered plant and animal species that inhabit different, mosaically distributed habitats such as: forests, pasture-meadows, rock habitats and rocky terrains, as well as aquatic habitats. • integrity – the nature park represents a whole in the geographical and geomorphological terms. it is mostly bounded by natural watersheds that separate it from adjacent river basins, which makes it a geomorphologically well-individualized area, and thus the defining of protection borders is made much easier. • beauty/aesthetics criterion – the beauty/aesthetics of the area of šargan and mokra gora is evidenced with the exceptional seasonal dynamics and vividness of colors, the mosaic shift of the diverse rural-pastoral ambiental sequences and the specific architecture of each of the railway station complexes, as well as by drvengrad. an additional beauty of this area is given by the narrow-gauge heritage railway “šarganska osmica” (šargan eight), which has become famous for its technical solution of overcoming a great altitude difference at a short distance. • conservation – natural landscapes and forest complexes in the protected area are an important factor in the conservation of ecosystem services, environmental elements, and are a significant renewable natural resource. this picture is complemented by the exceptionally conserved watercourses of the area, which is confirmed by the specific fish fauna that inhabits them (brown trout, southern barbel/gudgeon, european bullhead, chub, etc.). • the monumental heritage of šargan and mokra gora is unique in the wider region. the ethno settlement drvengrad on mećavnik, which although recently built, seems to have been here for centuries, also gives an exceptional cultural value to the area. the concept of protection and protection regimes taking into account the state in the field, protection objectives and legislation, the concept of protection of the nature park “šargan mokra gora” is based on conservation of natural and created values. for this reason, protection regimes i, ii and iii have been established in this area. the total protected area is 11.379,78 ha, within which special spatial units with protection regimes i, ii and iii have been established. protection regimes i and ii cover the most valuable and conserved natural values and specific features of the area at 3,889.70 ha. as a starting point for establishing the protected area, a study on protection was prepared as a professionally documented basis for initiating protection. the mentioned study was submitted to the ministry of environmental protection, which informed the public about the procedure for initiating protection of the natural area on the ministry’s website. with this, the area of 11,379.78 17 ha is considered protected according to the law on nature protection (official gazette of the rs, nos. 36/09, 88/10, 91/10 correction and 14/16) (fig. 7). following the revision of the natural values of the protected area, in 2015, the area under protection regime i was increased from 414.84 ha to 774.50 ha (6.81% of the total protected area). the regime of protection is prescribed in five isolated sites within the protected natural area, of which 4 sites have previously been selected. these are: site “jelovac” is situated in the eastern part of protected area. upon revision, its acreage has been 18 decreased and is now 11.31 ha. the site is overgrown with tall, medium-age, welltended forest of european black pine (erico-pinetum nigrae (z. pavlović 1951) b. jovanović 1972), which is a typical representative of the pine forest on the serpentinic bedrock of šargan mountain. “klisura dubošca-skakavac” (dubošac gorgeskakavac) covers an area of 271.34 ha. part of this site is within the previously designated site in the protection regime i “klisura dubošca” (dubošac gorge) (area 106.01 ha). the gorge is located in the southeast part of the protected area and it is deeply cut into the serpentinitic massif. the skakavac waterfall, in the kamišna river bed, about 12 m high, is particularly prominent in this area. the steep sections of the entrance to this gorge contain imposing, solitary and very old specimens of european black pine, which together with hop hornbeam build the association ostryopinetum nigrae čolić 1965. site “ograđenica” is a site that previously covered 73.12 ha and was expanded to 164.67 ha after the revision. it is located in the southern part of the protected area. it is a limestone ridge surrounded by serpentinites, and in some way, has an island character regarding the distribution of a number of flora and fauna species that are ecologically related to thermophillic limestone forests, carbonate rocky terrains and talus slopes. site “međedova ljeska” covers an area of 8.35 ha, prior to the revision of the protection regime i it was established at 123.98 ha. it is located in the southern part of the protected area and at the far southwestern point it is in contact with the site “tusto brdo” in the protection regime ii, which is in the immediate vicinity of the plateau of ograđenica, from which it differs in orographic terms and therefore in vegetation. it is a typical example of biologica nyssana ● 11 (1) september 2020: 9-22 ostojić et al. ● natural values and concept of protection of the nature park “šargan-mokra gora” fig. 7: area proposed for protection in 2015, with the external border, protection regimes and sites under certain regimes, map: d. kaličanin a combination of thermophilic oak and mesophilic beech habitats in a relatively small area. site “debela kosa” is a newly selected site under the protection regime i, which covers an area of 318.83 ha. it is located in the southeast part of the protected area. it is overgrown with tall forest of european black pine (erico-pinetum nigrae (z. pavlović 1951) b. jovanović 1972 and euphorbio glabriflorae-pinetum nigrae pavlović, 1952) on the primeval humus-silicate soils of the peridotite and serpentinite bedrock. according to the decree on the protection of outstanding natural landscape “šargan mokra gora” (“official gazette of rs”, no. 52/05), the area under protection regime ii covered 1,187.46 ha. upon revison in 2015, this area was increased to 3,124.70 ha (27.46% of the total protected area) and the protection regime ii was established at ten isolated sites within the protected area: site “šišatovac-jelovac” has previously been called šišatovac, which now covers 105.69 ha. it is located in the eastern part of the protected area, and is overgrown with high forests of black pine (ericopinetum nigrae (z. pavlović 1951) b. jovanović 1972), which are young, of an extremely dense canopy cover and with a large amount of residual felling (timber stock). site “đoga” is part of the former site “đoga kozja stena”, covering an area of 148.66 ha. this site is in the far southeast of the protected area. the steep slopes and the massive remnant rocks of the đogo ridge are dominated by giant black pine trees of umbrella-like tree crowns. site “krečnjačko brdo vao” (limestone hill vao) covers an area of 130.61 ha. it is located in the central part of the protected area and has been reduced in size upon the revision. it encompasses the deep-cut and heavily accessible valleys of the grubiševac and suvodol streams. the foothill of this site is covered with communities of hay meadows (festuco-brometea), which are characterized by the presence of different species of clover and legumes, which makes them economically important. at higher altitudes, meadow enclaves are replaced by the beech forests of fagetum moesiacae montanum bleč. et lakušić 1970 of good quality with trees of seed origin and pronounced dendrometric characteristics. site “tusto brdo” formerly part of the site “tusto brdo božurica”, which upon the revision has been divided into “tusto brdo” and “božurica”, covers 298.73 ha. it is located in the southern part of the protected area. in terms of vegetation, this site is covered with man-grown cultivated mixed stands of black pine and scots pine, which at their age of about 60 years according to structure, cenotic composition and stability, are very similar to the natural forests of scots pine and black pine (pinetum sylvestris-nigrae typicum pavlović, 1952). site “božurica” covers 64.04 ha, and it is located in the southern part of the protected area. one part of this landscape unit is covered with man-grown black pine (pinetum nigrae) stands on different brown soils, while the other part is covered by mountain beech forest (fagetum moesiacae montanum bleč. et lakušić 1970) on different brown soils. site “ljuto polje” is a newly selected site located in an extended part of the protected area, in the far northern part of the protected area, with a total area of 503.53 ha. it is a karst field with pronounced forms of karst micro-relief, the natural characteristics of which are contained in the mixed forests of spruce and scots pine that are unique in the mokra gora area. also, numerous mires are important as habitats of priority for protection at the national and international levels. site “bratešina” is a newly declared site of 1,396.52 ha. this is the largest site in the protected area, which is located in its central part, whereas in its southeastern part there is a site of the first degree of protection “jelovac”. the basic vegetation of this site consists of forests of black pine (erico-pinetum nigrae (z. pavlović 1951) b. jovanović 1972 and euphorbio glabriflorae-pinetum nigrae pavlović, 1952) on initial humus-silicate soils on peridotites and serpentinites, as well as pine forests (ornoericion horvat, 1959 and orno-pinion em, 1978) on soils formed on base rocks. site “ograđenica 2” is a new site measuring 79.93 ha. part of this site was within the previously declared site in protection regime ii “tusto brdo božurica”, and is surrounded on the north, west and southwest side by a limestone massif, that is, the site in the protection regime i “ograđenica”. considering the geomorphological, vegetational and ambient features of the part of ograđenica, it represents a buffer zone around the area under protection regime i and it physically encircles the entire area of the massif. site “kanjon belog rzava” (beli rzav canyon) is a new site of 279.77 ha. it is located in the southernmost part of the protected area. in terms of vegetation, this site is very diverse, at higher elevations, there are the specimens of magnificent, single, black pine trees, as remnants of former natural black pine forests. site “šarganska osmica” (šargan eight) is a new site of 117.22 ha. it is located in the southeast part of the protected area. this site is overgrown with pine forests (orno-ericion horvat, 1959 and orno-pinion em, 1978) on soils formed on base rocks. the forests in this site are in one part natural biologica nyssana ● 11 (1) september 2020: 9-22 ostojić et al. ● natural values and concept of protection of the nature park “šargan-mokra gora” 19 tall black pine forest, while the rest of these forests are cultivated. the protected area under the protection regime iii occupies a total of 7,480.58 ha (65.73% of the total protected area). compared to the initial protection of the area from 2005, the areas under the protection regime iii were increased from 2,138.77 ha to 5,341.81 ha. conclusion due to its geographical location, geological bedrock, hydrographic features, orography, climate and historical development, the mokra gora and šargan area is characterized by a diversity of species and ecosystems of exceptional originality. orographic plasticity and the alternation of different geological bedrocks in a rather small area are certainly the most important reasons for the pronounced richness of flora and vegetation. the relatively small area of šargan and mokra gora is inhabited by 722 species of vascular flora, of which 6.2% of taxa belong to endemic and subendemic flora. there are 14 species in this area, which represent strictly protected species, as well as 88 protected species. from a scientific point of view, particular traits of this area are very rare endemic taxa adapted to the highly unfavorable serpentinitic bedrock. the largest number of endemics found in this area belongs to the group of balkan endemics. in terms of vegetation, the šargan and mokra gora area is a natural habitat of black pine and scots pine. these pines build pure and mixed stands, significant within large forest complexes. forest ecosystems of šargan mokra gora nature park consist of 10 representative communities, namely: alder forests and willows of the salicion albae soo (1930) 1940 alliance, gray willow community (salicetum eleagni moor 1958 em. oberdorfer 1962), turkey oak forests on thermophilic slopes (quercetum cerris e. vukićević1966), mesophilic forests of sessile oak and hornbeam (querco-carpinetum moesicae rudski (1940) 1949), type of sessile oak forest (quercetum montanum serpentinicum b. jovanović 1959), as well as the community of south european flowering ash and hop hornbeam (ostryo carpinifoliae-fraxinetum orni aichinger 1933). considering the pine forests, the most important are the two associations: pine forests with winter heath (erico-pinetum nigrae serpetinicum krause 1957) on serpentinite and black pine forest with hop hornbeam (ostryo-pinetum nigrae čolić 1965) on limestone. beech forests of the types fagetum moesiacае montanum bleč. et lakušić 1970 and fagetum submontanum (rudski 1949) b. jovanović 1976 are distributed on the limestones of tusto brdo. the mokra gora and šargan area is home to a rich and diverse wildlife. in the waters of this area, a total of six (6) fish species have been recorded, with five (5) species having protection status. only european bullhead has the status of a strictly protected species, while four (4) protected species are: brown trout, spirlin/common bleak, southern barbel/gudgeon and chub. in addition, 17 species of amphibians and reptiles have been recorded in the area of šargan and mokra gora. of these, 12 species are strictly protected, two species are protected, while three species do not have protection status. the ornithofauna significance is reflected in the presence of 120 bird species, of which 101 species are strictly protected and 18 species are protected. the protected area is also characterized by 49 species of mammals, which makes up about half the number of species registered so far in serbia. the šargan-mokra gora nature park, with its conserved natural landscapes, is a recognizable landscape through which runs the narrow-gauge railway šargan eight, which is widely known for its remarkable technical solution of overcoming a great altitude difference at a short distance. 10 years upon the establishment of spatial protection, the conducted research, revision and revaluation of the natural values of šargan and mokra gora, the boundaries of the protected area were expanded, the borders under the protection regimes were revised, new sites with the protection regimes i and ii were established, with the proposed total protected area of 11,379.78 ha. with this proposal, the area under protection regime i is 774.50 ha (6.81% of the total protected area) and is located in five isolated sites. under the protection regime ii, the proposed area occupies 3,124.70 ha (27.46% of the total protected area) and is located in ten isolated sites, while the area with the protection regime iii amounts to 7,480.58 ha (65.73% of the total protected area). the concept of protection of the area is aimed at conserving the overall diversity, autochthonous character and originality of species and habitats, the sustainable and controlled use of natural values and rarities. the concept is implemented through three protection regimes and legal measures defining certain regimes. the conservation guidelines defined in the conservation study (ostojić et al., 2015) aims to preserve and enhance the authenticity and autochthonous character of this area, which requires the cooperation of public institutions and civil society sectors. all the mentioned features of the šargan – mokra gora nature park make it one of the most important natural resources for biodiversity conservation in serbia. based on the valorization of the state of natural values, the area meets the criteria of the protected natural area in the nature park category. biologica nyssana ● 11 (1) september 2020: 9-22 ostojić et al. ● natural values and concept of protection of the nature park “šargan-mokra gora” 20 references cvijić, j. 1924: geomorfologija. morphologie terrestre, državna štamparija kraljevine srba, hrvata i slovenaca, beograd. cvijić, j. 1991: geomorfologija i. reprint državne štamparije iz 1924 godine, beograd, 1-558. dimitrijević, m. 1995: geologija jugoslavije. geoinstitut i barex, beograd, 1-205. dinić, a., tomić, z., mišić, v., tatić. b., janković, m., m., jovanović, b. 2006: vegetacija srbije ii 2. sanu, odeljenje hemijskih i bioloških nauka, beograd. jovanović, b. et al. (eds.) 1986: prodromus phytocenosum yugoslaviae ad mappam vegetationis 1:200.000. naučno veće vegetacijske karte jugoslavije, bribir-ilok. jovanović, b., mišić, v., dinić, a., diklić, n., vukićević, e. 1997: vegetacija srbije ii 1, sanu, odeljenje prir.-matem. nauka, beograd. jovanović, s., stevanović, v., jovanovićdunjić, r. 1992: contribution to the knowledge on the serpentine vegetation of serbia. glasnik prirodnjačkog muzeja u beogradu, b47: 43-51. jović, n., knežević, m. 1992: zemljišta u šumama sa drugim vrstama goča. manuskript. šumarski fakultet, str. 36-55, beograd. kalezić, m. i sar. 2015: crvena knjiga faune srbije i – vodozemci. zavod za zaštitu prirode srbije, beograd. komatina, m. 1977: oblast zapadne srbije, u geologija srbije; knj. hidrogeologija. rudarsko geološki fakultet, beograd, 122-147. komatina, m., čubrilović p. 1996: hidrogeološke odlike zlatibora; geologija zlatibora. geoinstitut, posebna izdanja , knj.18, beograd, 113-120. komatina, m., zlokolica-mandić m. 1998: bilans rezervi podzemnih voda teritorije srbije; 13. kongres geologa jugoslavije, knj. 5, hidrogeologija i inženjerska geologija, herceg novi, 469-478. kryštufek, b., milenković, m., rapaić ž., tvrtković, n. 1997: wild sheep and goats and their relatives (former yugoslavia). in: shackleton, d.m. (ed.): wild sheep and goats and their relatives status survey and conservation action plan for caprinae. 138-143. iucn/ssc caprinae specialist group. marinović, lj. 1997: potencijalna staništa za uzgoj divokoza u brdsko-planinskom područjima srbije. savetovanje savremeni aspekti gajenja, zaštite i korišćenja divljači u funkciji razvoja brdskoplaninskih područja jugoslavije. zbornik radova, 179-184. lovački savez jugoslavije, beograd. mojsilović, s., baklajić, d., đoković, i. 1977: tumač ogk 1:100 000 list titovo užice k34-4. savezni geološki zavod, beograd. mojsilović, s., baklajić, d., đoković, i., avramović, v. 1978: tumač ogk 1:100 000 list titovo užice k34-4, savezni geološki zavod, beograd.olujić, j, karović j. 1986a: ogk 1:100 000 i tumač za ogk sfrj, list višegrad. savezni geološki zavod, beograd. olujić, j, karović j. 1986b: ogk 1:100 000 i tumač za ogk sfrj, list višegrad. savezni geološki zavod, beograd. ostojić, d. 2007: šumski ekosistemi predela izuzetnih odlika „šargan-mokra gora“, zaštita, očuvanje, unapređenje i korišćenje. prirodne i društvene vrijednosti ekosistema dinarida, posvećen životu i djelu profesora dr. radomira lakušićameđunarodni naučni skup, berane, andrijevica, plav, 1-25. ostojić, d. 2017: zaštićena prirodna dobra srbije, iii izdanje: prilog o parku prirode šargan-mokra gora, str.55-57. zavod za zaštitu prirode srbije, beograd. ostojić, d., krasulja, s., đorđević, z., belij, zlatković, b., bjedov, v., sekulić, n., andonović, j., mijović, d., s., grubač, b., sekulić, g., jović, d., jovanović, b., ajtić, r. 2005: predlog za zaštitu prirodnog dobra „šargan-mokra gora“ kao predela izuzetnih odlika, beograd. ostojić, d., krvavac, m. 2017: park prirode šargan-mokra gora. zavod za zaštitu prirode srbije, beograd. ostojić, d., nikolić, v., krvavac, m., radaković, m., stojanović,v., jelić, i., tošić, s., pejić, m., bjedov, v., kličković, m. 2015: studija zaštite park prirode šargan-mokra gora, zavod za zaštitu prirode srbije, beograd. pančić, j. 1859: die flora der serpentinberge in mittel serbien. verhandlungen der zoologischbotanischen gesellschaft in österreich, wien, 9: 139-150. pančić, j. 1874: flora kneževine srbije. državna štamparija beograd. pančić, j. 1884: dodatak flori kneževine srbije. kraljevsko-srpska državna štamparija beograd. paunović m., milenković m. 1996: the current status and distribution of the otter lutra lutra biologica nyssana ● 11 (1) september 2020: 9-22 ostojić et al. ● natural values and concept of protection of the nature park “šargan-mokra gora” 21 l., 1758 in serbia and montenegro. iucn otter specialist group bulletin, 13 (2): 71-76. paunović, m., karapandža, b., ivanović, s. 2011: slepi miševi i procena uticaja na životnu sredinu – metodološke smernice za procenu uticaja na životnu sredinu i stratešku procenu uticaja na životnu sredinu. društvo za očuvanje divljih životinja „mustela“, beograd, 142 p. petrov, b. 1992: mammals of yugoslavia insectivores and rodents. natural history museum in belgrade, special issues, 37, belgrade. 186 p. puzović, s., sekulić, g., stojnić, n. grubač, b., tucakov, m. 2009: značajna područja za ptice u srbiji. ministarstvo životne sredine i prostornog planiranja. zavod za zaštitu prirode srbije. beograd. radovanović, m. 1951: vodozemci i gmizavci naše zemlje. naučna knjiga, beograd. rajzer, o. 1906: izveštaj o uspjehu ornitoloških putovanja u srbiji godine 1899. i 1900. glasnik zemaljskog muzeja u bosni i hercegovini, sarajevo, xvi: 125–152. savić, i., paunović, m., milenković, m., stamenković, s. 1995: diverzitet faune sisara (mammalia) jugoslavije, sa pregledom vrsta od međunarodnog značaja. u: stevanović, v., vasić, v. (eds.): biodiverzitet jugoslavije sa pregledom vrsta od međunarodnog značaja, 517-554. biološki fakultet i ecolibri. beograd. savić, i.r., milenković, m., paunović, m., ćirović, d., stamenković, s. 1997: diversity of carnivora in serbia. biodiversity and ecological problems of balkan fauna abstracts, 43. sofia. stevanović, v. (ed.) 1999: crvena knjiga flore srbije 1. ministarstvo za životnu sredinu republike srbije, biološki fakultet u beogradu, zavod za zaštitu prirode srbije. beograd. stevanović, v. (ed.) 2002: preliminarna crvena lista vaskularne flore srbije i crne gore – prema kriterijumima iucn-a iz 2001 godine, beograd. [manuscript]. stevanović, v. (ed.) 2002: preliminarna lista ugroženih taksona flore srbije. biološki fakultet, ecolibri, beograd. stevanović, v., vasić, v. (eds.) 1995: biodiverzitet jugoslavije sa pregledom vrsta od međunarodnog značaja. biološki fakultet i ekolibri, beograd. biologica nyssana ● 11 (1) september 2020: 9-22 ostojić et al. ● natural values and concept of protection of the nature park “šargan-mokra gora” šćiban, m., rajković, d., radišić, d., vasić, v., pantović, u. 2015: ptice srbije – kritički spisak vrsta. pokrajinski zavod za zaštitu prirode i društvo za zaštitu i proučavanje ptica srbije, novi sad. tomović, lj. i sar. 2015: crvena knjiga faune srbije ii – gmizavci. zavod za zaštitu prirode srbije, beograd. visiani, r., pancic, j. 1862: plantae serbicae rariores aut novae. memorie dell i.r. instituto veneto di scienze, lettere ed arti, 10: 3-26. visiani, r., pancic, j. 1862: plantae serbicae rariores aut novae ii. memorie dell i.r. instituto veneto di scienze, lettere ed arti, 12: 1-18. visiani, r., pancic, j. 1862: plantae serbicae rariores aut novae iii. memorie dell i.r. instituto veneto di scienze, lettere ed arti, 15: 3-21. pravilnik o proglašenju i zaštiti strogo zaštićenih i zaštićenih divljih vrsta biljaka, životinja i gljiva („službeni glasnik rs“ br. 5/2010, 47/2011, 32/2016 i 98/2016). pravilnik o kriterijumima vrednovanja i postupku kategorizacije zaštićenih područja („službeni glas rs“, br. 103/2013 i br. 97/2015). pravilnik o prekograničnom prometu i trgovini zaštićenim vrstama („službeni glas rs“, br. 99/2009 i br. 6/2014). prostorni plan područja posebne namene nacionalni parka „tara“ („službeni glasnik rs“, br. 100/2010). uredba o stavljanju pod kontrolu korišćenja i prometa divlje flore i faune („službeni glasnik rs“, br. 31/2005, 45/2005, 22/2007, 38/2008, 9/2010, 69/2011 i 95/2018-drugi zakon). uredba o zaštiti prirodnih retkosti („službeni glasnik rs“, br. 53/93, 93/93) uredba o zaštiti predela izuzetnih odlika „šargan mokra gora” („službeni glasnik rs“, br. 52/2005). uredba o zaštiti parka prirode „šargan mokra gora” („službeni glasnik rs“, br. 81/2008). zakon o divljači i lovstvu („službeni glasnik rs“, br. 18/2010). zakon o zaštiti prirode („službeni glasnik rs“, br. 36/09, 88/10, 91/10ispravka i 14/16). 22 xhulaj, 2019, biologica nyssana 10(2) 10 (2) december 2019: 155-158 doi: 10.5281/zenodo.3600193 preliminary data on lichens from albanian alps (razëm locality, northern albania) original article skerdilaid xhulaj research center of flora and fauna, faculty of natural sciences, university of tirana, bulevardi zog i, nr. 25/1, tirana, albania skerdilaid.xhulaj@fshn.edu.al (corresponding author) received: october 7, 2019 revised: december 3, 2019 accepted: december 6, 2019 abstract: a list of 82 taxa of lichens collected in the albanian alps in two nearby localities is presented here. more than half of them (58 taxa) represent new record for the investigation area. key words: albanian alps, biodiversity, lichens, new records apstract: preliminarni podaci o lišajevima sa albanskih alpa (razëm locality, northern albania) prikazan je spisak od 82 vrste lišajeva prikupljenih u albanskim alpima na dva obližnja lokaliteta. više od polovine njih (58 vrsta) predstavlja novi nalaz za područje istraživanja. ključne reči: albanski alpi, biodiverzitet lišajevi, novi nalazi introduction studies on albanian lichen flora have been and remained low in number. most of these studies have been conducted by foreign authors, increasingly highlighting the need for local specialists in this field. based on historical data, the earliest works belong to körber (1867) and zahlbruckner (1897), who provided information on areas now outside the borders of albania and belonging to montenegro, kosovo and northern macedonia (hafellner & kashta, 2003). data on some specimens of lichens, collected by j. b. kümmerle in the northeastern part of the country, are published by szatala & timko (1926). during his visit in 1924 and 1928, markgraf, while studying albanian vegetation, also collected lichens, most of which were identified by j. hillmann and mentioned by markgraf (1927, 1931) as part of his herbarium. the first important work on albanian lichen flora comes from hafellner & kashta (2003), which provides about 137 taxa of lichens, collected mainly in the north of the country. hafellner (2007), summarizes a list of species reported up to that time for albania by various authors. his list included about 191 taxa of lichens. this list has occasionally been enriched with scarce data from various authors such as: llop et al. (2007), mayrhofer & sheard (2007), hafellner (2009), obermayer (2009), kukwa (2011) and hafellner & zimmermann (2012) (svoboda et al., 2012). the most complete information so far on albanian lichen flora appears to be that of svoboda et al. (2012). of the 43 areas visited within this study, a list of 333 lichen taxa was compiled, 205 of which reported for the first time for albania (svoboda et al., 2012). despite studies and reports to date, the lichen flora of our country, based on climatic and geological conditions, is represented by an even greater number of species. in this aspect, the paper is a contribution to the recognition and enrichment of albanian lichen flora. materials and methods specimens were collected during field trips carried out in kastrat, razëm (malësi e madhe district). the collected specimens were initially divided by growth form and within the growth form were further grouped by the type of their fruiting bodies. lichens were determined by standard microscopic methods (stereo and microscope) and relevant literature: poelt (1969), poelt & vězda (1977, 1981), clauzade & roux (1985), wirth (1995), nimis & © 2019 xhulaj. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 155 13th symposium on the flora of southeastern serbia and neighboring regions martellos (2004), nimis & martellos (2008), smith et al. (2009) and wirth et al. (2013). the collected specimens are deposited in the author’s personal herbarium. the following provides more detailed data on two of six localities where the study was conducted. specimens from other localities, specifically 1, 2, 3 and 6 are still being analyzed in the laboratory and identification is ongoing, so therefore they have not been included here. data about localities locality 4: kastrat, northern albania, malësia e madhe district, c. 3 km from razëm village, beech forest, 42°20’45”n/19°32’52”e, alt. c. 1080 m, 23.04.2018. locality 5: kastrat, northern albania, malësia e madhe district, c. 1.5 km from vila alpini, beech forest, 42°20’24.47’’n/ 19°32’43.56’’e, alt. c. 1112m, 23.04.2018. list of substrates and their abbreviations cor: on bark (used in the case of phorophyte not identified) cal: on limestone ter-cal: on calcareous soil bry: on bryophytes fag: on fagus sylvatica results preliminary results concluded in 82 taxa which have been determined so far. 61 taxa were recorded on trees. a few stones, comprising 14 saxicolous lichens, were picked up together with common terricolous species, 21 in all. species are arranged alphabetically. numbers 4 and 5 correspond to the localities listed above. the abbreviations following this number correspond to the substrates listed also above. author’s initials and numbers in brackets show individual consecutive numbering systems used by the author to identify and locate individual specimens. an asterisk (*) indicates a new record for the investigation area. list of species anaptychia ciliaris (l.) körb. 4, 5 cor (sx 4021, 5001) *arthonia lapidicola (taylor) branth & rostr. 5 cal (sx 5003) *arthonia radiata (pers.) ach. 4, 5 cor (sx 4002, 5004) *aspicilia calcarea (l.) mudd 5 cal (sx 5005) *athallia holocarpa (hoffmann) arup, frödén & søchting 4 cor (sx 4003) *bagliettoa marmorea (scop.) gueidan & cl. roux 4, 5 cal (sx 4004, 5006) *buellia poeltii schauer (turner & borrer ex sm.) almb. 4, 5 fag (sx 4005, 5007) *caloplaca aurantia (pers.) hellb. 5 cal (sx 5008) caloplaca cerina (ehrh. ex hedw.) th.fr. var. cerina 4, 5, cor (sx 4005, 5008) *caloplaca cerinelloides (erichsen) poelt 4, 5 cor (sx 4006, 5010) caloplaca ferruginea (huds.) th. fr. 4 cor (sx 4007) *caloplaca flavorubescens (huds.) j.r. laundon 5 cor (sx 5012) *caloplaca haematites (chaub. ex st.amans) zwackh 5 cor (sx 5013) candelariella vitellina (hoffm.) müll. arg. 4, 5 cor (sx 4007, 5018) candelariella xanthostigma (ach.) lettau 4, 5 cor (sx 4009, 5020) candelariella sp. 4 cor (sx 4010) *catillaria nigroclavata (nyl.) schuler 4 cor (sx 4014) *catinaria atropurpurea (schaer.) vězda & poelt 4 fag (sx 4015) *circinaria contorta (hoffm.) a. nordin, s. savic & tibell 5 cal (sx 5022) *cladonia convoluta (lam.) p. caut. 5 ter-cal (sx 5023) cladonia furcata (huds.) schrad. 5 ter-cal (sx 5024) cladonia rangiformis hoffm. 4 ter-cal (sx 4020) collema subflaccidum degel. 4 cor (sx 4025) *collema subnigrescens degel. 4, 5 cor (sx 4025, 5031) *collema tenax (sw.) ach. 4, 5 cal (sx 4026, 5032) *haematomma ochroleucum var. ochroleucum (neck) j.r. laundon 4, 5 fag (sx 4027, 5034) *hypogymnia tubulosa (schaer.) hav. 4 cor (sx 4028) lathagrium cristatum (l.) otálora, p.m. jørg. & wedin 4, 5 cal (sx 4029, 5035) *lecania cyrtella (ach.) th. fr. 4, 5 cor (sx 4041, 5038) *lecania koerberiana j. lahm 4 cor (sx 4042) *lecanora allophana nyl. 4 cor (sx 4043) 156 biologica nyssana ● 10 (2) december 2019: 155-158 xhulaj ● preliminary data on lichens from albanian alps (razëm locality, northern albania) *phaeophyscia endophoenicea (harm.) moberg 4, 5 cor (sx 4071, 5061) *phaeophyscia orbicularis (neck.) moberg 4, 5, cor (sx 4072, 5062) *phaeophyscia poeltii (frey) clauzade & cl. roux 5 cor (sx 5063) *phlyctis argena (sprengel) flotow 4, 5 cor (sx 4082, 5065) *physcia adscendens (fr.) h.olivier 4, 5, cor (sx 4083, 5066) physcia aipolia (ehrh. ex humb.) fürnr. 4 cor (sx 4084) *physcia biziana (a.massal.) zahlbr. 4 cor (sx 4085) *physcia dubia (hoffm.) lettau 5 cor (sx 5067) *physcia semipinnata (j.f.gmel.) moberg. 4 cor (sx 4086) *physma omphalarioides (anzi) arnold 4, 5 cor (sx 4087, 5068) pseudevernia furfuracea (l.) zopf 5 cor (sx 5081) *ramalina farinacea (l.) ach. 5 cor (sx 5085) *ramonia luteola vězda 4, 5 fag (sx 4092, 5087) *rinodina albana (a. massal.) a. massal. 5 cor (sx 5088) *rinodina pyrina (ach.) arnold 4, 5 cor (sx 4094, 5090) *rinodina sophodes (ach.) massal. 4, 5 cor (sx 4095, 5091) *scoliciosporum umbrinum (ach.) arnold 4, 5 fag (sx 4096, 5092) squamarina cartilaginea (with.) p. james 5 ter-cal (sx 5100) *staurothele caesia (arnold) arnold 5 cal (sx 5101) *variospora velana (a. massal.) arup, søchting & frödén 4, 5 cal (sx 4100, 5102) verrucaria muralis ach. 5 cal (sx 5103) verrucaria nigrescens pers. 4, 5 cal (sx 4101, 5104) xanthoria parietina (l.) th. fr. 4, 5 cor (sx 4105, 5108) *xanthoria polycarpa (hoffm.) rieber 4, 5 cor (sx 4106, 5109) discussion the findings show many aspects. pectenia atlantica, peltigera collina, nephroma laevigatum, nephroma parile, leptogium saturnium, lecanora intumescens, ramonia luteola, catinaria artropurpurea show that the forest is primeval. the two last mentioned lecanora carpinea (l.) vain. 4 cor (sx 4044) *lecanora chlarotera nyl. 5 cor (sx 5040) *lecanora coiliocarpa (ach.) nyl. 4, 5 cor (sx 4045, 5041) *lecanora hagenii (ach.) ach. 5 cor (sx 5042) *lecanora intumescens (rebent.) rabenh. 4 fag (sx 4046) *lecanora leptyrodes (nyl.) degel. 5 cor (sx 5043) *lecanora muralis (schreb.) rabenh. 5 cal (sx 5044) *lecanora subfuscata f. variolosa (körb.) grummann 4, 5 cor (sx 4047, 5045) *lecidella elaeochroma (ach.) m. choisy 5 cor (sx 5046) lecidella euphorea (flörke) hertel 4 cor (sx 4048) *leptogium massiliense nyl. 4 cal (sx 4049) leptogium saturninum (dickson) nyl. 4, 5 cor (sx 4050, 5047) *lobothallia radiosa (hoffm.) hafellner 5 cal (sx 5048) melanelixia glabra (schaer.) o.blanco et al. 4 cor (sx 4051) *nephroma laevigatum ach. 4, 5 bry (sx 4061, 5050) nephroma parile (ach.) ach. 5 cor (sx 5051) *ochrolechia alboflavescens (wulfen) zahlbr. 4, 5 cor (sx 4062, 5052) *parmelia atlantica ach. 4 cor (sx 4063) *parmelia exasperata de not. 5 cor (sx 5053) parmelina pastillifera (harm.) hale 5 cor (sx 5054) *parvoplaca cf. suspiciosa (nylander) arup, søchting & frödén 4, 5 cor (sx 4064, 5055) pectenia atlantica (degel.) p.m. jørg. 4, 5 cor (sx 4065, 5056) *peltigera collina (ach.) schrad. 4, 5 fag (4066, 5057) *pertusaria albescens var. corallina (zahlbr.) j.r. laundon 4, 5 cor (sx 4067, 5058) *pertusaria hymenea (ach.) schaer. 4 cor (sx 4068) pertusaria pertusa (l.) tuck. 4, 5 cor (sx 4069, 5059) *phaeophyscia ciliata (hoffm.) moberg 4, 5 cor (sx 4070, 5060) 157 biologica nyssana ● 10 (2) december 2019: 155-158 xhulaj ● preliminary data on lichens from albanian alps (razëm locality, northern albania) species also show an alpine feature together with lecanora leptyrodes, buellia poeltii, phaeophyscia poeltii, and lecanora coiliocarpa. an oceanic feature is disclosed by physma omphalarioides, collema subnigrescens and parmelia atlantica. especially the habitation, but also a road through the area bring about the presence of physcia, xanthoria and rinodina species, and lecanora allophana. everywhere in the mountain area a rather common species on deciduous trees was a parvoplaca cf. suspicosa which is a scandinavian species (hitherto found only in the north of sweden). presumably the specimens found in razëm represent some mediterranean alpine species. some taxa have not been finally determined. by defining all the taxa found, a more complete interpretation of the lichen flora of this area can be made. references clauzade, g., roux, c. 1985: likenoj de okcidenta eŭropo. ilustrita determinlibro. bulletin de la société botanique du centre-ouest. nouvelle série. numéro spécial, 7: 1-893. hafellner, j., kashta, l. 2003: miscellaneous records of lichens and lichenicolous fungi from albania. herzogia, 16: 135-142. hafellner, j. 2007: checklist and bibliography of lichenized and lichenicolous fungi so far reported from albania (version 05-2007). fritschiana (graz), 59: 1-18. hafellner, j. 2009: phacothecium resurrected and the new genus phacographa (arthoniales) proposed. bibliotheca lichenologica, 100: 85-121. hafellner, j., zimmermann, e. 2012: a lichenicolous species of pleospora (ascomycota) and a key to the fungi invading physcia species. herzogia, 25: 47-59. körber, g. w. 1867: lichenen aus istrien, dalmatien u. albanien. gesammelt vom k. k. corvettenarzte dr. em. weiss. verhandlungen der kaiserlichköniglichen zoologisch-botanischen gesellschaft in wien, 17: 611-618. kukwa, m. 2011: the lichen genus ochrolechia in europe. gdánsk: fundacja rozwoju uniwersytetu gdánskiego. 309 pp. llop, e., ekman, s., & hladun, n. l. 2007: bacidia thyrrenica (ramalinaceae, lichenized ascomycota), a new species from the mediterranean region, and a comparison of european members of the bacidia rubella group. nova hedwigia, 85: 445-455. markgraf, f. 1927: an den grenzen des mittelmeergebietes. pflanzengeographie von mittelalbanien. feddes repertorium beiheft, 45: 1-217, vegetationskarte. 158 markgraf, f. 1931: pflanzen aus albanien 1928. denkschriften der kaiserlichen akademie der wissenschaften / mathematisch-naturwissenschaftliche classe, 102: 317-360, tab. mayrhofer, h., sheard, j. w. 2007: rinodina archaea (physciaceae, lichenized ascomycetes) and related species. bibliotheca lichenologica, 96: 229246. nimis, p. l., martellos, s. 2004: keys to the lichens of italy. vol. 1: terricolous species. edizioni goliardiche, trieste: 346 pp. nimis, p. l., martellos, s. 2008: italic the information system on italian lichens. version 4.0. university of trieste. deparrtment of biology, in4.0/1 (http://dbiodbs.univ.trieste.it/) obermayer, w. 2009: dupla graecensia lichenum (2009, nos 581-680). fritschiana (graz), 65: 7-32. poelt, j. 1969: bestimmungsschlüssel europäischer flechten. lehre: cramer, 757 pp. poelt, j., vězda, a. 1977: bestimmungsschlüssel europäischer flechten. ergänzungsband i. bibliotheca lichenologica, 9: 1-258 poelt, j., vězda, a. 1981: bestimmungsschlüssel europäischer flechten. ergänzungsband ii. bibliotheca lichenologica 16: 1-390 smith, c.w., aptroot, a., coppins, b.j., fletcher, a., gilbert, o.l., james, p.w., wolse-ley, p.a. 2009: the lichens of great britain and ireland. the british lichen society & the natural history museum, london, 1046 pp. svoboda, d., bouda, f., malíček, j., hafellner, j. 2012: a contribution to the knowledge of lichenized and lichenicolous fungi in albania. herzogia, 25: 149-165. szatala, ö., timko, g. 1926: additamenta ad floram albaniae. iv. lichenes. magyar tudományos akadémia balkán-kutatásainak tudományos eredményei, budapest, 3: 159-179 wirth, v. 1995: flechtenflora. bestimmung und ökologische kennzeichnung der flechten südwestdeutschlands und angrenzender gebiete. 2. auflage. stuttgart: eugen ulmer: 661 pp wirth, v., hauck, m., schultz, m. 2013: die flechten deutschlands. 2 bände. stuttgart: eugen ulmer: 1244 pp zahlbruckner, a. 1897: lichenes albanici a cl. j. dörfler anno 1893 lecti. rep. krypt. lit. beibl. hedwigia 36: 1-4. biologica nyssana ● 10 (2) december 2019: 155-158 xhulaj ● preliminary data on lichens from albanian alps (razëm locality, northern albania) pejčić et al. 2021, biologica nyssana 12(1) 12 (1) september 2021: 47-54 doi: 10.5281/zenodo.5522995 antimicrobial efficacy of basil and sage essential oils against pseudomonas aeruginosa: time-lapse kinetics and type of interaction with ciprofloxacin original article milica pejčić department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, niš, serbia milicapejcic1991@gmail.com (corresponding author) zorica stojanović-radić department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, niš, serbia zstojanovic@pmf.ni.ac.rs marina dimitrijević department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, niš, serbia dimitrijevicmarina92@yahoo.com niko radulović department of chemistry, faculty of sciences and mathematics, university of niš, višegradska 33, niš, serbia nikoradulovic@yahoo.com received: october 21, 2020 revised: february 16, 2021 accepted: february 17, 2021 abstract: the increasing resistance of pseudomonas aeruginosa strains to used antibiotics is a big problem of modern medicine. finding antimicrobial substances which will increase the antibiotics effects is necessary. therefore, the aim of the present study was to evaluate the time-lapse activity of the basil and sage oils, to evaluate the combined oils effect against p. aeruginosa clinical isolates and to determine the oils potential to enhance the activity of ciprofloxacin. the obtained growth curves showed a reduction in the number of bacterial cells in the range of 10.5-94% when basil oil was applied. in the case of the sage oil, higher reduction has been observed (48.5-100%). the basil oil achieved a synergistic interaction with ciprofloxacin in 8 isolates, while the same effect for sage/ciprofloxacin combination was observed in 6 isolates. indifferent effect was noticed in 64% of the tested isolates for basil/sage combination. these data showed potential benefits of the oils and ciprofloxacin combination therapy in the treatment of p. aeruginosa infections. key words: basil oil, sage oil, time-lapse activity, synergy, ciprofloxacin, pseudomonas aeruginosa apstract: antimikrobna efikasnost etarskih ulja bosiljka i žalfije na pseudomonas aeruginosa: vremenska kinetika i tip interakcije sa ciprofloksacinom povećanje rezistencije sojeva pseudomonas aeruginosa na dosad korišćene antibiotike predstavlja veliki problem u medicini. neophodno je pronaći antimikrobne supstance koje će povećati efikasnost antibiotika. zbog toga je cilj ovog istraživanja bio analiza vremenske kinetike ulja bosiljka i žalfije, analiza kombinovanog efekta ispitivanih ulja na kliničke izolate p. aeruginosa i određivanje potencijala ulja u povećanju aktivnosti ciprofloksacina. dobijene krive rasta pokazale su smanjenje broja bakterijskih ćelija u rasponu od 10,5-94% prilikom primene ulja bosiljka. u slučaju ulja žalfije primećeno je smanjenje broja ćelija u rasponu 48,5-100%. ulje bosiljka pokazalo je sinergističku interakciju sa ciprofloksacinom na 8 testiranih izolata, dok je isti efekat kombinacije žalfija/ciprofloksacin primećen kod 6 izolata. indiferentni efekat uočen je kod 64% testiranih izolata za kombinaciju bosiljak/žalfija. ovi rezultati su pokazali potencijalne koristi kombinovane terapije ulja i ciprofloksacina u lečenju infekcija izazvanih vrstom p. aeruginosa. ključne reči: ulje bosiljka, ulje žalfije, vremenska kinetika, sinergizam, ciprofloksacin, pseudomonas aeruginosa introduction the increase of resistance between the bacterial strains caused by incorrect or over used antimicrobial therapy is becoming a big problem in modern medicine these days. antimicrobial resistance is a natural evolutional phenomenon where microbes develop resistance against commonly used antibiotics or antimicrobial drugs by different adaptation mechanisms. owing to increased spreading, the world health organization placed antimicrobial resistance among the top 10 urgent threats for the year 2019 (yu et al., 2020). among these bacteria, pseudomonas aeruginosa is one of the most frequent multiresistant species and becomes a major concern in public health (el-hosseiny et al., 2014; alhazmi, 2015). therefore, it is necessary to find same antimicrobial substances which will increase © 2021 pejčić et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 47 the antimicrobial potential of existing drugs, and help in overcoming the bacterial resistance. the last few decades are marked by a large number of studies on antimicrobial (akthar et al., 2014; calo et al., 2015; tariq et al., 2019), antibiofilm and anti-virulence (artini et al., 2018; reichling, 2020) activity of essential oils (eos) and their components. the use of drug combinations in antibacterial treatment rather than single antimicrobial agents provides better results, because the use of a single agent is highly associated with resistance occurrence. due to the complexity in chemical composition, no particular bacterial adaptation to essential oils has been described (mikulášová et al., 2016). owing to the existing antimicrobial potential, combinations between different antibiotics and other antimicrobial substances, such as eos, represent one of the most promising advances against drugresistant microorganisms. the interaction between antimicrobials in combinations can result in three different outcomes: synergistic, additive, or antagonistic effect (chouhan et al., 2017). in recent years, there have been many studies that investigated the increasing effect of eos and their components to antimicrobial potential of conventional drugs. these studies have been performed on a number of pathogenic bacteria and p. aeruginosa was among them (hemaiswarya & doble, 2009; van vuuren et al., 2009; langeveld et al., 2014; yap et al., 2014; aelenei et al., 2016; chouhan et al., 2017). the antimicrobial potential of salvia officinalis (sage) and ocimum basilicum (basil) essential oils against different pathogenic bacteria is already well known (delamare longaray et al., 2007; bassolé et al., 2010; fournomiti et al., 2015; tardugno et al., 2018). furthermore, different interactions between the sage oil and some conventional antibiotics (elhosseiny et al., 2014; adrar et al., 2016), as well as interactions between the basil oil and antibiotics (mahmoud et al., 2016; silva et al., 2016) against p. aeruginosa have been reported. based on our previous work (pejčić et al., 2020), we decided to broaden our investigations on the basil (ocimum basilicum l.) and sage (salvia officinalis l.) essential oils, which demonstrated the antibacterial efficacy, as well as potential to reduce the virulence factors (biofilm formation, mature biofilm resistance, motility, and pyocyanin production) of the p. aeruginosa clinical isolates. therefore, the aim of the present study was: a) to evaluate the time-lapse activity of the tested oils; b) to evaluate the combined effects of basil and sage essential oils, used in mixture against p. aeruginosa clinical isolates; and c) to determine the potential of the tested oils to enhance the activity of antibiotic ciprofloxacin. materials and methods essential oils the essential oils of sage (salvia officinalis l. (lamiaceae), montes, leskovac) and basil (ocimum basilicum l. (lamiaceae), mygreenway, niš) were purchased at a local herbal pharmacy (beyond, niš). the minimal inhibitory concentrations (mics) and chemical composition of the oils were studied in detail and compared to the literature data in our previous work (pejčić et al., 2020). used microorganisms strains used in this study were isolated from different human material, identified as p. aeruginosa, phenotypically characterized and presented in our previous work (pejčić et al., 2020). total of 13 isolated p. aeruginosa strains were stored at the microbiology laboratory, department of biology and ecology, faculty of sciences and mathematics, university of niš. for the experiments, the investigated strains were grown on nutrient agar (na) for 18 h and used to prepare suspensions in physiological saline (0.85% nacl) that corresponded to the 0.5 mcfarland turbidity standard (108 cells/ ml). the suspension turbidity was adjusted by using a mcfarland densitometer (den-1 mcfarland densitometer, biosan). time-kill curve the activity of essential oils against the bacterial strains over time was determined by using the time-kill assay. the initial bacterial suspension was adjusted in mueller hinton broth to achieve the final cell concentrations of 5x106 cfu/ml. the effect of oil in mics concentrations previously determined (pejčić et al., 2020) on bacterial growth was monitored via measurement of turbidity changes 37 °c during 48 h at 600 nm (stojanović-radić et al., 2012). checkerboard assay the antimicrobial agents (basil oil, sage oil and ciprofloxacin) were combined with each other and their interaction against p. aeruginosa clinical isolates was investigated using a checkerboard method (stojanović-radić et al., 2020). three combinations were tested: basil oil/ciprofloxacin; sage oil/ciprofloxacin, and basil oil/sage oil. each antibacterial agent was tested in mics (previously determined) and several concentrations below the mics. the test was performed in 96-well microtiter plates, where serial dilutions of each agent were made in horizontal and vertical rows, which resulted in various combinations of both agents. the stocks of tested essential oils were prepared in 100% dmso 48 biologica nyssana ● 12 (1) september 2021: 47-54 pejčić et al. ● antimicrobial efficacy of basil and sage essential oils against pseudomonas aeruginosa: time-lapse kinetics... 49 and then diluted with mhb. the combination of essential oils and antibiotics was prepared by adding 25 µl of each to the same well of microtiter plate and inoculating with 150 µl of bacterial suspension (5x106 cfu/ml), afterwards the plates were incubated at 37 °c for 24 h. the interaction between the two antimicrobial agents was estimated by calculating the fractional inhibitory index (fici). the calculation of fici was done using the following equations: fica = mica-b/ mica ficb = micb-a/ micb fici = fica + ficb where fica is the fractional inhibitory concentration of antimicrobial agent a, mica-b is mic of the agent a in combination with agent b, mica is mic of agent a alone, ficb is the fractional inhibitory concentration of agent b, micb-a is mic of agent b in combination with the agent a, and micb is mic of agent b alone. fici was interpreted as a synergistic effect when its value was ≤ 0.5, as an additive 0.5 < fici ≤ 1, indifferent effect when fici was 1 < fici ≤ 2, and as an antagonistic interaction when it was < 2 (stojanović-radić et al., 2020). statistical analysis all experiments were done in triplicate and data are given as average values ± standard deviations. statistical comparison was done using two way anova followed by tuckey post hoc test (graphpad prism version 5.03, san diego, ca, usa). probability values (p) less than 0.05 were considered to be statistically significant. results and discussion as a continuation of our previous investigation (pejčić et al., 2020), where the basil and sage oils antibacterial and anti-virulence potential were determined, the present work reports the timelapse activity of the tested oils and interactions of combined antimicrobial agents (basil, sage oil and ciprofloxacin) against p. aeruginosa clinical isolates. growth curve our previous investigation (pejčić et al., 2020) of the herein tested oils antimicrobial activities showed the moderate action of both oils in the range of 5.0–20.0 mg/ml, depending on the isolated strain. the mics were further tested to investigate the activities of oils during the total incubation period of 48 h, and the results are presented as a growth curve (fig. 1). inhibition of bacterial growth was observed during the first 21 h of the treatments, after which very slight recovery was detected, but the oils significantly reduced total population number. following the incubation period of 48 h in total, the basil oil exhibited growth inhibition in the range 10.5-94%. the highest activity has been achieved against isolate no. 7, where reduction of 93.95% was observed after 48 h. on the other hand, the isolate no. 4 showed somewhat higher resistance in comparison to the others, where inhibition of only 10.5% was measured. in the case of the sage oil, higher reduction has been observed (48.5100%). the most prominent action of the sage oil was detected against several isolates, namely no. 4, no. 7, no. 11 and no. 13, where 100% of growth reduction was measured. the sage oil effectively inhibited regrowth of isolates no. 3, no. 4, no. 6, no. 7 and no. 11 (bacteriostatic effect). after total incubation period, ld50 (reduction over 50% of the total population size) was achieved in 92% (sage oil) and 53% (basil oil) of the tested isolates, which demonstrated higher time-lapse efficacy of the sage oil. according to our previous results, the oils showed similar potential considering antimicrobial action, as well as virulence factors-inhibiting potential (pejčić et al., 2020). however, in the present study, higher effect over time is visible in the case of the sage essential oil. studies reported that the antimicrobial activities of the essential oils were due to the presence of some major and minor constituents and some type of interaction between many components (miticculafic et al., 2005; van vuuren & viljoen, 2007; bassolé et al., 2010). salvia officinalis essential oils containing α-thujone (24%) and camphor (10%) (delamare longaray et al., 2007) or 1,8 cineol as a major compounds (el-hosseiny et al., 2014) failed to show activity against p. aeruginosa. in another study, where time-lapse effect of the sage oil was investigated, a very low efficacy against pseudomonas aeruginosa (atcc 27853) was observed after the total incubation period of 24 h. each tested concentration (5, 10 and 20 µl) affected only limited number of cells after 10 minutes and 1 h, which resulted in unchanged population size after total incubation period (khalil & li, 2011). the mentioned oil was characterized by 1,8cineole (62%) as a major component while the herein tested oil had different chemical composition with α-thujone (29.7%), camphor (16.6%), and 1, 8 cineole (9.2%) as the dominant compounds (pejčić et al., 2020). this is probably the reason for better activity of the herein tested oil. on the other hand, salvia officinalis essential oil with very similar content (α-thujone 37%, camphor 14%, and 1,8 cineole 14%) to the oil used in the present study biologica nyssana ● 12 (1) september 2021: 47-54 pejčić et al. ● antimicrobial efficacy of basil and sage essential oils against pseudomonas aeruginosa: time-lapse kinetics... 50 showed promising results on the kinetics of survival of staphylococcus aureus and bacillus subtilis. sage oil and fractions with high content of α-thujone exhibited a strong bactericidal effect on s. aureus, and within 4 hours the bacterial population was completely inactivated. on the other hand, fractions containing other dominant compounds (camphor and 1,8 cineole) exhibited lower effect, demonstrating that α-thujone presents the carrier of antimicrobial activity (mitic-culafic et al., 2005). previous studies on the basil oil efficacy against p. aeruginosa reported mics from 5-40 mg/ml, (hammer et al., 1999; niculae et al., 2009; pejčić et al., 2020). however, these studies have not performed the time kill assays which would allow us to compare the basil’s efficacy against p. aeruginosa strains over the time. on the other hand, data for the time-kill kinetic against one s. aureus mrsa clinical isolate in the presence of basil oil showed decreased cell number in the first 30 min and this reduction continues until the end of incubation period of 24 h (opalchenova & obreshkova, 2003). the same results were obtained for basil oil in mic concentration against candida albicans attc 10231, where after 2 h of incubation the oil reduced the number of living cells (gucwa et al., 2018). when compared to the literature data, the commercial essential oils tested in the present study showed higher activity during a longer application time. also, having in mind that the oils that the oils showed efficiency against clinical isolates with higher resistance to antibiotics, it is another confirmation of their higher activity. type of interaction between antimicrobial agents oils combination with ciprofloxacin resistance of p. aeruginosa to fluoroquinolones, the group of antibiotics that ciprofloxacin belongs to, can be due to mutations in regulation of the efflux systems, or to mutations of the topoisomerase targets (gyra and also parc). efflux and target mutations can cooperate to increase the resistance level to other drugs (rossolini & mantengoli, 2005). the use of combined therapy has been shown as a better solution in comparison to the use of individual antimicrobial agents in the treatment of bacterial resistance. to investigate the potential of the tested oils for enhancing the activity of ciprofloxacin and to determine the type of interaction between the tested essential oils against p. aeruginosa clinical isolates, checkerboard assay was performed. the three combinations were tested: basil/sage oil (bs), basil oil/ciprofloxacin (bc), sage oil/ciprofloxacin (sc) and the results are presented in tab. 1. the synergy was found in 61.5% of the tested isolates and additive effect against the two tested isolates when bc combination was applied. isolates no. 7, no. 11 and no. 13 expressed somewhat higher resistance to the tested combination in comparison to other isolates, where calculated ficis showed indifferent biologica nyssana ● 12 (1) september 2021: 47-54 table 1. interaction between antimicrobial agents against clinical isolates of pseudomonas aeruginosa basil oil/ciprofloxacin sage oil/ciprofloxacin basil oil/sage oil strains fici outcome fici outcome fici outcome no.1 0.38 s 0.38 s 2.00 i no.2 0.25 s 2.00 i 2.00 i no.3 0.09 s 0.16 s 0.56 ad no.4 0.13 s 0.17 s 0.63 ad no.5 0.12 s 0.15 s 0.53 ad no.6 0.53 ad 0.53 ad 0.75 ad no.7 1.03 i 0.53 ad 0.53 ad no.8 0.38 s 1.25 i 2.00 i no.9 0.38 s 0.38 s 1.50 i no.10 0.75 ad 0.56 ad 2.00 i no.11 1.06 i 1.13 i 2.00 i no.12 0.13 s 0.16 s 2.00 i no.13 1.03 i 0.53 ad 2.00 i fici -fractional inhibitory index s-synergistic effect ≤ 0.5; ad-additive effect 0.5 >1; i-indifferent effect 1 ≥2; a-antagonistic effect >2. pejčić et al. ● antimicrobial efficacy of basil and sage essential oils against pseudomonas aeruginosa: time-lapse kinetics... 51 biologica nyssana ● 12 (1) september 2021: 47-54 fig. 1. growth of pseudomonas aeruginosa clinical isolates in the presence of salvia officinalis and ocimum basilicum commercial essential oils. data are given as mean ± sd. the analysis was done using two way anova followed by tukey’s post hoc test (p < 0.05) vs. control. pejčić et al. ● antimicrobial efficacy of basil and sage essential oils against pseudomonas aeruginosa: time-lapse kinetics... interaction between the basil oil and ciprofloxacin. on the other hand, sc indicated somewhat weaker activity in comparison to the bc combination. the sc combination expressed synergistic effect in 46.15% and additive in 30.7% of tested isolates. isolates no. 8 and no. 11 manifested the same sensitivity to sc combination in comparison to the results obtained for these agents alone. these results are very promising and point to the very high potential of basil and sage essential oil as agents that would increase the effectiveness of ciprofloxacin used in antipseudomonal therapy. the somewhat lower potential of herein tested sage oil in enhancing the ciprofloxacin activity in comparison to the basil may be due to the chemical composition of the oil. the results of chemical characterization of herein tested oils showed α-thujone (29.7%), camphor (16.6%), and eucalyptol (9.2%) as the major constituents of the sage oil and (e)-anethole (39.4%), linalool (34.3%), eugenol (5.8%) and longifolene (6.2%) of the basil oil (pejčić et al., 2020). it has been reported that terpenoid ethers (1,8-cineole) have a lower capacity to sensitize the pathogen to the antibiotic activity as they lack free hydroxyl groups in comparison to both phenolic (eugenol) and alcoholic terpenoids (linalool) which are characterized by the presence of free hydroxyl groups. the hydroxyl group of eugenol is responsible for the inhibition of some enzymes, such as atp-ase, histidine decarboxylase or proteases (griffin et al., 1999). herein obtained results for basil oil are in agreement with silva et al. (2016) who reported the synergism for o. basilicum oil associations with ciprofloxacin against the clinical isolate p. aeruginosa 1662339, while effect of the same combination against p. aeruginosa attc 25852 was classified as indifferent. mahmoud et al. (2016) also reported that the basil/ciprofloxacin combination was effective in 88.89% of the total of 9 tested p. aeruginosa isolates. the only study on the sage oil in combination with antibiotics activity reported its potential to significantly increase the efficacy of several conventional antibiotics against p. aeruginosa (el-hosseiny et al., 2014). in the mentioned study (el-hosseiny et al., 2014) although the sage oil displayed inactivity against the p. aeruginosa strain, it potentiated the activity of antibiotics piperacillin, meropenem, and gentamicin, while in combination with antibiotic norfloxacin displayed antagonistic interaction. another study showed the potential of the sage oil to enhance the activity of ciprofloxacin in control of infections caused by mrsa strains (milenković et al., 2015). sage/basil oil combination sb combination expressed indifferent effect in 61.54% and additive in 38.46% of tested isolates. there was not synergistic effect noticed between the basil and sage oils. on the other hand, calculated ficis showed values very close to antagonistic interaction in 7 of the total 13 tested isolates. according to the chemical compositions of investigated oils, these results are expected. the study of faleiro et al. (2003) reported both antagonistic and synergistic effects of the pure essential oil components in combination, depending on the tested microorganisms. the mixture of linalool plus 1,8-cineole showed antagonistic effect in the case of the escherichia coli strain, while the same mixture showed synergistic interaction when applied to c. albicans. similar results were reported by adrar et al. (2016), where the sage oil when combined with thymus numidicus essential oil (reach in linalool (8.62–9.26%)), expressed antagonistic effect against e. coli 161. conclusions the observed results showed time-lapse efficacy of the basil and sage oils. however, higher effect over time is visible in the case of the sage essential oil. tested oils have also shown promising effect on increasing activity of antibiotic ciprofloxacin against p. aeruginosa clinical isolates. on the other hand, basil and sage oils used in combination require caution due to possible antagonism, and therefore additional research needs to be done on this topic. according to the presented results, basil and sage commercial essential oils can be used as an adjunctive therapy in combination with ciprofloxacin for the treatment of chronical infections caused by pseudomonas aeruginosa. references adrar, n., oukil, n., bedjou, f. 2016: antioxidant and antibacterial activities of thymus numidicus and salvia officinalis essential oils alone or in combination. industrial crops and products, 88: 112–119. aelenei, p., miron, a., trifan, a., bujor, a., gille, e., aprotosoaie, a. 2016: essential oils and their components as modulators of antibiotic activity against gram-negative bacteria. medicines, 3(3): 19. akthar, m., degaga, b., azam, t. 2014: antimicrobial activity of essential oils extracted from medicinal plants against the pathogenic microorganisms: a review. issues in biological sciences and pharmaceutical research, 2(1): 1–7. 52 biologica nyssana ● 12 (1) september 2021: 47-54 pejčić et al. ● antimicrobial efficacy of basil and sage essential oils against pseudomonas aeruginosa: time-lapse kinetics... alhazmi, a. 2015: pseudomonas aeruginosa – pathogenesis and pathogenic mechanisms. international journal of biology, 7(2): 44–67. artini, m., patsilinakos, a., papa, r., boović, m., sabatino, m., garzoli, s., vrenna, g., tilotta, m., pepi, f., ragno, r., selan, l. 2018: antimicrobial and antibiofilm activity and machine learning classification analysis of essential oils from different mediterranean plants against pseudomonas aeruginosa. molecules, 23(2): 482. bassolé, i. h. n., lamien-meda, a., bayala, b., tirogo, s., franz, c., novak, j., nebié, r. c., dicko, m. h. 2010: composition and antimicrobial activities of lippia multiflora moldenke, mentha x piperita l. and ocimum basilicum l. essential oils and their major monoterpene alcohols alone and in combination. molecules, 15(11): 7825–7839. calo, j. r., crandall, p. g., o’bryan, c. a., ricke, s. c. 2015: essential oils as antimicrobials in food systems a review. food control, 54: 111–119. chouhan, s., sharma, k., guleria, s. 2017: antimicrobial activity of some essential oilspresent status and future perspectives. medicines, 4(4): 58. delamare longaray, a. p., moschen-pistorello., t., i., artico, l., atti-serafini, l., echeverrigaray, s. 2007: antibacterial activity of the essential oils of salvia officinalis l. and salvia triloba l. cultivated in south brazil. food chemistry, 100(2): 603–608. el-hosseiny, l., el-shenawy, m., haroun, m., & abdullah, f. 2014: comparative evaluation of the inhibitory effect of some essential oils with antibiotics against pseudomonas aeruginosa. international journal of antibiotics, 2014: 1–5. faleiro, m. l., miguel, m. g., ladeiro, f., venâncio, f., tavares, r., brito, j. c., figueiredo, a. c., barroso, j. g., pedro, l. g. 2003: antimicrobial activity of essential oils isolated from portuguese endemic species of thymus. letters in applied microbiology, 36(1): 35–40. fournomiti, m., kimbaris, a., mantzourani, i., plessas, s., theodoridou, i., papaemmanouil, v., kapsiotis, i., panopoulou, m., stavropoulou, e., bezirtzoglou, e. e., alexopoulos, a. 2015: antimicrobial activity of essential oils of cultivated oregano (origanum vulgare), sage (salvia officinalis), and thyme (thymus vulgaris) against clinical isolates of escherichia coli, klebsiella oxytoca, and klebsiella pneumoniae. microbial ecology in health & disease, 26: 23289–23295. griffin, s. g., wyllie, s. g., markham, j. l., leach, d. n. 1999: the role of structure and molecular properties of terpenoids in determining their antimicrobial activity. flavour and fragrance journal, 14(5): 322–332. gucwa, k., milewski, s., dymerski, t., szweda, p. 2018: investigation of the antifungal activity and mode of action of thymus vulgaris, citrus limonum, pelargonium graveolens, cinnamomum cassia, ocimum basilicum, and eugenia caryophyllus essential oils. molecules, 23(5): hammer, k. a., c.f. carson, riley, t. v. 1999: antimicrobial activity of essential oils and other plant extracts. journal of applied microbiology, 9071(86): 985–990. hemaiswarya, s., doble, m. 2009: synergistic interaction of eugenol with antibiotics against gram negative bacteria. phytomedicine, 16 (11): 997– 1005. khalil, r., li, z. g. 2011: antimicrobial activity of essential oil of salvia officinalis l. collected in syria. african journal of biotechnology, 10(42): 8397–8402. langeveld, w. t., veldhuizen, e. j. a., burt, s. a. 2014: synergy between essential oil components and antibiotics: a review. critical reviews in microbiology, 40(1): 76–94. mahmoud, a. m., el-baky, r. m. a., ahmed, a. b. f., gad, g. f. m. 2016: antibacterial activity of essential oils and in combination with some standard antimicrobials against different pathogens isolated from some clinical specimens. american journal of microbiological research, 4(1): 16–25. mikulášová, m., chovanová, r., vaverková, š. 2016: synergism between antibiotics and plant extracts or essential oils with efflux pump inhibitory activity in coping with multidrug-resistant staphylococci. phytochemistry reviews, 15(4): 651– 662. milenković, m. t., božić, d. d., slavkovska, v. n., lakušić, b. s. 2015: synergistic effects of salvia officinalis l. essential oils and antibiotics against methicillin-resistant staphylococcus aureus. archives of biological sciences, 67(3): 949–956. mitic-culafic, d., vukovic-gacic, b., knezevicvukcevic, j., stankovic, s., simic, d. 2005: comparative study on the antibacterial activity of volatiles from sage (salvia officinalis l.). archives of biological sciences, 57(3): 173–178. niculae, m., spînu, m., şandru, c. d., cadar, d., szakacs, b., scurtu, i., bolfă, p., mateş, c. i. 2009: antimicrobial potential of some lamiaceae essential oils against animal multiresistant bacetria. lucrari ştiinłifice meducina veterinara, 42(1): 170–175. 53 biologica nyssana ● 12 (1) september 2021: 47-54 pejčić et al. ● antimicrobial efficacy of basil and sage essential oils against pseudomonas aeruginosa: time-lapse kinetics... opalchenova, g., obreshkova, d. 2003: comparative studies on the activity of basil an essential oil from ocimum basilicum l. against multidrug resistant clinical isolates of the genera staphylococcus, enterococcus and pseudomonas by using different test methods. journal of microbiological methods, 54(1): 105–110. pejčić, m., stojanović-radić, z., genčić, m., dimitrijević, m., radulović, n. 2020: antivirulence potential of basil and sage essential oils: inhibition of biofilm formation, motility and pyocyanin production of pseudomonas aeruginosa isolates. food and chemical toxicology, 141(5): 111431. reichling, j. 2020: anti-biofilm and virulence factor-reducing activities of essential oils and oil components as a possible option for bacterial infection control. planta medica, 86(8): 520–537. rossolini, g. m., mantengoli, e. 2005: treatment and control of severe infections caused by multiresistant pseudomonas aeruginosa. clinical microbiology and infection, supplement, 11(4): 17–32. silva, v. a., da sousa, j. p., de luna freire pessôa, h., de freitas, a. f. r., coutinho, h. d. m., alves, l. b. n., lima, e. o. 2016: ocimum basilicum: antibacterial activity and association study with antibiotics against bacteria of clinical importance. pharmaceutical biology, 54(5): 863–867. stojanović-radić, z., čomić, l., radulović, n., blagojević, p., mihajilov-krstev, t., rajković, j. 2012: commercial carlinae radix herbal drug: botanical identity, chemical composition and antimicrobial properties. pharmaceutical biology, 50(8): 933–940. stojanović-radić, z., dimitrijević, m., genčić, m., pejčić, m., radulović, n. 2020: anticandidal activity of inula helenium root essential oil: synergistic potential, anti-virulence efficacy and mechanism of action. industrial crops and products, 149: 112373. tardugno, r., pellati, f., iseppi, r., bondi, m., bruzzesi, g., benvenuti, s. 2018: phytochemical composition and in vitro screening of the antimicrobial activity of essential oils on oral pathogenic bacteria. natural product research, 32(5): 544–551. tariq, s., wani, s., rasool, w., shafi, k., bhat, m. a., prabhakar, a., shalla, a. h., rather, m. a. 2019: a comprehensive review of the antibacterial, antifungal and antiviral potential of essential oils and their chemical constituents against drug-resistant microbial pathogens. microbial pathogenesis 134: 103580. van vuuren, s. f., suliman, s., viljoen, a. m. 2009: the antimicrobial activity of four commercial essential oils in combination with conventional antimicrobials. letters in applied microbiology, 48(4): 440–446. van vuuren, s. f., viljoen, a. m. 2007: antimicrobial activity of limonene enantiomers and 1,8-cineole alone and in combination. flavour and fragrance journal, 22(6): 540–544. yap, p. s. x., yiap, b. c., ping, h. c., lim, s. h. e. 2014: essential oils, a new horizon in combating bacterial antibiotic resistance. the open microbiology journal, 8(1): 6–14. yu, z., tang, j., khare, t., kumar, v. 2020: the alarming antimicrobial resistance in eskapee pathogens: can essential oils come to the rescue? fitoterapia 140: 104433. biologica nyssana ● 12 (1) september 2021: 47-54 54 pejčić et al. ● antimicrobial efficacy of basil and sage essential oils against pseudomonas aeruginosa: time-lapse kinetics... stefanović et al. 2021, biologica nyssana 12(2) 12 (2) december 2021: 167-176 doi: 10.5281/zenodo.5759829 melatonin affects gene expression of noradrenaline transporter and enzymes in the hearts of stressed rats original article bojana stefanović laboratory of molecular biology and endocrinology, vinča institute of nuclear sciences, the national institute of the republic of serbia, university of belgrade, mike petrovića alasa 12-14, 11351 belgrade, serbia stefanovic_bojana@vin.bg.ac.rs (corresponding author) nataša spasojević laboratory of molecular biology and endocrinology, vinča institute of nuclear sciences, the national institute of the republic of serbia, university of belgrade, mike petrovića alasa 12-14, 11351 belgrade, serbia predrag jovanović cedars-sinai, centre for neural science and medicine, 8700 beverly blvd., los angeles, ca 90048, usa harisa ferizović laboratory of molecular biology and endocrinology, vinča institute of nuclear sciences, the national institute of the republic of serbia, university of belgrade, mike petrovića alasa 12-14, 11351 belgrade, serbia milica janković laboratory of molecular biology and endocrinology, vinča institute of nuclear sciences, the national institute of the republic of serbia, university of belgrade, mike petrovića alasa 12-14, 11351 belgrade, serbia perica vasiljević department of biology and ecology, faculty of sciences and mathematics, university of nis, višegradska 33, 18000 niš, serbia slađana dronjak laboratory of molecular biology and endocrinology, vinča institute of nuclear sciences, the national institute of the republic of serbia, university of belgrade, mike petrovića alasa 12-14, 11351 belgrade, serbia received: april 28, 2021 revised: september 01, 2021 accepted: november 01, 2021 abstract: the role of stress is important in the etiology of depression which subsequently affects cardiovascular regulation. there has been a lot of evidence of the beneficial effects of melatonin in various cardiovascular pathologies. we examined the effect of chronic melatonin treatment on noradrenaline content, synthesis, transport and degradation in the left atria and ventricle of rats exposed to chronic mild unpredictable stress (cums). our results show that cums decreased expression of mrna th and net in the left atria and comt in the left ventricles, whereas increased mao-a enzymes in the left ventricles. melatonin treatment induced a significant increase in gene expression of net in the atria and a decrease in mao-a in the ventricle. the observed beneficial effects of enhanced uptake and reduced degradation during melatonin treatment were most probably a compensatory mechanism which protects cardiomyocytes from the deleterious effects of noradrenaline overstimulation. key words: melatonin, noradrenaline, heart, stress, transporter, enzymes apstrakt: efekat melatonina na ekspresiju gena za noradrenalinski transporter i enzime degradacije u srcu hronično stresiranih pacova uloga stresa je važna u etiologiji depresije koja posledično može uticati na kardiovaskularnu regulaciju. postoji mnogo dokaza o blagotvornom dejstvu melatonina u različitim kardiovaskularnim oboljenjima. iz tih razloga smo ispitivali uticaj hroničnog tretmana melatoninom na količinu noradrenalina, njegovu sintezu, transport i degradaciju u levoj pretkomori i komori pacova izlaganih hroničnom blagom nepredvidivom stresu (cums). naši rezultati pokazuju da je cums smanjio količinu irnk za th i net u levoj pretkomori i comt u levoj komori, dok je povećao gensku ekspresiju mao-a enzima u levoj komori. tretman melatoninom je značajno povećao gensku ekspresiju net u pretkomorama i smanjio količinu enzima mao-a u komori. uočeni korisni efekti melatonina na ponovno preuzimanje i smanjenu degradaciju noradrenalina predstavljaju kompenzacione mehanizme koje štite kardiomiocite od štetnih efekata njegove prekomerne stimulacije. ključne reči: melatonin, noradrenalin, srce, stres, transporter, enzimi introduction stress is an underlying trigger that leads to the development of depression as well as cardiovascular disease. depression is found in 20– 30% of cardiovascular patients (whooley, 2006). depression is associated with the autonomic nervous system dysfunction which could subsequently lead to cardiovascular disregulation (carney et al., 2005). several studies have demonstrated an exaggerated cardiac noradrenaline response in major depression (veith et al., 1994; yehuda et al., 1998; mausbach et al., 2005). the methods inducing depression-like states in experimental animals are generally accepted as valuable tools for the studies of affective disorders. among these methods, chronic unpredictable mild stress (cums) model of depression has been frequently employed (willner et al. 1992). grippo et al. (2006) reported that cums model of depression is characterized by © 2021 stefanović et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 167 anhedonia and elevated sympathetic cardiac tone. functional noradrenergic transmission consists of a balance between noradrenaline synthesis, secretion and reuptake. noradrenergic activity is dependent on the synthesis of noradrenaline as determined by the rate limiting enzyme tyrosine hydroxylase (th). noradrenaline released from cardiac sympathetic nerve terminals is removed from the neuroeffector junction by the neuronal noradrenaline transporter (net) and metabolized to dihydroxyphenylglycol (dhpg) via monoamine oxidase-a (mao-a) (esler et al., 1990). jovanović et al. (2019) reported that exposure of the animals to social isolation for 12 weeks induced hypertrophy of cardiomyocytes in the wall of the left ventricle and a significant increase in noradrenaline levels in the left atria and the ventricle. melatonin (n-acetyl-5-methoxytryptamine) is a neuroendocrine hormone, which is synthesized primarily by the pineal gland (hardeland et al., 2006). melatonin easily crosses all morphophysiological barriers, and it can enter all cells of the body, affecting the function of a variety of tissues (tan et al. 1999). additionally, by virtue of its ability to detoxify free radicals and related oxygen derivatives, melatonin affects the molecular physiology of cells and contributes to improved cellular and organismal physiology (reiter et al., 2010). the experimental data obtained from rodent studies suggest that melatonin has beneficial effects in the treatment of cardiovascular diseases (reiter et al., 2010). melatonin is also a well-established antioxidant molecule with many beneficial effects in various cardiovascular pathologies (sun, et al., 2016). in humans, melatonin production not only diminishes with age (sack et al., 1986) but is also significantly lower in many age-related diseases, including cardiovascular disease (sakotnik et al., 1999; altun et al., 2002; dominguez-rodriguez et al., 2002; yaprak et al., 2003). the link between the heart and melatonin and the effect of endogenous melatonin on cardiovascular function is well established (dominguez-rodriguez et al., 2010). melatonin interacts with the heart and blood vessels indirectly via the nervous system (sewerynek, 2002; pechanova et al., 2014), and directly, through its receptor-dependent and independent activities as a signaling molecule and a free radical scavenger (paulis et al., 2012; galano & reiter, 2018). there is also an increasing amount of evidence regarding the beneficial effects of melatonin via exogenous administration (tarocco et al., 2019). recently, we have reported that melatonin treatment enhanced noradrenaline content and protein expression of th, dbh, pnmt, and net in the adrenal medulla of rats exposed to cums (stefanovic et al., 2019). although many studies have indicated its cardioprotective actions, the underlying mechanistic links remain unclear. hence, it is important to examine the regulation of more specific variables, such as the gene expression of noradrenaline biosynthetic and degrading enzymes and transporter in the left atria and ventricles of rats exposed to cums model of depression. materials and methods animals and treatment the experiments were performed on adult (twomonth-old) male wistar rats. the weight of males at the beginning of the experiment was 250-310 g. all animals were housed in a temperature-controlled room (20±2.0 °c) and synchronised to a 12-hour light/dark regime. four rats were kept in a standard plastic cages and given ad libitum access to standard laboratory food (commercial rat pellets) and tap water. care was taken to minimise the pain and discomfort of the animals in accordance with the guide for the care and use of laboratory animals (8th edition, national academies press). all procedures with animals were approved by the ethical committee for the use of laboratory animals of the vinča institute and the ministry of agriculture and environmental protection, authority for veterinary, permission no. 323-07-04657/2015-05/02. in this experiment, we used 24 animals, which were allocated to control (unstressed) and chronic unpredictable mildly stressed groups. these groups were further divided into two subgroups each and were receiving daily injection of vehicle (5% ethanol) or melatonin in dose of 10 mg kg-1 body weight by intraperitoneally (i.p.) route one hour before the dark phase (bassani et al., 2014). this time was chosen to avoid disruption of circadian rhythms that occurs if melatonin is administered throughout the day. melatonin (q-1300, bachem, switzerland) was dissolved in nacl (0.9%) containing 5% ethanol. the dose of 10 mg kg-1 is a supraphysiological dose chosen with the aim of assessing the drug’s therapeutic effect and not for replacing or restorative purposes. the dosage of 10 mg/kg/day was regularly adjusted according to the body weight for the entire period of treatment. exposure to cums and the vehicle, i.e. drug administration started on the same day and continued for 4 weeks. the stressors were presented for 1 week and repeated during the following 4 weeks. the same stress method was not continuously applied to ensure the rats would be unable to anticipate the next type of stress that would be applied. control animals were left undisturbed in their home cages with the exception of general handling (i.e. regular 168 biologica nyssana ● 12 (2) december 2021: 167-176 stefanović et al. ● melatonin affects gene expression of noradrenaline transporter and enzymes in the hearts of stressed rats 169 cage cleaning and body weight measuring). chronic unpredictable mild stress the cums procedure was carried out according to the methods described by grippo et al. (2002), and it was designed to maximise the unpredictable nature of the stressors. the cums groups of animals were exposed to the following stressors in random order: forced running (15 mins., 10 m/ min); soiled cage (500 ml 22 °c water spilled into bedding for 5 h); 45° cage tilt along the vertical axis for 7 h; 17 h food deprivation, 5 h cold room (4 °c) and water deprivation (water bottles were removed from cage during this time), 5 h grouped housing (8 rats per cage) and individual housing (48 h). control animals were left undisturbed in the home cage with the exception of general handling (i.e. regular cage cleaning and body weight measuring), which was matched to that of the cums group. immediately after the cums procedure, all animals were sacrificed, between 10:00–11:00 a.m., by rapid decapitation with a guillotine (harvard-apparatus, usa). left atria and ventricles were extracted quickly on ice, frozen in liquid nitrogen and stored at -70 °c until sample preparation for further molecular analysis. high-performance liquid chromatography assay ethylene glycol tetraacetic acid (egta) and dlnorepinephrine hydrochloride (na) were purchased from sigma–aldrich. ammonium formate was supplied by fisher scientific (loughborough, uk); formic acid (49–51%) by fluka analytical (switzerland); methanol by j.t. baker (deventer, the netherlands), perchloric acid (70%) by sigma– aldrich and magnesium chloride (mgcl2×5h2o) by sigma–aldrich. purified water was obtained via a blueclearro600p reverse osmosis water cleaning system with integrated bluesoft07-mb mixed bed salt remover (euro-clear ltd., hungary). the stock standard solution of na [1 mg/ ml] was prepared in methanol and kept at -20 °c. standard solutions in concentrations of 1, 2, 4, 8, 16, 32, 40 and 50 µg/ml were prepared by dilution of the stock standard solution in deprot (2% egta; 0,1n hclo4; 0.2% mgcl2). the ammonium formate buffer (100 mm, ph 3.6) was used as one of the mobile phase components. tissue samples were homogenised in deprot (1 mg:10 µl) using an ultra-turrax homogeniser (t10 basic), sonicated (branson sonic power company, danbury, connecticut; 3×10 s) and centrifuged (sorvall supert21; 30 mins, 18.000 rpm, +4 °c). supernatants were transferred in separate tubes and placed in the autosampler of the hplc system. data were obtained using a thermo scientific (dionexultimate 3000) hplc system consisting of degasser unit, binary pump (hpg3200sd), autosampler (wps-3000 splitloop), column oven (col. comp. tcc-3000) and rs electrochemical detector (ecd-3000rs) equipped with the 6041rs ultra amperometric analytical cell and glassy carbon working electrode. a hibar 125-4 licrospher100 rp-18 (5 µm) hplc column (merck millipore, darmstadt, germany) was used. instrument control and data acquisition was carried out by the chromeleon7 chromatography data system (thermo scientific). the following chromatographic conditions were obtained after the method optimisation. the mobile phase consisting of the ammonium formate buffer (100 mm, ph 3.6) as an a solution and methanol as a b solution was pumped at a flow rate of 500 µl/mins with the following step gradient. the run started with a mobile phase consisting of 98% a and 2% b solution. at the 9th minute of the run the part of b solution started to rise, reaching the 50% in the 15th mins and 80% in the 18th mins. starting from the 20th mins until the end of the run (30th mins) the column was re-equilibrated with the initial mixture of mobile phase solutions (2% of a and 98% of b solution). the applied potential for electrochemical measurements was +850 mv and the separation temperature was set at 25 °c. the 50 µl of samples and standard solutions were applied to the system. rna isolation and real-time rt-pcr the total rna from left atria and ventricles was extracted using trizol® reagent (invitrogen, ca, usa). total rna was isolated using chloroformisopropanol extraction and quantification and rna quality was carried out using spectrophotometer (nanodrop 1000 spectrophotometer, thermo scientific). tissue samples were homogenised in 1 ml trizol® reagent per 100 mg of tissue, using electrical homogeniser (ika-werke, gmbh & co, germany). reverse transcription was performed using ready-to-go you-prime first-strand bead (ge healthcare life sciences, pa usa) and pd(n)6 primer according to the manufacturer’s protocol. real-time rt-pcr assay was done exactly as previously described by gavrilović et al. (2008). pcr reaction was performed in the abi prism 7000 sequence detection system at 50 °c for 2 mins., 95 °c for 10 mins., followed by 40 cycles at 95 °c for 15 s and 60 °c for 1 min. taqman pcr reaction was carried out using assay-on-demand gene expression products (thermo fischer scientific, ma usa) for th (id: rn00562500_m1), for net (id: rn00580267_m1), for comt (id:rn01404927_g1) and for mao-a (id:rn01430950_m1). a reference endogenous control was included in each analysis to biologica nyssana ● 12 (2) december 2021: 167-176 stefanović et al. ● melatonin affects gene expression of noradrenaline transporter and enzymes in the hearts of stressed rats 170 correct the differences in the inter-assay amplification efficiency and all transcripts were normalised to cyclophilin a (id:rn00690933) expression. the obtained results were analyzed by rq study add on software for 7000 v 1.1 sds instrument (abi prism sequence detection system) with a confidence level of 95% (p<0.05). the relative expression of the target gene was normalized to cyclophyline a and expressed in relation to the calibrator, i.e. the control sample. western blot analysis left atria and ventricles were homogenised in ripa lysis buffer system (santa cruz biotechnology, inc., santa cruz, ca, sc-24948). after centrifugation (12 000 r.p.m., 20 mins at 4 °c), the supernatant was taken and protein concentration was determined by the method of lowry et al. (1951). samples were incubated for 5 mins at 100 °c in the appropriate amount of denaturing buffer according to laemmli (1970). 30 µg of protein extract from tissue samples separated by 12% sds-poly-acrylamide gel electrophoresis were transferred to a supported pvdf membrane (immobilon-p membrane, merck millipore, massachusetts, usa). the membranes were blocked in 5% non-fat dry milk in tris-buffered saline-tween 20 (tbst) for 1 h. all following washes (three times for 15 mins.) and antibody incubation (overnight at 4 °c for primary antibody and 1 h at ambient temperature for secondary antibody) were also performed in tbst at ambient temperature on a shaker. for measuring th, net, comt and mao-a, a rabbit polyclonal anti-th primary antibody (ab51191, dilution 1:1000, abcam, cambridge, uk), a rabbit polyclonal anti-net primary antibody (ab41559, dilution 1:1000, abcam, cambridge, uk), a rabbit polyclonal anti-comt primary antibody (ab126618, dilution 1:5000, abcam, cambridge, uk) and a rabbit polyclonal anti-mao-a primary antibody (ab126751, dilution1:1000, abcam, cambridge, uk), was used, respectively. washed membrane was further incubated in the horseradish peroxidaseconjugated secondary anti-rabbit antibody for luminol-based detection (ab6721, dilution 1:5000, abcam, cambridge, uk). the secondary antibody was then visualized by immobilon western chemiluminescent hpr substrate (merck millipore, massachusetts, usa) and exposed to x-ray film for western blot detection. image j analysis pc software (nih, bethesda, md) was used for quantification densitometry of protein bands on x-ray film. amounts of all analysed proteins were normalised to β-actin levels. statistical analysis the results are reported as means ± s.e.m. the significance of the differences in the catecholamine concentration, gene expression and protein levels of the examined th, net, comt and mao-a in the left atria and ventricles of the four groups of rats was estimated by a two-way anova test. the tukey post-hoc test was used to evaluate the differences between the groups. statistical significance was set at p<0.05. results the concentration of noradrenaline in the left atria and ventricles the results (fig. 1) indicate that chronic exposure of rats to unpredictable stressors, as well as melatonin treatment, did not alter the noradrenaline concentration in the left atria and ventricles. the gene expression of th enzyme in the left atria and ventricles two-way anova showed that chronic stress biologica nyssana ● 12 (2) december 2021: 167-176 stefanović et al. ● melatonin affects gene expression of noradrenaline transporter and enzymes in the hearts of stressed rats fig. 1. the effect of chronic melatonin treatment on noradrenaline content in the left atria (a) and ventricle (b) of controls and rats exposed to cums. the results are presented as mean ± s.e.m. for a sample of 6 rats 171 biologica nyssana ● 12 (2) december 2021: 167-176 stefanović et al. ● melatonin affects gene expression of noradrenaline transporter and enzymes in the hearts of stressed rats fig. 2. the effect of chronic melatonin treatment on th mrna (a, b) and protein level (c, d) in the left atria and ventricle of controls and rats exposed to cums. the results are presented as mean ± s.e.m. for a sample of 6 rats. statistical significance: *p<0.05 control vs. cums (tukey test) fig. 3. the effect of chronic melatonin treatment on net mrna (a, b) and protein level (c, d) in the left atria and ventricle of controls and rats exposed to cums. the results are presented as mean ± s.e.m. for a sample of 6 rats. statistical significance: **p<0.01 control vs. cums; #p<0.05 placebo vs. melatonin (tukey test) affects th mrna levels (f(1.23) = 4.72, p<0.05) in the left atria (fig. 2a). exposure to cums significantly reduced mrna levels for th (by 55%, p<0.05), but did not alter the protein levels of this enzyme in the left atria. there was not a significant effect of melatonin treatment on the gene expression of th enzyme in the left atria. cums did not change the expression of the th gene in the left ventricles. the analysis of the results showed no significant effect of melatonin treatment on the th mrna and protein levels in the left ventricles. the gene expression of net transporter in the left atria and ventricles the analysis of the results showed in fig. 3 indicated the significant effect of both stress and melatonin treatment on the net mrna levels (f(1.23) = 16.77, p<0.001), as well as the protein content of this transporter (f(1.23) = 4.63, p<0.05) in the left atria. chronic stress led to a decrease in both the mrna level (by 55%, p<0.01), as well as the protein level of this transporter (by 30%, p<0.01). on the other hand, melatonin treatment induced an increase in net gene expression (p<0.05) in animals exposed to cums, returning mrna levels to control values. the obtained results in fig. 3 showed that neither cums nor melatonin changed net gene expression in the left ventricles. the gene expression of mao-a enzyme in the left atria and ventricles changes as a result of the interaction between chronic stress and melatonin treatment on mao-a mrna (f(1.23) = 48.13, p <0.001) and protein levels (f(1.23) = 69.28, p<0.001) in the left ventricles are presented in fig. 4. cums increased mao-a gene expression in the left ventricles (p<0.01), while melatonin decreased mrna (p<0.01) and protein levels (p<0.05) of mao-a enzyme in the left ventricles of stressed rats. the gene expression of comt enzyme in the left atria and ventricles the results showed in fig. 5 indicate that chronic exposure to unpredictable stressors, as well as melatonin treatment, did not significantly alter comt gene expression in the left atria of examined animals. the analysis of the results showed that cums 172 biologica nyssana ● 12 (2) december 2021: 167-176 stefanović et al. ● melatonin affects gene expression of noradrenaline transporter and enzymes in the hearts of stressed rats fig. 4. the effect of chronic melatonin treatment on mao-a mrna (a, b) and protein level (c, d) in the left atria and ventricle of controls and rats exposed to cums. the results are presented as mean ± s.e.m. for a sample of 6 rats. statistical significance: **p<0.01 control vs. cums; ##p<0.01 placebo vs. melatonin (tukey test) reduced comt mrna levels (by 45%, p<0.05), while melatonin failed to change the mrna levels of comt in the left ventricles. there was also no significant change in comt protein levels in the left ventricles under the stress protocol and melatonin treatment. discussion the changes in autonomic regulation of the heart, such as the activation of the sympathetic nervous system, the reduction in vagal tone and the increase in heart rate (hr) is often observed in depressed patients. the cums model of stress increases the sympathetic tone (grippo et al., 2002), suggesting that autonomic changes may mediate changes in the heart rate of rats exposed to cums. noradrenaline contribution to an increase in cardiac output is well established. our results showed that cums as well as melatonin treatment does not change the concentration of noradrenaline in the left atria and ventricles (fig. 1). ganguly et al. (1997) also reported that the level of noradrenaline remained unchanged in rats with myocardial infarction. induction of gene expression of the noradrenalinesynthesizing enzyme has a very important role in responding to stress and in the regulation of the cardiovascular system. our results show that cums decreased the expression of mrna th in the left atria whereas gene expression of th remained unchanged in the ventricle (fig. 2). these findings are in agreement with gavrilović et al. (2009) who have demonstrated that long-term social isolation stress also produced a decrease in th mrna level in left atria. the cardiac neuronal net in sympathetic neurons is responsible for the uptake of released noradrenaline from the neuroeffector junction (esler et al., 1990). there are studies that indicated reduced uptake in the diseased heart (habecker et al., 2006). we showed a decrease in the gene expression of the net transporter in the left atria, while the expression of this transporter is unchanged in the left ventricle under stress protocol (fig. 3). in the study doca-salt hypertension, net mrna was reduced in the left atria (wehrwein et al., 2013). decreased sympathoneural uptake of noradrenaline has been associated with cardiovascular disease while faulty net activity is a contributing factor to the increase in cardiac pathology (shanks et al., 2013). catechol o-methyltransferase (comt) and mao-a contributes to the removal of noradrenaline in the myocardium and it is believed to exert degradative action at high noradrenaline levels. in our experimental model there was no change in the 173 biologica nyssana ● 12 (2) december 2021: 167-176 stefanović et al. ● melatonin affects gene expression of noradrenaline transporter and enzymes in the hearts of stressed rats fig. 5. the effect of chronic melatonin treatment on comt mrna (a, b) and protein level (c, d) in the left atria and ventricle of controls and rats exposed to cums. the results are presented as mean ± s.e.m. for a sample of 6 rats. statistical significance: *p<0.05 control vs. cums (tukey test) level of mrna and the comt protein in the atria but the level of mrna for the same enzyme decreased in the left ventricles (fig. 5). these effects of cums were not observed at the levels of comt protein in the left ventricle (fig. 5). changes caused by stress in comt degrading enzyme certainly depend on the type and duration of the stressor and numerous other factors. kuroko et al. (2005) have shown that comt contributes to the inactivation of high levels of noradrenaline during myocardial ischemia and it has been assumed that this degrading enzyme is more active in more severe heart damages. namely, higher levels of catecholamine are necessary for the activation of comt. in the study of kaludercic et al. (2014), mao activity has been pointed out as an another mechanism potentially implicated in heart failure. in peripheral tissues, mao is involved in oxidative catabolism of amines, which generates aldehyde, which is in its turn metabolized to the corresponding acid by aldehyde dehydrogenase, and also ammonia and h2o2, products that are toxic in high concentration (kaludercic et al., 2011). in our experimental cums model, the gene expression of mao-a enzymes in the left ventricles has been increased (fig. 4). mao-a contributes to heart failure progression via enhanced noradrenaline catabolism and oxidative stress. based on our results we could suggest that enhanced gene expression of mao-a might results in augmented ros generation, contributing to left ventricular dysfunction in hearts rats subjected to cums. among endogenous substances with great success against cardiac illness certainly is melatonin. that is also a hormone with marked antioxidant properties. studies reports that circulating levels of melatonin and his synthesis are reduced in patients with different cardiovascular diseases (dominguezrodriguez et al., 2012). effects of melatonin on noradrenaline biosynthesis, uptake and degradation in the heart of depression model rats are still poorly understood. the melatonin treatment in our experiment induced a significant increase in gene expression of net in the atria (fig. 3) and a decrease in mao-a in the ventricle (fig. 4). we failed to observe any effects of the melatonin treatment in the gene expression of th (fig. 2) and comt (fig. 5) in the heart of cums rats. given that net is responsible for rapidly eliminating noradrenaline from the cardiac synaptic cleft, the elevated expression of net with the melatonin treatment may explain the greater capacity of noradrenaline uptake in the left atria; a fact which may contribute to the maintenance of atrial function in a stressful situation. an aberrantly high level of cytoplasmic noradrenaline has been linked to oxidative stress and neuronal toxicity. increased sequestration and reduced deamination of noradrenaline by mao-a during melatonin treatment produced a decreased generation of reactive oxygens species. thus, the observed beneficial effects of enhanced uptake and reduced degradation during melatonin treatment were most probably a compensatory mechanism which protects cardiomyocytes from the deleterious effects of noradrenaline overstimulation. conclusions additional experiments are needed for a better understanding of the role of melatonin, by identifying the molecular markers involved in chronic stress and thereby ensuring the use of melatonin for therapeutic purposes. the implications of this study may provide insights into the mechanisms of cardiovascular dysfunction that frequently accompany depression. although melatonin is an effective cardioprotective agent, further investigation is needed to expand its range of therapeutic applications. these findings may provide an insight into the mechanisms underlying depression and cardiovascular disease in humans. acknowledgements. this work was supported by the ministry of education, scientific and technological development of the republic of serbia. references altun, a., yaprak, m., aktoz, m.,vardar, a., betul, u.a., ozbay, g. 2002: impaired nocturnal synthesis of melatonin in patients with cardiac syndrome x. neuroscience letters, 327(2): 143-145. bassani, t.b., gradowski, r.w., zaminelli, t., barbiero, j.k., santiago, r.m., boschen, s.l., da cunha, c., lima, m.m., andreatini, r., vital, m.a. 2014: neuroprotective and antidepressant like effects of melatoninin a rotenone-induced parkinson’s disease model in rats. brain research, 1593: 95-105. carney, r.m., freedland, k.e., richard c veith, r.c. 2005: depression, the autonomic nervous system, and coronary heart disease. psychosomatic medicine, 67: 29-33. dominguez-rodriguez, a., abreu-gonzalez, p., avanzas, p. 2012: the role of melatonin in acute myocardial infarction. frontiers in bioscience, 17: 2433-2441. dominguez-rodriguez, a., abreu-gonzalez, p., garcia, m.j., sanchez, j., marrero, f., armastrujillo, d. 2002: decreased nocturnal melatonin levels during acute myocardial infarction. journal of pineal research, 33(4): 248-252. biologica nyssana ● 12 (2) december 2021: 167-176 174 stefanović et al. ● melatonin affects gene expression of noradrenaline transporter and enzymes in the hearts of stressed rats dominguez-rodriguez, a., abreu-gonzalez, p., sanchez-sanchez, j.j., kaski, j.c., reiter, r.j. 2010: melatonin and circadian biology in human cardiovascular disease. journal of pineal research, 49(1): 14-22. esler, m., jennings, g., lambert, g., meredith, i., horne, m., eisenhofer, g. 1990: overflow of catecholamine neurotransmitters to the circulation: source, fate, and functions. physiological reviews, 70(4): 963-985. galano, a., reiter, r.j. 2018: melatonin and its metabolites versus oxidative stress: from individual actions to collective protection. journal of pineal research, 65(1): e12514. ganguly, p.k., dhalla, k.s., shao, q., beamish, r.e., dhalla, n.s. 1997: differential changes in sympathetic activity in left and right ventricles in congestive heart failure after myocardial infarction. american heart journal, 133(3): 340-345. gavrilovic, l., spasojevic, n., tanic, n., dronjak, s. 2008: chronic isolation of adult rats decreases gene expression of catecholamine biosynthetic enzymes in adrenal medulla. neuroendocrinology letters, 29(6): 1015-1020. gavrilovic, l., spasojevic, n., zivkovic, m., dronjak, s. 2009: effect of immobilization stress on gene expression of catecholamine biosynthetic enzymes in heart auricles of socially isolated rats. brazilian journal of medical and biological research, 42(12): 1185-1190. grippo, a.j., beltz, t.g., weiss, r.m., johnson, a.k. 2006: the effects of chronic fluoxetine treatment on chronic mild stress-induced cardiovascular changes and anhedonia. biological psychiatry, 59(4): 309-316. grippo, a.j., moffitt, j.a., johnson, a.k. 2002: cardiovascular alterations and autonomic imbalance in an experimental model of depression. american journal of physiology. regulatory, integrative and comparative physiology, 282(5): r1333-1341. habecker, b.a., willison, b.d., shi, x., woodward, w.r. 2006: chronic depolarization stimulates norepinephrine transporter expression via catecholamines. journal of neurochemistry, 97(4): 1044-1051. hardeland, r., pandi-perumal, s.r., cardinali, d.p. 2006: melatonin. the international journal of biochemistry and cell biology, 38(3): 313-316. jovanović, p., spasojević, n., puskaš, n., stefanović, b., dronjak, s. 2019: oxytocin modulates the expression of norepinephrine transporter, β3 adrenoceptors and muscarinic m2 receptors in the heart of socially isolated rats. peptides, 111: 132-141. kaludercic, n., carpi, a., menabò, r., di lisa, f., paolocci, n. 2011: monoamine oxidases (mao) in the pathogenesis of heart failure and ischemia/ reperfusion injury. biochimica et biophysica acta, 1813(7): 1323-1332. kaludercic, n., mialet-perez, j., paolocci, n., parini, a., di lisa, f. 2014: monoamine oxidases as sources of oxidants in the heart. journal of molecular and cellular cardiology, 73: 34-42. kuroko, y., fujii, t., yamazaki, t., akiyama, t., ishino, k., sano, s., mori, h. 2005: contribution of catechol o-methyltransferase to the removal of accumulated interstitial catecholamines evoked by myocardial ischemia. neuroscience letters, 388(2): 61-64. laemmli, u.k. 1970: cleavage of structural proteins during the assembly of the head of bacteriophage t4. nature, 227: 680-685. lowry, o.h., rosebrough, n.j., farr, a.l., randall, r.j. 1951: protein measurement with the folin phenol reagent. the journal of biological chemistry, 193(1): 265-275. mausbach, b.t., dimsdale, j.e., ziegler, m.g., mills, p.j., ancoli-israel, s., patterson, t.l., grant, i. 2005: depressive symptoms predict norepinephrine response to a psychological stressor task in alzheimer’s caregivers. psychosoatic medicine, 67(4): 638-642. paulis, l., simko, f., laudon, m. 2012: cardiovascular effects of melatonin receptor agonists. expert opininion on investigational drugs, 21(11): 1661-1678. pechanova, o., paulis, l., simko, f. 2014: peripheral and central effects of melatonin on blood pressure regulation. international journal of molecular science, 15(10): 17920-17937. reiter, r.j., tan, d.x., fuentes-broto, l. 2010: melatonin: a multitasking molecule. progress in brain research, 181: 127-151. reiter, r.j., tan, d.x., paredes, s.d., fuentesbroto, l. 2010: beneficial effects of melatonin in cardiovascular disease. annals of medicine, 42(4): 276-285. sack, r.l., lewy, a.j., erb, d.l., vollmer, w.m., singer, c.m. 1986: human melatonin production decreases with age. journal of pineal research, 3(4): 379-388. 175 biologica nyssana ● 12 (2) december 2021: 167-176 stefanović et al. ● melatonin affects gene expression of noradrenaline transporter and enzymes in the hearts of stressed rats biologica nyssana ● 12 (2) december 2021: 167-176 176 stefanović et al. ● melatonin affects gene expression of noradrenaline transporter and enzymes in the hearts of stressed rats sakotnik, a., liebmann, p.m., stoschitzky, k., lercher, p., schauenstein, k., klein, w., eber, b. 1999: decreased melatonin synthesis in patients with coronary artery disease. european heart journal, 20(118): 1314-1317. sewerynek, e. 2002: melatonin and the cardiovascular system. neuro endocrinology letters, 23: 79-83. shanks, j., mane, s., ryan, r., paterson, d.j. 2013: ganglion-specific impairment of the norepinephrine transporter in the hypertensive rat. hypertension, 61(1): 187-193. stefanovic, b., spasojevic, n., jovanovic, p., dronjak, s. 2019: melatonin treatment affects changes in adrenal gene expression of catecholamine biosynthesizing enzymes and norepinephrine transporter in the rat model of chronic-stress-induced depression. canadian journal of physiology and pharmacology, 97(7): 685-690. sun, h., gusdon, a.m., qu, s. 2016: effects of melatonin on cardiovascular diseases: progress in the past year. current opinion in lipidology, 27(4): 408-413. tan, d.x., manchester, l.c., reiter, r.j., qi, w.b., zhang, m., weintraub, s.t., cabrera, j., sainz, r.m., mayo, j.c. 1999: identification of highly elevated levels of melatonin in bone marrow: its origin and significance. biochimica et biophysica acta, 1472(1-2): 206-214. tarocco, a., caroccia, n., morciano, g., wieckowski, m.r., ancora, g., garani, g., pinton. p. 2019: melatonin as a master regulator of cell death and inflammation: molecular mechanisms and clinical implications for newborn care. cell death and disease, 10(4): 317. veith, r.c., lewis, n., linares, o.a., barnes, r.f., raskind, m.a., villacres, e.c., murburg, m.m., ashleigh, e.a., castillo, s., peskind, e.r., pascualy, m., halter, j.b.1994: sympathetic nervous system activity in major depression. basal and desipramine-induced alterations in plasma norepinephrine kinetics. arcives of general psychiatry, 51(5): 411-422. wehrwein, e.a., novotny, m., swain, g.m., parker, l.m., esfahanian, m., spitsbergen, j.m., habecker, b.a., kreulenet, d.l. 2013: regional changes in cardiac and stellate ganglion norepinephrine transporter in doca-salt hypertension. autonomic neuroscience: basic & clinical, 179(1-2): 99-107. whooley, m.a. 2006: depression and cardiovascular disease: healing the broken-hearted. journal of the american medical association, 295(24): 2874-2881. willner, p., muscat, r., papp, m. 1992: chronic mild stress-induced anhedonia. a realistic animal model of depression. neuroscience and biobehavioral reviews, 16(4): 525-534. yaprak, m., altun, a., vardar, a., aktoz, m., ciftci, s., ozbay, g. 2003: decreased nocturnal synthesis of melatonin in patients with coronary artery disease. international journal of cardiology, 89(1): 103-107. yehuda, r., siever, l.j., teicher, m.h., levengood, r.a., gerber, d.k., schmeidler, j., yang, r.k. 1998: plasma norepinephrine and 3-methoxy4-hydroxyphenylglycol concentrations and severity of depression in combat posttraumatic stress disorder and major depressive disorder. biological psychiatry, 44(1): 56-63. ciftci, erol 2020, biologica nyssana 11(2) 11 (2) december 2020: 85-91 doi: 10.5281/zenodo.4393951 micromorphological studies on inflorescence and seeds of some plantain (plantago l.) taxa in turkey original article almila ciftci biology department, science faculty, istanbul university, istanbul, turkey almila.ciftci@istanbul.edu.tr (corresponding author) osman erol biology department, science faculty, istanbul university, istanbul, turkey erol@istanbul.edu.tr received: julz 14, 2020 revised: august 28, 2020 accepted: september 15, 2020 abstract: plantago l. (plantaginaceae) is one of the largest genera of the plantaginaceae family. there are relatively fewer morphological studies on the genus. seed surface micromorphology has become popular in plant taxonomy in recent years and found useful in most groups. the inflorescence and seed surface characteristics of 22 plantago taxa collected from turkey were examined using a scanning electron microscope (sem). cluster analysis performed in r. we observed that the micromorphology of the inflorescence parts and seeds of genus plantago are very variable, and do not support the classification based on general morphology. this study shows that these characteristics are highly variable and micromorphological characteristics of inflorescence parts are not good characters for making a specific distinction between different taxa. additionally, some groups have been to possess a stable seed surface characteristics. however, these characters are not reliable among all taxa, which means that they should be used carefully, especially when identifying widespread taxa. key words: inflorescence, micromorphology, plantago, plantaginaceae, seed apstract: mikromorfološka studija cvast i semena nekih taksona bokvica (plantago l.) u turskoj plantago l. (plantaginaceae) je jedan od najvećih rodova porodice plantaginaceae. postoji relativno malo morfoloških studija o ovom rodu. mikromorfologija površine semena postala je popularna u biljnoj taksonomiji poslednjih godina i smatra se korisnom u većini grupa. karakteristike cvasti i površine semena 22 taksona roda plantago sakupljenih iz turske ispitane su pomoću skenirajućeg elektronskog mikroskopa (sem). klaster analiza je izvršena u r. primetili smo da su mikromorfologija delova cvasti i semena roda plantago veoma promenljive i ne podržavaju klasifikaciju zasnovanu na opštoj morfologiji. ova studija pokazuje da su ove karakteristike veoma promenljive, a mikromorfološke karakteristike delova cvasti nisu dobri karakteri za pravljenje posebne razlike između različitih taksona. pored toga, neke grupe su pokazale da imaju stabilne karakteristike površine semena. međutim, ovi karakteri nisu pouzdani kod svih taksona, što znači da ih treba pažljivo koristiti, posebno pri identifikaciji rasprostranjenih vrsta. ključne reči: cvasti, mikromorfologija, plantago, plantaginaceae, seed introduction plantaginaceae is a very heterogeneous family with diverse evolutionary trends, and plantago l. is one of the greatest genera of the plantaginaceae family with 200 species, 56 subspecies, 188 varieties and 9 subvarieties according to albach et al. (2005). it is probably the most widespread genus of the family plantaginaceae (albach et al., 2005). plantago genus is divided into nineteen sections and two subgenera (pilger, 1937). 9 of these sections including 23 taxa, two of which are endemic to turkey, have been recorded in the flora of turkey and the east aegean islands (tutel, 1982). although rahn (1996) claimed that plantago can be divided into six genera, he divided the genus into six subgenera and twelve sections in order to prevent synonym pollution. these two classification suggestions for the genus are summarized in tab. 1. here in this study, we used pilger’s (1937) classifica© 2020 ciftci, erol. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 85 tion in order to be able to follow the ranking in the flora of turkey (tutel, 1982). plantago taxa are annual or perennial herbs or dwarf shrubs with alternate rosettes or opposite leaves. the inflorescence is a spike with 4-merous flowers. the sepals are imbricate and keeled. the flowers have bracts. the corolla is sympetalous and forms a cylindrical tube (tutel, 1993). even though various studies have been made with plantago taxa, morphological and systematic studies on plantago taxa are relatively limited (sagar & harper, 1964; sharma et al., 1990; tutel et al., 2005). micromorphological studies have been made in china (jun-zhe et al., 1992) and poland (klimko et al., 2004) which were both limited to the taxa in their region. the most extensive study on micromorphology of plantago seeds composed by shehata and loutfy (2006) included 31 taxa. seed surface micromorphology has become very popular in plant taxonomy in recent years. one of the main reasons for that is that structure of the seed surface seems to be the most stable character due to less contact with environment. flower elements are also one of the most stable characters in flowering plants (endress, 2001). for this reason, we decided to include the surface investigations of inflorescence elements in this study. considering the lack of the studies about plantago taxa, this study aims to clarify the relationships within plantago taxa in turkey; using seed and inflorescence features. moreover, sem was used to investigate further the micromorphology of the seed and the inflorescence surfaces of these taxa in turkey for the first time. materials and methods 22 turkish plantago taxa including four varieties, which were not presented in the flora of turkey and east aegean islands (tutel, 1982), are examined in this study (tab. 2). these taxa were obtained mostly from istf (istanbul university faculty of sciences herbarium) as well as from ank (ankara university faculty of science herbarium). we used 15 individuals’ inflorescence elements and seeds from each 86 sensu pilger sensu rahn subgen. sect. taxa sect. subgen. euplantago plantago plantago major subsp. major plantago plantago p. major subsp. intermedia gentianoides p. gentianoides lamprosantha p. media coronopus p. crassifolia coronopus coronopus p. coronopus p. weldenii p. holosteum p. maritima maritima oreades p. atrata montana albicans leucopsyllium p. albicans p. loeflingii arnoglossum p. lanceolata lanceifoliap. argentea p. lagopus hymenopsyllium p. cretica hymenopsyllium p. bellardii psyllium psyllium p. squarrosa psyllium psyllium p. afra p. scabra p. sempervirens p. euphratica table 1. classification of the turkish plantago taxa according to pilger [2] and rahn [4] biologica nyssana ● 11 (2) december 2020: 85-91 ciftci, erol ● micromorphological studies on inflorescence and seeds of some plantain (plantago l.) taxa in turkey 87 taxon when possible. apart from the other studies (jhun-zhe et al., 1992; klimko et al., 2004; shehata & loutfy, 2006) on plantago micromorphology, this study includes some other areas from the inflorescence parts (e.g. bract, anterior and posterior sepals, and corolla) (fig. 1). for the examination of the inflorescence characteristics, the lowest and the most mature flowers of the inflorescences have been chosen, and the macro morphological observations have been made using olympus szx7 stereomicroscope. the micromorphology of the inflorescence parts and seeds fig. 1. general view of a plantago flower (a). the observed areascorolla tube (1), petal (2), anterior sepal base (3), bract margin (4), posterior sepal carina (5) and margin (6), seed ventral (7) and dorsal (8) surfaces are shown with squares (b). scale bars 1 mm. table 2. the examined taxa in the study with voucher specimens taxa voucher no location p. major l. subsp. major istf 24301 rize p. major l. subsp. intermedia (gilib) lange istf 23423 i̇stanbul p. coronopus l. subsp. coronopus istf 557 i̇stanbul p. weldenii rchb. istf 2306 i̇stanbul p. crassifolia forsskal istf 40190 istanbul p. maritima l. istf 26779 kirklareli p. holosteum scop. istf 27341 ankara p. media l. istf 23998 erzurum p. atrata hoppe istf 23710 erzurum p. gentianoides sm. subsp. gentianoides istf 32242 bursa p. lanceolata l. istf 23372 istanbul p. argentea chaix istf 63 bursa p. lagopus l. istf 22271 istanbul p. albicans l. ank ankara p. loeflingii l. istf 35420 sanliurfa p. cretica l. istf 24743 antalya p. bellardii all. istf 27212 i̇stanbul p. squarrosa murray istf 9790 antalya p. scabra moench istf 32258 i̇stanbul p. afra l. istf 36362 mersin p. sempervirens crantz alm 27 tekirdağ p. euphratica decne. ex barnéoud istf 37227 malatya biologica nyssana ● 11 (2) december 2020: 85-91 ciftci, erol ● micromorphological studies on inflorescence and seeds of some plantain (plantago l.) taxa in turkey 88 have been observed and photographed by a jeol benchtop scanning electron microscope jcm-5000 under 10 kv of voltage. the nomenclature of the surface characteristics is adapted from stearn (1991) and barthlott et al. (1998). the categorical variables of surface characters in various anatomical compartments of plantago were used as variables in the cluster analysis. hierarchical cluster analysis of 26 categorical variables across 22 taxa was performed using a clustering algorithm via ward.d method. in brief, a mean distances matrix was generated and subjected to clustering with the ward.d method of the hclust function. following the clustering, the relationships between the sections were visualised as dendrograms using the tree functions in r v3.4.3. results the dissimilarity cluster analyses formed two main groups and four subgroups according to the surface characters (fig. 2). both of the groups included different taxa from different sections. the macro-morphological observations showed that the seed shapes and sizes of plantago taxa are very variable. the micromorphological aspects of the examined parts using sem are shown in tab. 3. the sem photos of all the examined areas are given in fig. 3. it can be seen from the fig. 3 that hairs, and epidermal cell arrangements are variable among the studied taxa. observations showed that the surface type of corolla tube was the most stable feature appearing to be lineate in all of the examined taxa but only differing in terms of cell size and presence of hairs (tab. 3, fig. 3). this result shows that the surfaces of the petals and the posterior sepals seem to have more or less stable features through sections (tab. 3, fig. 3). being the outermost part of a flower structure, bract has shown a great variety in terms of surface micromorphology among all the examined parts (tab. 3, fig. 3). in section coronopus bract surfaces are differing among the taxa whereas the seed surface characteristics are very stable. all taxa in this section has reticulate seed surface ornamentation and all but one taxa (p. holosteum) has undulated anticlinal walls. section coronopus also show stability in terms of inflorescence parts’ surface characters generally except anterior sepal and bract surfaces (tab. 3). similarly, taxa in section arnoglossum have very stable seed surface characteristics (smooth). the smooth seed surfaces have also been observed in some taxa of section psyllium. section psyllium, has the most variable seed surface characteristics. the inflorescence surface characters vary among these taxa, as well (tab. 3). sections lamprosantha, oreades fig. 2. cluster dendrogram of plantago taxa. the sections marked with different colors biologica nyssana ● 11 (2) december 2020: 85-91 ciftci, erol ● micromorphological studies on inflorescence and seeds of some plantain (plantago l.) taxa in turkey and gentianoides are represented by one taxon each, and these taxa have similar characteristics with each other, and in the terms of seed surface characters they resemble section plantago. in p. major subsp. major and in p. major subsp. intermedia seed surfaces are rugose. other examined parts (i.e. anterior sepal, posterior sepal carina, corolla tube, petal, and dorsal and ventral sides of seeds) show parallel characteristics within these two taxa except anterior sepals. the bases of anterior sepals are both rugose, but cell shapes and anticlinal wall nature is different (tab. 3). the seed surfaces are reticulate in section leucopsyllium and section hymenopsyllium. section leucopsyllium has diverse inflorescence surface characters. section hymenopsyllium taxa have more or less stable characters on micromorphological level with rugose posterior sepal carina surfaces, striate corolla tube, and striate petal surfaces but there is only two taxa available in this study. discussion the two subspecies of p. major appear in two different main groups. concurrently, the observation of the seed surfaces of p. major were controversial in previous studies focusing on the seed micromorphology of plantago taxa (klimko et al., 2004; shehata & loutfy, 2006; hoghoughi et al., 2016; verma & bharti, 2017). klimko et al. (2004) reported the seed surfaces of p. major as “not slightly rugose as it has been reported in previous studies” (rymkiewicz, 1979; rothmaler et al., 1984). on the contrary, other authors identified the same area as reticulate (shehata & loutfy, 2006), tuberculate (verma & bharti, 2017), and areolate with wrinkled surface (hog89 fig. 3. sem micrographs of studied areas. posterior sepal carina surfaces of (a) reticulate, plantago major subsp. major; (b) rugose, p. weldenii; (c) striate, p. gentianoides subsp. gentianoides; petal surfaces of (d) granulate, p. weldenii; (e) striate, p. gentianoides subsp. gentianoides; (f) rugose, p. sempervirens; anterior sepal base surfaces of (g) rugose, p. major subsp. major; (h) rugulose, p. coronopus subsp. coronopus; (i) granulate, p. albicans; bract margin surfaces of (j) reticulate, p. coronopus subsp. coronopus; (k) rugose, p. maritima; (l) smooth, p. argentea; p. squarrosa; (m) fissured, p. afra; (n) granulate, p. sempervirens; corolla tube surface of (o) lineat, p. gentianoides subsp. gentianoides; seed surfaces of (p) rugose, plantago major subsp. major; (q) reticulate, p. crassifolia; (r) smooth, p. lagopus. biologica nyssana ● 11 (2) december 2020: 85-91 ciftci, erol ● micromorphological studies on inflorescence and seeds of some plantain (plantago l.) taxa in turkey houghi et al, 2016). in this study, seed surfaces of both p. major subsp. major and p. major subsp. intermedia were identified as rugose. plantago lanceolata is another taxon which shows a great variety in the sense of seed micromorphology like p. major. verma et al. (2017) reported the seed surface of p. lanceolata as scalariform to reticulate with spindle shaped striations whereas hoghoughi et al. (2016) identifies it as negative reticulate, and the samples in this study showed no ornamentation (smooth surface). these taxa are widespread and adapted to a variety of habitats, therefore it is an expected outcome. similarly, p. maritima and p. gentianoides were reported as having regulate or areolate ornamentation and reticulum cristatum respectively (hoghoughi et al., 2016), but in this study they are identified as reticulate and rugose. it has been known that plantago taxa are very variable in morphology, and that this variation may be due to environmental differences (meudt, 2012). however, some taxa show very consistent surface characteristics. for instance, p. coronopus has found to be reticulate in both this study and others (hoghoughi et al., 2016). plantago major is a widespread species with capability to adapt in a variety of habitats, thus its positioning in the clustering may not be considered meaningful. section psyllium taxa have representatives in both main groups in dendrogram. some of these taxa have shown different seed surface characters than previously studied (shehata & loutfy, 2006). the comparison of these results shows that seed surfaces may show variation on an intraspecific level among sect. psyllium and also some other taxa (p. major and p. lanceolate) section coronopus taxa have a very distinct surface structure. all of the studied taxa in this section have shown undulated anticline membranes and ruminate seed surfaces. these findings are in accordance with the previous studies (klimko et al., 2004; shehata & loutfy, 2006). however, the classification of the coronopus section, including p. maritima, is problematic. rahn (1978); dietrich (1980) and klimko et al. (2004) have divided the section coronopus into two sections as section coronopus and section maritima. shehata & loutfy (2006) have separated the p. maritima taxon from subgenus coronopus. in this study, p. maritima emerged in the second main group with the other section coronopus (p. weldenii, p. crassifolia and p. holosteum) except p. coronopus. the dendrogram separated the taxa of sect. coronopus into four subgroups; p. maritima grouped with p. crassifolia; p. weldenii which was formerly assigned as a subspecies of p. coronopus. however, these findings don’t agree with the molecular data completely (serrano et al., 2017). in tutel et al. (2005), it has been suggested that hymenopsyllium section in subgenus euplantago is closer to subgenus psyllium based on morphological traits. in this study sect. hymenopsyllium is clustered completely in first main group, whereas most of the sect. psyllium taxa are placed in the second main group. therefore, this study does not support the initial statement. however, the results showed the latter section is very diverse in terms of micromorphologic traits and the study of molecular markers already showed p. bellardii and p. cretica are in the subgenus psyllium (ronsted et al., 2002). this shows the micromorphology of inflorescence parts of these taxa are not very reliable in this subgenus. the plantago taxa are known to be wind polinated but we observed that the flowers attract some insects (syrphidae) in field and alfred heilbronn botanical garden. by the reason of the fact that they do not have flashy flowers, it may be due to the uv reflection characteristics of the epicuticular waxes (whitney et al., 2009). some taxa having more ability to draw the insects (p. maritima, p. lanceolata, p. argentea) have granulate and striate petal surfaces. however, this finding needs further investigations. in the overall results of the examinations, we have pointed out that the inflorescence parts’ surfaces are variable among taxa except for the corolla tube, and the differences are probably not distinctive characteristics for these taxa. this may be due to its protective position. this work shows that hairs, epidermal cell arrangements and surface characters are very variable across the genus. therefore, analysis of inflorescence parts is not conclusive to make a specific distinction between different taxa. although some groups have very stable seed surface characteristics, they are not reliable among all taxa, which means it should be used with extreme care especially with the widespread taxa. further studies may be conducted to understand how variable the seed characteristics of the widespread taxa, and whether it is a result of different habitat types. acknowledgements. we would like to thank dr. vahap eldem for performing the data processing in r, and prof. dr. orhan kucuker for sharing his knowledge. this work was supported by the research fund of istanbul university, with project number 10413. references albach, d. c., meudt, h.m., oxelman, b. 2005: piecing together the “new” plantaginaceae. american journal of botany, 92: 297-315. barthlott, w., neinhuis, c., cutler, d., ditsch, f., meusel, i., theisen, i., wilhelmi, h. 1998: classification and terminology of plant epicuticular waxes. botanical journal of the linnean society, 126 90 biologica nyssana ● 11 (2) december 2020: 85-91 ciftci, erol ● micromorphological studies on inflorescence and seeds of some plantain (plantago l.) taxa in turkey (3): 237-260. dietrich, h. 1980: zytologische untersuchung innerhalb der familie der plantaginaceae. naturwissenschaften, 29: 559-587. endress, p.k. 2001: origins of flower morphology. journal of experimental zoology, 291 (2): 105-115. hoghoughi, t., jafari, a., mahmoodzadeh, h. 2016: palynological and seed micromorphological studies on plantago l. species in north eastern of iran. journal of biology and environmental science, 8 (1): 133-138. jun-zhe, l., qing-min, z., shu-hua, g., xingdi, z. 1992: seed morphology of plantago in china and its taxonomic significance. journal of systematics and evolution, 30 (2): 118-125. klimko, m., idzikowska, k., truchan, m., kreft, a. 2004: seed sculpture of polish species of the genus plantago l. acta societatis botanicorum poloniae, 73 (2): 103-111. meudt, h. m. 2012: a taxonomic revision of native new zealand plantago (plantaginaceae). new zealand journal of botany, 50 (2): 101-178. pilger, r. 1937: plantaginaceae. in: engler, a. (ed.), das pflanzenreich, 1-466. engelmann verlag. berlin. rahn, k. 1978: nomenclatorial changes within the genus plantago l., intraspecific taxa and subdivisions of the genus. botanisk tidsskrift, 73: 106-111. rahn, k. 1996: a phylogenetic study of the plantaginaceae. botanical journal of the linnean society, 120 (2): 145-198. rønsted, n., chase, m. w., albach, d. c., bello, m. a. 2002: phylogenetic relationships within plantago (plantaginaceae): evidence from nuclear ribosomal its and plastid trnl-f sequence data. botanical journal of the linnean society, 139 (4): 323-338. rothmaler, w., vent, w., jäger, e., meusel, h., werner, k., pankow, h., ... & schubert, r. 1984: exkursionsflora für die gebiete der ddr und der brd. volk und wissen. rymkiewicz, a. 1979: studies on the species of the genus plantago l. with reference to carpology and chemotaxonomy. monographiae botanicae, 57: 71103. (in polish with english summary). sagar, g., harper, j. 1964: biological flora of the british isles: plantago major l., plantago media l., and plantago lanceolata l. journal of ecology, 52 (1): 189-221. serrano, h. c., cotrim, h., pinto, m. j., martinsloução, m. a., branquinho, c. 2017: metal hyperaccumulation patterns within plantago phylogeny (plantaginaceae). plant and soil, 411 (1-2): 227-41. sharma, n., koul, p., koul, a. k. 1990: reproductive biology of plantago l. iii. floral adaptation to wind pollination in plantago lagopus l. proceedings: plant sciences, 100 (6): 393-398. shehata, a. a., loutfy, m.h.a. 2006: on the taxanomy of plantaginaceae juss. sensu lato: evidence from sem of the seed coat. turkish journal of botany, 30 (2): 71-84. stearn, w. t. 1991: botanical latin history, grammar, syntax, terminology and vocabulary. timber press. london. tutel, b. 1982: plantago l. in: davis, p. h. (ed.), flora of turkey and the east aegean islands, 7: 504521, edinburgh university press. edinburgh. tutel, b. 1993: türkiye florası atlası (atlas flora turcicae) plantago 5-7. i̇. ü. yay. 3689 fen fak 225. i̇stanbul. tutel, b., kandemir, i., kus, s., kence, a. 2005: classification of turkish plantago l. species using numerical taxonomy. turkish journal of botany, 29 (1): 51-61. verma, a., kumar avinash bharti, n.g. 2017: macro and micro-morphological characteristics of plantago seeds and its implication for species identification. current botany 8: 159-163. whitney, h. m., kolle, m., andrew, p., chittka, l., steiner, u., glover, b. j. 2009: floral iridescence, produced by diffractive optics, acts as a cue for animal pollinators. science, 323: 130-133. 91 biologica nyssana ● 11 (2) december 2020: 85-91 ciftci, erol ● micromorphological studies on inflorescence and seeds of some plantain (plantago l.) taxa in turkey i̇rez et al. 2021, biologica nyssana 12(1) 12 (1) september 2021: 55-62 doi: 10.5281/zenodo.5523017 fomes fomentarius (l.) fr. extracts as sources of an antioxidant, antimicrobial and antibiofilm agents original article eylül i̇rem i̇rez department of biology, faculty of arts and sciences, çanakkale onsekiz mart university, çanakkale, turkey eyluliremirez@gmail.com nurcihan hacioğlu doğru department of biology, faculty of arts and sciences, çanakkale onsekiz mart university, çanakkale, turkey nurcihan.n@gmail.com (corresponding author) neslihan demir department of biology, faculty of arts and sciences, çanakkale onsekiz mart university, çanakkale, turkey neslihandemir@comu.edu.tr received: mart 03, 2020 revised: december 22, 2020 accepted: february 08, 2021 abstract: this paper evaluated antioxidant, antimicrobial and antibiofilm activities of ethanol, methanol, acetone and chloroform extracts of lignicolous fungal species fomes fomentarius (l.) fr. (polypodiaceae). antiradical activity was evaluated by using dpph (2,2-diphenyl-1-picrylhydrazyl) assay. the antimicrobial screening was carried out via disc diffusion and microdilution methods in order to estimate minimum inhibitory (mic) and minimum bactericidal concentration (mbc) of analyzed extracts against seven standard bacteria and one yeast: escherichia coli nrrl b-3704, pseudomonas aeruginosa atcc 27853, proteus vulgaris atcc 13315, acinetobacter baumanii atcc 19606, bacillus subtilis atcc 6633, staphylococcus aureus atcc 25923, s. haemolyticus atcc 43252 and candida albicans atcc 10231. in vitro antibiofilm activities were investigated based on crystal violet binding assay. all the extracts showed higher antioxidant activity compared to bht (butylated hydroxytoluene) which was used as a standard. ethanol, methanol and acetone extracts of the tested macrofungus also showed higher antimicrobial effect against p. aeruginosa atcc 27853 in comparison to the antibiotic penicillin (p10). the lowest mic was recorded by ethanol extract against s. haemolyticus atcc 43252 (0.625 µg/ml). the highest antibiofilm activity was also noticed against biofilm formed by s. haemolyticus atcc 43252. the results indicated that the f. fomentarius tested represent potential source of natural bioactive compounds with respect to antimicrobial, antibiofilm and antioxidant activities. key words: antimicrobial, antibiofilm, antioxidant activity, fomes fomentarius apstract: fomes fomentarius (l.) fr. ekstrakti kao izvori antioksidanasa, antimikrobnih i antibiotskih sredstava ovaj rad se bavi procenom antioksidativne, antimikrobne i antibiofilm aktivnosti etanolnog, metanolnog, acetonskog i hloroformnog ekstrakta lignikolne gljive fomes fomentarius (l.) fr. (polyporaceae). antiradikalna aktivnost je utvrđena korišćenjem dpph (2,2-difenil-1-pikrilhidrazil) eseja. antimikrobni skrining je izvršen putem disk difuzione i mikrodilucione metode kako bi se utvrdile minimalna inhibitorna (mik) i minimalna baktericidna koncentracija (mbk) analiziranih ekstrakata u odnosu na sedam standardnih bakterija i jednu gljivu: escherichia coli nrrl b-3704, pseudomonas aeruginosa atcc 27853, proteus vulgarisatcc 13315, acinetobacter baumaniiatcc 19606, bacillus subtilis atcc 6633, staphylococcus aureus atcc 25923, s. haemolyticus atcc 43252 and candida albicans atcc 10231. in vitro antibiofilm aktivnost je ispitana korišćenjem kristal violet eseja. svi ekstraktu pokazali su veću antioksidativnu aktivnost u poređenju sa bht (butilovanim hidroksitoluenom) koji je korišćen kao standard. takođe, etanolni, metanolni i acetonski ekstrakti testirane makrogljive su u poređenju sa antibiotikom penicilinom (p10) pokazali veći antimikrobni efekat na p. aeruginosa atcc 27853. najniža mik vrednost je zabeležena zza etano0lni ekstrakt i to protiv s. haemolyticus atcc 43252 (0.625 µg/ml). najviša antibiofilm aktivnost uočena je kod biofilma formiranog od strane s. haemolyticus atcc 43252. rezultati su ukazalu da testirana vrsta f. fomentarius predstavlja potencijalni izvor prirodnih bioaktivnih jedinjenja u smislu antimikrobne, antibiofilm i antioksidativne aktivnosti. ključne reči: antimikrobna, antibiofilm, antioksidativna aktivnost, fomes fomentarius © 2021 i̇rez et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 55 introduction in recent years, the problem of antibiotic resistance, where bacterial and fungal pathogens developed numerous defense mechanisms against antimicrobial agents is increased, and scientific studies presented new and more powerful agents as an alternative to antibiotic therapy (heleno et al., 2013). the interest in natural compounds has been a common point for most biotechnology companies to be used in the production of new antimicrobial drugs (butler, 2004). microbial biofilms are communities of bacteria, embedded in a self-producing matrix, forming on living and nonliving solid surfaces (vasudevan, 2014). they are considered as an important virulence factor that causes persistent chronic and recurrent infections; they are highly resistant to antibiotics and host immune defenses. biofilm resistance is due to several reasons, like restricted diffusion of antibiotics into biofilm matrix, expression of multidrug efflux pumps, type iv secretion systems, decreased permeability, and the action of antibioticmodifying enzymes (alekshun and levy, 2007). the increased biofilm resistance to conventional treatments enhances the need to develop new control strategies (simões et al., 2007; sánchez et al., 2016). it is common to believe that it is difficult to prevent and treat biofilm infections. current findings regarding the fact that biofilms have a highly heterogeneous structure necessitates the development of new treatment strategies unlike the treatment strategies used in the destruction of microorganisms in the planktonic phase (clatworthy et al., 2007). besides its antimicrobial effects, some natural product extracts are known to prevent biofilm formation. due to the inadequate antimicrobial therapies in combating biofilms, researchers are directed towards the discovery and identification of new antimicrobial agents, especially natural (karaca et al., 2017). antioxidants have the ability to protect the body against damage caused by oxidative stress. the interest in natural antioxidants is increasing day by day. for example, polyphenols, found in medicinal plants and foods, can help prevent oxidative damage (silva et al., 2005). mushrooms are rich sources of antioxidant compounds such as phenolic compounds (phenolic acids and flavonoids) and tocopherols (cheung et al., 2003; ferreira et al., 2009; heleno et al., 2010; sun et al., 2011; dündar et al., 2016). fomes fomentarius (l.) fr., polyporaceae, tinder fungus is a woody, perennial fungus, large in size which develops as a parasite or saprophyte on the beech (fagus sylvatica l.) and other deciduous species. it is a white root fungus causing heart rot of the wood. in recent years, the active compounds of f. fomentarius have been studied extensively and most of its biological activities have been put forward. it is found that f. fomentarius have significant effects such as antioxidant (lee, 2005; vazirian et al., 2014; dündar et al., 2016; bal et al., 2017), antimicrobial (kolundžić et al., 2016), antifungal (dresch et al., 2015), anti-inflammatory (vazirian et al., 2014), cytotoxic (kolundžić et al., 2016), antitumor (chen et al., 2008), antiviral (aoki et al., 1993), dna protective (bal et al., 2017), fibrinolytic activity (sánchez-santillán et al., 2015). this extended study was to evaluate the antimicrobial, antibiofilm and antioxidant activities of ethanol, methanol, acetone and chlorofom extracts of f. fomentarius. although there have been extensive researches to reveal antioxidant and antimicrobial activity of f. fomentarius, studies on antibiofilm activity have not been found in the literature. we also aimed to determine antibiofilm potential of the four extracts obtained from f. fomentarius against important pathogen microorganisms for the first time. materials and methods the macrofungal material fomes fomentarius was collected from çanakkale, turkey on decaying stump in january, 2018 and identified by dr. ersin karabacak. preparation of extracts the sample of the dried macrofungus (15 g) was extracted with four solventsethanol, methanol, acetone and chloroform (150 ml) using the soxhlet apparatus (isolab). the extraction was continued until the final extract was colorless according to khan et al. (1988). the solvents were removed under reduced pressure and dried using a rotary evaporator (heidolp, laborota 4001, germany) at 55 °c. dry extract was taken into glass bottles with dark lid and stored at +4 °c. for the experiments, dissolution in dimethyl sulfoxide (dmso) (10%) and different concentrations of the prepared extract were sterilized by a membrane filter (0.2 µm) and prepared for screening. evaluation of antiradical activity dpph assay the antioxidant activity of the extracts was measured by using commercial free radical 2,2-diphenyl-1-picrylhydrazyl (dpph) following the procedure described by brand-williams et al. (1995), with slight modifications. the extracts were evaporated to dryness and re-suspended in dmso. butylated hydroxytoluene (bht) solution was used as a positive control. the absorbance 56 biologica nyssana ● 12 (1) september 2021: 55-62 i̇rez et al. ● fomes fomentarius (l.) fr. extracts as sources of an antioxidant, antimicrobial and antibiofilm agents 57 values of the samples were measured on a uv-vis spectrophotometer (pg instruments t+80) at 517 nm against a blank. the experiment was repeated three times and the arithmetic mean of the readings was taken. the radical scavenging activity of each sample was calculated using the following equation and the results were expressed as % inhibition. inhibition (%) = [(a0 a1) / a0] x 100 a0: extract or non-standard control absorbance, a1: extract or standard absorbance evaluation of antimicrobial activity sterilized antibiotic discs 6 mm in diameter (schleicher and schull no. 2668, dassel, germany) were impregnated with 50 μl of each extract (10 mg/disc) at concentration of 200 mg/ml. gram negative bacteria escherichia coli nrrl b-3704, pseudomonas aeruginosa atcc 27853, proteus vulgaris atcc 13315, acinetobacter baumanii atcc 19606, gram positive bacteria bacillus subtilis atcc 6633, staphylococcus aureus atcc 25923, s. haemolyticus atcc 43252 and yeast culture candida albicans atcc 10231 were used as the test microorganisms. all the bacteria were incubated at 35 ± 0.1 °c for 24 h by inoculation into nutrient broth (difco laboratories, mi, usa) and the yeast culture studied was incubated in malt extract broth (difco laboratories, mi, usa) at 25 ± 0.1 °c for 48 h. an inoculum containing 106 bacterial cells or 108 yeast cells/ml was spread on mueller hinton agar (mha) (oxoid ltd., hampshire, uk) plates (1 ml inoculum/plate). the discs injected with extracts were placed on the inoculated agar by pressing slightly. petri dishes were placed at ±4 °c for 2 h and incubated for 24 h and 48 h for bacteria and yeast, respectively (collins et al., 1989). at the end of the period, inhibition zones formed on the medium were evaluated in millimeters. studies were performed in triplicate. treatments with penicillin (p10), and nystatin (nys30) served as positive controls and treatments with ethanol, methanol, acetone and chloroform without fungal materials served as negative controls. for quantitative antimicrobial analyses, minimum inhibitory concentration (mic) values of all samples were determined. mic and mbc were investigated as recommended instruction of the clinical and laboratory standards institute (clsi, 2006). briefly, stock solution of each extract was diluted in the muller hinton broth (mhb) in two-fold serial dilutions to obtain final concentrations range of 200.156 mg/ml at a total volume of 100 μl per well in 96-well microtiter plates. the lowest concentration of extracts inhibiting visible growth of each test microorganisms was taken as the mic. the medium, 0.1% (w/v) streptomycin (st), nystatin (nys30) and 10% dmso were used as the non-treated, positive and negative controls, respectively (teanpasian et al., 2017). confirmation of mic and establishment of the minimum bactericidal concentration (mbc) and minimum fungicidal concentration (mfc), 10 µl of the following dilutions (40 to 2.5 µg/ml) were inoculated into plates with mha to evaluate microbial growth. after 24 h of incubations at 35 ± 0.1 °c for bacteria and 48 h at 25 ± 0.1 °c for the yeast culture, the plates with no apparent cfu of surviving microorganisms were determined. each experiment was repeated three times (pacheco-ordaz et al., 2018). biofilm inhibition assay microplate biofilm method (merrit et al., 2005) was used to evaluate the inhibition of biofilm formation by f. fomentarius extracts against tested microorganisms. cultures were incubated in 5 ml tryptic soy broth (tsb) medium containing 5% glucose. cultures were diluted 1:100 in tsb and loaded into each well in 4 sterile microplate. different concentrations of fungal extracts (mic and submic concentrations: 50, 25, 12.5% of mic) were prepared and transferred to each microplate well. after incubation at 37 ± 0.1 °c for 48 h planktonic bacteria were removed from the wells and wells were washed twice with distilled water. crystal violet solution (0.1%, 200 μl) was added to each well and incubated for 20 minutes. the crystal violet bounded extracts were poured and washed until the crystal violet was finally removed. the microtiter plates were inverted and the remaining liquid was drained and dried in room heat. finally, the adhered biofilm bounded crystal violet was eluted in ethanol (95%) and the absorbance was measured at 550 nm by using an automated elisa reader (biotek, uk). all experiments were repeated in triplicate. the calculation of the antibiofilm effect of the extract was made by the reduction formulation percentage. % inhibition = (acontrol – asample / acontrol) x 100 acontrol: absorbance of the control (containing 100 µl tbs instead of fungal extract) reaction, asample: absorbance of the tested compound results and discussion two polar (ethanol and methanol), one intermediate polar (acetone) and one nonpolar (chloroform) solvent were selected for comparison of biological activity of f. fomentarius. the choice of solvents depends on the type of macrofungi, part of the materials to be extracted, nature of the bioactive biologica nyssana ● 12 (1) september 2021: 55-62 i̇rez et al. ● fomes fomentarius (l.) fr. extracts as sources of an antioxidant, antimicrobial and antibiofilm agents 58 compounds, the availability of solvent and the literature. polar solvents such as ethanol, methanol and acetone were used in extraction of polar compounds, whereas nonpolar solvents such as chloroform were used in extraction of nonpolar compounds such as terpenoids, flavonoids, fats and oils (abubakar and hague, 2020). the findings showed that polar solvents, especially ethanol, are more successful in extraction of secondary metabolites with antioxidant, antimicrobial and antibiofilm activity. dpph assay is widely used in determination of the antioxidant activity of compounds and different fungal extracts. in this work, it was found that the inhibition percentage of ethanol extracts from f. fomentarius was slightly higher (89.95±0.21) than when acetone, methanol and chloroform solvents were applied (88.88±0.18, 87.34±0.11 and 80.93±0.07, respectively) (fig. 1). bht which was used as a standard showed lower antioxidant activity (75.81±0.03) compared with all other extracts, obtained by different solvents. studies have shown that the biological activities of the extracts can vary depending on the season, type of extract, dose and duration of application. depending on the extraction method, the level of bioactive substances can be observed (anlas et al., 2017). mircea et al. (2015) and kolundžić et al. (2016) reported that dpph radical scavenging activity of f. fomentarius methanol extract results correlated with the polyphenol content. in addition, f. fomentarius methanol and ethanol extracts have been reported to have the strongest dpph activity comparing to other solvents (bojin et al., 2020). the present study indicated that ethanol extract showed slightly higher antioxidant activities than the other solvents. the antimicrobial activities of the macrofungal extracts against different test strains were assessed according to inhibition zones diameter and mic and mbc or mfc values (tab. 1). all extracts showed higher antimicrobial effect against p. aeruginosa, p. vulgaris, s. haemolyticus, c. albicans except chloroform extract with inhibition zone of 10-13 mm. ethanol, methanol and acetone extracts of macrofungi also showed higher antimicrobial effect against p. aeruginosa in comparison to antibiotic p10. the lowest mic value was recorded by f. fomentarius ethanol extract against s. haemolyticus (0.625 μg/ml). also, ethanol extract showed higher mic against e.coli, b. subtilis, s. aureus and s. haemolyticus bacteria when compared to antibiotic streptomycin probably due to the polar component that the ethanol extract contain. there are many scientific reports about f. fomentarius antimicrobial activity (zhao et al., 2013; dündar et al., 2016; kolundžić et al., 2016; gedik et table 1. antimicrobial activity of f. fomentarius extracts fig. 1. dpph free scavenging activity of f. fomentarius extracts test microorganisms *disc diffusiona control antibiotics mic (µg/ml) control antibiotics mbc (mfc) mbc (mfc)/ mic e1 e2 e3 e4 p10 ny 100 e1 e2 e3 e4 st ny 100 e1 e2 e3 e4 e1 e2 e3 e4 e.coli 7.0 7.0 10.0 8.0 16.0 nt 2.5 2.5 10 20 4.0 nt 40 2.5 40 40 16 1 4 2 p. aeruginosa 10.0 11.0 10.0 7.0 8.0 nt 10 10 10 20 1.0 nt 40 40 40 40 4 4 4 2 p. vulgaris 13.0 10.0 10.0 7.0 13.0 nt 20 5 10 20 4.0 nt 40 40 40 40 2 8 4 2 a. baumanii 7.0 11.3 8.6 9.0 12.0 nt 5 5 10 20 2.0 nt 40 5 40 40 4 1 4 2 b. subtilis 13.0 9.0 8.3 8.0 14.0 nt 2.5 2.5 10 20 4.0 nt 40 2.5 40 40 16 1 4 2 s. aureus 13.0 10.6 8.3 9.0 15.0 nt 2.5 5 10 20 4.0 nt 40 5 40 40 16 1 4 2 s. haemolyticus 11.6 11.6 10.3 8.0 14.0 nt 0.6 5 10 20 5.0 nt 40 10 40 40 64 2 4 2 c. albicans 10.0 11.0 10.0 7.0 nt 16.0 5 5 5 20 nt 5.0 40 40 40 40 4 8 8 2 biologica nyssana ● 12 (1) september 2021: 55-62 i̇rez et al. ● fomes fomentarius (l.) fr. extracts as sources of an antioxidant, antimicrobial and antibiofilm agents al., 2019). similar to the present study, kolundžić et al. (2016) tested the antimicrobial activity of f. fomentarius extracts of different polarity, especially against gram-negative and gram-positive bacteria. they indicated that their f. fomentarius extracts (cyclohexane, dichloromethane, methanol and aqueous) displayed strong antimicrobial activity. gedik et al. (2019) also showed that ethanol and aqueous extracts of f. fomentarius have higher antibacterial activity against k. pneumoniae, a. baumanii, s. aureus, vancomycin-resistant enterococci (vre+), e. coli against standard antibiotics. in our study, a similar situation was obtained against p. vulgaris, b. subtilis, s. aureus bacteria. the results of zhao et al. (2013) demonstrated weak antimicrobial activity in isolated phenyl-ethanediols from the fruiting bodies of f. fomentarius. moreover, methanolic extracts of f. fomentarius have been found to be effective against m. luteus, but no activity was detected against s. aureus, e. coli, p. aeruginosa, b. subtilis, enterococcus faecalis (kolundžić et al., 2016). in the present study we obtained different results for antimicrobial activity of f. fomentarius against the tested microorganisms; that is possibly due to differences of fungal species habitat factors, ecological status, seasonal differences and variety of extraction methods. the results of potential inhibition of test microorganism’s biofilm formation by four extracts of f. fomentarius were shown in fig. 2. the mic values of f. fomentarius extracts were used to determine antibiofilm rates. it can be seen that antibiofilm rates of four extracts ranged from 5.03 to 95.2%. the results indicated that ethanol and methanol extracts of f. fomentarius could inhibit the biofilm formation of all the tested strains, significantly. the highest antibiofilm activity was noticed against biofilm formed by s. haemolyticus. both acetone and chloroform extracts showed very low antibiofilm effects compared to ethanol and methanol, except for c. albicans (fig. 2). although recent studies on the evaluation of medically important fomes species in this direction are few in the literature (bin et al., 2012; solmaz et al., 2013; alves et al., 2014; petrović et al., 2014; signoretto et al., 2014; carvalho et al., 2016; karaca et al., 2017), there is no study on the antibiofilm effects of the f. fomentarius on the related pathogen strains. alves et al. (2014) tested the antibiofilm capacity of different macrofungal species against e. coli, p. aeruginosa, a. baumannii and p. mirabilis strains. they found that all macrofungi methanol extracts showed strong antibiofilm activity against pathogens. petrović et al. (2014) also reported that hot water extract from agaricus blazei could influence on biofilm formation, twitching and swimming activity, pyocyanin production which are part of anti-quorum sensing activity. karaca et al. (2017) investigated antibiofilm potentials of methanolic and ethanolic extracts of the three medicinal macrofungi species and found that the highest antibiofilm activity was observed with ganoderma lucidum methanolic extract. therefore, the findings obtained from the present study are presenting the important contributions to the literature data on this subject. conclusions in the present study, it was determined that f. fomentarius demonstrated high antioxidant, 59 fig. 2. inhibition (%) of biofilms formation of f. fomentarius extracts (e1: ethanol, e2: methanol, e3: acetone, e4: chloroform) biologica nyssana ● 12 (1) september 2021: 55-62 i̇rez et al. ● fomes fomentarius (l.) fr. extracts as sources of an antioxidant, antimicrobial and antibiofilm agents antimicrobial and antibiofilm activities. in conclusion, it was determined that analyzed f. fomentarius extracts could be used after chemical characterization and toxicity testing as natural resources in the fields of medicine, pharmacology, nutrition and cosmetics due to their strong antioxidant and antimicrobial activities. acknowledgment. authors would like to thank assoc. prof. dr. ersin karaback for defining macrofungus species. this research is a part of mrs. eylül i̇rem i̇rez’s tübi̇tak-2209 a project which is supported with the frame of undergraduate students grant in biological science. references abubakar, a.r., hague, m. 2020: preparation of medicinal plants: basic extraction and fractionation procedures for experimental purposes, journal of pharmacy & bioallied sciences, 12(1): 1-10. alekshun, m.n., levy, s.b. 2007: molecular mechanisms of antibacterial multidrug resistance, cell, 128(6): 1037-1050. alves, m. j., ferreira, i. c., lourenço, i., costa, e., martins, a., pintado, m. 2014: wild mushroom extracts as inhibitors of bacterial biofilm formation. pathogens, 3(3): 667-679. anlas, c., ustuner, o., ustun alkan, f., bakirel, t., aydogan, m.n., baykan, erel, s. 2017: a comparative study on the antioxidant activities and phenolic contents of different extracts of achillea nobilis subsp. sipylea and alcea apterocarpa (fenzl) boiss, endemic plants in turkey. fresenius environmental bulletin, 26(2): 1423-1430. aoki, m., tan, m., fukushima, a., hieda, t., kubo, s., takabayashi m., ono, k., mikami, y. 1993: antiviral substances with systemic effects produced by basidiomycetes such as fomes fomentarius. bioscience, biotechnology, and biochemistry, 57(2): 278-82. bal, c., akgul, h., sevindik, m., akata, i., yumrutas, ö. 2017: determination of the antioxidative activities of six mushrooms. fresenius environmental bulletin, 26(10): 6246-6252. brand-williams, w., cuvelier, m.e., berset, c. 1995: use of a free radical method to evaluate antioxidant activity. lebensmittel-wissenschaft undtechnologie/food science and technology, 28:2530. bin, l., wei, l., xiaohong, c., mei, j., mingsheng, d. 2012: in vitro antibiofilm activity of the melanin from auricularia auricula, an edible jelly mushroom. annals of microbiology, 62(4): 1523-1530. bojin, l., serb, a., pascariu, m., moaca, a., kostici, r., purcarea, v.l., penescu, m. ivan, m.v., georgescu, m., sisu, e. 2020: assessment of antioxidant properties of different fomes fomentarius extracts. farmacia, 68(2): 322-328. butler, m.s. 2004: the role of natural product chemistry in drug discovery. journal of natural products, 67: 2141–2153. carvalho, m. p., gulotta, g., do amaral, m. w., lünsdorf, h., sasse, f., abraham, w. r. 2016: coprinus lactone protects the edible mushroom coprinus comatus against biofilm infections by blocking both quorum sensing and mura. environmental microbiology, 18(11): 4254-4264. chen, w., zhao, z., chen, s.f. 2008: optimization for the production of exopolysaccharide from fomes fomentarius in submerged culture and its antitumor effect in vitro. bioresource technology, 99(8): 3187–3194. cheung, l.m., cheung, p.c.k., ooi, v.e.c. 2003: antioxidant activity and total phenolics of edible mushroom extracts. food chemistry, 81: 249–255. clatworthy, a.e., pierson, e., hung, d.t. 2007: targeting virulence: a new paradigm for antimicrobial therapy. nature chemical biology, 3(9): 541-548. clsi, 2006. clinical and laboratory standarts institute. methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard-seventh edition. m07a7, villanova, pa, usa. collins, c.h., lyne, p.m, grange, j.m. 1989: microbiological methods, sixth edition. london, butterworths & co ltd. dresch, p., d ́aguanno, m.n., rosam, k., grienke, u., rollinger, j.m., peintner, u. 2015: fungal strain matters: colony growth and bioactivity of the european medicinal polypores fomes fomentarius, fomitopsis pinicola and piptoporus betulinus. amb express, 5(4): 1-14. dülger, b., şen, f., gücin f. 1999: russula delica fr. makrofungusunun antimikrobiyal aktivitesi. turkish journal of biology, 23: 127-133. dündar, a., okumus, v., ozdemir, s., celik, k.s., boğa, m., ozcagli, e. 2016: determination of cytotoxic, anticholinesterase, antioxidant and antimicrobial activities of some wild mushroom species cogent food & agriculture, 2: 1178060. ferreira, i.c.f.r., barros, l., abreu, r.m.v. 2009: antioxidants in wild mushrooms. current medicinal chemistry, 16: 1543–1560. 60 biologica nyssana ● 12 (1) september 2021: 55-62 i̇rez et al. ● fomes fomentarius (l.) fr. extracts as sources of an antioxidant, antimicrobial and antibiofilm agents gedik, g., dülger, g., asan, h., ozyurt, a., allı, h., asan, a. 2019: the antimicrobial effect of various formulations obtained from fomes fomentarius against hospital isolates. the journal of fungus, 10(2): 103-109. halliwell, b., gutteridge, j.m.c. 1996: free radicals in biology and medicine. oxford: oxford u heleno, s.a., barros, l., sousa, m.j., martins, a., ferreira, i.c.f.r. 2010: tocopherols composition of portuguese wild mushrooms with antioxidant capacity. food chemistry, 119: 1443–1450. karaca, b., akata, i., çöleri-cihan, a. 2017: lentinus edodes, lactarius delicious ve ganoderma lucidum’un antibiyofilm ve antimikrobiyal etkinlikleri. kastamonu univ., orman fakültesi dergisi, 17(4): 660-668. khan, n.h., kamal, m.s.a., rahman, m. 1988: antibacterial activity of euphorbia thymifolia linn. indian journal of medical research, 87: 395-397. kolundžić, m., grozdanić, n., dodevska, m., milenković, m., sisto, f., miani, f., farronato, g., kundaković, t. 2016: antibacterial and cytotoxic activities of wild mushroom fomes fomentarius (l.) fr., polyporaceae. industrial crops and products, 79: 110–115. lee, j.s. 2005: effects of fomes fomentarius supplementation on antioxidant enzyme activities, blood glucose, and lipid profile in streptozotocininduced diabetic rats. nutrition research, 25(2):187195 merritt, j.h., kadouri, d.e., o’toole, g.a. 2005: growing and analyzing static biofilms. current protocols in microbiology, 1(1b):1-17. mircea, c., cionca, o., iancu, c., tataringa, g., hancianu, m. 2015: in vitro antioxidant activity of some extracts obtained from agaricus bisporus brown, pleurotus ostreatus and fomes fomentarius. farmacia, 63(6): 927-933. pacheco-ordaz, r., wall-medrano, a., goñi, m.g., ramos-clamont-montfort, g., ayalazavala, j.f., gonzález-aguilar, g.a. 2018: effect of phenolic compounds on the growth of selected probiotic and pathogenic bacteria. letters in applied microbiology, 66(1): 25-31. petrović, j., glamočlija, j., stojković, d., nikolić, m., ćirić, a., fernandes, a., soković, m. 2014: bioactive composition, antimicrobial activities and the influence of agrocybe aegerita (brig.) sing on certain quorum-sensing-regulated functions and biofilm formation by pseudomonas aeruginosa. food & function, 5(12): 3296-3303. sánchez-santillán, p., meneses-mayo, m., miranda-romero, l., santellano-estrada, e., p alarcón-zúñiga, b. 2015: fribrinolytic activity and gas production by pleurotus ostreatus-ie8 and fomes fomentarius eum1 in bagasse cane. rev. mvz córdoba, 20(supl): 4907-4916. sánchez, e., morales, c.r., castillo, s., leosrivas, c., garcía-becerra, l., martínez, d.m.o. 2016: antibacterial and antibiofilm activity of methanolic plant extracts against nosocomial microorganisms. evidence-based complementary and alternative medicine, 2016: 1-8. signoretto, c., marchi, a., bertoncelli, a., burlacchini, g., papetti, a., pruzzo, c., spratt, d. a. 2014: the anti-adhesive mode of action of a purified mushroom (lentinus edodes) extract with anticaries and antigingivitis properties in two oral bacterial pathogens. bmc complementary and alternative medicine, 14(1): 75-84. silva, c.g., herdeiro, r.s., mathias, c.j., panek, a.d., silveira, c.s., rodrigues, v.p., renno, m.n., falcao, d.q., cerqueira, d.m., minto, ab., nogueira, f.l., quaresma, c.h., silva, j.f., menezes, f.s., eleutherio, e.c. 2005: evaluation of antioxidant activity of brazilian plants. pharmacological research, 52(3): 229-233. simões, l.c., simões, m., vieira, m.j. 2007: biofilm interactions between distinct bacterial genera isolated from drinking water. applied and environmental microbiology, 73(19): 6192–6200. soković, m.; ćirić, a.; glamočlija, j.; nikolić, m.; van griensven, j.l.d.l. 2014: agaricus blazei hot water extract shows anti quorum sensing activity in the nosocomial human pathogen pseudomonas aeruginosa. molecules, 19: 4189–4199. solmaz, g., ozen, f., ekinci, y., bird, p. s., korachi, m. 2013: inhibitory and disruptive effects of shiitake mushroom (lentinula edodes) essential oil extract on oral biofilms. jundishapur journal of microbiology, 6(9): 1-6. sun, l. p., zhuang, y. l., bai, x. 2011: effects of boiling and microwaving treatments on nutritional characteristics and antioxidant activities of agaricus blazei murril. international journal of food science & technology, 46: 1209–1215. teanpaisan, r., kawsud, p., pahumunto, n., puripattanavong, j. 2017: screening for antibacterial and antibiofilm activity in thai medicinal plant extracts against oral microorganisms. journal of traditional and complementary medicine, 7: 172-177. 61 biologica nyssana ● 12 (1) september 2021: 55-62 i̇rez et al. ● fomes fomentarius (l.) fr. extracts as sources of an antioxidant, antimicrobial and antibiofilm agents vasudevan, r. 2014: biofilms: microbial cities of scientific significance. journal of microbiology & experimentation, 1(3): 84‒98. vazirian, m., dianat, s., manayi, a., ziari, r., mousazadeh, a., habibi, e., saeidnia, s., amanzadeh, y. 2014: anti-inflammatory effect, total polysaccharide, total phenolics content and antioxidant activity of the aqueous extract of three basidiomycetes. research journal of pharmacognosy (rjp), 1: 13-19. větrovský, t., voříšková, j., snajdr, j., gabriel, j., baldrian, p. 2011: ecology of coarse wood decomposition by the saprotrophic fungus fomes fomentarius. biodegradation, 22 (4): 709–718. zhao, j.y., ding, h., li, z.h., dong, j., feng, t., zhang, b., liu, j.k. 2013: three new phenylethanediols from the fruiting bodies of the mushroom fomes fomentarius. journal of asian natural products research, 15(3): 310–314. 62 biologica nyssana ● 12 (1) september 2021: 55-62 i̇rez et al. ● fomes fomentarius (l.) fr. extracts as sources of an antioxidant, antimicrobial and antibiofilm agents rajicic et al. 2021, biologica nyssana 12(2) 12 (2) december 2021: 141-150 doi: 10.5281/zenodo.5759859 yield components and genetic potential of winter wheat on pseudogley soil of western serbia original article vera rajičić university of niš, faculty of agriculture, kosančićeva 4, 37000 kruševac, republic of serbia verarajicic@yahoo.com (corresponding author) dragan terzić university of niš, faculty of agriculture, kosančićeva 4, 37000 kruševac, republic of serbia violeta babić university of niš, faculty of agriculture, kosančićeva 4, 37000 kruševac, republic of serbia vesna perišić university of niš, faculty of agriculture, kosančićeva 4, 37000 kruševac, republic of serbia marijana dugalić university of niš, faculty of agriculture, kosančićeva 4, 37000 kruševac, republic of serbia dragoslav đokić university of niš, faculty of agriculture, kosančićeva 4, 37000 kruševac, republic of serbia snežana branković university of kragujevac, faculty of science, department of biology and ecology, radoja domanovića 12, kragujevac, republic of serbia received: july 09, 2021 revised: november 19, 2021 accepted: december 02, 2021 abstract: in order to determine the effect of genotype and vegetation season on the yield and components of the winter wheat yield an experiment was performed in the secondary agricultural-chemical school “dr đorđe radić” in kraljevo. five wheat varieties (kruna, renesansa, pobeda, ns 40s and takovčanka) were examined during two growing seasons in the agro-ecological conditions of western serbia. trials were arranged according to a randomized scheme in five repetitions. grain yield (gy), 1000 grain weight (tgw) and test weight (tw) in winter wheat grain were analyzed. the study was performed as an experiment based on extremely acidic pseudogley soil. on average, for all wheat varieties, during the two-year trial, the highest yield of winter wheat 4.673 t/ha was obtained in the variety renesansa. the renesansa variety showed the highest yield and 1000 grain weight. the highest 1000 grain weight had been established for wheat cultivar renesansa (43.72 g). the highest two-year average value of test weight was found in the varieties kruna and pobeda (77.52 kg/hl and 77.31 kg/hl). analysis of variance revealed a highly significant effect of the growing season on the yield and test weight of wheat. a highly significant effect of genotype on 1000 grain weight and test weight was determined. key words: yield components, grain yield, wheat apstrakt: komponente prinosa i genetski potencijal ozime pšenice na pseudoglejnom zemljištu zapadne srbije u cilju utvrđivanja uticaja genotipa i vegetacione sezone na prinos i komponente prinosa ozime pšenice izveden je ogled u srednjoj poljoprivrednohemijskoj školi „dr đorđe radić“ u kraljevu. ispitivano je pet sorti pšenice (kruna, renesansa, pobeda, ns 40s i takovčanka) tokom dve vegetacione sezone u agroekološkim uslovima zapadne srbije. ogledi su postavljeni po slučajnom blok sistemu u pet ponavljanja. analizirani su prinos zrna (pz), masa 1000 zrna (mhz) i hektolitarska masa (hm) u zrnu ozime pšenice. ogled je zasnovan na izuzetno kiselom zemljištu tipa pseudoglej. u proseku, za sve ispitivane sorte pšenice, tokom dvogodišnjeg ogleda, najveći prinos ozime pšenice od 4.673 t/ha dobijen je kod sorte renesansa. najveći prinos i masa 1000 zrna ustanovljena je kod sorte renesansa. najveća masa 1000 zrna ustanovljena je kod sorte pšenice renesansa (43.72 g). najveća dvogodišnja prosečna vrednost hektolitarske mase utvrđena je kod sorti kruna i pobeda (77.52 kg/hl i 77.31 kg/hl). analiza varijanse pokazala je veoma značajan uticaj vegetacije na prinos i hektolitarsku masu pšenice. utvrđen je visoko značajan uticaj genotipa na masu 1000 zrna i hektolitarsku masu. ključne reči: komponente prinosa, prinos zrna, pšenica introduction winter wheat (triticum aestivum l.) is one of the most important field crops in world and republic of serbia. world’s current average wheat production in tested period (2017-2018), was 752.841.037 tons and in republic of serbia 2.608.612 tons. the average area under wheat in the world in tested period was 216.141 mill. ha and in republic of serbia was 599.599 ha. the average wheat yield in the world was 3.483 t/ha and in republic of serbia was 4.333 t/ha (fao, 2021). the average yield of wheat in 2017 in the world was 3.538 t/ha and in the republic of serbia 4.092 t/ha. in 2018, the average yield of wheat in the world was 3.427 t/ha, and in the republic of serbia 4.574 t/ha (fao, 2021). © 2021 rajičić et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 141 wheat production in serbia depends to a large extent on the environmental factors. the grain yield and yield components of winter wheat vary considerably depending on the cultivation system, the applied doses of nitrogen, variety and conditions during the growing year, as well as their complex interactions (jaćimović et al., 2011; đekić et al., 2014; jelic et al., 2015; terzić et al., 2018; đekić et al., 2019a; popović et al., 2020; ljubičić et al., 2021). the interaction between the genotype and environment greatly limits the effectiveness of selection, if it is performed only on the basis of the average yield (dimitrijević et al., 2011; laghari et al., 2011; perišić et al., 2011; milovanović et al., 2012; đekić et al., 2013; hristov et al., 2014; jocković et al., 2014; đurić et al., 2016; terzić et al., 2018). varieties that have a smaller contribution to interaction are less sensitive to changing environmental conditions, such varieties are stable. genetic variation plays a significant role in the adaptability of wheat to stress caused by external environment (mladenov et al., 2007; milovanović et al., 2011; luković et al., 2014; đurić et al., 2018; rajičić et al., 2020). understanding the genetic basis of adaptation and its physiological causes is important for understanding the interactions, leading to more efficient choices of superior and stable genotypes (perišić, 2016; djuric et al., 2018; luković et al., 2020). the yield per unit area is the result of the action of factors of variety fertility in interaction with environmental factors. the main goal in breeding wheat is to create varieties with high yield potential and good grain quality suitable for animal feed or brewing industry (milovanović et al., 2011; 2012; hristov et al., 2014; perišić, 2016; đurić et al., 2018). improved production technology has contributed to the increase in yields in recent decades, because improved production technologies enable better realization of the potential for yield. high and stable yield of wheat is the main goal of breeding, especially in institutions where it is not possible to adequately test the breeding material on quality traits. yield increase is the result of improving germplasm and improvement of production technology (đurić et al., 2018). the need for a variety of short or long growing season depends on the environmental conditions of a particular area. in winter wheat often variations in yield can be detected, also between the years and between locations (đekić et al., 2015; đurić et al., 2018; jevtić and đekić, 2018; terzic et al., 2018; luković et al., 2020). the 1000 grain weight and test weight are direct components of grain yield and change under the influence of environmental factors, but primarily varietal characteristics. they indicate the grain size and are important criterions in breeding wheat. the 1000 grain weight in addition to the number of grains per spikes is the most important criterion in breeding wheat for yield increase (perišić, 2016). production of winter wheat with high grain yield and appropriate quality is possible only by choosing varieties of good quality with appropriate cultivation conditions and appropriate production technology. the aim of this study was the determination of the varieties and ecological environmental factors influence on differences in stability and adaptability of varieties regard the grain yield, 1000 grain weight and test weight of tested winter wheat varieties. during the vegetation years (2016-2018), in the field trials, five winter wheat varieties were examined in order to determine the selection of the better varieties for the production conditions in western serbia. materials and methods materials and field trials field experiments were performed on the experimental field of the secondary agriculturalchemical school “dr. đorđe radić” in kraljevo (43°43´00´´n, 20°40´60´´e, 198 m above sea level) during two growing seasons (2016/17 and 2017/18) in dry crop conditions, with the aim of analysing the yield and quality of winter wheat grains in western serbia. the experiments were conducted in randomized block systems, with a plot size of 50 m2 (5 m x 10 m) in five replications. wheat has been preceded by maize in all seasons. the basic tillage was done classically, up to 25 cm deep. the sowing was carried out using a machine with row spacing of 12 cm. the soil on which the trial was conducted was uniform and well prepared. the amount of seed per square meter amounted to 400-450 viable seeds, depending on the characteristics of varieties. it was sown in the third decade of october, with 300 kg/ha of fertilizer npk 15:15:15, which was added in the fall, while during the spring fertilization soil was carried out in 2-3 leaf stage was supplemented with 300 kg/ha (can-calcium-ammonium-nitrate, 27% n). the winter wheat varieties kruna and takovčanka originated from the centre for small grains in kragujevac and renesansa, pobeda and ns 40s originated from the institute of field and vegetable crops, novi sad were selected as the material for the experiment. the following properties were analyzed: grain yield (t/ha), 1000 grain weight (g) and test weight (kg/hl). grain yield was measured for each plot and converted to grain yield in t/ha based on 14% grain moisture, after which a sample was taken for analysis of 1000 grain weight and test weight of the wheat. 142 biologica nyssana ● 12 (2) december 2021: 141-150 rajičić et al. ● yield components and genetic potential of winter wheat on pseudogley soil of western serbia 143 soil conditions the trial at the secondary agricultural-chemical school “dr. đorđe radić” in kraljevo, was performed on soil that is characterized as pseudogley. the trial was performed on pseudogley soil, of unfavourable physical characteristics, of poor water-air regime with frequent water or air deficiencies. this soil has very bad physical properties (compacted, having a high content of silt and clay particles, with slow water percolation) and an extremely acid ph value (ph<4.5). its total humus content is relatively fair, but microbiological activity is low, because of its poor physical properties. it is characterized by low content of available phosphorus (6.70-6.90 mg/100g of soil) and potassium (7.80-9.80 mg/100g of soil). meteorological conditions this study was conducted over a two-year period in the kraljevo, located in the čačak-kraljevo valley that belongs to the western morava river region (tab. 1). it is surrounded by a large number of mountain ranges and their hills through which the wide river valleys of the west morava, the lower ibar and the lower gruža intersect. it is located at 20°40´60´´e longitude and 43°43´00´´n latitude. the soil of the studied area is in a zone of temperate continental climate, with uneven distribution of precipitation by months. based on the data of meteorological stations in kraljevo, in the years in which the researches were carried out, the conditions differed from the long-term average characteristic of the area. the data in tab. 1 for the investigated period (2016-2018) clearly indicate that the average air temperature was higher than the annual average by 0.8 ºc in 2017/18 and lower by 0.4 ºc in 2016/17 (tab. 1). average monthly air temperatures in 2016/17 were slightly lower, especially in january (-5.0 оc) than in 2017/18. however, in both years, temperatures were within optimal limits and had no negative effects on yield. the data presented in tab. 1 the wheat growing season analyzed (2016-2018) clearly suggest differences in weather conditions between the years of the study and the long-term mean for the region. the total amount of precipitation in the vegetation in 2016/17 was 524.4 mm and 758.7 mm in 2017/18. the total amounts of rainfall were above the perennial average in the surveyed growing seasons (2017/18), with a rather uneven monthly distribution. weather conditions in the growing period in 2017/18 were marked by high rainfall during march, while rainfall in april was significantly lower. variable and moderately warm weather, with less precipitation than average, marked may 2016/17 and 2017/18 (tab. 1). during the run of the experiment, the differences between the mean precipitation values and the perennial average in the second years of the study were the highest in march and june. higher precipitation and their better distribution in 2017/18, especially in october, march and june, contributed to better fertilizer decomposition and higher wheat grain yield. statistical analysis on the basis of achieved research results the usual variational statistical indicators were calculated: average values. experimental data were analyzed by descriptive and analytical statistics using the statistics module analyst program genstat (2013) for pc/windows 7. all evaluations of significance were made on the basis of the anova test at 5% and 1% significance levels. relative dependence was defined through correlation analysis (pearson’s correlation coefficient), and the coefficients that were obtained were tested at the 5% and 1% levels of significance. biologica nyssana ● 12 (2) december 2021: 141-150 rajičić et al. ● yield components and genetic potential of winter wheat on pseudogley soil of western serbia table 1. mean monthly air temperatures and precipitations in kraljevo, serbia (2016-2018), in relation to many years average months interval x xi xii i ii iii iv v vi average mean monthly air temperature (oc) 2016/17 7.6 6.0 5.8 -5.0 4.5 10.3 11.3 16.2 22.4 8.8 2017/18 11.9 6.8 4.0 2.7 2.0 6.5 16.6 19.1 20.9 10.0 average 11.6 7.7 2.0 0.6 3.3 7.9 12.5 16.3 20.9 9.2 the amount of precipitation (mm) 2016/17 84.1 77.6 9.4 22.1 35.3 57.7 82.1 99.9 56.2 524.4 2017/18 133.3 32.4 55.6 51.0 80.9 111.2 40.6 84.4 169.3 758.7 average 73.4 37.8 56.0 48.6 51.5 80.8 80.3 112.1 82.4 622.9 144 results grain yield and yield components the average values of productive grain yield and yield components of the investigated winter wheat varieties grown in the secondary agricultural-chemical school “dr. đorđe radić” in kraljevo are shown in tab. 2. grain yield: in regard to grain yield, differences were established in the tested varieties and growing seasons. the variety with the lowest average yield in all years was variety renesansa (4.673 t/ha). the highest average grain yield in the first year of testing was recorded for the variety renesansa (4.273 t/ha) and the lowest for the variety ns 40s (3.894 t/ha). the average grain yield of all varieties in 2016/17 was 4.077 t/ha. in the second year of testing, the highest yield of all varieties was achieved, also in this year, with the variety renesansa (5.073 t/ha). average yield of winter wheat for all varieties in 2017/18 was 4.639 t/ha and the average grain yield was 4.358 t/ha. the highest average grain yield in all years was the variety renesansa (4.673 t/ha) and the slightly lower yield the varieties takovčanka and pobeda (4.386 t/ha and 4.379 t/ha). 1000 grain weight: average values for 1000 grain weight of all studied varieties are presented in tab. 2. the average of 1000 grain weight of all tested varieties and years of winter wheat was 40.38 g. the variety ns 40s had the lowest 1000 grain weight for all years (34.68 g) and the highest 1000 grain weight the variety renesansa (43.72 g). in 2016/17, the average of 1000 grain weight of all varieties was 39.48 g and in 2017/18 was 41.28 g. the growing period in 2016/17 at the time of grain loading was marked by drought and high temperatures, which reduced the weight of 1000 grains. test weight: the average value for all varieties and years was 76.02 kg/hl (tab. 2). the lowest test weight grain of winter wheat at two years was recorded for the variety ns 40s (74.01 kg/hl), while the highest value for the varieties pobeda and kruna (77.31 kg/hl and 77.52 kg/hl). the average value of test weight of grain for all varieties was significantly higher in 2017/18 (77.26 kg/hl) compared to 2016/17 (74.78 kg/hl). regardless of the year, varibiologica nyssana ● 12 (2) december 2021: 141-150 rajičić et al. ● yield components and genetic potential of winter wheat on pseudogley soil of western serbia table 2. average values of investigated winter wheat varieties traits varieties 2016/17 2017/18 average x s sx x s sx x s sx yield, t/ha kruna 4.014 0.634 0.284 4.614 0.117 0.052 4.314 0.534 0.169 renesansa 4.273 0.443 0.198 5.073 0.475 0.213 4.673 0.604 0.191 pobeda 4.073 0.464 0.207 4.686 0.551 0.246 4.379 0.579 0.183 ns 40s 3.894 0.353 0.158 4.178 0.207 0.093 4.036 0.311 0.098 takovčanka 4.129 0.465 0.208 4.643 0.573 0.256 4.386 0.561 0.177 average 4.077 0.457 0.091 4.639 0.486 0.097 4.358 0.546 0.077 1000 grain weight, g kruna 39.49 0.905 0.405 40.94 1.781 0.797 40.21 1.536 0.486 renesansa 41.64 0.689 0.308 45.79 1.728 0.773 43.72 2.512 0.794 pobeda 40.98 0.655 0.293 43.84 1.427 0.638 42.41 1.838 0.581 ns 40s 34.45 0.636 0.284 34.91 0.578 0.259 34.68 0.622 0.197 takovčanka 40.82 1.192 0.533 40.94 1.781 0.797 40.88 1.430 0.452 average 39.48 2.772 0.554 41.28 4.008 0.801 40.38 3.530 0.499 test weight, kg/hl kruna 75.42 0.335 0.150 79.62 0.228 0.102 77.52 2.230 0.705 renesansa 74.70 0.400 0.179 78.03 0.480 0.215 76.36 1.804 0.570 pobeda 75.82 0.415 0.185 78.81 0.431 0.193 77.31 1.625 0.514 ns 40s 73.26 0.865 0.387 74.77 0.228 0.102 74.01 0.994 0.314 takovčanka 74.70 0.400 0.179 75.06 0.817 0.365 74.88 0.636 0.201 average 74.78 1.010 0.202 77.26 2.069 0.414 76.02 2.040 0.289 145 biologica nyssana ● 12 (2) december 2021: 141-150 rajičić et al. ● yield components and genetic potential of winter wheat on pseudogley soil of western serbia eties pobeda and kruna had the highest test weight. however, observed by years, there is considerable disagreement on their differences. the highest test weight in the first year of testing had variety pobeda (75.82 kg/hl) and the lowest variety kruna (75.42 kg/hl). in the second year of testing, variety kruna (79.62 kg/hl) had the highest test weight. analysis of variance between observed traits of wheat the analysis of yield variance, 1000 grain weight and test weight of winter wheat varieties during two growing seasons 2016/17 and 2017/18, are shown in tab. 3. analysis of variance in winter wheat genotypes tested showed statistically highly significant differences in yield and test weight in relation to the growing season (p<0.01). in regard to the 1000 grain weight and test weight, statistically highly significant differences were determined between the varieties of winter wheat. statistically highly significant difference in 1000 grain weight and test weight is determined under the influence of the interaction year x genotype. correlation dependence between tested wheat traits the average values of the pearson’s coefficient of correlation (r) of investigated winter wheat traits are shown in tab. 4. correlation coefficients based on all traits tested during 2016/17 and 2017/18 had positive. highly significant and positive correlation coefficients, during 2016/17, were found between 1000 grain weight and test weight (r=0.673**). during 2017/18, highly significant positive correlation coefficients were found between 1000 grain weight and test weight (r=0.596**) and significant and positive dependence was determined between yield and 1000 grain weight (r=0.496*). over a two-year study period (20162018), significant positive correlation coefficients were found between yields and 1000 grain weight (r=0.467*) and test weight (r=0.459*). also, highly significant positive correlations were found between 1000 grain weight and test weight (r=0.623**). discussion wheat grain yield depends on the type of soil and its fertility, weather conditions during the vegetation period, the genetic potential of the variety and the levels applied agrotechnical measures, especially mineral nutrition of plants with nitrogen (zafaranaderi et al., 2013; đekić et al., 2015; luković et al., 2016; perišić et al., 2016; đurić et al., 2018). small grains are very sensitive to soil acidity, although some genotypes show different tolerances. therefore, there is a need to grow wheat and other cereals on acid soils to isolate genotypes adapted to acid soils and different climatic conditions (jelić et al., 2016). numerous studies in our country and the world indicate that the combined application of organic and mineral fertilizers is the most efficient way to eliminate unfavorable production properties of acid table 3. analysis of variance of the tested parameters (anova) effect of year on the traits analyzed traits mean sqr effect mean sqr error f (1. 48)8 p-level grain yield (t/ha ) 3.948 0.223 17.733 0.000** 1000-grain weight (g) 40.825 11.874 3.438 0.070ns test weight (kg/hl) 76.756 2.651 28.951 0.000** effect of genotype on the traits analyzed traits mean sqr effect mean sqr error f (4. 45) p-level grain yield (t/ha ) 0.515 0.279 1.842 0.137 ns 1000-grain weight (g) 120.115 2.896 41.480 0.000** test weight (kg/hl) 23.414 2.452 9.547 0.000** effect of the year x genotype interaction traits mean sqr effect mean sqr error f (4. 40) p-level grain yield (t/ha ) 0.088 0.207 0.425 0.789 ns 1000-grain weight (g) 7.119 1.525 4.667 0.003** test weight (kg/hl) 5.860 0.254 23.069 0.000** *statistically significant (p<0.05); **statistically high significant (p<0.01) soils, which multiplies the yields of cultivated plants (đekić et al., 2013; jelić et al., 2014; biberdžić et al., 2014; rajičić et al., 2019). the results of our tests show that, on average for the investigated factors, the yield wheat grains amounted to 4.358 t/ha (tab. 2). the studied genotypes achieved the lowest yield values in 2016/17 years of research (4.077 t/ha) and the highest yield values in 2017/18 years (4.639 t/ha). živanović et al. (2017) gained similar wheat yields on the vertisol of šumadija (4.310-4.440 t/ha). the expressed differences in yield can be the result of the chemical properties of the given soil, since vertisol is of the acidic reaction and has lower nitrogen, phosphorus and potassium content compared to the alluvium. thus stanković (2009) pointed out that the yield of wheat grains depends on the type of soil and its fertility, weather conditions during the growing period, the genetic potential of the variety and the level of applied cultural practices measures. according to the authors đekić et al. (2019a), varieties of winter wheat tested to low soil ph values show very good results. based on the level of yield achieved, winter wheat varieties are known to be the most tolerant the kruna variety proved to be more adaptable. the same authors point out that grain yield of wheat cultivars ranged from 3.743 t/ha to 6.275 t/ha. newly created high-yielding wheat varieties have been found to be less responsive to temperature deviation than is the case with rainfall (đurić et al., 2013; hristov et al., 2013; đekić et al., 2015; jelic et al., 2015; luković et al., 2020; rajičić et al., 2020). đekić et al. (2019) pointed out that intensive preharvest rainfall in 2010 led to crop lodging in case of wheat cultivars (toplica and vizija), which reduced yield. jevtić & đekić (2018) pointed out that the yield of wheat varieties varied significantly by years of research and amounted from 3.125 to 3.518 t/ha in 2012/13, while a much higher yield of 4.395 to 4.729 t/ha was established in 2011/12. according to the authors terzić et al. (2018), average yield of wheat in the experiment was 5.091 t/ha, with a variation of 4.630 t/ha in the 2011/12 to 5.553 t/ha in the first year of the study (2010/11). biberdžić et al. (2020) investigated different wheat genotypes over a two-year study and reported that yields ranged from 3.090 to 3.745 t/ha in 2016/17 and from 3.839 to 4.770 t/ha in 2017/18. the same authors pointed out that grain yield of wheat cultivars ranged from 3.464 t/ha (the variety daria on vertisol) to 6.865 t/ha (the variety avenue on the alluvial). the 1000 grain weight is a very important qualitative characteristic of grain that has a strong impact on grain yield. the 1000 grain weight during the two-year study period was 40.38 g. the values obtained are slightly lower than the results obtained by biberdžić et al. (2020), jevtić & đekić (2018) and terzić et al. (2018). jevtić & đekić (2018), examined different wheat genotypes and stated that the 1000 grain weight ranged from 42.72 to 44.05 g. terzić et al. (2018) performed a two-year study and the results showed that the 1000 grain weight ranged from 41.98 g to 45.0 g. biberdžić et al. (2020) pointed out that the average weight of 1000 grains in the optimal sowing period was 41.8 g, while in the delayed time it was 40.08 g, which does not represent a statistically significant difference. test weight is an indicator of grain size and filling, and it is a very important parameter when buying 146 biologica nyssana ● 12 (2) december 2021: 141-150 rajičić et al. ● yield components and genetic potential of winter wheat on pseudogley soil of western serbia table 4. analysis of variance of the tested parameters (anova) grain yield, gy 1000 grain weight, gw test weight, tw correlations between the traits analysed in the 2016/17 grain yield, gy 1.00 0.272 0.183 1000 grain weight, gw 0.673** test weight, tw 1.00 correlations between the traits analysed in the 2017/18 grain yield, gy 1.00 0.496* 0.234 1000 grain weight, gw 0.596** test weight, tw 1.00 correlations between the traits analysed in the 2016-2018 grain yield, gy 1.00 0.467* 0.459* 1000 grain weight, gw 0.623** test weight, tw 1.00 *statistically significant (p<0.05); **statistically high significant (p<0.01) 147 biologica nyssana ● 12 (2) december 2021: 141-150 rajičić et al. ● yield components and genetic potential of winter wheat on pseudogley soil of western serbia wheat. the average two-year value of test weight was 76.02 kg/hl and the highest were obtained for the genotype kruna (77.52 kg/hl). the values obtained were slightly higher than the test weight obtained by jevtić & đekić (2018) and biberdžić et al. (2020). a significant influence of the growing season on test weight in wheat is also established by jevtić & đekić (2018). during a two-year study, jevtić & đekić (2018), have found that the test weight ranged from 69.5 to 71.03 kg/hl. according to the authors biberdžić et al. (2020), in the optimal sowing period, test weight was 71.42 kg/hl, while in the delayed time, it was 69.96 kg/hl, which does not represent a statistically significant difference. based on the analysis of variance, it can be concluded that the influence of growing season on grain yield was highly significant (fexp=17.733**). also, biberdžić et al. (2020), jevtić & đekić (2018) and terzić et al. (2018), state that differences in yields between years occur due to environmental conditions. the 1000 grain weight in the study showed highly significant dependence on the genotypes (fexp=41.480**) and very significant in the interaction of growing seasons and genotypes (fexp=4.667**). the present results confirm the opinion of many authors that the 1000 grain weight are strongly modified by the nutrient status of the environment and weather conditions (đekić et al. 2014; jevtić & đekić, 2018; terzić et al., 2018; rajičić et al., 2020). correlation analysis shows the intensity of dependence between studied traits. from the genetic aspect, correlation indicates links between genes, or the appearance of pleiotropic effects of genes. gene linkage pertains to their position on the same chromosome, while pleiotropy appears when one gene regulates the expression of several traits. a strong positive correlation between small grains yields and 1000 grain weight has been found by many researchers (terzic et al., 2018; rajičić et al., 2020) and medium positive dependence has been identified by jelic et al. (2013), đekić et al. (2014) and terzić et al. (2018). positive and weak dependence of yield and 1000 grain weight (r=0.31) was found by đekić et al. (2019b). iftikhar et al. (2012) have established that the 1000 grain weight have a positive and statistically significant correlation with grain yield. rajičić et al. (2020) found a highly significant and positive correlation between yield and 1000 grain weight in 2013/14 (r=0.683**) and highly significant and positive correlation coefficients in 2014/15 (r=0.733**). the duration of the grain filling period is significantly influenced by environmental factors, above all the high temperatures that shortened this period. selection of early-flowering varieties can influence the formation of more flowers and a longer grain filling period, i.e. the selection of higher yielding varieties (djuric et al., 2018). based on the results of the research it can be concluded that a greater number of traits have a decisive role in the formation of grain yield. the contribution of each individual trait may be different in various varieties and in various environmental conditions so that the correlation between two quantitative traits is not fixed. this results from the interaction between the traits of each genotype and the interaction of varieties with external factors. conclusions the highest yield of grain, 1000 grain weight and test weight in all wheat varieties were in the second vegetation period with moderate temperatures at the time of grain filling and a large amount of precipitation. grain yields in wheat varieties ranged from 4.036 t/ha (ns 40s) to 4.673 t/ha (renesansa). the average 1000 grain weight in the study was 40.38 g, with a variation of 39.48 g in the vegetation year 2016/17 to 41.28 g in the vegetation year 2017/18. the test weight for all examined wheat varieties in the study was 76.02 kg/hl and varied from 74.01 kg/ hl in variety ns 40s to 77.52 kg/hl in variety kruna. grain yield shows a tendency to grow in years with a larger amount and better distribution of precipitation during the critical phases of plant development. analysis of variance revealed a very significant influence of the growing season on grain yield and a highly significant influence of the year x variety interaction on grain yield. based on these results, it can be concluded that several traits have a decisive role in the formation of grain yield. the contribution of each individual feature can be different for different varieties and the various environmental conditions so that this results from the interaction between the features within each genotype and genotype interactions with environmental factors. acknowledgements. ph.d. vera rajičić and authors sincerely thanks to the secondary agricultural-chemical school “phd đorđe radić” in kraljevo in serbia, which has enabled the use and analysis of traits of the wheat. investigations necessary for this paper are part of the projects agreement number 451-03-09/2021-14/200383 financed by the ministry of education, science and technology development of republic of serbia. biologica nyssana ● 12 (2) december 2021: 141-150 rajičić et al. ● yield components and genetic potential of winter wheat on pseudogley soil of western serbia references biberdžić, m., jelić, m., knežević, b., barać, s., lalević, d. 2014: yield of wheat in pseudogley in dependence of fertilisation with mineral fertilisers, lime fertiliser and manure. agriculture & forestry, 60(4): 7-13. biberdžić, m., barać, s., deletić, n., stojković, s., madić, m., lalević, d., đekić, v., stojiljković, j. 2020: the effect of sowing time on the yield of some wheat varieties. proceedings of the 2st international symposium “modern trends in agricultural production and environmental protection”, 01-04 july, tivat, montenegro, 66-75. dimitrijević, m., knežević, d., petrović, s., zečević, v., bošković, j., belić, m., pejić, b., banjac, b. 2011: stability of yield components in wheat (triticum aestivum l.). genetika, 43(1): 2939. đekić, v., staletić, m., jelić, m., popović, v., branković, s. 2013: the stability properties of wheat productionon acid soil. proceedings, 4th international symposium “agrosym 2013”, 03-06. oktober, jahorina, 84-89. đekić, v.; milovanović, m.; popović, v.; milivojević, j.; staletić, m.; jelić, m.; perišić, v. 2014: effects of fertilization on yield and grain quality in winter triticale. romanian agricultural research, 31: 175-183. đekić, v., milovanović, m., milivojević, j., staletić, m., popović, v., simić, d., mitrović, m. 2015: impact of year and grain quality of winter wheat. proceedings of research papers pkb agroekonomik, belgarde, 21(1-2): 79-86. đekić. v., popović. v., terzić. d., đurić. n., perišić. v., perišić. v., luković. k. 2019a: the impact of climate change on the grain yeald of wheat. proceedings of research papers pkb agroekonomik, belgarde, 25(1-2): 9-18. đekić, v., milivojević, j., madić, m., popović, v., branković, s., perišić, s., terzić, d. 2019b: grain yield and its components in winter barley grown. journal of central european agriculture, 20(1): 238-250. đurić, n., trkulja, v., simić, d., prodanović, s., đekić, v., dolijanović, ž. 2013: analiza prinosa zrna i kvaliteta brašna nekih sorata ozime pšenice u proizvodnoj 2011-2012. godini. zbornik naučnih radova instituta pkb agroekonomik, 19(1-2): 1521. đurić, n., cvijanović, g., dozet, g., matković, m., branković, g., đekić, v. 2016: correlation analysis of more significant production traits of certain winter wheat pkb varieties. agronomy journal, 78(2-3): 85-96. đurić, n., trkulja, v., cvijanović, v., branković, g., đekić, v., spasić, m., ivanović, d. 2018: imperija-nova sorta ozime pšenice stvorena u institutu pkb agroekonomik. zbornik naučnih radova instituta pkb agroekonomik, 24(1-2): 5964. djuric, n., prodanovic, s., brankovic, g., djekic, v., cvijanovic, g., zilic, s., dragicevic, v., zecevic, v., dozet, g. 2018: correlation-regression analysis of morphological-production traits of wheat varieties. romanian biotechnological letters, 23(2): 13457-13465. faostat 2021. available online: http://faostat. fao.org (accessed on 8 jul 2021). genstat release 16.2 (pc/windows 7) 2013: genstat procedure library. release pl24.2. vsn international ltd. rothamsted, uk. hristov, n., mladenov, n., kondić-špika, a., jocković, b. 2013: uticaj padavina i temperature na prinos ozime pšenice pri različitim gustinama setve. zbornik naučnih radova instituta pkb agroekonomik, 19(1-2): 31-38. hristov, n., mladenov, n., jocković, b., kondićšpika, a. 2014: uticaj sorte, lokaliteta i godine na prinos ozime pšenice. zbornik naučnih radova instituta pkb agroekonomik, 20(1-4): 33-40. iftikhar, r., khaliq, i., ijaz, m., rashid, rahman, a.m. 2012: association analysis of grain yield and its components in spring wheat (triticum aestivum l.). american-eurasian journal of agricultural and environmental science, 12(3): 289-392. jaćimović, g., malešević, m., aćin, v., marinković, b., crnobarac, j., latković, d., bogdanović, d., pejić, b. 2011: efikasnost mineralne ishrane pšenice u zavisnosti od intenziteta đubrenja. letopis naučnih radova, 35(1): 75-86. jelić, m., milivojević, j., đekić, v., paunović, a., tmušić, n. 2014: impact of liming and fertilization on grain yield and utilization of nitrogen and phosphorus in wheat plant grown on soil type pseudogley. proceedings of research papers pkb agroekonomik, belgarde, 20(1-4): 49-56. jelic, m., milivojevic, j., nikolic, o., djekic, v., stamenkovic, s. 2015: effect of long-term fertilization and soil amendments on yield, grain quality and nutrition optimization in winter wheat on an acidic pseudogley. romanian agricultural research, 32: 165-174. 148 jelić, m., milivojević, j., paunović, a., madić, m., đekić, v. 2016: adaptation of winter wheat genotypes to low ph and high mobile al content in the soil. proceedings of vii international scientific symposium “agrosym jahorina 2016”, agrosym, 06-09. october, jahorina, 762-767. jevtić, a. and đekić v. 2018: influence of growing season on some agronomic characteristics of winter wheat cultivars. biologica nyssana, 9(2): 133-139. jocković, b., mladenov, n., hristov, n., aćin, v., đalović, i. 2014: interrelationship of grain filling rate and other traits that affect the yield of wheat (triticum aestivum l.). romanian agricultural research, 31: 1-7. laghari, g.m., oad, f.c., tunio, s., chachar, q., gandahi, a.w., siddiqui, m.h. 2011: growth and yield attributes of wheat at different seed rates. sarhad journal of agriculture, 27: 177-183. luković, k., milovanović, m., perišić, v., bratković, k., staletić, m. 2014: variety perfektaanother contribution to biodiversity of winter wheat in serbia. proceedings, xviii international ecoconference 2014, 8th eco-conference® on safe food, 24-27. september 2014, novi sad, 173-180. luković, k., milovanović, m., prodanović, s., perović, d., perišić, v., staletić, m., đekić v. 2016: kg variety olimpija contribution to biodiversity of spring wheat in serbia. proceedings, xx international eco-conference® 2016, 9th ecoconference® on safe food, 28-30. september 2016, novi sad, 73-82. luković, k., prodanović, s., perišić, v., milovanović, m., perišić, v., rajičić, v., zečević, v. 2020: multivariate analysis of morphological traits and the most important productive traits of wheat in extreme rainfall conditions. applied ecology and environmental research, 18 (4), 58575871. ljubičić, n., popović, v., ćirić, v., kostić, m., ivošević, b., popović, d., pandžić, m., el musafah, seddiq, janković, s. 2021: multivariate interaction analysis of winter wheat grown in environment of limited soil conditions. plantsbasel, 10(3): 604. milovanović, m., staletić, m., đekić, v., nikolić, o., luković, k. 2011: seed production and contribution of kg varieties to biodiversity of small grains in the period 2006-2010. economics of agriculture, ii(58): 103-111. milovanović, m., staletić, m., rajičić, v., nikolić, o., perišić, v. 2012: actualities of hard winter wheat breeding in center for small grains in kragujevac. xvi international eco-conference, novi sad, proceedings safe food, 115-123. mladenov, n., denčić, s., hristov, n. 2007: oplemenjivanje na prinos i komponente prinosa zrna pšenice. zbornik radova instituta za ratarstvo i povrtarstvo, 43: 21-27. perišić, v., milovanović, m., đekić, v., staletić, m. 2011: nasleđivanje dužine klasa i broja zrna u klasu kod hibrida pšenice. zbornik pkb, 17(1-2): 19-26. perišić, v., perišić, v., živanović, t., milovanović, m., đekić, v., staletić, m., luković, k. 2016: comparative stability analysis of yield components of wheat genotypes. proceedings, xx international eco-conference® 2016, 9th eco-conference® on safe food, 28-30. september 2016, novi sad, 63-72. perišić, v. 2016: varijabilnost osobina i stabilnost prinosa i komponenti rodnosti ozime pšenice. doktorska disertacija, poljoprivredni fakultet, beograd. popović, v., ljubičić, n., kostić, m., radulović, m., blagojević, d., ugrenović, v., popović, d., ivošević, b. 2020: genotype x environment interaction for wheat yield traits suitable for selection in different seed priming conditions. plants, 9(12): 1804. rajičić, v., milivojević, j., popović, v., branković, s., đurić, n., perišić, v., terzić, d. 2019: winter wheat yield and quality depending on the level of nitrogen, phosphorus and potassium fertilization. agriculture and forestry, 65(2): 79-88. rajičić, v., terzić, d., perišić, v., dugalić, m., madić, m., dugalić, g., ljubičić, n. 2020: impact of long-term fertilization on yield in wheat grown on soil type vertisol. agriculture & forestry, 66(3): 127-138. sas/stat 2000: user’s guide, version 9.1.3. sas institute inc. stanković s. 2009: uticaj azota na proizvodnju pšenice na različitim tipovima zemljišta. doktorska disertacija, poljoprivredni fakultet, beogradzemun. terzic, d., đekić, v., jevtic, s., popovic, v., jevtic, a., mijajlovic, j., jevtic, a. 2018: effect of long term fertilization on grain yield and yield components in winter triticale. the journal of animal and plant sciences, 28(3): 830-836. terzić, d., đekić, v., milivojević, j., branković, s., perišić, v., perišić, v., đokić, d. 2018: yield components and yield of winter wheat in different years of research. biologica nyssana, 9(2): 119-131. rajičić et al. ● yield components and genetic potential of winter wheat on pseudogley soil of western serbia biologica nyssana ● 12 (2) december 2021: 141-150 149 zafaranaderi, n., aharizad, s., moha-mmadi, s.a. 2013: relationship between grain yield and related agronomic traits in bread wheat recombinant inbred lines under water deficit condition. annals of biological research, 4(4): 7-11. biologica nyssana ● 12 (2) december 2021: 141-150 rajičić et al. ● yield components and genetic potential of winter wheat on pseudogley soil of western serbia živanović, lj., popović v., ikanović j., kolarić, lj. 2017: uticaj količine i oblika azota na produktivnost ozime pšenice. “xxii savetovanje o biotehnologiji”, zbornik radova, knjiga 1. čačak. 150 aydin & kadioğlu 2022, biologica nyssana 13(1) 13 (1) september 2022: 33-40 doi: 10.5281/zenodo.7117537 total phenolic content and antioxidant activity of thymus vulgaris, curcuma longa, propolis and their mixtures original article sinem aydin faculty of science and arts, giresun university, department of biology, 28200, giresun, turkey sinem.aydin@giresun.edu.tr (corresponding author) gülşah kadioğlu faculty of science and arts, giresun university, department of biology, 28200, giresun, turkey received: may 23, 2022 revised: july 19, 2022 accepted: september 16, 2022 abstract: the current study, we investigated phenolic content and antioxidant activity of ethyl acetate and dichloromethane extracts of thyme, turmeric, propolis and their mixtures. the highest and the lowest phenolic contents were found in ethyl acetate extract of propolis (214.94±0.023 µg gae/ml) and dichloromethane extract of thyme (21.02±0.013 µg gae/ml). total antioxidant capacity of ethyl acetate extracts ranges from 127.15±0.031 µg aae/ml and 232.2±0.028 µg aae/ml; dichloromethane extracts ranges from 61.6±0.019 µg aae/ml and 159.95±0.035 µg aae/ml. cuprac activity and dpph radical scavenging activity of ethyl acetate extracts are higher than dichloromethane extracts. according to the obtained results, it can be said that propolis, thyme and turmeric could be an alternative to synthetic antioxidants. key words: thyme, turmeric, propolis, antioxidant activity apstrakt: ukupan sadržaj fenola i antioksidativna aktivnost thymus vulgaris, curcuma longa, propolisa i njihovih mešavina u ovoj studiji, ispitivali smo sadržaj fenola i antioksidativnu aktivnost etil acetatnih i dihlormetanskih ekstrakata majčine dušice, kurkume, propolisa i njihovih mešavina. najviši sadržaj fenola ustanovljen je u etil acetatnom ekstraktu propolisa (214.94±0.023 µg gae/ml), a najniži u dihlormetanskom ekstraktu majčine dušice (21.02±0.013 µg gae/ml). ukupna antioksidativna aktivnost etil acetatnih ekstrakata kreće se u opsegu od 127.15±0.031 µg aae/ ml do 232.2±0.028 µg aae/ml, a dihlormetanskih ekstrakata od 61.6±0.019 µg aae/ml do 159.95±0.035 µg aae/ml. cuprac aktivnost i aktivnost uklanjanja dpph radikala viša je kod etil acetatnih ekstrakata u odnosu na dihlormetanske ekstrakte. na osnovu dobijenih rezultata, može se zaključiti da bi propolis, majčina dušica i kurkuma mogli biti alternativa sintetičkim antioksidansima. ključne reči: majčina dušica, kurkuma, propolis, antioksidativna aktivnost introduction medicinal plants are utilized worldwide for the cure of many illnesses such as asthma, gastrointestinal symptoms, skin disorders, respiratory and urinary problems and cardiovascular diseases. plants synthesize various biologically active compounds which are crucial for them to survive in the natural environment and protect them against abiotic stresses derived from temperature, water and mineral nutrient supply (egamberdieva et al., 2017). plants have been utilized as therapeutic resources such as herbal teas, crude extracts or pharmaceutical preparations (tinctures, pills and capsules) for many years. the world health organization (who) predicts that 65% of the world’s population still use plants as traditional medicine (karakaş et al., 2012). medicinal plants has been investigated for their antioxidant capacities by many researchers. natural antioxidants are very effective to hinder the devastating effects caused by oxidative stress. plants, vegetables and fruits have natural antioxidants such as phenolics, flavonoids, tannins and proanthocyanidins. antioxidants present in plants may protect plants from diseases (saeed et al., 2012). © 2022 aydin & kadioğlu. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 33 thymus vulgaris l. (thyme) growing wild in turkey belongs to labiatea family and possess many advantageous effects such as carminative, antiseptic, antioxidant and antimicrobial activities. thymol and carvacrol are major components of thyme essential oil. thymol and other phenolic components in thymus inhibit microorganisms by increasing permeability of the cell membrane and reduction of vital intracellular substances or by disruption of bacterial enzyme systems (tural & turhan, 2017). curcuma longa l. (turmeric) belongs to zingiberaceae family generally utilized in indian and chinese systems of medicine. turmeric spice are obtained from plant rhizome known as “yellow root”. it has been also utilized for the treatment of many diseases. also, c. longa reduces risk of cancer (schaffer et al., 2011)and has antiinflammatory, antioxidant and wound healing properties (maheshvari et al., 2006). propolis is a natural resinous mixture produced from substances collected from some parts of plants, buds and secretions by honey bees. propolis is one of the “natural medicines” utilized since ancient times. more than 300 active compounds were defined in propolis. propolis has antibacterial, antiviral and antioxidant properties. moreover, propolis is utilized in apitheraphy, cosmetic and food industry for its antioxidant and antibacterial features (çoşkun & i̇nci, 2020). in the current study, antioxidant activity and total phenolic contents of dichlorometane and ethanol extracts of thyme, turmeric, propolis and their mixtures (thyme/propolis, turmeric/propolis) at 1/1 ratio have been evaluated. we also targeted to reveal antagonistic and synergistic effects of the combination of thyme, turmeric and propolis extracts. materials and methods providing of the samples thyme, turmeric and propolis were bought from a herbal shop in giresun, turkey. preparation of extracts 20 g of thyme, turmeric and propolis were extracted in a shaker for 24 h utilizing 200 ml ethyl acetate and dichloromethane, separetely. thyme:propolis and curcumin:propolis were extracted with 200 ml ethyl acetate and 200 ml dichloromethane in a shaker for 24 h, separetely. the extracts were filtered through whatman filter paper no. 1 and residues were evaporated (40 °c) with rotary evaporator (murugan & parimelazhagan, 2014). antioxidant activity total phenolic content total phenolic contents of the extracts were determined in accordance with the method of slinkard & singleton (1977) utilizing gallic acid standard. shortly, 0.1 ml extract was diluted with 4.5 ml distilled water. then, 0.1 ml of the folin– ciocalteu reagent (previously diluted 3‐fold with distilled water) was put into the mixture. after 3 minutes, 0.3 ml na2co3 (2%) was added. the absorbance was measured at 760 nm after incubating the mixture for 90 min. total phenolic content of the extracts was expressed as µg gallic acid equivalents (gae)/ml by using the calibration curve. the tests were performed in triplicate (slimkard & singleton, 1977). total antioxidant capacity phosphomolybdenum method was used to determine total antioxidant capacity of the extracts. 0.3 ml extract and 3000 µl reagent (contains 0.6 m sulfuric acid, 28 mm sodium phosphate and 28 m ammonium molybdate) was mixed and incubated at 95 °c for 90 min. then, absorbance was read at 695 nm. ascorbic acid was used as the standard (prieto et al., 1999). the total antioxidant capacity was expressed as µg ascorbic acid equivalent (aae)/ml. the tests were performed in triplicate. cupric reducing antioxidant capacity (cuprac) test 0.5 ml extract (250-1000 µg/ml concentration), 1.0 ml cucl2 solution (1x10 -2), 1.0 ml neocuproine solution (7.5x10-3 m) and 1.0 ml ammonium acetate buffer (1.0 m, ph: 7.0) were mixed in a test tube. then, the tube was vortexed and stored in a dark place for 30 min. after this period, the absorbance was read at 450 nm. butylated hydroxytoluene (bht) was used as a standard antioxidant agent (özyürek et al., 2009). dpph radical scavenging activity dpph radical scavenging activity of the extracts was established by dpph. appropriate dilution series (250-1000 µg/ml) were prepared for ethanolic extracts in dmso. 0.75 ml of each solution was added to 1.5 ml of a 6x10-5 m methanolic solution of dpph. the mixture was stirred vigorously and allowed to stand in the dark at the room temperature for 30 min. decrease in absorbance of the solution against methanol was measured at 517 nm with a shimadzu 1240 uv-vis spectrophotometer (williams et al., 1995). rutin and butylated hydroxytoluene (bht) were used as standard antioxidants. the dpph radical scavenging activity was calculated using the following equation: 34 biologica nyssana ● 13 (1) september 2022: 33-40 aydin & kadioğlu ● total phenolic content and antioxidant activity of thymus vulgaris, curcuma longa, propolis and their mixtures 35 biologica nyssana ● 13 (1) september 2022: 33-40 aydin & kadioğlu ● total phenolic content and antioxidant activity of thymus vulgaris, curcuma longa, propolis and their mixtures dpph radical scavenging activity (%)=[(a0a1)/a0]x100 a0: absorbance of control a1: absorbance of extract or standard results and discussion total phenolic content phenolic compounds are significant plant metabolites which have redox properties responsible for antioxidant activity (aryal et al., 2019). total phenolic content was determined by utilizing the folin–ciocalteu reagent. the results were calculated from a calibration curve (y = 0.013x, r2 = 0.9934) of gallic acid and expressed as µg gallic acid equivalent (gae)/ml (tab. 1). the highest and the lowest phenolic contents were found in ethyl acetate of propolis (214.94±0.023 µg gae/ml) and dichloromethane extract of thyme (21.02±0.013 µg gae/ml). ethyl acetate extracts exhibited higher total phenolic content than dichloromethane extracts except for ethyl acetate extract of thyme/ propolis. total phenolic contents of ethyl acetate and dichloromethane extracts of thyme/propolis and turmeric/propolis were decreased when compared with ethyl acetate and dichloromethane extracts of thyme, turmeric and propolis except for dichloromethane extract of thyme/propolis. total phenolic content of thyme, turmeric and propolis was also searched by many authors. for example, bulut et al. (2020) found total phenolic content of ethanol extracts of thyme leaves as 7.01±0.13 mg gae/g (bulut et al., 2020). köksal et al. (2017) determined total phenolic content of lyophilized water extract and ethanol table 1. total phenolic contents of the extracts (µg gae/ml) extract total antioxidant capacity ethyl acetate extract of thyme 73.25±0.030 ethyl acetate extract of turmeric 175.74±0.050 ethyl acetate extract of propolis 214.94±0.023 ethyl acetate extract of thyme/propolis 129.2±0.007 ethyl acetate extract of turmeric/propolis 173.61±0.008 dichloromethane extract of thyme 21.02±0.013 dichloromethane extract of turmeric 93.66±0.013 dichloromethane extract of propolis 107.82±0.011 dichloromethane extract of thyme/propolis 174.74±0.029 dichloromethane extract of turmeric/propolis 82.89±0.025 extracts of thyme as 256 µg gae/mg and 158 µg gae/mg, respectively (köksal et al., 2017). erdoğan & erbaş (2021) stated that total phenolic content of ethanol extract of turmeric was 82.47±2.70 mg gae/g (erdoğan & erbaş, 2021). yan & asmar (2010) declared that total phenolic content of methanol extract of fresh and powder of turmeric was 348.±1.26 mg gae/100 g and 2013.09±5.13 mg gae/100 g, respectively (yan & asmah, 2010). keskin & kolayli (2019) reported that the total phenolic substance amount of anatolian propolis ranged between 16.13-178.34 mg gae/g (keskin & kolayli, 2019). özdal et al. (2019) reported that the total phenolic substance amount of propolis obtained from different regions of anatolia varies between 2,748 mg gae/100 g and 19,969 mg gae/100 g (özdal et al., 2019). collecting plants from different locations, using different extraction methods and solvents may cause discrepancy in results. total antioxidant capacity total antioxidant capacity method is based on the reduction of mo (vi) to mo (v) by the extract and subsequent formation of green phosphate/mo (v) complex at acid ph. ascorbic acid was utilized to compare total antioxidant capacity of the extracts (aliyu et al., 2012). tab. 2 shows total antioxidant capacity of extracts. while total antioxidant capacity of ethyl acetate extracts ranges from 127.15±0.031 µg aae/ml and 232.2±0.028 µg aae/ml; dichloromethane extracts ranges from 61.6±0.019 028 µg aae/ml and 159.95±0.035 µg aae/ ml. ethyl acetate extracts exhibited higher total antioxidant capacity than dichloromethane extracts except for ethyl acetate extract of thyme/propolis. total phenolic contents of ethyl acetate and dichloromethane extracts of thyme/propolis and turmeric/ propolis were decreased when compared with ethyl acetate and dichloromethane extracts of thyme, turmeric and propolis except for dichloromethane extract of thyme/propolis. this situation might be arised by the interactions among the active substances in propolis and thyme or turmeric. the presence of phenolic compounds could be attributable to the observed high total antioxidant capacity. many surveys were done by other researchers about total antioxidant capacity of propolis, thyme and turmeric. yılmaz et al. (2017) investigated total antioxidant capacity of propolis collected from sakyatan (ks) and kızılören biologica nyssana ● 13 (1) september 2022: 33-40 36 (kk) regions of konya and they found total antioxidant capacities of ks propolis and kk propolis as 2.21±0.11 mmol tes/g extract and 2.40±0.15 mmol tes/g extract, respectively (yılmaz et al., 2017). özcan & özkan (2018) investigated total antioxidant activity of different extracts of thyme and they found that total antioxidant activity of thyme ranges from 91.14±0.87 -123.34±0.95 mg aae/g. bulus et al. (2017) determined that total antioxidant capacity of butanol extract of turmeric was 370 aae/g. our results and literature results are different. this differences can be explained with collecting sample from different locations, using different solvents and extraction techniques. table 2. total antioxidant capacity of the extracts (µg aae/ml) extract total antioxidant capacity ethyl acetate extract of thyme 127.15±0.031 ethyl acetate extract of turmeric 177.16±0.021 ethyl acetate extract of propolis 232.2±0.028 ethyl acetate extract of thyme/propolis 147.52±0.031 ethyl acetate extract of turmeric/propolis 174.6±0.046 dichloromethane extract of thyme 61.6±0.019 dichloromethane extract of turmeric 113.85±0.019 dichloromethane extract of propolis 101±0.010 dichloromethane extract of thyme/propolis 159.95±0.035 dichloromethane extract of turmeric/propolis 85.04±0.038 extract concentration (µg/ml) cuprac activity ethyl acetate extract of thyme 2501.4479±0.031 5002.2108±0.0006 7502.3390±0.020 10002.3562±0.088 ethyl acetate extract of turmeric 2501.7463±0.039 5001.9936±0.003 7502.0067±0.022 10002.0151±0.017 ethyl acetate extract of propolis 2501.9856±0.073 5001.9761±0.024 7502.0374±0.019 10002.0573±0.109 ethyl acetate extract of thyme/propolis 2501.801±0.043 5001.8249±0.030 7501.9752±0.075 10002.0055±0.049 ethyl acetate extract of turmeric/propolis 2501.8199±0.018 5002.1059±0.0462 7502.0493±0.019 10001.9689±0.041 dichloromethane extract of thymus 2500.5513±0.032 5000.6634±0.049 7501.1269±0.018 10001.3479±0.037 table 3. cuprac activity of the extracts extract concentration (µg/ml) cuprac activity dichloromethane extract of turmeric 2501.3326±0.042 5001.8600±0.029 7501.8966±0.017 10001.9426±0.055 dichloromethane extract of propolis 2501.6693±0.059 5001.8658±0.005 7502.0466±0.021 10002.1393±0.013 dichloromethane extract of thyme/propolis 2501.8020±0.015 5001.9255±0.011 7501.9895±0.011 10002.0625±0.078 dichloromethane extract of turmeric/propolis 2500.9922±0.016 5001.4591±0.051 7501.7229±0.044 10001.7569±0.007 bht 2500.6635±0.023 5000.7016±0.021 7500.8283±0.024 10000.9716±0.014 aydin & kadioğlu ● total phenolic content and antioxidant activity of thymus vulgaris, curcuma longa, propolis and their mixtures aydin & kadioğlu ● total phenolic content and antioxidant activity of thymus vulgaris, curcuma longa, propolis and their mixtures biologica nyssana ● 13 (1) september 2022: 33-40 37 cuprac test tab. 3 presents cuprac activity of the extracts. ethyl acetate extracts had better cuprac activity than dichloromethane extracts at 1000 µg/ml concentration. cuprac activity of ethyl acetate and dichloromethane extracts of thyme/propolis and turmeric/propolis were decreased when compared with ethyl acetate and dichloromethane extracts of thyme, turmeric and propolis except for dichloromethane extract of thyme/propolis at concentration of 1,000 µg/ml. this situation might be a consequence of the interactions among active substances in propolis and thyme or turmeric. the results indicate a concentration dependent cuprac activity. all extracts had higher activity than bht. dpph radical scavenging activity tab. 4 demonstrates dpph radical scavenging potentials of extracts at different concentrations (250-1000 µg/ml) measured as a degree of discoloration displayed the extracts’ scavenging potential. dichloromethane extract of thyme showed no activity, while all the extracts exhibited lower activity than bht and rutin. table 4. dpph radical scavenging activity of the extracts and standards (% inhibition) extract concentration (µg/ml) dpph radical scavenging activity ethyl acetate extract of thyme 250 not activity 500 7.54±0.002 750 39.04±0.0009 1000 67.56±0.004 ethyl acetate extract of turmeric 250 10.3±0.002 500 38.53±0.0013 750 55±0.004 1000 75.25±0.006 ethyl acetate extract of propolis 250 66.4±0.005 500 67.99±0.004 750 73.36±0.001 1000 79.53±0.001 ethyl acetate extract of thyme/propolis 250 45.63±0.008 500 73.19±0.005 750 77.57±0.005 1000 81.64±0.003 ethyl acetate extract of turmeric/propolis 250 38.02±0.036 500 61.68±0.009 750 65.23±0.008 1000 74.16±0.004 dichloromethane extract of thymus 250 not activity 500 not activity 750 not activity 1000 not activity dichloromethane extract of turmeric 250 50.94±0.001 500 55.22±0.002 750 59.94±0.001 1000 70.39±0.014 biologica nyssana ● 13 (1) september 2022: 33-40 aydin & kadioğlu ● total phenolic content and antioxidant activity of thymus vulgaris, curcuma longa, propolis and their mixtures dpph radical scavenging activity of ethyl acetate and dichloromethane extracts of thyme/ propolis and turmeric/propolis were increased when compared with ethyl acetate and dichloromethane extracts of thyme, turmeric and propolis at 1,000 µg/ml concentration. the best activity was detected in ethyl acetate extract of thyme/propolis (81.64%) and the worst activity was detected in ethyl acetate extract of thyme (67.56%) concentration of 1,000 µg/ml. ethyl acetate extracts generally showed better activity than dichloromethane extracts. dpph radical scavenging activity was searched by many authors. can et al. (2015) concluded that dpph scavenging activity of propolis from azerbaijan ranges from 15±1.00-198±3.40 (can et al., 2015). köksal et al. (2017) found dpph scavenging activity (ic50 value) of lyophilized water extract and ethanol extract of thyme as 13.4 and 12.1, respectively (köksal et al., 2017). priyanka et al. (2017) investigated dpph scavenging activity (% inhibition) of turmeric cultivars and they found that activity ranges from 49.63±2.97 to 59.58±2.95 (priyanka et al., 2017). these differences might be a consequence of used solvent and different location of material collection. conclusions the results suggest that the thyme, turmeric and propolis utilized in the current study possess antioxidant properties. thyme, turmeric and propolis also can be used as ingredients for development of a new antioxidant agents. further work should be focused on the isolation and elucidation of secondary metabolites in thyme, turmeric and propolis responsible for the antioxidant activity. antioxidant activity of mixtures are lower than thyme, turmeric and propolis because of interactions of active substances in mixtures. since the antioxidant activity of plant mixtures with propolis is lower than the antioxidant activities of these plants and propolis alone, plants and propolis should be consumed individually, not as a mixture. references aliyu, a.b., ibrahim, m.a., ibrahim, h., musa, a.m., lawal, a.y., oshanimi, j.a., usman, m., abdulkadir, i.e., oyewale, a.o., amupitan, j.o. 2012: free radical scavenging and total antioxidant capacity of methanol extract of ethulia conyzoides growing in nigeria. romanian biotechnological 38 extract concentration (µg/ml) dpph radical scavenging activity dichloromethane extract of propolis 250 26.41±0.033 500 30.69±0.033 750 41.67±0.023 1000 69.08±0.005 dichloromethane extract of thyme/propolis 250 52.9±0.066 500 68.57±0.002 750 70.1±0.001 1000 74.81±0.002 dichloromethane extract of turmeric/propolis 250 59.5±0.018 500 69.81±0.014 750 71.77±0.016 1000 76.48±0.019 bht 250 88.85±0.012 500 89.55±0.005 750 90.27±0.011 1000 91.55±0.008 rutin 250 86.80±0.008 500 87.91±0.003 750 90.60±0.004 1000 91.89±0.011 biologica nyssana ● 13 (1) september 2022: 33-40 aydin & kadioğlu ● total phenolic content and antioxidant activity of thymus vulgaris, curcuma longa, propolis and their mixtures letters, 17(4): 7458-7465. aryal, s., baniya, m.k., danekhu, k., kunwar, p., gurung, r., koirala, n. 2019: total phenolic content, flavonoid content and antioxidant potential of wild vegetables from western nepal. plants (basel, switzerland), 8(4): 96. brand-williams, w., cuvelier, m., berset, c. 1995: use of a free radical method to evaluate antioxidant activity. lebensmittel-wissenschaft und technologie food science and technology, 28: 2530. bulus, t., david, s.i., bilbis, l.s., babando, a. 2017: in vitro antioxidant activity of n-butanol extract of curcuma longa and its potentıal to protect erythrocytes membrane agaınst osmotıc-induced haemolysıs. science world journal, 12(1): 13-17. bulut, m., akpolat, h., tunçtürk, y., alwazeer, d., türkhan, a. 2020: determination of optimum ethanolic extraction conditions and phenolic profiles of thyme, mint, uckun, grape seeds and green tea waste fiber. international journal of agriculture and wildlife science, 6(3): 605-614. can, z., yıldız, o., şahin, h., asadov, a., kolaylı, s. 2015: phenolic profile and antioxidant potential of propolis from azerbaijan. mellifera, 15(1): 16-28. çoşkun, p., i̇nci, h. 2020: antibacterial, antiviral, antioxidant activity and chemical content of propolis. ispec journal of agricultural sciences, 4(4): 10531070. egamberdieva, d., wirth, s., behrendt, u., ahmad, p., berg, g. 2017: antimicrobial activity of medicinal plants correlates with the proportion of antagonistic endophytes. frontiers in microbiology, 8: 199-210. erdoğan, ü., erbaş, s. 2021: phytochemical profile and antioxidant activities of zingiber officinale (ginger) and curcuma longa l. (turmeric) rhizomes. bilge international journal of science and technology research, 5(1-6): 1-6. karakaş, f.p., yıldırım, a., türker, a. 2012: biological screening of various medicinal plant extracts for antibacterial and antitumor activities. turkish journal of biology, 36: 641-652. keskin, m., kolaylı, s. 2019: ticari propolis ekstraktlarının kalite parametreleri açısından karşılaştırılması. uludağ bee journal, 19(1): 43-49. köksal, e., bursal, e., gülçin, i̇., korkmaz, m., çağlayan, c., gören, a.c., alwaseld, s.h. 2017: antioxidant activity and polyphenol content of turkish thyme (thymus vulgaris) monitored by liquid chromatography and tandem mass spectrometry. international journal of food properties, 20(3): 514–525. maheshwari, r.k., singh, a.k., gaddipati, j., srimal, r.c. 2006: multiple biological activities of curcumin: a short review. life sciences, 78(18): 2081-2087. murugan, r., parimelazhagan, t. 2014: comparative evaluation of different extraction methods for antioxidant and anti-inflammatory properties from osbeckia parvifolia arn. an in vitro approach. journal of king saud university science, 26(4):267-275. özcan, m.m., özkan, g. 2018: the total phenol, flavonol amounts, antioxidant capacity and antiradical actıvıty of some thyme species growıng in turkey. journal of the chilean chemical society, 63(3): 4051-4056. özdal, t., ceylan, f. d., eroglu, n., kaplan, m., olgun, e. o., capanoglu, e. 2019: investigation of antioxidant capacity, bioaccessibility and lcms/ms phenolic profile of turkish propolis. food research international, 122: 528-536. özyürek, m., bektaşoğlu, b., güçlü, k., apak, r. 2009: measurement of xanthine oxidase inhibition activity of phenolics and flavonoids with a modified cupric reducing antioxidant capacity (cuprac) method. analytica chimica acta, 636(1): 42-50. prieto, p., pineda, m., anguilar, m. 1999: spectrophotometric quantitation of antioxidant capacity through the formation of a phosphomolybdenum complex: specific application to the determination of vitamin e. analytical biochemistry, 269: 337-341. priyanka, r., vasundhara, m., rao, g.g.e., thara, b.s., radhika, b., marappa, n. 2017: antioxidant activity of turmeric (curcuma longa l.) cultivars. medicinal plants, 9(3): 189-194. saeed, n., khan, m.r., shabbir, m. 2012: antioxidant activity, total phenolic and total flavonoid contents of whole plant extracts torilis leptophylla l. bmc complementary medicine and therapies, 12: 221-233. slinkard, k., singleton, v.l. 1977: total phenol analyses: automation and comparison with manual methods. american journal of enology and viticulture, 28: 49-55. schaffer, m., schaffer, p. m., zidan, j., bar sela, g. 2011: curcuma as a functional food in the control of cancer and inflammation. current opinion in clinical nutrition and metabolic care, 14(6): 588– 597. 39 biologica nyssana ● 13 (1) september 2022: 33-40 aydin & kadioğlu ● total phenolic content and antioxidant activity of thymus vulgaris, curcuma longa, propolis and their mixtures tural s., turhan s. 2017: antimicrobial and antioxidant properties of thyme (thymus vulgaris l.), rosemary (rosmarinus officinalis l.) and laurel (lauris nobilis l.) essential oils and their mixtures. gıda the journal of food, 42(5): 588-596. yan, s.w. asmah, r. 2010: comparison of total phenolic contents and antioxidant activities of turmeric leaf, pandan leaf and torch ginger flower. international food research journal, 17: 417-423. yılmaz, s.c., azman, z.n., kosem, k., gündüz, e., grenman, r. 2017: evaluating antioxidant capacity of different propolis samples from konya, turkey and their inhibitory effect on head and neck cancer cells. biorxiv, 183913. 40 xhulaj 2022, biologica nyssana 13(2) 13 (2) december 2022: 173-178 doi: 10.5281/zenodo.7437326 various records of lichens from southeastern albania original article skerdilaid xhulaj research center of flora and fauna, faculty of natural sciences, university of tirana, bulevardi zog i, nr. 25/1, tirana, albania skerdilaid.xhulaj@fshn.edu.al (corresponding author) received: october 10, 2022 revised: november 16, 2022 accepted: november 23, 2022 abstract: 117 taxa of lichens from two neighboring districts (pogradec and korçë) in the southeast of albania are presented. 62 taxa are new for the study area and 27 taxa are new to albania. key words: biodiversity, lichens, flora of albania, new records apstrakt: različiti zapisi o lišajima iz jugoistočne albanije predstavljeno je 117 taksona lišaja iz dva susedna okruga (pogradec i korçë) na jugoistoku albanije. 62 taksona su nova za područje istraživanja i 27 taksona su nova za albaniju. ključne reči: biodiverzitet, lišaji, flora albanije, novi zapisi introduction hitherto, no thorough investigation about the lichens of this area has been carried out. as a result, publications are quite sporadic or lacking. markgraf (1927, 1931) has produced a few records from korçë district as part of his expedition focused on the flora and vegetation of albania. hafellner (2007) provides a checklist of lichens based on historical records by several authors. this list does not include any species of lichen from the area specifically covered in this investigation, except those reported by markgraf (1927, 1931). svoboda et al. (2012) have reported a greater number of lichens from 5 localities visited in this area (1 in the district of pogradec and 4 in the district of korça) compared to the above-mentioned authors. it should be noted that their localities in the district of korça were positioned within the bredhi i drenovës national park, leaving other areas or habitats of this district uncovered. the results presented below are based on fieldwork undertaken by author encouraged by the need to increase and improve knowledge on the diversity of lichens in albania. materials and methods specimens were collected by the author during field trips carried out in both districts. the sampling localities were chosen according to diversity of the habitats paying attention to the substrates. specimens were determined using lichenological routine light microscopy methods and based on relevant literature according to poelt (1969), poelt & vězda (1977, 1981), clauzade & roux (1985), wirth (1995), nimis & martellos (2004), smith et al. (2009), wirth et al. (2013). the nomenclature follows nimis & martellos (2008) and reflects a more modern concept of genus. the specimens are deposited in the author’s personal herbarium. list of sampling localities 1: pogradec district, calcareous hills w of ohrid lake c. 20 km n of pogradec, 41°03’40”n/20°36’36.2”e, alt. c. 700 m, 10.06.2020. 2: pogradec district, calcareous rocks on carpinus scrub, near the village rëmenj, c. 16 km s of pogradec, an area characterized by drought but influenced by human activity, 40°51’05.6”n/20°36’49.4”e, alt. c. 830 m, 10.06.2020. 3: pogradec district, near the village peshkëpi, c. 20 km e of pogradec, 40°53’45.9”n/20°46’45.7”e, alt. c. 940 m, 11.06.2020. 4: pogradec district, near the village guri i bardhë, c. 22 km w of pogradec, hedges between pastures, 40°52’53.8”n/20°33’03.6”e, alt. c. 1050 m, 11.06.2020. 5: korçë district, on roadside trees (populus), c. 5 km nw of korça, 40°39’13.8”n/20°44’48”e, alt. c. 850 m, 12.06.2020. 6: korçë district, on robinia behind the memorial of martyrs in korçë, 40°37’10.3”n/20°47’22.4”e, alt. 950 m, 12.06.2020. © 2022 xhulaj. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 173 14th symposium on the flora of southeastern serbia and neighboring regions 174 biologica nyssana ● 13 (2) december 2022: 173-178 xhulaj ● various records of lichens from southeastern albania 7: korçë district, bozdovec, bredhi i drenovës national park, c. 7 km e of the village drenovë, on hill wooded by middle-aged mixed forest with dominance of abies borisii-regis in the northern part of the park, 40°34’36.8”n/20°50’34.5”e, alt. 1360 m, 13.06.2020. 8: korçë district, korçë, bredhi i drenovës national park, tourist route in the southern part of the park, 40°32’33.3”n/20°49’18.2”e, alt. 1240-1300 m, 14.06.2020. 9: korçë district, near the village vithkuq, c. 32 km sw of korçë, limestone outcrops, mixed deciduous forest, 40°31’05.3”n/20°35’09.4”e, alt. c. 1230 m, 15.06.2020. 10: korçë district, near the village dardhë, c. 18 km se of korçë, mixed forest, 40°30’54”n/20°49’25”e, alt. c. 1250 m, 16.06.2020. list of substrates and their abbreviations: cor: on bark of trees cal: on limestone ter-cal: on calcareous soil sil: on siliceous rocks bry: on bryophytes abb: abies borisii-regis aps: acer pseudoplatanus car: carpinus sp. fag: fagus sylvatica pop: populus sp. qce: quercus cerris rps: robinia pseudoacacia sal: salix sp. results a total of 117 taxa of lichens were recorded, including 62 lichens new to the investigation area and 27 new to the country. species are arranged alphabetically. numbers from 1 to 10 correspond to the localities listed above; the abbreviations following this number correspond to the substrates listed above. author’s initials and numbers in brackets show individual consecutive numbering systems used by the author to identify and locate individual specimens. new records for the investigation area are indicated with an asterisk (*), and new records for albania with a ring (o). acarospora cervina a. massal. 1, 2, 5, 9 cal (sx 7108, 7211, 8521, 8964) *acarospora glaucocarpa (ach.) körb. 1 cal (sx 7133) anaptychia ciliaris (l.) flot. 1 car, 6 rps (sx 7113, 8656) *oarthonia apatetica (a. massal.) th. fr. 3, 4, 5, cor (sx 7314, 7404, 8593) arthonia didyma körb. 7, 8, 10 abb (sx 8772, 8811, 8123) athallia alnetorum (giralt, nimis & poelt) arup, frödén & søchting 4 qce, 5 pop (sx 7415, 8542) *oathallia cerinella (nyl.) arup, frödén & søchting 6 cor (sx 8619) athallia holocarpa (hoffm.) arup, frödén & søchting 5 cor (sx 8565) *oathallia pyracea (ach.) arup, frödén & søchting 4, 7, 8, 10 aps (sx 7423, 8735, 8809, 8176) *bagliettoa marmorea (scop.) gueidan & cl. roux 1 cal (sx 7186) *obagliettoa parmigera (j. steiner) vězda & poelt 1 cal (sx 7153) *obagliettoa parmigerella (zahlbr.) vězda & poelt 1 cal (sx 7104) blastenia crenularia (with.) arup, søchting & frödén 1, 2, 4 cal (sx 7111, 7270, 7441) blastenia ferruginea (huds.) a. massal. 3, 4, 6, 9 cor (sx 7315, 7476, 8655, 8911) calicium salicinum pers. 7, 8 abb (sx 8768, 8832) caloplaca cerina (hedw.) th. fr. 1 cor, 7 fag, 8 fag, 9 sal (sx 7174, 8761, 8844, 8924) *caloplaca conversa (kremp.) jatta 1 cal (sx 7168) *candelariella aurella (hoffm.) zahlbr. 1 cal (sx 7156) *candelariella vitellina (hoffm.) müll. arg 6, 10 cor (sx 8660, 8111) candelariella xanthostigma (ach.) lettau 3, 4, 6 cor, 7, 8 abb (sx 7310, 7437, 8692, 8750, 8812) *circinaria calcarea (l.) a. nordin, savić & tibell 1, 2, 3, 6, 9, 10 cal (sx 7137, 7261, 7303, 8605, 8922, 8108) *circinaria contorta (hoffm.) a. nordin, savić & tibell 1, 2, 3, 6, 9 cal (sx 7157, 7224, 7302, 8613, 8957) *ocircinaria hoffmanniana (s. ekman & fröberg ex r. sant.) a. nordin 1, 2 cal (sx 7107, 7223) 175 biologica nyssana ● 13 (2) december 2022: 173-178 xhulaj ● various records of lichens from southeastern albania *cladonia pyxidata (l.) hoffm. f. pocillum (ach.) nyl. 1, 2, ter-cal (sx 7105, 7219) *cladonia pyxidata (l.) hoffm. f. pyxidata 1, 2, 3, 4 ter-cal, 7, 9, 10 bry (sx 7152, 7261, 7308, 7430, 8708, 8964, 8152) collema flaccidum (ach.) ach. 1, 2, 3, 4 ter-cal (sx 7170, 7252, 7344, 7408) *oenchylium polycarpon (hoffm.) otálora, p.m. jørg. & wedin subsp. polycarpon 1, 2, 4 cal (sx 7114, 7216, 7426) enchylium tenax (sw.) gray 1, 2, 3, 4 ter-cal (sx 7175, 7246, 7322, 7467) *fuscopannaria praetermissa (nyl.) p.m. jørg. 1 ter-cal, 9 bry (sx 7169, 8913) *ogyalolechia flavorubescens (huds.) søchting, frödén & arup var. flavorubescens 2 car, 5 pop (sx 7266, 8590) hypogymnia farinacea zopf 7, 8 abb (sx 8718, 8875) *lathagrium auriforme (with.) otálora, p.m. jørg. & wedin 1 cal (sx 7103) *lathagrium cristatum (l.) otálora, p.m. jørg. & wedin 1, 2, 9 cal (sx 7161, 7230, 8959) lecanora allophana (ach.) nyl. f. allophana 3, 5, 6 pop, 7, 8, 10 fag (sx 7317, 8546, 8643, 8715, 8808, 8151) lecanora allophana f. sorediata vain. 3, 5 pop (sx 7347, 8513) *lecanora argentata (ach.) malme 7, 8, 9 fag (sx 8780, 8867, 8934) *lecanora carpinea (l.) vain. 3, 4, 10 cor (sx 7319, 7440, 8105) lecanora chlarotera nyl. subsp. chlarotera 3, 4 cor, 8 fag (sx 7324, 7449, 8833) lecanora intumescens (rebent.) rabenh. 7, 8 fag (sx 8762, 8843) lecanora leptyrodes (nyl.) degel. 1, 3, 4, 8, 9, 10 cor (sx 7102, 7366, 7465, 8812, 8918, 8116) lecanora salicicola h. magn. 4, 9 cor (sx 7409, 8912) lecanora subcarpinea szatala 7, 8, 10 abb (sx 8709, 8822, 8160) lecidea atrobrunnea (dc.) schaer. subsp. atrobrunnea 3, 10 sil (sx 7313, 8149) *lecidella elaeochroma (ach.) m. choisy var. elaeochroma 1, 3, 5, 6, 10 cor (sx 7127, 7329, 8597, 8617, 8121) *lecidella euphorea (flörke) hertel 1, 2, 7, 9 cor (sx 7106, 7255, 8731, 8961) lecidella flavosorediata (vězda) hertel & leuckert 5, 6, 9 cor (sx 8536, 8671, 8953) lepra albescens (huds.) hafellner 9, 10 cor (sx 8926, 8172) leptogium saturninum (dicks.) nyl. 4 cor, 8 fag (sx 7413, 8841) *lobothallia radiosa (hoffm.) hafellner 1, 2, 9 cal (sx 7119, 7279, 8948) melanohalea exasperata (de not.) o. blanco, a. crespo, divakar, essl., d. hawksw. & lumbsch 9, 10 cor (sx 8931, 8124) melanohalea laciniatula (h. olivier) o. blanco, a. crespo, divakar, essl., d. hawksw. & lumbsch 8 abb (sx 8847) *omicarea globulosella (nyl.) coppins 10 abb (sx 8131) *myriolecis dispersa (pers.) sliwa, zhao xin & lumbsch 2, 9 cal (sx 7253, 8938) myriolecis hagenii (ach.) sliwa, zhao xin & lumbsch 3, 5, 6 cor (sx 7316, 8594, 8612) nephroma resupinatum (l.) ach. 4, 9, 10 cor (sx 7432, 8925, 8137) ochrolechia pallescens (l.) a. massal. 7 aps, 8 abb (sx 8710, 8859) ochrolechia szatalaensis verseghy 10 abb (sx 8128) parmelia submontana hale 7 fag, 10 abb (sx 8724, 8162) phaeophyscia nigricans (flörke) moberg 2 cor, 5 pop (sx 7247, 8567) phaeophyscia orbicularis (neck.) moberg 1, 2, 5, 6 cor (sx 7158, 7271, 8525, 8644) *physcia adscendens h. olivier 2, 6, 9 cor (sx 7281, 8672, 8903) physcia aipolia (humb.) fürnr. 7 abb, 9 cor (sx 8704, 8962) *physcia biziana (a. massal.) zahlbr. var. biziana 176 biologica nyssana ● 13 (2) december 2022: 173-178 xhulaj ● various records of lichens from southeastern albania 4, 6, 9, 10 cor (sx 7405, 8640, 8958, 8136) *physcia dubia (hoffm.) lettau 2 cal, 6 cor (sx 7267, 8673) *physcia leptalea (ach.) dc. 9 cor (sx 8919) *physcia stellaris (l.) nyl. 5, 9 cor (sx 8553, 8914) *physciella chloantha (ach.) essl. 9 cor (sx 8933) physconia distorta (with.) j.r. laundon 7 fag, 8 aps, 9 abb (sx 8759, 8848, 8927) physconia perisidiosa (erichsen) moberg 8 abb (sx 8814) *oplacidium lachneum (ach.) b. de lesd. 1, 2 ter-cal, 9 bry (sx 7139, 7222, 8924) *oplacocarpus schaereri (fr.) breuss 1, 2 cal (sx 7199, 7286) *oplacolecis opaca (dufour) hafellner 2 cal (sx 7280) *placynthium nigrum (huds.) gray 2 cal (sx 7278) pleurosticta acetabulum (neck.) elix & lumbsch 7, 8 abb (sx 8725, 8846) *opolycauliona polycarpa (hoffm.) frödén, arup & søchting 5 cor (sx 8518) protoblastenia incrustans (dc.) j. steiner var. incrustans 1, 2, 9 cal (sx 7116, 7265, 8952) *oprotoparmeliopsis graeca (j.steiner) sipman & cl. roux 2 cal (sx 7251) *protoparmeliopsis muralis (schreb.) m. choisy var. muralis 1, 9 cal (sx 7177, 8906) psora rubiformis (ach.) hook. 7 sil, 10 bry (sx 8757, 8125) *opyrenodesmia chalybaea (fr.) a. massal. 1, 2, 4 cal (sx 7163, 7249, 7463) *pyrenodesmia variabilis (pers.) a. massal. 2, 4, 9 cal (sx 7245, 7433, 8923) ramalina calicaris (l.) fr. 3, 4 cor, 8, 10 abb (sx 7345, 7434, 8823, 8142) ramalina canariensis j. steiner 3, 4, 9 cor (sx 7328, 7481, 8907) ramalina fraxinea (l.) ach. 7, 8, 9 cor (sx 8792, 8836, 8951) *rinodina albana (a. massal.) a. massal. 3, 6 cor (sx 7320, 8606) *orinodina anomala (zahlbr.) h. mayrhofer & giralt 2 cor (sx 7204) *orinodina bischoffii (hepp) a. massal. 1, 2 cal (sx 7162, 7222) *rinodina exigua (ach.) gray 3, 9 cor (sx 7338, 8929) rinodina immersa (körb.) j. steiner 1, 2, 4, 9 cal (sx 7190, 7283, 7444, 8902) *orinodina pyrina (ach.) arnold 2 cor (sx 7286) *orinodinella dubyanoides (hepp) h. mayrhofer & poelt 1, 2 cal (sx 7193, 7232) sanguineodiscus haematites (chaub. ex st.-amans) i.v. frolov & vondrák 1, 2, 4, 8 cor (sx 7130, 7243, 7461, 8834) *sarcogyne regularis körb. var. regularis 2 cal (sx 7273) scoliciosporum umbrinum (ach.) arnold 7, 8, 10 abb (sx 8736, 8882, 8153) *oscytinium fragile (taylor) otálora, p.m. jørg. & wedin 1, 2 cal (sx 7144, 7213) scytinium lichenoides (l.) otálora, p.m. jørg. & wedin 1, 2 ter-cal, 6, 10 bry (sx 7164, 7248, 8639, 8117) scytinium pulvinatum (hoffm.) otálora, p.m. jørg. & wedin 1, 2, 7 ter-cal (sx 7165, 7250, 8732) scytinium tenuissimum (hoffm.) otálora, p.m. jørg. & wedin 1, 2 cor (sx 7166, 7254) squamarina cartilaginea (with.) p. james 1, 2 ter-cal (sx 7124, 7257) staurothele areolata (ach.) lettau 7, 8, 10 sil (sx 8734, 8845, 8143) *osynalissa ramulosa (bernh.) fr. 2 cal (sx 7264) *thalloidima candidum (weber) a.massal. 1, 2 cal (sx 7112, 7238) *othalloidima diffractum (a. massal.) a. massal. 1 cal (sx 7129) biologica nyssana ● 13 (2) december 2022: 173-178 xhulaj ● various records of lichens from southeastern albania thalloidima opuntioides (vill.) kistenich, timdal, bendiksby & s.ekman 2 cal, 9 bry (sx 7229, 8963) usnea barbata (l.) f.h. wigg. 8 abb (sx 8821) usnea perplexans stirt. 10 abb (sx 8104) *variospora aurantia (pers.) arup, frödén & søchting 1, 9 cal (sx 7146, 8928) *ovariospora dalmatica (a. massal.) 1, 2 cal (sx 7142, 7233) *ovariospora flavescens (huds.) arup, frödén & søchting 2 cal (sx 7236) *overrucaria caerulea dc. 1, 2 cal (sx 7191, 7260) *verrucaria nigrescens pers. 1, 2 cal (sx 7192, 7258) *xanthocarpia ochracea (schaer.) a. massal. & de not. 1, 2 cal (sx 7115, 7242) *oxanthomendoza fallax (hepp) søchting, kärnefelt & s.y. kondr. 5 cor (sx 8508) xanthomendoza fulva (hoffm.) søchting, kärnefelt & s.y. kondr. 8, 10 cor (sx 8863, 8133) *oxanthoparmelia glabrans (nyl.) o. blanco, a. crespo, elix, d. hawksw. & lumbsch 7 sil (sx 8755) xanthoparmelia pulla (ach.) o. blanco, a. crespo, elix, d. hawksw. & lumbsch 7, 10 sil (sx 8758, 8122) *xanthoria parietina (l.) th. fr. 1, 2 cal, 4, 5, 6, 9 cor (sx 7109, 7263, 7435, 8541, 8654, 8921) discussion thus far, 55 taxa of lichens were known from both districts (markgraf, 1927, 1931; svoboda et al., 2012). this study is able to add a further 62 taxa. more than 60% of the lichen taxa added by this contribution are crustose and many of them are widely distributed in the mediterranean region. regarding the substrate ecology of the newly recorded lichen taxa, 58% of the added taxa are saxicolous, 35% are corticolous and only 6% are terricolous or muscicolous. new lichen species for the albania are arthonia apatetica, athallia cerinella, a. pyracea, bagliettoa parmigera, b. parmigerella, circinaria hoffmanniana, enchylium polycarpon subsp. polycarpon, gyalolechia flavorubescens var. flavorubescens, micarea globulosella, placidium lachneum, placocarpus schaereri, placolecis opaca, polycauliona polycarpa, protoparmeliopsis graeca, pyrenodesmia chalybaea, rinodina anomala, r. bischoffii, r. pyrina, rinodinella dubyanoides, scytinium fragile, synalissa ramulosa, thalloidima diffractum, variospora dalmatica, v. flavescens, verrucaria caerulea, xanthomendoza fallax and xanthoparmelia glabrans. it should be noted: in korça town itself were only found three species, miserably developed (physcia biziana, physciella chloantha and phaeophyscia orbicularis). compared to the coastal areas visited so far this is poor, presumably because of the dry climate far from the coast. calcareous rocks on carpinus scrub south of pogradec, an area characterized by drought but influenced by human activity. to be noticed on carpinus: anaptychia ciliaris, gyalolechia flavorubescens, physcia leptalea, rinodina pyrina. on limestone: placocarpus schaereri, placolecis opaca, scytinium fragile, synalissa ramulosa and thalloidima diffractum. conclusions despite the considerable number of taxa found and localities visited, it is expected that the lichen flora should be richer due to the fact that some areas are difficult to reach and the large extent of both districts. although many factors influence the species composition of the investigated localities, it may be concluded that the general climate and the potential natural vegetation/vegetation zones adjust the general pattern of the lichenic flora, while the stand parameters and the conditions of the instant surroundings, especially the topography-influenced local climatic conditions and the present vegetation surrounding the habitats, may affect the species composition to a considerable extent. references clauzade, g., roux, c. 1985: likenoj de okcidenta eŭropo. ilustrita determinlibro. bulletin de la société botanique du centre-ouest. nouvelle série. numéro spécial, 7: 1-893. hafellner, j. 2007: checklist and bibliography of lichenized and lichenicolous fungi so far reported from albania (version 05-2007). fritschiana (graz), 59: 1-18 177 biologica nyssana ● 13 (2) december 2022: 173-178 xhulaj ● various records of lichens from southeastern albania poelt, j., vězda, a. 1981: bestimmungsschlüssel europäischer flechten. ergänzungsband ii. bibliotheca lichenologica, 16: 1-390. smith, c.w., aptroot, a., coppins, b.j., fletcher, a., gilbert, o.l., james, p.w., wolse-ley, p.a. 2009: the lichens of great britain and ireland. the british lichen society & the natural history museum. london. 1046 p. svoboda, d., bouda, f., malíček, j., hafellner, j. 2012: a contribution to the knowledge of lichenized and lichenicolous fungi in albania. herzogia, 25: 149-165. wirth, v. 1995: flechtenflora. bestimmung und ökologische kennzeichnung der flechten südwestdeutschlands und angrenzender gebiete. 2. auflage. stuttgart: eugen ulmer. 661 p. wirth, v., hauck, m., schultz, m. 2013: die flechten deutschlands. 2 bände. stuttgart: eugen ulmer. 1244 p. markgraf, f. 1927: an den grenzen des mittelmeergebietes. pflanzengeographie von mittelalbanien. feddes repertorium beiheft, 45: 1-217, vegetationskarte. markgraf, f. 1931: pflanzen aus albanien 1928. denkschriften der kaiserlichen akademie der wissenschaften/mathematischnaturwissenschaftliche classe, 102: 317-360, tab. nimis, p.l., martellos, s. 2004: keys to the lichens of italy. vol. 1: terricolous species. edizioni goliardiche. trieste. 346 p. nimis, p.l., martellos, s. 2008: italic the information system on italian lichens. version 4.0. university of trieste. deparrtment of biology, in4.0/1 (http://dbiodbs.univ.trieste.it/). poelt, j. 1969: bestimmungsschlüssel europäischer flechten. lehre: cramer. 757 p. poelt, j., vězda, a. 1977: bestimmungsschlüssel europäischer flechten. ergänzungsband i. bibliotheca lichenologica, 9: 1-258. 178 microsoft word 0101_randjelovic_et_al biologica nyssana 1 (1-2) december 2010: 1-7 ranđelović, v. et al. phytogeographical and phytocoenological… 1 original article ! phytogeographical and phytocoenological analysis of the threatened plant taxa in the flora of the vlasina plateau (se serbia) vladimir ranđelović1*, bojan zlatković1, danijela dimitrijević1, tijana vlahović2 1 university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia 2 national library of serbia, belgrade, serbia * e-mail: vladar@pmf.ni.ac.rs abstract: ranđelović, v., zlatković, b., dimitrijević, d., vlahović, t.: phytogeographical and phytocoenological analysis of the threatened plant taxa in the flora of the vlasina plateau (se serbia). biologica nyssana, 1 (1-2), december 2010: 1-7. in analysing the flora of the vlasina plateau, it was determined that this area contains 956 species, 23 subspecies, 32 varieties and 28 forms of vascular plants. the data shows the exceptional floristic riches of this territory. among these species, there are 91 species (9.52%) which may be categorized as under threat in the flora of serbia. critically endangered taxa (cr) are especially interesting because their sanctuary in serbia is only in the vlasina plateau. this group includes the following species: betula pubescens, elatine triandra, utricularia minor, cirsium helenioides and carex limosa. all these species have the boreal type of distribution and live in wetlands. key words: flora, serbia, threatened plant taxa, vlasina plateau introduction ! the vlasina plateau is situated in southeastern serbia between vardenik, čemernik, plana and bukova glava mountains (fig. 1). geologically, the greater part of vlasina plateau is uniform area and it is made of metamorphic rocks, the most frequent of them being schists, rarely gneisses, andesites and dacites (p e t k o v i ć et al., 1977, d r a g i š i ć , 1997). vlasina plateau is in the zone of brown forest land (dystric cambisol), but characteristic soil is peat, which is differentiated in four types: phragmitetum-type, caricetum-type, equisetetumtype and mossy peat (r a n đ e l o v i ć 1994). the climate of this plateau is a typical continental one (đ u k a n o v i ć , 1967, r a n đ e l o v i ć & z l a t k o v i ć , 2010). floristically and phytocoenologicaly, vlasina plateau is relatively well documented in the monograph "flora and vegetation of the vlasina plateau" (r a n đ e l o v i ć & z l a t k o v i ć , 2010). also, in this monograph threatened plant taxa are processed. however, phytogeographical and phytocoenological analysis of vlasina plateau is not made. material and methods list of flora of the vlasina plateau from r a n đ e l o v i ć & z l a t k o v i ć (2010) was used for the analysis. the nomenclature follows medchecklist (g r e u t e r et al., 1984-1989) and flora europaea (t u t i n et al., eds. 1964-1980). all taxa that occur in the area of the vlasina are assessed according to iucn version 3.1 (iucn, 2001) methods for determining vulnerability at the local level and in serbia. iucn red list criteria were used to classify species according to one of the 10th sfses • 17-20 june 2010, vlasina lake1 (1-2) • december 2010: 1-7 biologica nyssana 1 (1-2) december 2010: 1-7 ranđelović, v. et al. phytogeographical and phytocoenological… 2 five categories of threat: extinct in serbia (ew), critically endangered (cr), endangered (en), vulnerable (vu) and near threatened (nt). if there was insufficient information to make an assessment, the category data deficient (dd) was assigned, especially in cases of taxonomic uncertainty. area-types, area-groups and life forms are taken from r a n đ e l o v i ć & z l a t k o v i ć (2010). phytocoenological relevance of taxa is determined by r a n đ e l o v i ć & z l a t k o v i ć (2010), h o r v a t et al. (1974) and a p o s t o l o v a & s l a v o v a (1997). results and discussion flora of vlasina plateau contains 956 species, 23 subspecies, 32 varieties and 28 forms of vascular plants. the data shows the exceptional floristic riches of this territory. among these species, there are 90 species (9.41%) which may be categorized as under threat in the flora of serbia (tab. 1) (r a n đ e l o v i ć & z l a t k o v i ć , 2010). the high degree of presence of endangered species that make flora of the vlasina plateau and the fact that every eleventh species in some way is endangered and that sooner or later it is in danger of disappearing from the territory of serbia, shows the biodiversity importance of this area. among other things, it was one of the reasons for classifying the vlasina plateau in botanical significant areas of europe(ipa) (r a n đ e l o v i ć in s t e v a n o v i ć , ed. in r a d f o r d , o d é , eds. 2009). the red data list of flora of serbia ( s t e v a n o v i ć , ed. 1997) states 53 taxa from the vlasina plateau. the first volume of the red data book of flora of serbia (s t e v a n o v i ć , ed. 1999), which contains extinct and critically endangered taxa, lists 11 taxa from the vlasina plateau. as extinct species from the flora of serbia, the following species are processed: polemonium coeruleum (r a n đ e l o v i ć , 1999a), dracocephalum ruyschiana (d i k l i ć , 1999), caldesia parnassifolia (v u k o j i č i ć , j a n k o v i ć , 1999) and juncus capitatus (r a n đ e l o v i ć , 1999). however, later investigations showed that species juncus capitatus (t o m o v i ć et al, 2009) and dracocephalum ruyshiana (l a z a r e v i ć et al., 2009) are not extinct, because they were found on the other localities in serbia. the species ranunculus lingua (s t o j š i ć , p a n j k o v i ć , 1999), betula pubescens (j o v a n o v i ć , 1999), elatine triandra (b l a ž e n č i ć , b l a ž e n č i ć , 1999), utricularia minor (b l a ž e n č i ć , b l a ž e n č i ć , 1999a), cirsium helenioides (r a n đ e l o v i ć , 1999c), carex limosa (r a n đ e l o v i ć , 1999b) and sparganium natans (b l a ž e n č i ć , b l a ž e n č i ć , 1999b) are classified as critically endangered. phytogeographical analysis according to phytogeographical analysis (fig. 2), highest proportions of threatened taxa have boreal (21 taxa) or euroasian mountain (27) type of distribution. within euroasian mountain area-type, middle-south-european mountain area-group (19) stands out with the number of taxa, which includes most of the balkan endemic species (11). phytocoenological analysis the highest proportion of threatened taxa is characteristic of peat vegetation (21), and another 39 for other types of wet vegetation. high presence of threatened taxa (11) is found in the forest vegetation. (fig. 3). analysis of life forms according to analysis of life forms (fig. 4) the dominance of hemicryptophytes (33 taxa) is established. fig 1. geographical position of investigated area biologica nyssana 1 (1-2) december 2010: 1-7 ranđelović, v. et al. phytogeographical and phytocoenological… 3 0 5 10 15 20 25 cs m ho l ph olptr op aa bo r ea mt ms em t se mt ea s ea sw me m me mp ms m me me d n um be r of ta xa fig. 2. phytogeographical analysis of the threatened plant taxa (balkan endemic taxa (b) are marked in black). area types and area groups: csm – cosmopolitan, hol – holarctic, phol-ptrop – palaeoholarcticpalaeotropic, aa arcto-alpian, bor boreal, eamt euroasian mountain, msemt middle-southeuropean mountain, semt south-european mountain, eas euroasian, easw european west asian, mem middle european-mediterranean, memp middle european-mediterranean-pontic, msm merridional-submerridional, me middle-european, medp mediterranean pontian, med mediterranean 0 5 10 15 20 25 ch ar le mn po ta ph ra iso e mo li mu lg mo nt sc he fe st ca ll ju nc as pl va cc qu er ru de n um be r of ta xa fig. 3. phytocoenological analysis of the threatened plant taxa (balkan endemic taxa are marked in black). vegetation classis: char charetea, lemn lemnetea, pota potametea, phra phragmitetea, isoe isoeto-nanojuncetea, moli molinio-arrhenatheretea, mont montio-cardaminetea, mulg mulgedioaconitetea, sche scheuchzerio-caricetea fuscae, fest festuco-brometea, call calluno-ulicetea, junc juncetea trifidi, aspl – asplenietea trichomanis, vacc vaccinio-piceetea, quer querco-fagetea, rude ruderalis biologica nyssana 1 (1-2) december 2010: 1-7 ranđelović, v. et al. phytogeographical and phytocoenological… 4 0 2 4 6 8 10 12 14 16 p ch hs hc hr p hr o hs r hs g hr og gb g r gt hy dg hy dt t th n um be r of ta xa fig. 4. life forms of the threatened plant taxa (balkan endemic taxa are marked in black): p phanerophyta, ch chamaephyta, hs hemicryptophyta scaposa, hc hemicryptophyta caespitosa, hrp hemycryptophyta reptosa, hro hemycryptophyta rosulata, hsr hemycryptophyta semirosulata, hsg hemycryptophyta scaposa/geophyta, hrog hemicryptophyta rosulata/geophyta, gb geophyta tuberosa, gb geophyta bulbosa, gr geophyta rhyzomatosa, hydg hydrogeophyta, hydt hydrotherophyta, t therophyta (including: tc therophyta caespitosa, ts therophyta scaposa, tsb therophyta scaposa biennis), th therophyta/hemicryptophyta scaposa table 1. contribution of threatened taxa in the flora of vlasina plateau with categories of endangered (ce) in the flora of serbia, area-type (at), area-group (ag), life form (lf) and phytocoenological relevance of taxa (p) (abbreviations are shown in the legends of appropriate figures) name of taxon ce at ag lf p caldesia parnassifolia ew csm csm hydg pota polemonium coeruleum ew bor borea hs mulg trisetum sibiricum ew bor borh hc sche betula pubescens cr bor borea p sche cirsium helenioides cr bor borea hs mulg eleocharis mammillata cr bor borea hydg isoe sparganium natans cr bor borh hydg pota utricularia minor cr bor borh hydt pota carex limosa cr bor borh gr sche elatine triandra cr bor borh tsb isoe hypericum humifusum cr csm csm hrp isoe ranunculus lingua cr eamt eamt hydg phra ranunculus auricomus var. biformis cr eas eas hrp sche dracocephalum ruyschiana cr eas eas hs fest gentiana pneumonanthe cr eas memp hs moli sisyrinchium montanum cr hol hol gt moli juncus capitatus cr hol phol-ptrop tc isoe campanula hemschinica cr me meb tsb fest sagina saginoides en aa aa hc mont pirola chlorantha en bor borh hsr quer lycopodium clavatum en bor borh ch mont cardamine raphanifolia ssp. acris en eamt msemt hro mont alchemilla cinerea en eamt msemt hrog fest alchemilla connivens en eamt msemt hrog sche biologica nyssana 1 (1-2) december 2010: 1-7 ranđelović, v. et al. phytogeographical and phytocoenological… 5 table 1. continued alchemilla crinita en eamt msemt hrog fest alchemilla heterophylla en eamt msemtb hrog fest alchemilla viridiflora en eamt msemtb hrog mulg geum rivale x rhodopaeum en eamt msemtb gr mulg pedicularis hoermaniana en eamt msemtb hs fest gentiana acaulis en eamt semt hro call pirola rotundifolia en eas eas hsr quer dactylorhiza sambucina en eas mem gt quer scleranthus neglectus en eas mem tc aspl eriophorum gracile en hol hol gr sche stellaria alsine en hol hol hc mont potentilla palustris en hol hol hsg sche potamogeton obtusifolius en hol hol hydt pota ranunculus fontanus en med med ths mont ranunculus ophioglossifolius en msm msm ths isoe pseudorchis albida vu aa aa gt sche sagittaria sagittifolia vu bor borea hydt pota arctostaphylos uva-ursi vu bor borh ch vacc corallorhiza trifida vu bor borh gr quer drosera rotundifolia vu bor borh hro sche orthilia secunda vu bor borh hsr quer zanichellia palustris vu csm csm hydt pota salix rosmarinifolia vu eamt eamt p sche dactylorhiza cordigera vu eamt msemtb gt sche pseudorchis friwaldii vu eamt msemt gt sche geum rhodopaeum vu eamt msemtb gr mulg alchemilla glabra vu eamt msemt hrog mulg alchemilla reniformis vu eamt msemt hrog moli silene asterias vu eamt semtb hs sche salix pentandra vu eas eas p sche cephalanthera longifolia vu eas eas gt quer epipactis palustris vu eas eas gt sche dianthus superbus vu eas eas hs sche stellaria palustris vu eas eas hs sche dactylorhiza incarnata vu eas easw gt sche trollius europaeus vu eas eas hs moli callitriche verna vu hol hol hydt lemn menyanthes trifoliata vu hol hol hrp sche ranunculus aquatilis vu hol hol hydg pota najas minor vu hol phol-ptrop hydt char saxifraga stellaris ssp. alpigena nt aa aa hro mont schoenus ferrugineus nt bor bore hc phra pirola media nt bor borea hsr quer pinguicula vulgaris nt bor boren hro sche pirola minor nt bor borh hsr quer pedicularis palustris nt bor borh tsb sche alchemilla acutiloba nt eamt eamt hrog call alchemilla gracilis nt eamt eamt hrog mulg allium melanantherum nt eamt msemtb gb call silene lerchenfeldiana nt eamt msemtb hc aspl peucedanum oligophyllum ssp. aequiradium nt eamt msemtb hs junc trifolium trichopterum nt eamt msemtb ts fest biologica nyssana 1 (1-2) december 2010: 1-7 ranđelović, v. et al. phytogeographical and phytocoenological… 6 table 1. continued verbascum adamovicii nt eamt msemtb tsb call achillea lingulata nt eamt semt hro call butomus umbelatus nt eas eas hydg phra euphorbia villosa nt eas eas hs rude ribes alpinum nt eas easw p quer cardamine amara nt eas easw hs mont ranunculus flammula nt hol hol hydg isoe gratiola officinalis nt hol hol hs moli lilium jankae dd eamt msemt gb call ranunculus serbicus dd eamt semt hsg mulg silene viridiflora dd eas memp hs quer anagalis minimus dd me me ts mont malus praecox dd msm msm p quer conclusion phytogeographical analysis showed that representatives of the boreal flora are the most endangered, which indicates that the vlasina plateau is one of the most important refuges of postglacial floristic elements characteristic for far north of europe and asia. in support of this statement is the fact that the largest number of threatened taxa (60) occurs in wet habitats, especially in the peat vegetation (21). of 18 extinct and extremely vulnerable taxa, 16 are characteristic of wetlands. the highly fragile are forest ecosystems which are the habitat for 11 endangered taxa. based on the performed analysis we can conclude that it is necessary to protect all wetlands on the vlasina plateau, especially peat vegetation. also, it is necessary to prevent the irrational exploitation of forest cover, which is partly achieved by the declaration of the entire area of outstanding natural landscape features. acknowledgements. this study was supported by the project of ministry of science and technological development of republic of serbia (project no. 143015 – diversity of flora and vegetation in the central balkan peninsula – ecology, chorology and conservation). references апостолова, и., славова, л., 1997: конспект на растителните съобщества в българия (compendium of bulgarian plant community). бан, инст. по ботан., софия. 340 стр. (in bulgarian) blaženčić, j., blaženčić, ž., 1999: elatine triandra schkuhr. in: v. stevanović, ed.: crvena knjiga flore srbije 1, ministarstvo za životnu sredinu r srbije, biološki fakultet univ. u beogradu i zavod za zaštitu prirode r srbije, 286-287. blaženčić, j., blaženčić, ž., 1999a: utricularia minor l. in: v. stevanović, ed.: crvena knjiga flore srbije 1, ministarstvo za životnu sredinu r srbije, biološki fakultet univ. u beogradu i zavod za zaštitu prirode r srbije, 233-234. blaženčić, j., blaženčić, ž., 1999b: sparganium natans l. in: v. stevanović, ed.: crvena knjiga flore srbije 1, ministarstvo za životnu sredinu r srbije, biološki fakultet univ. u beogradu i zavod za zaštitu prirode r srbije, 380-382. diklić, n., 1999: dracocephalum ruyschiana l. in: v. stevanović, ed.: crvena knjiga flore srbije 1, ministarstvo za životnu sredinu r srbije, biološki fakultet univ. u beogradu i zavod za zaštitu prirode r srbije, 70-71. dragišić, v., 1997: geološko-hidrogeološke karakteristike sliva vlasinskog jezera. in: j. blaženčić, ed.: vlasinsko jezero – hidrobiološka studija. biološki fakultet univ. u beogradu, 2535. đukanović, d., 1967: klima sreza leskovac. beograd. greuter, w., burdet, h. m. & long, g., 1984-89: med-checklist. a critical inventory of vascular plants of the circum-mediterranean countries, 1, 3 and 4. optima, genève & berlin. horvat, i., glavač, v., ellenberg, h., 1974: vegetation südosteuropas. geobotanica selecta, band 4. gustav fischer verlag. stuttgart. 768 p. (in german) iucn, 2001. iucn red list categories and criteria: version 3.1. prepared by the iucn species survival commission. iucn, gland, switzerland and cambridge, u. k. jovanović, b., 1999: betula pubescens ehrh. ssp. carpatica (willd.) ascherson & graebner. in: v. stevanović, ed.: crvena knjiga flore srbije 1, ministarstvo za životnu sredinu r srbije, biološki fakultet univ. u beogradu i zavod za zaštitu prirode r srbije, 239-242. biologica nyssana 1 (1-2) december 2010: 1-7 ranđelović, v. et al. phytogeographical and phytocoenological… 7 lazarević, p., lazarević, m., krivošej, z., stevanović, v., 2009: on the distribution of dracocephalum ruyschiana (lamiaceae) in the balkan peninsula. phytologia balcanica, 15 (2): 175-179. petković, k., ed., 1977: geologija srbije iii-2: metamorfizam. zavod za regionalnu geologiju i paleontologiju rudarsko geološkog fakulteta. univ. u beogradu. radford, e. a., odé, b., eds., 2009: conserving important plant areas: investing in the green gold of south east europe. plantlife international, salisbury. ranđelović, v., 1994: geobotanička studija vlasinske tresave. magistarska teza. biološki fakultet. beograd. ranđelović, v., 1999: juncus capitatus weigel. in: v. stevanović, ed.: crvena knjiga flore srbije 1, ministarstvo za životnu sredinu r srbije, biološki fakultet univ. u beogradu i zavod za zaštitu prirode r srbije, 74-75. ranđelović, v., 1999a: polemonium coeruleum l.. in: v. stevanović, ed.: crvena knjiga flore srbije 1, ministarstvo za životnu sredinu r srbije, biološki fakultet univ. u beogradu i zavod za zaštitu prirode r srbije, 85-86. ranđelović, v., 1999b: carex limosa l.. in: v. stevanović, ed.: crvena knjiga flore srbije 1, ministarstvo za životnu sredinu r srbije, biološki fakultet univ. u beogradu i zavod za zaštitu prirode r srbije, 247-249. ranđelović, v., 1999c: cirsium helenoides (l.) hill. in: v. stevanović, ed.: crvena knjiga flore srbije 1, ministarstvo za životnu sredinu r srbije, biološki fakultet univ. u beogradu i zavod za zaštitu prirode r srbije, 331-333. ranđelović, v., zlatković, b., 2010: flora i vegetacija vlasinske visoravni (flora and vegetation of vlasina plateau). prirodnomatematički fakultet univerziteta u nišu. 448 str. (in serbian) stevanović, v., ed., 1997: crvena lista flore srbije i crne gore (the red data list of flora of serbia and montenegro). manuscript. biological faculty, university of belgrade. stevanović, v., ed., 1999: crvena knjiga flore srbije, 1: iščezli i krajnje ugroženi taksoni (the red data book of flora of serbia, 1: extinct and critically endangered taxa). ministarstvo za životnu sredinu, biološki fakultet univ. u beogradu i zavod za zaštitu prirode r srbije. (in serbian and english) stojšić, v., panjković, b., 1999: ranunculus lingua l. in: v. stevanović, ed.: crvena knjiga flore srbije 1, ministarstvo za životnu sredinu r srbije, biološki fakultet univ. u beogradu i zavod za zaštitu prirode r srbije, 305-307. tomović, g., zlatković, b., niketić, m., perić, r., lazarević, p., duraki, š., stanković, m., lakušić, l., anačkov, g., knežević, j., szabados, k., krivošej, z., prodanović, d., vukojičić, s., stojanović, v., lazarević, m., stevanović, v., 2009: threat status revision of some taxa from “the red data book of flora of serbia 1”. botanica serbica, 33 (1): 33-43. tutin, t.g., v.h. heywood, n.a. burges, d.m. moore, d.h. valentine, s.m. walters & d.a. webb, eds., 1964-1980: flora europaea, i-v. cambridge university press. london. vukojičić, s., janković, m., 1999: caldesia parnassifolia (l.) parl. in: v. stevanović, ed.: crvena knjiga flore srbije 1, ministarstvo za životnu sredinu r srbije, biološki fakultet univ. u beogradu i zavod za zaštitu prirode r srbije, 62-63. savić et al. 2022, biologica nyssana 13(2) 13 (2) december 2022: 157-164 doi: 10.5281/zenodo.7437290 spatial and temporal distribution of the macrozoobenthos community in ponds of southeastern serbia original article ana savić department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, niš, serbia ana.savic@pmf.edu.rs (corresponding author) nenad ilić department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, niš, serbia jelena grozdanović department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, niš, serbia miodrag đorđević department of mathematics, faculty of sciences and mathematics, university of niš, višegradska 33, niš, serbia mrđan đokić department of geography, faculty of sciences and mathematics, university of niš, višegradska 33, niš, serbia received: october 31, 2022 revised: november 09, 2022 accepted: november 15, 2022 abstract: ponds are a prevalent type of freshwater ecosystems worldwide, but most studies have been traditionally focused on deep stratified lakes. the region of southeastern serbia is showcasing the fact that research on this type of small water bodies is scarce. in present study was compared macroinvertebrates community in ponds in southeastern serbia, at different altitudes but with similar other parameters. the goals of this study include: determining the composition and structure of macrozoobenthos communities in ponds at different altitudes; determining the seasonal changes in macrozoobenthos communities in these ponds; and determining the microdistribution of macroinvertebrate taxa in these ponds along the gradient of depth. in studied ponds, structuring of communities was mostly contributed to by insect groups. in the lower-altitude pond these were chironomidae and coenagrionidae, while in the higher-altitude pond chironomidae and dytiscidae. our study has shown that in most cases the sample of macroinvertebrate community included a greater number of families in autumn than in spring season. the greatest number of families was recorded in samples collected in the shallowest part of the pond, both in the pond with a low and in the pond with a high altitude. key words: macroinvertebrates, ponds, southeastern serbia apstrakt: prostorna i vremenska distribucija makroinvertebratske zajednice u barama jugoistočne srbije iako bare predstavljaju široko rasprostranjene, česte ekosisteme u celom svetu, veliki deo istraživanja je koncentrisan na duboka, stratifikovana jezera. u oblasti jugoistočne srbije takodje postoji nedostatak istraživanja malih vodenih tela. u ovom istraživanju je uporedjivana makroinvertebratska zajednica u barama koje se karakterišu različitim nadmorskim visinama, dok su sa druge strane, one slične po drugim karakteristikama. ciljevi ovog istraživanja su bili da se odredi sastav i struktura zajednice makroinvertebrata u barama sa različitom nadmorskom visinom, da se proprate sezonske promene u ovim zajednicama i da se odredi mikrodistribucija predstavnika ove zajednice duž gradijenta dubine. sastavu zajednice najviše doprinose insekatske grupe. u bari sa nižom nadmorskom visinom najveći doprinos struktuiranju imaju chironomidae i coenagrionidae, dok u bari sa većom nadmorskom visinom chironomidae i dytiscidae. u istraživanju je konstatovano da u najvećem broju uzoraka zajednica ima u sastavu veći broj familija u jesenjem aspektu nego u prolećnom. najveći broj familija je konstatovan u najplićim delovima bara koje su bile predmet ovog istraživanja. ključne reči: makroinvertebrate, bare, jugoistočna srbija introduction over a long period of time, conservation activities have mostly been focused on extensive ecosystems, especially those that contain large portions of the earth’s biodiversity (brooks et al., 2006; hunter et al., 2017; savić et al., 2022a). on the other side of the spatial gradient, there are some conservation efforts focused on small natural features in contrast to the large-scale conservation (hunter et al., 2017). small natural features (snfs) are analogous to keystone species because they have an ecological im© 2022 savić et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 157 portance that is disproportionate to their size; sometimes because they provide resources that constrain key populations or processes that affect a much larger area; sometimes because they support unusually high diversity, abundance, or productivity (hunter et al., 2017; savić et al., 2022a). ponds can be defined as small (1 m2 to max 5 ha), natural or man-made shallow waterbodies (normally fresh water, but occasionally brackish), which holds water for at least three months of the year or more (céréghino et al., 2008). this is just one of the possible definitions found in scientific literature, and there are almost as many definitions as there are authors of papers on these ecosystems. ponds (and shallow lakes also) are a prevalent type of freshwater ecosystems worldwide, but most studies have been traditionally focused on deep stratified lakes (hilt et al., 2017). the region of southeastern serbia is also showcasing the fact that research on this type of small waterbodies is scarce. they attracted more attention by botanists (ranđelović et al., 2007), while there are only a few (more recent) papers on macroinvertebrate community of ponds (đorđević, 2017; petrović, 2017). knowledge of spatial and temporal distribution patterns reduces bias in the analysis of data and allows the elaboration of sampling strategies. this type of information is particularly needed for the communities of macrozoobenthos in ponds because they are poorly studied (oertli, 1995). space and time may be perceived as the two axes along which the changes in macroinvertebrate community come about. these changes may pertain to composition and structure of the community, its diversity, biomass and productivity, etc. the spatial distribution of macroinvertebrates depends on various factors, depending on which spatial scale is observed. regarding the micro-distribution, the key factors recognized in the previous studies included heterogeneity of substrate (heino, 2000), depth of water body and oxygen concentration (brinkhurst, 2002), interaction with other organisms and presence of aquatic plants (waters & san giovanni, 2002) etc. temporal variations in fauna can exist at scales ranging from minutes to years (oertli, 1995). in the present study we will investigate temporal variations in macroinvertebrate communities in terms of seasonal changes. considering mentioned, the goals of this study include: 1) determining the composition and structure of macrozoobenthos communities in ponds at different altitudes; 2) determining the seasonal changes in macrozoobenthos communities in these ponds; and 3) determining the microdistribution of macroinvertebrate taxa in these ponds along the gradient of depth. materials and methods the selected study sites included two ponds in southeastern serbia. one is situated in the close vicinity of city of niš (43°17′40.2’’n, 21°55′45.9’’e) at the altitude of 294 m above sea level, while the other is at vlasina plateau (42°40′42.2’’n, 22°21′17.2’’e) at the altitude of 1212 m (fig. 1). average annual temperature on localities are 11.65 oc and 5.72 oc, respectively. 158 biologica nyssana ● 13 (2) december 2022: 157-164 savić et al. ● spatial and temporal distribution of the macrozoobenthos community in ponds of southeastern serbia fig. 1. map of the study area samples were collected in two seasons: in late may and late september, according to the following procedure: five spots were selected at each pond and one sample was taken at each point. samples were taken during a transect, first sample at depth of 30 cm and each following sample at distance of 2 m toward the deepest part of the pond (fig. 2a and 2b). each sample of macrozoobenthos was collected with a net with square frame 30 x 30 cm, mesh size 300 μm and size of sampled surface 0.30 m x 0.30 m. during the sampling process, the frame was placed vertically at the bottom of the pond and pulled along the bottom in the same length as the side of the frame (30 cm). the collected material was transferred to plastic bags and then conserved biologica nyssana ● 13 (2) december 2022: 157-164 savić et al. ● spatial and temporal distribution of the macrozoobenthos community in ponds of southeastern serbia 159 in 70% ethyl alcohol. identification of material was performed by using following keys: belfiore (1983), nilsson (1997), vallenduuk & pillot (2007). simper analysis was used for analysing the year and seasonal structuring of the community, as well as for analysis and comparison of community structure along the gradient of depth. in other words, simper analysis was performed to test differences within faunal composition of groups, dissimilarities between and similarities within the above groups. this analysis was performed using statistical program primer 7.0 (clarke & gorley, 2015). results the samples at the pond in vicinity of niš included representatives of 14 families (550 individuals), with 9 families recorded during the spring sampling and 10 families in the autumn sampling. at the annual level, the best-represented family was coenagrionidae (fig. 3a). the spring aspect was dominated by family chironomidae and the autumn aspect by family coenagrionidae (fig. 3b and 3c). the comparison of localities along the gradient of depth at the annual level has shown that the greatest fig. 2. sampling transects in: a pond in the vicinity of niš; b – pond of the vlasina plateau fig. 3. percentages of macrozoobenthos groups (at family level) in pond in vicinity of niš: a annual; b – in spring period; c – in autumn period 160 biologica nyssana ● 13 (2) december 2022: 157-164 savić et al. ● spatial and temporal distribution of the macrozoobenthos community in ponds of southeastern serbia fig. 4. annual number of benthic macroinvertebrate families in pond in vicinity of niš, their seasonal dynamic and distribution along depth gradient (where loc 1a is the shallowest and loc 5a is the deepest) fig. 5. percentages of macrozoobenthos groups (at family level) in pond on vlasina plateau: a annual; b – in spring period; c – in autumn period number of families was recorded at the locality with shallowest water, while the smallest number was recorded at the locality with deepest water (fig. 4). in four out of five localities, the number of families was greater in autumn that during the spring period. the samples from the pond at the vlasina plateau included representatives of 8 families (241 1ndividuals) (fig. 5a), where both spring and autumn samples were represented by 5 families each. at the annual level, the bestrepresented family was chironomidae, fig. 6. annual number of benthic macroinvertebrate families in pond on vlasina plateau, their seasonal dynamic and distribution along depth gradient (where 1b is the shallowest and loc 5b is the deepest) both in spring and autumn periods (fig. 5b, fig. 5c), respectively. just as in the pond in vicinity of niš, at the annual level the sample from the shallowest locality included the greatest number of families and the sample from the deepest locality included the lowest number of families (fig. 6). in four of the five localities, the number of families was greater in autumn that during the spring period, just as in the first pond. the simper analysis at the annual level has shown that samples from the 161 biologica nyssana ● 13 (2) december 2022: 157-164 savić et al. ● spatial and temporal distribution of the macrozoobenthos community in ponds of southeastern serbia the two ponds have shown high percent of differences according to simper analysis (70.04). regarding the seasonal aspects of the community, by far the greatest impact on community structure at the vicinity of niš during the spring aspect was shown by chironomidae (89.96) with negligible presence of ceratopogonidae (4.41). the difference between samples from the two ponds was not particularly high (45.95%). during the autumn aspect, the community structure of the pond in vicinity of niš was mostly pond in vicinity of niš did not show high average similarity (41.46), while the samples from the pond at vlasina plateau have shown somewhat higher levels of similarity (58.24). the structuring of the community at the pond in vicinity of niš was mostly contributed to by families chironomidae (34.64%) and coenagrionidae (25.6%). the structuring of community in the pond at vlasina plateau was also mostly contributed to by family chironomidae (75.23%) while the next family was dytiscidae (14.27%). at the annual level, the communities of table 1. simper analysis of five groups of macroinvertebrates divided along the gradient of pond depth group i: average similarity=27.38 group ii: average similarity=20.00 group iii: average similarity=24.88 group iv: average similarity=37.91 group v: average similarity=31.58 group i and ii: average dissimilarity=65.08 group i and iii: average dissimilarity=52.47 group i and iv: average dissimilarity=54.29 group i and v: average dissimilarity=53.07 group ii and iii: average dissimilarity=77.01 group ii and iv: average dissimilarity=64.18 group ii and v: average dissimilarity=63.82 group iii and iv: average dissimilarity=53.87 group iii and v: average dissimilarity=60.49 group iv and v: average dissimilarity=54.64 family av. abund. contrib. % cum. % group i chironomidae 21 65.22 65.22 corixidae 4 17.39 82.61 dytiscidae 9.5 13.04 95.65 group ii dytiscidae 7.5 45.49 45.45 chironomidae 21 27.27 72.73 corixidae 4.5 27.27 100 group iii chironomidae 21 66.67 66.67 elmidae 6.5 18.52 85.19 ceratopogonidae 4 7.41 92.59 group iv chironomidae 32.5 86.21 86.21 dytiscidae 4 13.79 100 group v chironomidae 23 100 100 162 biologica nyssana ● 13 (2) december 2022: 157-164 savić et al. ● spatial and temporal distribution of the macrozoobenthos community in ponds of southeastern serbia determined by families coenagrionidae (42.43), tabanidae (20.71) and caenidae (16.79). on the other hand, the most dominant family in the community at vlasina plateau was chironomidae (50.9), but elmidae (39.6) were also significant. for the needs of analysing microdistribution of families, the samples were divided into five groups along the gradient of pond depth. the simper analysis has shown that family chironomidae is significantly contributing to structuring at all depths. the value was highest in the first sample from the shallowest water (65.22%). in the second group of samples, taken at slightly greater depth, the most dominant family was dytiscidae (45.45%) while chironomidae was only second. in the third group, chironomidae (66.6%) were joined by elmidae (18.52%), while in the fourth and fifth groups chironomidae became dominant again (tab. 1). discussion the variation of water temperature across spatiotemporal gradients is one of the key abiotic drivers that shape the distribution, ecology and biology of freshwater macroinvertebrates (vannote et al., 1980). latitudinal and altitudinal gradients are recognized as ecological analogues and drivers of environmental gradients, with both being considered proxies of temperature variation (dos santos et al., 2018; rendoll cárcamo et al., 2019). several studies have focused on temperature and elevation gradient effects on freshwater macroinvertebrates in central europe (novikmec et al., 2015), south america (nieto et al., 2016; shah et al., 2017; dos santos et al., 2018) etc. nevertheless, region of balkans, have received little attention in this regard. considering mentioned, here was compared macroinvertebrates community in ponds at different altitudes but with similar other parameters (similar pond size, way of water supply and hydrological regime). in our research greater number of familia was detected in the pond with lower altitude which is in concordance with the claim that different types of diversity are showing a similar pattern with values decreasing as elevation increased (rendoll cárcamo et al., 2019). the samples at the pond in vicinity of niš included representatives of 14 families, while samples from the pond at the vlasina plateau included representatives of 8 families. although inhabited by a smaller number of taxa, ponds at high elevations are a specific type of small waterbodies leading to high regional diversity (hamerlík et al., 2014; rendoll cárcamo et al., 2019). in the studied ponds, the total number of individuals (550 vs. 241) and abundance decreased with increase of altitude, matching the results by rendoll cárcamo et al. (2019). the simper analysis at the annual level has shown that samples from the pond in vicinity of niš did not show high average similarity (41.46), while the samples from the pond at vlasina plateau have shown somewhat higher levels of similarity (58.24). in other words, this fact indicates a more uniform community at high altitude, similar to some similar investigation in other geographic regions (rendoll cárcamo et al., 2019). in both studied ponds, structuring of communities was mostly contributed to by insect groups. in the lower-altitude pond these were chironomidae and coenagrionidae, while in the higher-altitude pond chironomidae and dytiscidae. the predominance of chironomids is a common pattern in european high altitude stagnant water bodies (fjellheim et al., 2009). role of representatives of order odonata in community structuring is somewhat less commonly observed in ponds (thornhill et al., 2017). our study has shown that in most cases the sample of macroinvertebrate community included a greater number of families in autumn than in spring season. there is still an ongoing debate in scientific circles on the best season to sample aquatic macroinvertebrates in ponds for biodiversity assessment, but our results match those from studies in northern europe where autumn is the most suitable period (hill et al., 2016). in both ponds, the greatest number of families was recorded in samples collected in the shallowest part of the pond. one possible reason is the fact that more different microhabitats are situated in this zone than in others. the second possible reason may pertain to development of riparian vegetation. therefore, conservation of riparian vegetation in both lotic and lentic ecosystems is seemingly crucial as it supports high values of macroinvertebrates diversity (savić et al., 2022b). conclusions our results have shown that ponds at higher altitudes were inhabited by a more uniform community of macroinvertebrates. although hosting a smaller diversity of macroinvertebrate communities than ponds at lower altitudes, the high-altitude ponds significantly contribute to regional diversity and therefore should be given more attention. this study also contributes to the debate on the best season to sample aquatic macroinvertebrates in ponds for biodiversity assessment: it is the autumn aspect. the future research will likely be directed toward studies of a larger number of small waterbodies of this and similar types. 163 biologica nyssana ● 13 (2) december 2022: 157-164 savić et al. ● spatial and temporal distribution of the macrozoobenthos community in ponds of southeastern serbia references belfiore, c. 1983: efemerotteri (ephemeroptera). in: ruffo s. (ed.) guide per il riconoscimento delle specie animali delle acque interne italiane, 24. consiglio nazionale delle ricerche. roma. 113 p. brinkhurst, r.o. 2002: the benthos of lakes. blackburn press. brooks, t.m., mittermeier, r.a., da fonseca, g.a.b., gerlach, j., hoffmann, m., lamoreux, j.f., mittermeier, c.g., pilgrim, j.d., rodrigues, a.s.l. 2006: global biodiversity conservation priorities. science, 313: 58–61. céréghino, r., biggs, j., oertli, b., declerck, s. 2008: the ecology of european ponds: defining the characteristics of a neglected freshwater habitat. hydrobiologia, 597: 1–6. clarke, k.r., gorley, r.n. 2015: primer v7: user manual/tutorial. plymouth, uk: primer‐e. dos santos, d.a., molineri, c., nieto, c., zúñiga, m.c., emmerich, d., fierro, p., pessaq, p., riostouma, b., márquez, j., gómez, d., salles, f.f., encalada, a.c., príncipe, r., gómez, g.c., valdovinos, c., domínguez, e. 2018: cold/warm stenothermic freshwater macroinvertebrates along altitudinal and latitudinal gradients in western south america: a modern approach to an old hypothesis with updated data. journal of biogeography, 00: 1–11. đorđević, a. 2017: komparacija asambleje mollusca u barama različitog tipa. master thesis. univerzitet u nišu, prirodno-matematički fakultet, niš. fjellheim, a., raddum, g.g., vandvik, v., cogalniceanu, d., boggero, a., brancelj, a., galas, j., šporka, f., vidinova, y., bitušík, p., dumnicka, e., gâldean, n., kownacki, a., krno, i., preda, e., rîsnoveanu, g., stuchlík, e. 2009: diversity and distribution patterns of benthic invertebrates along alpine gradients. a study of remote european freshwater lakes. advances in limnology, 62: 159–176. hamerlík, l., svitok, m., novikmec, m., očadlík, m., bitušík, p. 2014: among-site and regional diversity patterns of benthic macroinvertebrates in high altitude waterbodies: do ponds differ from lakes? hydrobiologia, 723(1): 41–52. heino, j. 2000: lentic macroinvertebrate assemblage structure along gradients in spatial heterogeneity, habitat size and water chemistry. hydrobiologia, 418(1): 229-242. hill, m.j., sayer, c.d., wood, p.j. 2016: when is the best time to sample aquatic macroinvertebrates in ponds for biodiversity assessment? environmental monitoring and assessment, 188. hilt, s., brothers, s., jeppesen, e., veraart, a.j., kosten, s. 2017: translating regime shifts in shallow lakes into changes in ecosystem functions and services. bioscience, 67: 928–936. hunter, m.l.jr., acuna, v., bauer, d.m., bell, k.p., calhoun, a.j.k., felipe-lucia, m.r., fitzsimons, j.a., gonzalez, e., kinnison, m., lindenmayer, d., lundquist, c.j., medellin, r.a., nelson, e.j., poschlod, p. 2017: conserving small natural features with large ecological roles: an introduction and definition. biological conservation, 211: 88-95. nieto, c., malizia, a., carilla, j., izquierdo, a., rodríguez, j., cuello, s., zannier, m., grau, h.r. 2016: patrones espaciales en comunidades de macroinvertebrados acuáticos de la puna argentina. revista de biología tropical, 64(2): 747–762. nilsson, a. 1997: aquatic insects of north europe. a taxonomic handbook vol. 2. apollo books, steenstrup. 440 p. novikmec, m., veselská, m., bitušík, p., hamerlík, l., matúšová, z., reduciendo klementová, b., svitok, m. 2015: checklist of benthic macroinvertebrates of high altitude ponds of the tatra mountains (central europe) with new records of two species for slovakia. check list, 11(1): 1522. oertli, b. 1995: spatial and temporal distribution of the zoobenthos community in a woodland pond (switzerland). hydrobiologia, 300(1): 195-204. petrović, t. 2017: asambleja diptera u barama različitog tipa u okolini niša. master thesis. univerzitet u nišu, prirodno-matematički fakultet, niš. ranđelović, v., matejić, j., zlatković, b. 2007: flora i vegetacija batušinačkih bara kod niša. proceedings of 9th symposium on flora of southeastern serbia and neighbouring regions, 1940. niš. rendoll cárcamo, j., contador, t., gañán, m., pérez troncoso, c., maldonado márquez, a., convey, p., kennedy, j., rozzi, r. 2019: altitudinal gradients in magellanic sub-antarctic lagoons: the effect of elevation on freshwater macroinvertebrate diversity and distribution. peerj, 7: e7128. savić, a., djordjević, m., djordjević, m., randjelović, v., dmitrović, d., pešić, v. 2022a: biologica nyssana ● 13 (2) december 2022: 157-164 savić et al. ● spatial and temporal distribution of the macrozoobenthos community in ponds of southeastern serbia 164 springs of southeastern serbia with a focus on the vlasina plateau: different types of challenges for the macroinvertebrate community. in: pešić v. et al. (eds.), small water bodies of the western balkans, springer water. savić, a., zawal, a., stępień, e., pešić, v., stryjecki, r., pietrzak, l., filip, e., skorupski, j., szlauer-łukaszewska, a. 2022b: main macroinvertebrate community drivers and niche properties for characteristic species in urban/rural and lotic/lentic systems. aquatic sciences, 84: 1. shah, a., gill, b., encalada, a., flecker, a., funk, w., chris, a., guayasamin, j., kondratieff, b., leroy, poff, w., thomas, s., zamudio, k., ghalambor, c. 2017: climate variability predicts thermal limits of aquatic insects across elevation and latitude. functional ecology, 31(11): 2118–2127. thornhill, i., batty, l., deith, r.g., friberg, n.r., ledger, m.e. 2017: local and landscape scale determinants of macroinvertebrate assemblages and their conservation value in ponds across an urban land-use gradient. biodiversity and conservation, 26: 1065–1086. vallenduuk, h.j., pillot, h.k.m. 2007: chironomidae larvae of the netherlands, adjacent lowlands: general ecology, tanypodinae. knnv publishing, zeist. 270 p. vannote, r.l., minshall, g.w., cummins, k.w, sedell, j.r., cushing, c.e. 1980: the river continuum concept. canadian journal of fisheries and aquatic sciences, 37(1): 130–137. waters, n.m., san giovanni, c.r. 2002: distribution and diversity of benthic macroinvertebrates associated with aquatic macrophytes. journal of freshwater ecology, 17(2): 223-232. anticancer compounds from medicinal plants biologica nyssana 2 (2)  december 2011: 00-00 ljupković r.b. et al..  removal cu(ii) ions from water... 43 short communication melanoides tuberculata (müller, 1774), a new species for the territory of serbia (gastropoda: thiaridae) miodrag milenković* 1 , vladimir gligorijević 2 1 partizanska 25, 18300 pirot, serbia 2 dragiše cvetkovića 31d/11, 18000 niš, serbia * e-mail: mjouri@gmail.com abstract: milenković, m.,gligorijević, v.: melanoides tuberculata (müller, 1774), a new species for the territory of serbia (gastropoda: thiaridae), biologica nyssana, 3 (1), september 2012: 43-45. this article relates to the first record of melanoides tuberculata (müller, 1774) for the territory of serbia. its distribution in the region of "niška banja", eastern serbia is a consequence of its release by aquarists at thermal water stream called "topla voda". short habitat description and the areal are given. key words: melanoides tuberculata, first record, niška banja, serbia, thiaridae the freshwater benthic snail m. tuberculata belongs to the family thiaridae and many of its native varieties were distributed all over the east africa, tropical asia and southeast asia (m a l e k & c h e n g , 1974). today, they are extensively dispersed in the tropical and subtropical regions of the world (b e d e , 1992; p o i n t i e r , 1993). regions that this snail invaded include north america (m u r r a y , 1964) and south america (d e m a r c o , 1999). also, the most widely used taxonomy in europe, "fauna europaea" reveals presence of mentioned thiarid snail in austria, france, germany, hungary, italy, malta, netherlands, poland, slovakia and spain (fauna europaea, 2011). according to available literature data, m. tuberculata was never recorded in serbia. usually named "malayan snail", commonly reaches a length of 30-36 mm (t h o m p s o n , 1984). he is ovoviviparous (b e n -a m i & h o d g s o n , 2005) and parthenogenetic species (p o i n t i e r and g u y a r d , 1992; p o i n t i e r et al., 1993), with diet that mostly consists of fine detritus and epiphytic algae (m a d s e n , 1992). the authors visited "topla voda" (fig. 1) stream on 27. november 2011, near "niška banja" settlement (latitude: 43, 17' 46"; latitude reference: north latitude, longitude: 22, 0' 0" longitude reference: east longitude). on this occasion, autors observed a single specimen of gastropods and realized that it represents m. tuberculata, thiarid snail who is well known to aquarists. this allochtone species was noted in a small, fast flowing, thermal stream called "topla voda" which is composed of two thermal water sources: "suvo vrelo" and "školska česma". this habitat is under the influence of temperate climate at the altitude of 210 m (d r a g i ć at al., 1997). 3 (1) • september 2012: 43-45 biologica nyssana 3 (1)  september 2012: 43-45 milenković, m., gligorijević, v.  melanoides tuberculata (müler, 1774)… 44 figure 1. "topla voda" stream, located near settlement "niška banja" photo: vladimir gligorijević the riverbed was covered in sludge and sand with some smaller rocks in the middle. the specimens were photographed on a stream bottom (fig. 2) during that survey and then fixed in alcohol for later identification according to http://el.erdc.usace.army.mil/ansrp/ansis/html/mel anoides_tuberculata_red_rimmed_melania.htm#mtu berculata_identification and http://www.gastropods.com/9/shell_11379.shtml). ten specimens, 25.3mm to 34.1mm in length were measured with vernier calliper and then collected and used for mentioned identification. the actual distribution of m. tuberculata in "niška banja" termal stream called "topla voda" is certainly the consequence of its inadequate manipulation by local acquarists. the finding of m. tuberculata increases the number of known species of gastropoda for the territory of serbia. acknowledgments. the authors want to express their gratitude to miloš popović for image processing and dr vladimir žikić. figure 2. m. tuberculata in nature habitat at "topla voda" stream., photo: miodrag milenković references bank, ruud a., fauna europaea, 2011. group coordinator audisio, p. fauna europaea version 2.4, web service available online at http://www.faunaeur.org bede, l.c., 1992. dinâmica populacional de melanoides tuberculata (prosobranchia: thiaridae) no reservatório da pampulha, belo horizonte, m.g., brasil. dissertação de mestrado. universidade federal de minas gerais, belo horizonte, 112 p. ben-ami, f. and a.n. hodgson., 2005. ovoviviparity and the structure of the brood pouch in melanoides tuberculata (gastropoda: prosobranchia: thiaridae). journal of morphology, 263:322-329. dragić, s., garovnikov, b., joksimović, a., komatina, m., marić, ž., đukanović, m., jovanović, v., čuk, m. s., mitekoš, d. 1997. predlog za utvrđivanje područja niške banje. beograd, 1-30. madsen, h., 1992. food selection by freshwater snails in the gezira irrigation channels, sudan. hydrobiologia, 228: 203-217. malek, e.a. and cheng, t.c., 1974. medical and economic malacology. academic press, london, 398 p. marco, de p. j., 1999. invasion by the introduced aquatic snail melanoides tuberculatus (müller, 1774) (gastropoda: prosobranchia: thiaridae) of http://el.erdc.usace.army.mil/ansrp/ansis/html/melanoides_tuberculata_red_rimmed_melania.htm#mtuberculata_identification http://el.erdc.usace.army.mil/ansrp/ansis/html/melanoides_tuberculata_red_rimmed_melania.htm#mtuberculata_identification http://el.erdc.usace.army.mil/ansrp/ansis/html/melanoides_tuberculata_red_rimmed_melania.htm#mtuberculata_identification http://www.gastropods.com/9/shell_11379.shtml biologica nyssana 3 (1)  september 2012: 43-45 milenković, m., gligorijević, v.  melanoides tuberculata (müler, 1774)… 45 the rio doce state park, minas gerais, brazil. studies on neotropical fauna and environment, tübinge, 34: 186-189. murray, h. d., 1964. tarebia granifera and melanoides tuberculatus in texas. american malacological union inc., annual report, pp. 1516, 1964. pointier, j.-p. and guyard, a. 1992. biological control of the snail intermediate hosts of schistosoma mansoni in martinique, french west indies. tropical medicine and parasitology, 43:98-101. pointier, j.-p., 1993. the introduction of melanoides tuberculata (mollusca: thiaridae) to the island of saint lucia (west indies) and its role in the decline of biomphalaria glabrata, the snail intermediate host of schistosoma mansoni. acta tropicalis, 54:13-18. pointier, j.-p., thaler, l., pernot, a.f. and delay, b., 1993 – invasion of the martinique island by the parthenogenetic snail melanoides tuberculata and the succession of morphs. acta ecologica, 14(1):33-42. thompson, f.g., 1984. the freshwater snails of florida: a manual for identification. university of florida press, gainesville. 94p. http://el.erdc.usace.army.mil/ansrp/ansis/html/mel anoides_tuberculata_red_rimmed_melania.htm# mtuberculata_identification http://www.gastropods.com/9/shell_11379.shtml http://www.gastropods.com/9/shell_11379.shtml 46 jenačković gocić et al. 2020, biologica nyssana 11(2) 11 (2) december 2020: 71-84 doi: 10.5281/zenodo.4393949 insight into the chorology of some endangered, rare and potentially invasive plant species in serbia original article dragana jenačković gocić department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, 18000 niš, serbia draganaj@pmf.ni.ac.rs (corresponding author) ljiljana bolbotinović department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, 18000 niš, serbia ljiljana.bolbotinovic@pmf.edu.rs marina jušković department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, 18000 niš, serbia marinaju@pmf.ni.ac.rs danijela nikolić department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, 18000 niš, serbia danid@pmf.ni.ac.rs vladimir ranđelović department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, 18000 niš, serbia vladar@pmf.ni.ac.rs received: december 07, 2020 revised: december 14, 2020 accepted: december 14, 2020 abstract: this paper presents the distribution of nine endangered, rare or potentially invasive plant species present in the serbian flora, based on field investigations, literature and herbarium data. the chorology of the following vascular plants: ranunculus lateriflorus, elatine alsinastrum, sedum stefco, galium boreale, ornithogalum narbonense, cyperus serotinus, cyperus odoratus, typha laxmannii and typha shuttleworthii, is shown on 10 x 10 km2 utm maps. the data on the distribution of these endangered and rare species provide a valuable base for estimating and revising their threatened status in serbia, as well as for conducting adequate conservation measures with regard to their habitats. chorological data on the cyperus odoratus species in serbia are significant for getting to know its naturalization history and spreading pathways in both serbia and its neighbouring countries. key words: distribution, endangered plants, flora, invasive species, new chorological data, rare species, serbia apstract: uvid u horologiju pojedinih ugroženih, retkih i potencijalno invazivnih biljnih vrsta u srbiji u ovom radu, prikazana je distribucija devet ugroženih, retkih i potencijalno invazivnih vrsta prisutnih u flori srbije na osnovu terenskih, literaturnih i herbarijumskih podataka. horologija sledećih vaskularnih biljaka: ranunculus lateriflorus, elatine alsinastrum, sedum stefco, galium boreale, ornithogalum narbonense, cyperus serotinus, cyperus odoratus, typha laxmannii i typha shuttleworthii, prikazana je na 10x10 km2 utm kartama. podaci o distribuciji ugroženih i retkih vrsta, koji su ovde prikazani, predstvaljaju dragocenu osnovu za procenu i reviziju njihovog statusa ugroženosti na području srbije, i sprovođenje adekvatnih mera zaštite njihovih staništa. podaci o distribuciji vrste cyperus odoratus u srbiji su od velikog značaja za upoznavanje istorije procesa naturalizacije i puteva širenja njenih individua u našoj i susednim zemljama. ključne reči: rasprostranjenje, ugrožene biljke, flora, invazivne vrste, novi horološki podaci, retke vrste, srbija introduction it is well-known that continuous monitoring of the floristic composition and abundance of plant species, especially those that are threatened, alien or weed, has enormous significances for biodiversity conservation. monitoring the population of threatened species has a significant role in conducting protection measures in order to prevent their eventual extinction, while collecting data on the presence and spreading pathways of new, mostly invasive species, is significant for controlling the size of their populations and protecting autochthonous flora. despite the intensive study of serbian flora over the last decades, new species are still being found (ranđelović et al., 2002; niketić et al., 2009; đorđević et al., 2010; đorđević et al., 2014; tomović et al., 2016; stojanović et al., 2017), as well as new localities for rare and endemic plant species (tomović et al., 2007; zlatković et al., 2007; niketić © 2020 jenačković gocić et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work noncommercially under the same license as the original. 71 & tomović, 2008; zlatković & bogosavljević, 2014; jenačković et al., 2015; đorđević et al., 2017), which supports the fact that the flora of serbia has not yet been, and probably never will be, fully explored. during intensive phytocoenological research conducted in the areas of ne, e and se serbia on diverse habitat types, including rivers, ponds, seasonal flooded habitats, wet meadows and rocks over the last three years, new chorological data on some threatened, rare, endemic and potentially invasive plant species have been collected. this research confirms the already known fact that the previously mentioned habitat types are refuges for numerous rare and endangered plants (stojanović et al., 1994), so they are significant for the conservation of genetic, species and ecosystem diversity. in the regions of ne, e and se serbia, they are, unfortunately, poorly investigated, both floristically (bogosavljević et al., 2007; petrović et al., 2007; stanković et al., 2009; jenačković et al., 2015) and phytocoenologically (ranđelovć et al., 2007; ranđelović & zlatković, 2010; jenačković et al., 2010; jenačković, 2017). generally, publishing new chorological data for the previously mentioned groups of plants enriches knowledge regarding their distribution and provides adequate material for writing and publishing new editions of flora of serbia, as well as red list and red books of the flora of serbia and the serbian list of invasive plant species. in this paper, the authors give detailed information on the distribution pattern of nine vascular plants in serbia: ranunculus lateriflorus dc., elatine alsinastrum l., sedum stefco stefanov, galium boreale l., ornithogalum narbonense l., cyperus serotinus rottb., cyperus odoratus l., typha laxmannii lepechin and typha shuttleworthii koch & sonder in koch. material and methods the distribution of each of the species presented in this study is showed on a 10 x 10 km2 utm grid based on field, literature and herbarium data. field investigation of different habitats was carried out in the period 2017-2020 in the areas of ne, e and se serbia. relevant floristic and phytocoenological literature published in the last seventy years was also used as an adequate source of chorological data. georeferenced data on the distribution of specimens deposited in the herbarium of the faculty of sciences and mathematics, department of biology and ecology, university of niš (hmn) and herbarium of the institute of botany and botanical garden “jevremovac”, university of belgrade (beou) are showed on utm grids, too. plant material collected during field investigation was determined by using the dichotomic identification keys (josifović, 1970 – 1980; tutin et al., 1964 – 1980). the nomenclature of the species recorded was uniformed to the flora europaea (tutin et al., 1964 – 1980). the threatened status for the species mentioned in the section above is considered in terms of iucn (2000) criteria and categories. results and discussion ranunculus lateriflorus dc. general distribution: r. lateriflorus has been recorded in europe (spain, france, italy, austria, slovakia, hungary, romania and all countries of the balkan peninsula except albania), north africa (morocco and algeria) and asia (countries of the western asia, ukraine and russia). distribution in serbia: most published data on r. lateriflorus distribution in serbia are for the province of vojvodina (diklić, 1992; purger, 1993; panjković et al., 2012; šturc, 2014) (fig. 1). other parts of serbia have been poorly investigated, especially the territory of nw, w, se and c serbia, pomoravlje and regions of kosovo. outside of vojvodina province, r. lateriflorus was recorded in the šumadija region: rakovica and grocka (diklić, 1992); ne serbia: negotin and kladovo (diklić, 1992); e serbia: pirot (nikolić et al., 1986; diklić, 1992) and s serbia: leskovac (diklić, 1992). unpublished chorological data: ne serbia: krivelj – ep88 (leg. pančić, j. 2327, 1876, beou); šumadija region: jovanovac – dp97 (leg. pančić, j. 2309, 1870, beou); banat region: melenci – dr44 (leg. niketić, m., tomović, g. 48059, 23.04.2016, beou); farkaždin – dr50 (leg. lakušić, d., tomović, g. 47302, 19.04.2016, beou; leg. lakušić, d., tomović, g., kuzmanović, n., kabaš, e., đurović, s., janković, i., milekić, t. 47375, 17.05.2016, beou). new chorological data: it has been found in the surroundings of donje međurovo village (e serbia – en69, 43°18’22’’ n, 21°50’25’’ e, 185 m a.s.l., leg./det. jenačković, d. 14367, 29.05.2018, hmn). at this study site, on a seasonally flooded and salted habitat that borders with a cultivated area, a small population of r. lateriflorus was recorded, which is under strict protection. it was made up of less than a hundred individuals. most of the individuals recorded are growing in association with eleocharis palustris and oenanthe silaifolia, and they are part of the composition of the halophilous community, with beckmannia eruciformis being dominant. habitat characteristics: r. lateriflorus is an annual therophyte that grows and blooms during the spring on wet, sandy and seasonally flooded habitats. iucn threatened status in the world: least concern (lc). iucn threatened status in serbia: endangered – vulnerable (en b1; b2b, c; c1; c2a/vu a1b, c, e; 72 biologica nyssana ● 11 (2) december 2020: 71-84 jenačković gocić et al. ● insight into the chorology of some endangered, rare and potentially invasive plant species in serbia 73 a2c, e; d1; d2). according to király (2007), r. lateriflorus is a legally protected species in hungary, where it is classified in the category nt (near threatened). generally, its habitats are exposed to strong negative anthropogenic pressure, which can be seen in the decreasing size of its population, so applying adequate conservation measure is needed not only in serbia, but also globally. elatine alsinastrum l. general distribution: e. alsinastrum is a eurosiberian sub-mediterranean species that grows in temporary water bodies in europe (from the iberian peninsula, north to france and belgium, east to poland and ukraine, and in almost all countries in the mediterranean basin), asia (russia, ukraine, georgia, armenia, w siberia and kazakhstan) and north africa morocco, algeria and tunisia (popiela et al., 2013). distribution in serbia: although blečić (1972) stated that the species is often found in serbia, literature sources and herbarium collections provide little data on e. alsinastrum chorology in serbia (fig. 2). until now, it has been documented that e. alsinastrum grows in ditches in the bačka (panjković, 2005) and srem regions (perić et al., 2016). unpublished chorological data: during the 19th century, josif pančić collected specimens of e. alsinastrum at a few locations south of vojvodina province. he found this species in the area surrounding belgrade (rakovica – dq55, leg. pančić, j. 3170, 1877, beou), sobovica (bele bare – dp88, leg. pančić, j. 3168, 1851, beou), bubanj – en69 (leg. pančić, j. 3167, 1885, beou) and in the valley of the kolubara river (leg. pančić, j. 14593, beou, this data is not shown on fig. 2). new chorological data: two years ago, in the area surrounding the village of donje međurovo (se serbia – utm, 43°18’18’’ n, 21°50’10’’ e, 183 m a.s.l.) in seasonally flooded habitats, a community was recorded, with e. alsinastrum dominance (fig. 2). the following species: eleocharis palustris, alisma plantago-aquatica, veronica anagallisaquatica, mentha pulegium and beckmannia eruciformis were part of the composition of the elatine alsinastrum community. specimens of e. alsinastrum are stored in hmn (leg./det. jenačković, d. 14370, 29.05.2018). more than a hundred years ago, josif pančić found e. alsinastrum in the vicinity of donje međurovo (bubanj – en69), but he did not publish that record. habitat characteristics: e. alsinastrum inhabits temporary water bodies or seasonally flooded zones of permanent water bodies. iucn threatened status in the world: near threatened (nt) with decreasing population trend. iucn threatened status in serbia: not threatened. fig 1. distribution map of ranunculus lateriflorus in serbia (a). the photographs show it growing on seasonally flooded habitats in the vicinity of donje međurovo village (b). biologica nyssana ● 11 (2) december 2020: 71-84 jenačković gocić et al. ● insight into the chorology of some endangered, rare and potentially invasive plant species in serbia 74 elatine triandra schkuhr is the only species from the genus elatine that is protected in serbia. based on literature sources and herbarium data, as well as the authors’ field experiences, it can be concluded that e. alsinastrum is a rare species of serbian flora which requires some kind of protection. its small populations inhabit sensitive habitat types that are threatened by diverse anthropogenic factors, such as the drainage of seasonally inundated land, stabilization of hydrological regimes, agricultural improvement etc. the same problem also exists in other european countries, so e. alsinastrum could be considered as rare species throughout most countries in central europe (schnittler & günther, 1999). elatine alsinastrum is classified as a critically endangered species in bulgaria (petrova & vladimirov, 2009), and near threatened in hungary (király, 2007). in croatia, it is rarely recorded, and known only from old literature and herbarium sources, so it is described as an insufficiently known plant species (dd – data deficient) (prlić, 2015). sedum stefco stefanov general distribution: it is an endemic species of the balkan peninsula that has been recorded in four countries (serbia, bulgaria, macedonia and greece) so far. distribution in serbia: the first data published on the presence of s. stefco in serbian flora date from the end of the 20th century and refer to radan mountain (zlatković et al., 1993; zlatković & ranđelović, 1995) and the pčinja river gorge (zlatković & ranđelović, 1995). by publishing data on the geographical position of population of the species s. stefco in e serbia (the temštica gorge) and s serbia (kozjak mountain – monastery prohor pčinjski), tomović et al. (2003) made the previous knowledge of its distribution more complete (fig. 3). new chorological data: two populations of the strictly protected species, s. stefco, were found on stara planina mountain at a locality called strejnica (e serbia – fp20, 43°21’20.24’’ n, 22°35’31.91’’ e, 1501 m a.s.l., leg./det. ranđelović, v., 10.07.2020; e serbia – fp20, 43°21’20.32’’ n, 22°35’31.70’’ e, 1550 m a.s.l., leg./det. ranđelović, v., 02.08.2020). individuals of the s. stefco species inhabit fragments of red sandstone with other species characteristic for rocky vegetation, including sedum annuum, sedum album, silene lerchenfeldiana, allium carinatum subsp. pulchellum, scleranthus perennis, anthemis carpatica, etc. habitat characteristics: in serbia, s. stefco is found on rocks (silicate and red sandstones), rock fragments, and dry, rocky ground at a wide range of altitudes, from 300 m to 1550 m a.s.l. iucn threatened status in the world: not threatened. iucn threatened status in serbia: near threatened – least concern (data deficient) (nt-lc fig 2. utm map of the distribution of elatine alsinastrum in serbia (a). the photographs present its populations that inhabit shallow, ephemeral depressions in the vicinity of donje međurovo village (b). biologica nyssana ● 11 (2) december 2020: 71-84 jenačković gocić et al. ● insight into the chorology of some endangered, rare and potentially invasive plant species in serbia (dd)). sedum stefco is classified as near threatened in the greek red data book (eleftheriadou et al., 2009), and vulnerable, and it is a legally protected species in the republic of bulgaria (petrova & vladimirov, 2010). galium boreale l. general distribution: it is distributed over the subarctic and temperate regions of the northern hemisphere. in europe, g. boreale is rare in the mediterranean region. distribution in serbia: g. boreale is a legally protected species in serbia. it is recorded in the bačka region (boža & vasić, 1986); nw serbia: maljen mountain (gajić, 1973; popović, 2005); w serbia: stolovi mountain (pavlović, 1974), tara mountain (gajić, 1988), zlatibor mountain (pavlović, 1951; gajić, 1973; novaković-vuković, 2015), the uvac river gorge (veljić et al., 2006); sw serbia: ozren mountain (pavlović, 1955; rakonjac, 2002) and pešter plateau (tatić, 1988; rakonjac, 2002) (fig. 4). new chorological data: while mapping herbaceous habitat types on vlasina plateau, individuals of g. boreale were recorded at two sites: murina valley (se serbia – fn12, 42°42’51.52’’ n, 22°21’39.09’’ e, 1264 m a.s.l., leg. jenačković gocić, d., det. ranđelović, v. 14383, 29.06.2020, hmn) and bratašnica (se serbia – fn02, 42°41’37.39’’ n, 22°19’17.48’’ e, 1226 m a.s.l., leg./det. ranđelović, v., 20.07.2020). they were a part of the meadow vegetation composition, together with individuals from the following species: sanguisorba officinalis, briza media, holcus lanatus, centaurea jacea, filipendula hexapetala, galium verum, hypericum maculatum subsp. immaculatum, potentilla erecta and campanula patula. habitat characteristics: g. boreale is a heliophilous species that prefers moist and well drained soils. it can tolerate partial shade and dry conditions. it is found in wet meadows and open woods. iucn threatened status in the world: not threatened. iucn threatened status in serbia: vulnerable – near threatened (data deficient) (vu-nt (dd)). galium boreale is a vulnerable species in the bulgarian flora (petrova & vladimirov, 2009), too. ornithogalum narbonense l. general distribution: it occurs in europe (portugal, spain, france, italy, romania and countries on the balkan peninsula), asia (turkey, azerbaijan, armenia, georgia, syria, lebanon, israel and jordan) and africa (egypt, libya, tunisia, algeria and morocco). distribution in serbia: according to diklić (1975), o. narbonense is a species with sporadic occurrence in serbia. data on its distribution in 75 fig 3. distribution of sedum stefco in serbia (a). photographs of sedum stefco specimens (b) and a mixed community dominated by succulent plant species sedum stefco and sedum album (c), developed on rocky fragments situated on stara planina mountain. biologica nyssana ● 11 (2) december 2020: 71-84 jenačković gocić et al. ● insight into the chorology of some endangered, rare and potentially invasive plant species in serbia serbia are poor. until now, o. narbonense has been recorded at two localities in the province of kosovo: podujevo and orahovac (nikolić et al., 1986) (fig. 5). ranđelović et al. (2005) stated that o. narbonense inhabits cultivated land in southeastern serbia, but did not give precise information on the geographical position of its populations. further field investigations are needed in order to determine whether o. narbonense is a rare or insufficiently investigated species in serbia. new chorological data: o. narbonense was recorded within meadow vegetation on vlasina biologica nyssana ● 11 (2) december 2020: 71-84 jenačković gocić et al. ● insight into the chorology of some endangered, rare and potentially invasive plant species in serbia 76 fig 4. utm map of the distribution of galium boreale in serbia (a) and photographs of individual specimens (b). fig 5. map of distribution of ornithogalum narbonense in serbia (a) and photographs of its inflorescence (b). biologica nyssana ● 11 (2) december 2020: 71-84 jenačković gocić et al. ● insight into the chorology of some endangered, rare and potentially invasive plant species in serbia plateau, in a place named jarčev potok (se serbia – fn02, 42°40’11.73’’ n, 22°20’12.35’’ e, 1234 m a.s.l., leg./det. ranđelović, v., 21.07.2020). agrostis capillaris, briza media, achillea millefolium, campanula patula, crepis conyzifolia, dianthus deltoides, galium verum, hypericum maculatum subsp. immaculatum, hypochaeris maculata, potentilla argentea and pastinaca hirsuta are the most abundant species at the site where these individuals of o. narbonense were found. habitat characteristics: it grows in sunny, grassy and dry places such as grasslands, roadsides, crops and disturbed ground. iucn threatened status in the world: not threatened. iucn threatened status in serbia: vulnerable – near threatened (data deficient) (vu-nt (dd)). cyperus serotinus rottb. general distribution: c. serotinus is a native species on the european and asian continents while it is naturalized in north america and australia. on the eurasian continent, c. serotinus is distributed from the iberia peninsula through the countries of central europe and the mediterranean basin, caucasus, kazakhstan, middle east and the himalayas to japan, most of china, taiwan and vietnam. distribution in serbia: c. serotinus is a rare and endangered species that is recorded in the banat region: deliblato sands (budak, 1983), labudovo okno (polić, 2006), žilovo islet, dolnice bay and stevan lowlands (stevanović et al., 2004); šumadija: on the banks of the danube river near belgrade (čanak, 1976), nw serbia: loznica (čanak, 1976), and c serbia: batušinac ponds (ranđelović et al., 2007) (fig. 6). unpublished chorological data: in the 21st century, individuals of the c. serotinus species were found at a few locations in the danube river valley, downstream from belgrade (malo bavanište embankment – eq05, leg. stevanović, v. 49867, 22.09.2002, beou; mali lap – eq16, leg. stevanović, v. 22335, 26.07.2003, beou; zatonje – eq35, leg. stevanović, v. 50093, 23.11.2013, beou; prahovo (bay kusjaka) – fq20, leg. stevanović, v. 50073, 23.07.2013, beou). new chorological data: one population of c. serotinus was recorded on the right bank of the danube river near tekija (ne serbia – fq14, 44°41’28’’ n, 22°24’56’’ e, leg. bolbotinović, lj., det. ranđelović, v. & jenačković, d. 14368, 25.09.2018, hmn). the first record (unpublished) for this toponymy dates from the 19th century (cyperus monti, tekija – fq14, leg. pančić j. 15359, 1877, beou; cyperus monti, tekija – fq14, leg. pančić j. 12347, 1878, beou), but it refers to “old” tekija, which was submerged after construction of the đerdap i hydropower dam. habitat characteristics: c. serotinus grows in seasonally flooded habitats, bays, banks of rivers and standing water bodies. 77 fig 6. utm maps of the distribution of cyperus serotinus (a) and a photograph of its inflorescence (b). biologica nyssana ● 11 (2) december 2020: 71-84 jenačković gocić et al. ● insight into the chorology of some endangered, rare and potentially invasive plant species in serbia iucn threatened status in the world: least concern (lc). iucn threatened status in serbia: near threatened – least concern (data deficient) (nt – lc (dd)). according to nikolić & topić (2005), c. serotinus is a vulnerable plant species in croatian flora. cyperus odoratus l. general distribution: c. odoratus is a pantropic species which has been naturalized in europe (spain, italy, serbia, bulgaria and romania) (verloove, 2014). distribution in serbia: for a long time, c. odoratus has been confused with c. strigosus and other species from the cyperus genus, so its exact naturalization history in serbia is not known. after the revision of herbarium specimens deposited in beou, verloove (2014) concluded that the first record of c. odoratus, under the erroneous binomial c. strigosus, was published at the beginning of the 21st century by stevanović et al. (2004, 2005). it refers to mali lap marsh situated near the village of dubovac (banat region – eq15) (fig. 7). stevanović et al. (2005) stated that c. strigosus grows in several places in the danube basin (km 1090 – 1075): žilovo islet, mali lap marsh, dolnice bay and the stevan lowlands. after the revision of herbarium material deposited in beou, the authors confirmed the presence of c. odoratus at žilovo islet – eq15 (cyperus sp., leg. stevanović, v., stevanović, b., sekulić, n., šinžar-sekulić, j. 49877, 08.09.2002, beou), while the presence of c. odoratus at dolnice bay and stevan lowlands remains unconfirmed. verloove (2014) stated that individuals from the c. odoratus species form small populations at numerous localities along the danube river, from kovin to mihajlovac near đerdap ii hydropower dam, but he did not give precise information on the localities. unpublished chorological data: after revision of specimens from the cyperus genus stored in beou, the authors established that c. odoratus grows in one more place in the banat region (malo bavanište – eq05, leg. stevanović, v. 49860, 22.09.2002, beou; leg. stevanović, v. 49866, 22.09.2002, beou). during 2020, individuals from the c. odoratus species were recorded at localities situated northwest of the places mentioned in earlier published papers (stevanović et al. 2005, verloove 2014): ada ciganlija (dq56 – 44°47’43.05” n, 20°25’31.15” e, leg. pantović, j., stevanoski, i., ćosić, m. 69151, 16.10.2019, beou) and pančevački rit (dq66 – 44°49’53.41” n, 20°30’14.35” e, leg. pantović, j., stevanoski, i., ćosić, m. 69160, 24.10.2019, beou) (fig. 7). new chorological data: c. odoratus was found at three locations (44°41’27’’ n, 22°24’56’’ e; 44°41’20’’ n, 22°24’47’’ e; 44°40’34’’ n, 22°24’70’’ e) on seasonally flooded habitat positioned in surrounding of tekija, near the danube river (ne serbia – fq14, leg. bolbotinović, lj., det. 78 fig 7. map of distribution of cyperus odoratus in serbia (a) and a photograph of individual specimens (b). jenačković, d. & ranđelović, v. 14369, 25.09.2018, hmn). habitat characteristics: in europe, c. odoratus is mostly found on mud flats, gravelly or sandy river banks, estuaries, and in riparian woodland, drainage channels and ditches (verloove, 2014). they form pure or mixed communities with other emergent macrophytes, such as c. serotinus. otherwise, c. odoratus occupies new pitches very quickly and behaves like an invasive environmental weed (verloove, 2014), so the knowledge of its distribution pattern and population status is important for determining how it spreads and how it can eventually be controlled. iucn threatened status in the world: not threatened. iucn threatened status in serbia: not threatened. typha laxmannii lepechin general distribution: t. laxmannii is distributed from europe through siberia, the caucasus, the middle east, kazakhastan and mongolia to the russian far east, china and japan. distribution in serbia: the first chorological data for t. laxmannii in serbia are from budak (1986), and they refer to the banat and bačka regions (fig. 8). radulović (2005) stated that individuals from the t. laxmannii species compose a community with typha latifolia dominance in carska bara special nature reserve. in the 21st century, a few authors have published data on t. laxmannii distribution outside the borders of vojvodina province. until now, it has been recorded in the following localities: batušinac ponds (ranđelović et al., 2007), šarpance village, vražji kamen, the vicinity of dimitrovgrad town, vranjska banja (zlatković et al., 2007), rgotina lake (zlatković & bogosavljević, 2014), smilovsko lake (jenačković, 2017) and zlatibor mountain (tomović et al., 2020) (fig.8). new chorological data: during the summer of 2020, a small population of t. laxmannii was recorded in a canal, positioned east of the dam, through which water enters vlasina lake (se serbia – fn03, 42°44’59.01’’n, 22°19’43.34’’e, 1222 m a.s.l., leg. nikolić, d., jušković, m., savić, a., det. ranđelović, v. 14388, 01.08.2020, hmn). at this site, t. laxmannii grows mainly in association with typha latifolia, juncus effusus, centaurium erythraea, etc. habitat characteristics: it prefers permanently flooded habitats with shallow waters or seasonally flooded habitats, such as the margins of ponds, pools, canals, rivers, streams and lakes. iucn threatened status in the world: least concern (lc). iucn threatened status in serbia: near threatened (nt). typha laxmannii is classified as a critically 79 biologica nyssana ● 11 (2) december 2020: 71-84 jenačković gocić et al. ● insight into the chorology of some endangered, rare and potentially invasive plant species in serbia fig 8. utm maps of the distribution of typha laxmannii (a) and a photograph of the population recorded at vlasina plateau (b). endangered species of croatian flora (nikolić & topić, 2005), and it was recorded during summer 2000 for the first time (topić & ozimec 2001). typha shuttleworthii koch & sonder in koch general distribution: it occurs in europe (from france east through the czech republic and hungary to romania and the ukraine), the caucasus and asia (turkey). distribution in serbia: t. shuttleworthii was considered to be a critically endangered species in serbia during the 20th century (ranđelović, 1999) because it was known at only a few localities: the spring of jabukovačka river on kukavica mountain, the area surrounding novi sad, kragujevac and sjenica (jovanović, 1986) (fig. 9). its status changed from critically endangered to endangered after establishing new sites in eastern serbia: crna reka gorge, svrljiški timok gorge and the environs of bosilegrad (topli dol and musulj) (tomović et al., 2009). the first data on t. shuttleworthii distribution in c serbia (radan mountain – gornji gajtan and kosmača) and w serbia (tara mountain – kurjačine and kozulja; golija mountain – izubra) were published this year (tomović et al., 2020). new chorological data: a small population of the strictly protected species t. shuttleworthii was recorded in a flooded depression on stara planina mountain, next to the road leading to tupavica waterfall (e serbia – fn49, 43°15’42’’ n, 22°46’26’’ e, 975 m a.s.l., leg./det. ranđelović, v. & jenačković, d. 14384, 22.07.2018, hmn). habitat characteristics: t. shuttleworthii usually builds small populations that cover a few square meters, mostly in anthropogenic habitats such as roadside ditches (tomović et al., 2020), river and stream valleys, swamps and wet meadows (kozłowska et al. 2011). iucn threatened status in the world: data deficient (dd). iucn threatened status in serbia: endangered (en b1 ab(i,ii,iii); b2 ab(i,ii,iii); c1) (tomović et al. 2009). there is a need for additional field investigation due to the possibility of its wider distribution in serbia. it is considered that its significant morphological similarity with t. latifolia is the main reason for the insufficient study of its distribution in serbia (tomović et al., 2020). the same problem exists in bulgaria and croatia, so t. shuttleworthii is classed as data deficient in these countries (petrova & vladimirov, 2009; nikolić & topić, 2005). király (2007) stated that t. shuttleworthii is extinct in hungary. conclusion the chorological data presented in this paper are a valuable base for better understanding the distribution patterns in serbia of the vascular plants analyzed in this study, as well as estimating and potentially reevaluating their threatened status, and controlling 80 fig 9. map of distribution of typha shuttleworthii in serbia (a) and a photograph of its inflorescence (b). biologica nyssana ● 11 (2) december 2020: 71-84 jenačković gocić et al. ● insight into the chorology of some endangered, rare and potentially invasive plant species in serbia 81 the size of their populations. the strictly protected and endangered plant species, r. lateriflorus, is poorly investigated outside the borders of vojvodina province, especially in w serbia and the province of kosovo. it mostly grows in shallow, seasonally flooded depressions that are affected by negative anthropogenic factors, so continuous monitoring of its population is needed due to a possible decrease in its population size. similar habitat types are preferred by e. alsinastrum, a globally near threatened species whose distribution in serbia has not been researched enough. although it is not recognized as threatened in serbia, the authors suggest that its status in this country should be reconsidered in accordance with the data provided in this research. according to existing floristic data, the balkan endemic species s. stefco is distributed in the southern and eastern parts of serbia. the border of its geographic range in serbia is expanding to the north by establishing its existing population on stara planina mountain. individuals from the legally protected and vulnerable species g. boreale were mainly recorded within wet meadows and open woods in western serbia. the borders of its geographic range have moved significantly to the east after establishing its presence on vlasina plateau. the poorly studied, vulnerable species of serbian flora, o. narbonense, requires additional investigation in order to collect information with regard to its presence in other parts of serbia (except the province of kosovo and vlasina plateau) and to reevaluate its threatened status. populations of the near threatened plant species c. serotinus are recorded at only a few sites in serbia, mainly in the valleys of the great lowland rivers including the danube, drina and južna morava. a recently recorded species in serbia, c. odoratus spreads quickly on gravelly and sandy substrates along the danube and sava rivers, by forming a mixed community with the autochthonous macrophyte species. it is considered important to monitor its populations due to its huge potential to inhabit new pitches. based on literature data and field experience, the authors consider that t. laxmanii has wider distribution in serbia than currently known. its habitats are insufficiently researched, especially south of vojvodina province, so additional field investigations are needed in order to adequately estimate its threatened status. during the 21st century, the amount of chorological data on the strictly protected species, t. shuttleworthii, has significantly increased, so its threatened status has changed from critically endangered to endangered in serbian flora. collecting data on its distribution and population size is important, not only for reevaluating its threatened status at a national level, but also for globally estimating the category of its vulnerability. acknowledgements. this study was supported by the project of ministry of science and technological development of republic of serbia (contract no. 451-0368/2020-14/200124). the authors would like to thank snežana vukojičić phd (curator at the beou herbarium) and marija marković phd (curator at hmn herbarium) for their help during the collection of chorological data for the species studied. references blečić, v. 1972: rod elatine l. in: josifović, m. (ed.), flora sr srbije, 3: 126-128, srpska akademija nauka i umetnosti, beograd. bogosavljević, s., zlatković, b., ranđelović, v. 2007: flora klisure svrljiškog timoka. proceeding of 9th symposium on flora of southeastern serbia and neighbouring regions, niš, 41-54. boža, p., vasić, o. 1986: galium boreale l. f. hyssopifolium (hoffm.) dc. in: sarić, m., diklić, n. (eds.), flora sr srbije, 10: 161, srpska akademija nauka i umetnosti, beograd. budak, v. 1983: fam. cyperaceae j. st. hil. in: gajić, m. (ed.), flora deliblatske peščare: 362-372, prirodno-matematički fakultet, oour institut za biologiju, novi sad, šumsko-industrijski kombinat “pančevo“, oour specijalni prirodni rezervat “deliblatski pesak”, pančevo. budak, v. 1986: typha laxmannii lepech. in: sarić, m., diklić , n. (eds.), flora sr srbije, 10: 255-256, srpska akademija nauka i umetnosti, beograd. čanak, m. 1976: rod pycreus p. beauv. in: josifović, m. (ed.), flora sr srbije, 8: 153-158, srpska akademija nauka i umetnosti, beograd. diklić, n. 1975: rod ornithogalum l. in: josifović, m. (ed.), flora sr srbije, 7: 544-559, srpska akademija nauka i umetnosti, beograd. diklić, n. 1992: rod pulsatilla adans. in: sarić, m. (ed.), flora sr srbije, 1: 352-333, srpska akademija nauka i umetnosti, beograd. đorđević, v, tomović, g, lakušić, d. 2010: epipactis purpurata sm. (orchidaceae) – a new species in the flora of serbia. archives of biological sciences, 62: 1175–1180. đorđević, v., jovanović, s., stevanović, v. 2014. dactylorhiza fuchsii (orchidaceae), a new species in the flora of serbia. archives of biological sciences, 66 (3): 1227-1232. biologica nyssana ● 11 (2) december 2020: 71-84 jenačković gocić et al. ● insight into the chorology of some endangered, rare and potentially invasive plant species in serbia 82 đorđević, v., lakušić, d., jovanović, s., stevanović, v. 2017. distribution and conservation status of some rare and threatened orchid taxa in the central balkans and the southern part of the pannonian plain. wulfenia, 24: 143-162. eleftheriadou, e., raus, th., theodoropoulos k., tsiripidis i. 2009: sedum stefco stef. in: phitos, d., constantinidis, th., kamari, g. (eds.), vivlío erythrón dhedhoménon ton spánion & apeiloúmenon fitón tis elládhas, 2 [the red data book of rare and threatened plants of greece, 2], hellenic botanical society. gajić, m. 1973: rod galium l. in: josifović, m. (ed.), flora sr srbije, 5: 476-498, srpska akademija nauka i umetnosti, beograd. gajić, m. 1988: flora nacionalnog parka tara. ln: gajić, m. (ed.), flora nacionalnog parka tara, šumarski fakultet, univerzitet u beogradu, beograd. iucn world commission on protected areas and europark federation 2000. guidelines for protected area management categories. grafenau, brussels. http://www.iucnredlist.org jenačković, d., dimitrijević, d., ranđelović, v. 2010: macrophytic flora and vegetation of the rivers svrljiški and beli timok (eastern serbia). biologica nyssana, 1 (1-2): 23-26. jenačković, d., miljković, m., mitrović, d., ranđelović, v. 2015: contribution to the knowledge of distribution of certain macrophytes, invasive and threatened species in serbia. biologica nyssana, 6 (2): 59-65. jenačković, d. 2017: fitocenološko-ekološka studija močvarne vegetacije (phragmitetea communis r. tx. et preising 1942) centralnog balkana. phd thesis. univerzitet u beogradu, biološki fakultet. beograd. josifović, m. (ed.) 1970–1980: flora sr srbije, 1-10. srpska akademija nauka i umetnosti. beograd. jovanović, b. 1986: typha shuttleworthii koch & sonder. in: sarić, m., diklić, n. (eds.), flora sr srbije, 10: 256, srpska akademija nauka i umetnosti, beograd. király, g. (ed.) 2007: vörös lista: a magyarországi edényes flóra veszélyeztetett fajai [red list of the vascular flora of hungary]. saját kiadás, sopron. kozłowska, k., nobis, a., nobis, m. 2011: typha shuttleworthii (typhaceae), new for poland. polish botanical journal, 56 (2): 299-305. niketić, m., tomović, g. 2008: survey of some rare and endangered plants in serbia with new chorological data. bulletin of the natural history museum, 1: 113-148. niketić, m., perić, r., škondrić, s. 2009: cerastium subtetrandrum (caryophyllaceae), a new species to the flora of serbia. bulletin of the natural history museum, 2: 83-94. nikolić, v., sigunov, a., diklić, n. 1986: dopuna flori sr srbije novim podacima o rasprostranjenju biljnih vrsta. in: sarić, m., diklić, n. (eds.), flora sr srbije, 10: 259-336, srpska akademija nauka i umetnosti, beograd. nikolić, t., topić, j. 2005: crvena knjiga vaskularne flore hrvatske. ministarstvo kulture, državni zavod za zaštitu prirode. novaković-vuković, m. r. 2015: florističke karakteristike šuma crnog i belog bora na serpentinitu i peridotitima u zapadnoj i centralnoj srbiji. phd thesis. univerzitet u beogradu, šumarski fakultet. beograd. panjković, b. 2005: akvatična i semiakvatična vegetacija apatinskog i monoštorskog rita. phd thesis. univerzitet u novom sadu, prirodnomatematički fakultet. novi sad. panjković, b., perić, r., stojšić, v., batanjski, v. 2012: new data on the distribution of ranunculus polyphyllus waldst. & kit. ex willd. in serbia. archieves of biological sciences, 64 (2): 715 720. pavlović, z. 1951: vegetacija planine zlatibora. zbornik radova instituta za ekologiju i biogeografiju srpske akademije aauka, 2: 115-182. pavlović, z. 1955: o pašnjačkoj i livadskoj vegetaciji centralnog dela kopaonika. glasnik prirodnjačkog muzeja srpske zemlje, b 7 (1): 47-76. pavlović, z. 1974: livadska vegetacija na serpentinskoj podlozi brdsko-planinskog područja srbije. glasnik prirodnjačkog muzeja u beogradu, b29: 29-40. perić, r., stojšić, v., rilak, s., škondrić, s. 2016: the account of elatine ambigua wight, e. triandra schkuhr and e. hungarica moesz collected in vojvodina (serbia). bulletin of the natural history museum, 9: 81-93. petrova, a., vladimirov, v. (eds.) 2009: red list of bulgarian vascular plants. phytologia balcanica, 15 (1): 63-94. petrova, a., vladimirov, v. 2010: balkan endemics in the bulgarian flora. phytologia balcanica, 16 (2): 293-311. biologica nyssana ● 11 (2) december 2020: 71-84 jenačković gocić et al. ● insight into the chorology of some endangered, rare and potentially invasive plant species in serbia 83 petrović, b., ranđelović, v., zlatković, b. 2007: flora and vegetation of krupačko blato swamp in eastern serbia. proceeding of the 9th symposium on flora of southeastern serbia and neighbouring regions, niš, 63-72. polić, d. 2006: florističko-fitocenološko proučavanje labudovog okna. zadužbina andrejević, beograd. popiela, a., łysko, a., molnár, a. 2013: recent distribution of the euro-siberian-sub-mediterranean species elatine alsinastrum l. (elatinaceae). acta botanica croatica, 72 (2): 375-386. popović, i. 2005: vaskularna flora divčibara. magistarska teza. univerzitet u beogradu, biološki fakultet. beograd. prlić, d. 2015: novo nalazište nedovoljno poznate vrste elatine alsinastrum l. (elatinaceae) u hrvatskoj. glasnik hrvatskog botaničkog društva, 3 (3): 41-46. purger, d. 1993: vegetacija u okolini doroslova (zapadna bačka). magistarski rad. univerzitet u novom sadu, prirodno-matematički fakultet. novi sad. radulović, s. 2005: ekologija i disribucija akvatičnih fitocenoza carske bare u gis tematskom modelu. phd thesis. univerzitet u novom sadu, prirodno-matematički fakultet. novi sad. rakonjac, lj. 2002: šumska vegetacija i njena staništa na pešterskoj visoravni kao osnova za uspešno pošumljavanje. phd thesis. univerzitet u beogradu, šumarski fakultet. beograd. ranđelović, v. 1999: typha shuttleworthii koch & sonder. in: stevanović, v. (ed.), crvena knjiga flore srbije, 1: 384–386, ministarstvo za životnu sredinu republike srbije, biološki fakultet univerziteta u beogradu, zavod za zaštitu prirode republike srbije, beograd. ranđelović, v., zlatković, b., jušković, m. 2002: astragalus wilmottianus stoj. a new species of the serbian flora. proceeding of the 7th symposium on flora of southeastern serbia and neighboring regions, dimitrovgrad, 1-4. ranđelović, v., zlatković, b., jušković, m. 2005: analiza korovske flore jugoistočne srbije. proceeding of 8th symposium on flora of southeastern serbia and neighbouring regions, niš, 47-60. ranđelovć, v., matejić, j., zlatković, b. 2007: flora i vegetacija batušinačkih bara kod niša. 9th symposium on flora of southeastern serbia and neighbouring regions, niš, 19-40. ranđelović, v., zlatković, b. 2010: flora i vegetacija vlasinske visoravni. univerzitet u nišu, prirodno-matematički fakultet. niš. schnittler, m., günther, k. f. 1999: central european vascular plants requiring priority conservation measures – an analysis from national red lists and distribution maps. biodiversity and conservation, 8: 891-925. stanković, ž., borišev, m., simić, s., vučković, m., igić, r., vidović, m., miljanović, b. 2009: macrophytes of the grlište reservoir (serbia): fifteen years after its establishment. archives of biological sciences, 61 (2): 267-278. stevanović, v., sinžar-sekulić, j., stevanović, b. 2004: expansion of the adventive species paspalum paspaloides (michx) schribner, echinochloa oryzoides (ard.) fritsch and cyperus strigosus l. in the yugoslav part of the danube reservoir (km 1090-1075). in proceedings 35th iad conference, limnological reports, novi sad, serbia and montenegro, 399-405. stevanović, v., tan, k., tomašević, m., uotila, p. 2005: the occurrence of cyperus strigosus (cyperaceae) in serbia and montenegro. phytologia balcanica, 11 (2): 137-138. stojanović, s., butorac, b., vučković, m., stanković, ž., žderić, m., kilibarda, p., radak, lj. 1994: biljni svet kanala vrbas bezdan. univerzitet u novom sadu, prirodno-matematički fakultet. novi sad. stojanović, v., petrović, s., kovačević, j., stojanović, d., bjedov, i. 2017: heracleum sosnowskyi manden. (apiaceae): a new invasive species in the flora of serbia. glasnik šumarskog fakulteta, 116: 215-220. šturc, b. 2014: prirodna flora subotičko-horgoške peščare i pitanja njene zaštite. gradski muzej subotice. subotica. tatić, b., veljović, v., petković, b., stefanović, m., radotić, s. 1988: ass. lathyreto-molinietum coeruleae nova zajednica livadske vegetacije sa pešterske visoravni jugozapadna srbija. glasnik instituta za botaniku i botaničke bašte univerziteta u beogradu, 12: 31-38. tomović, g., ranđelović, v., niketić, m., vukojičić, s., zlatković, b. 2003: new distribution data of some pontic and submediterranean plant species in serbia. archives of biological sciences, 55 (1-2): 45-54. tomović, g., vukojičić, s., niketić, m., lakušić, d. 2007: new chorological data on some threatened biologica nyssana ● 11 (2) december 2020: 71-84 jenačković gocić et al. ● insight into the chorology of some endangered, rare and potentially invasive plant species in serbia and rare plants in serbia. archives of biological sciences, 59 (1): 63-73. tomović, g., zlatković, b., niketić, m., perić, r., lazarević, p., duraki, š., stanković, m., lakušić, d., anačkov, g., knežević, j., szabados, k., krivošej, z., prodanović, d., vukojičić, s., stojanović, v., lazarević, m., stevanović, v. 2009: threat status revision of some taxa from “the red data book of flora of serbia 1”. botanica serbica, 33 (1): 33–43. tomović, g., zlatković, b., lazarević, m., niketić, m. 2016. viola orbelica (violaceae), new species for the flora of serbia. bulletin of the natural history museum, 9: 67-79. tomović, g., sabovljević, m., đokić, i., petrović, p., đorđević, v., lazarević, p., mašić, e., barudanović, s., ştefănuţ, s., niketić, m., butorac, b., pantović, j., hajrudinovićbogunić, a., bogunić, f., kabaš, e., vukojičić, s., kuzmanović, n., đurović, s., buzurović, u. 2020: new records and noteworthy data of plants, algae and fungi in se europe and adjacent regions, 2. botanica serbica, 44 (2): 251-259. topić, j., ozimec, s. 2001: typha laxmannii lepechin (typhaceae), a new species in croatian flora. natura croatica: periodicum musei historiae naturalis croatici, 10 (1): 61-65. tutin, t. g., heywood, v. h., burges, n. a., moore, d. m., valentine, d. h., walters, s. m., webb, d. a. (eds.) 1964–1980: flora europaea, 1–4. cambridge university press. london. veljić, m., marin, p. d., krivošej, z., ljubić, b. 2006: vascular flora of the uvac river gorge in serbia. archives of biological sciences, 58 (2): 125133. verloove, f. 2014: a conspectus of cyperus s.l. (cyperaceae) in europe (incl. azores, madeira and canary islands), with emphasis on non-native naturalized species. webbia, 69 (2): 179-223. zlatković, b., ranđelović, v., ranđelović, n. 1993: građa za floru jugoistočne srbije. 3. simpozijum o flori jugoistočne srbije, zbornik radova, 1. flora i vegetacija, leskovac-pirot, 95-109. zlatković, b., ranđelović, v. 1995: distribution and ecology of sedum species (crassulaceae) in serbia: i. sedum tuberiferum stoj. et stef., s. stefco stef. and s. aetnense tineo. ekologija, 31 (1): 65-71. zlatković, b., ranđelović, v., jušković, m. 2007: reports 136-138. in: vladimirov, v., dane, f., stevanović, v., tan, k. (eds.): new floristic records in the balkans: 6. phytologia balcanica, 13 (3): 433455. zlatković, b., bogosavljević, s. 2014: report on the new floristic data from serbia. biologica nyssana, 5 (2): 123-129. 84 biologica nyssana ● 11 (2) december 2020: 71-84 jenačković gocić et al. ● insight into the chorology of some endangered, rare and potentially invasive plant species in serbia hasanović et al. 2020, biologica nyssana 11(1) 11 (1) september 2020: 65-69 doi: 10.5281/zenodo.4060309 contribution to the knowledge of invasive flora in kozara national park short communication mujo hasanović institute for genetic engineering and biotechnology, university of sarajevo, bosnia and herzegovina mujohh@gmail.com (corresponding author) emir delić association of biology students in bosnia and herzegovina, sarajevo, bosnia and herzegovina emir.delicc@otulook.com mensud šarić association of biology students in bosnia and herzegovina, sarajevo, bosnia and herzegovina saric994@hotmail.com received: december 26, 2019 revised: april 30, 2020 accepted: may 27, 2020 abstract: kozara national park is exposed to different anthropogenic influences even though it is categorized as iucn category ii. in this study, we investigated, identified and mapped invasive flora and their impact on the native plant communities. during summer 2018, the fieldwork was conducted at 6 locations using the braun-blanquet method for the investigation of plant communities. the total of 13 invasive alien species (ias) have been detected. most of the ias belong to the asteraceae family, originating from north america (76.92%). the most abundant life forms were therophytes and geophytes found in artemisietalia communities. conclusively, the ias are widespread in the examined area, particularly in the parts of the park designated for tourist recreational activities. key words: alien flora, bosnia and herzegovina, vegetation apstract: prilog poznavanju invazivne flore nacionalnog parka kozara nacionalni park kozara izložen je različitim antropogenim uticajima, iako prema iucn spada u kategoriju ii. u istraživanju smo identifikovali i mapirali invazivnu floru i njihov uticaj na autohtone biljne zajednice. tokom leta 2018. terenski rad je sproveden na 6 lokacija primenom braunblankuet-ove metode za ispitivanje biljnih zajednica. otkriveno je ukupno 13 invazivnih stranih vrsta (ias). većina ias pripada porodici asteraceae, poreklom iz severne amerike (76,92%). najzastupljenije životne forme su terofite i geofite, pronađene u zajednicama reda artemisietalia. zaključno, ias su široko rasprostranjeni u ispitivanom području, posebno u delovima parka predviđenim za turističke rekreativne aktivnosti. ključne reči: strana flora, bosna i hercegovina, vegetacija the invasive alien species (ias) in bosnia and herzegovina were observed in research in the previous century (slavnić, 1960; slavnić 1964; bjelčić & stefanović, 1986; abadžić, 1986/87). however, only since the verification of cbd (convention on biological diversity) in 2002, monitoring ias has become mandatory. according to the national cbd report (strategija i akcioni plan za zaštitu biološke raznolikosti bosne i hercegovine 2015-2020. podrška bosni i hercegovini za revidiranje strategije i akcionog plana za zaštitu biološke raznolikosti i izradu petog nacionalnog izvještaja prema konvenciji o biološkoj raznolikosti, 2016), the plan was to observe all ias and identify pathways of spreading by 2020. so far, no official list of invasive flora and fauna exists. in that light, every investigation which goes into the direction of study the ias is of great importance. every year, high biodiversity and ecological value of kozara national park attract many visitors, who can potentially endanger certain eco© 2020 hasanović et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 65 systems. it is worth noting that bucalo et al. (2007) detected 657 vascular plants in a relatively small area of the park (3910 ha). with this in mind, the main purpose of the study was to contribute to better understanding of distribution of invasive flora in the kozara national park. the investigated area included the central and south part of kozara massif. the fieldwork was performed on 6 locations through original phytocoenological research during the summer 2018. the analysis of the floristic composition was executed with the standard method zurich-montpellier school braun-blanquet (1964). description of each site was made using garmin gps and topographic, geological and pedological maps (tab. 1.) preliminary list of invasive plant flora for b&h (maslo, 2016) was used as a primary database. for the plant identification, we used following identification keys: domac (1979), javorka and csapody (1991), šarić (1991), šilić (2005), nikolić et al. (2014). the nomenclature 66 table 1. general locality information no. locality coordinates altitude (m) slope (º) exposition bedrock soil type 1 zečiji kamen viewpoint n 45° 1'3.13" e 16°52'16.15" 655 45 sw limestone calcomelanosol 2 ski plateau mrakovica n 45° 0'50.75" e 16°54'13.38" 785 5 w eocene flysch deposol 3 gumline viewpoint n 44°59'43.29" e 16°54'43.37" 520 10 sw limestone calcomelanosol 4 road for kotlovača n 45° 00'23.9" e 16°52'42.5" 655 0 w gabbro deposol 5 sledding site mrakovica n 45° 0'56.59" e 16°54'18.73" 790 5 se eocene flysch deposol 6 mountain hut mrakovica n 45° 00'50.8" e 16°54'20.6" 650 50 se eocene flysch deposol table 2. recorded invasive flora in kozara national park taxon family detected in community origin life ambrosia artemisiifolia l. asteraceae artemisietalia lohm. prsg. et tx. in tx. 1950; querco-ostryetum carpinifolie ht. 1938 north america t erigeron canadensis (l.) cronquist asteraceae artemisietalia lohm. prsg. et tx. in tx. 1950 north america h erigeron annus (l.) pers. asteraceae artemisietalia lohm. prsg. et tx. in tx. 1950; querco-ostryetum carpinifolie ht. 1938 north america h galinsoga parviflora cav. asteraceae artemisietalia lohm. prsg. et tx. in tx. 1950 north and south america t juncus tenuis willd. juncaceae artemisietalia lohm. prsg. et tx. in tx. 1950 north america h parthenocissus quinquefolia (l.) planch. vitaceae fagetalia sylvaticae pawlowski in pawlowski et al. 1928 north america ph phytolacca americana l. phytolaccaceae artemisietalia lohm. prsg. et tx. in tx. 1950 north america g reynoutria japonica houtt. polygonaceae adenostyletalia br.-bl. 1931 east asia g robinia pseudoacacia l. fabaceae fagetalia sylvaticae pawlowski in pawlowski et al. 1928 north america ph solidago gigantea aiton asteraceae artemisietalia lohm. prsg. et tx. in tx. 1950 northen america g sorghum halepense (l.) pers. poaceae artemisietalia lohm. prsg. et tx. in tx. 1950 middle east g veronica persica poir. artemisietalia lohm. prsg. et tx. in tx. 1950 south-western asia t xanthium strumarium l. subsp. italicum (moretti) d. love asteraceae artemisietalia lohm. prsg. et tx. in tx. 1950 north america t biologica nyssana ● 11 (1) september 2020: 65-69 hasanović et al. ● contribution to the knowledge of invasive flora in kozara national park 67 follows the euro+med plantbase (2006) and the international organization for plant information (iopi) database (2017). syntaxonomic position of the analyzed communities was determined based on the list of plant communities in bosnia and herzegovina (prodromus) (lakušić 1978, barudanović et al., 2015) and international code of phytosociological nomenclature (weber et al., 2000). the taxa are listed alphabetically and accompanied by life form data (ph-phanerophyte, ch-chamaephyte, h-hemicryptophyte, g-geophyte, hy-hydrophyte, t-therophyte) (raunkiaer, 1934). out of the 119 identified plant species in the kozara national park, 13 (10.9%) were ias (tab. 2). maslo (2016) listed 50 ias in the preliminary list of ias in bosnia and herzegovina. it is important to state that the overall number of ias in the park may not be definitive due to the scope of research. all identified plants are neophytes, originated from north america (76.92%) and asia (23.08%). the majority of ias belong to asteraceae (46%) which is, regarding invasiveness, one of the most abounding in the world (pyšek, 1998). the most abundant life forms in the examined flora of the investigated area are therophytes (30.76%) and geophytes (30.76%) followed by hemicryptophytes (23.1%) and phanerophytes (15.38%). even though the studied area is characterized by mesophilic and hydrophilic habitats with the larger portion of phanerophytes and hemicryptophytes (lakušić et al., 1991; bucalo et al., 2007), continuous anthropogenic impacts in the form of degradation resulted in expansion of nitrified, xerophytic and stomped habitats. hard and compact soil, high insolation and temperature, along with weak soil transparency enabled the development and domination of therophytes and geophytes (jovanović, 1993). the relatively high percentage of hemicryptophytes (23.1%) is not surprising because these plant species have extraordinary regenerative potential and can endure stomping and breaking (topalić-trivunović, 2006). continuous construction projects and expansion of tourist offer allow undisturbed spreading of invasive flora, both alien and native. with five marked recreational trails and the accompanying infrastructure, invasive flora is spreading on the ski and trim tracks near mrakovica where we detected nine ias: ambrosia artemisiifolia, erigeron canadensis, erigeron annuus, galinsoga parviflora, juncus tenuis, reynoutria japonica, robinia pseudoacacia, veronica persica and xanthium strumarium subsp. italicum. along the roads from mrakovica to gola planina, three ias were found, ambrosia artemisiifolia, reynoutria japonica and erigeron annuus. by the trails from mrakovica to kozarački kamen, benkovac and zečiji kamen we identified ambrosia artemisiifolia, erigeron canadensis, erigeron annuus, robinia pseudoacacia, solidago gigantea, galinsoga parviflora and veronica persica. phytolacca americana was detected in the northwestern and sorghum halepense in the southern part of the park. the gathered data suggest that all ias continuously spread alongside the roads and trails. many studies confirm that ias spread near roads (hansen & clevenger, 2005; meunier & lavoie, 2012; brisson et al., 2017). of the detected invasive flora, the most important is the spreading of ambrosia artemisiifolia and reynoutria japonica given their negative impact on human health (leru et al., 2015) and biodiversity (aguilera et al., 2010). the high abundance of a. artemisiifolia on the mrakovica plateau is of special concern. even though it was detected in only two localities near mrakovica, reynoutria japonica populations could possibly continue the spreading around the nearest pathways. growing in high-density populations (naiman et al., 2005), japanese knotweed disables the development of any other plant species through strong allelopathic effect (murrell et al., fig. 1. map of detected invasive flora in the kozara national park biologica nyssana ● 11 (1) september 2020: 65-69 hasanović et al. ● contribution to the knowledge of invasive flora in kozara national park 68 2011). the spreading of this species would be harmful particularly in the zone of pašini konaci due to unique swamp habitats with communities glycerietum plicatae kulczynski 1928 and lemnetum minoris (oberd.1957) th. mull. gors.1960. acknowledgements. this research was supported by bosnia and herzegovina federal ministry of education and science and embassy of the united states in bosnia and herzegovina. references abadžić, s. 1986-1987: prilog poznavanju horologije i ekologije dviju adventivnih vrsta – echinocystis lobata (michx) torrey et gray i bidens bipinnata l. u flori bosne i hercegovine. glasnik zemaljskog muzeja bosne i hercegovine (prirodne nauke, nova serija), 25-26: 71-77. aguilera, a. g., alpert, p., dukes, j. s., harrington, r. 2010: impacts of the invasive plant fallopia japonica (houtt.) on plant communities and ecosystem processes. biological invasions. 12(5): 1243-1252. barudanović, s., macanović, a., topalićtrivunović, lj., cero, m. 2015: ekosistemi bosne i hercegovine u funkciji održivog razvoja. prirodnomatematički fakultet, univerzitet u sarajevu, sarajevo. 249 p. bjelčić, ž., stefanović, v. 1986: phytolacca americana l. u flori i vegetaciji bosne i hercegovine. godišnjak biološkog instituta univerziteta u sarajevu, 5: 5-11. braun-blanquet, j. 1964: pflansensoziologie. third edition. springer, vienna, austria. brisson, j., blois, s., lavoie, c. 2017: roadside as invasion pathway for common reed (phragmites australis). invasive plant sceince and management. 3(4): 506-514. bucalo, v., brujić, j., travar j., milanović, đ. 2007: flora nacionalnog parka kozara. šumarski fakultet univerziteta u banjoj luci – regionalni zavod za upravljanje šumama i agrikulturom pokrajine lombardija milano, banja luka: 398 p. domac, r. 1979: mala flora hrvatske i susjednih područja. školska knjiga, zagreb: 543 p. euro+med 2006: euro+med plantbase the information resource for euro-mediterranean plant diversity. http://ww2.bgbm.org/europlusmed/ federalno ministarstvo okoliša i turizma 2016: strategija i akcioni plan za zaštitu biološke raznolikosti bosne i hercegovine 2015-2020. podrška bosni i hercegovini za revidiranje strategije i akcionog plana za zaštitu biološke raznolikosti i izradu petog nacionalnog izvještaja prema konvenciji o biološkoj raznolikosti, sarajevo: 176 p. http://vijeceministara.gov.ba/akti/prijedlozi_ zakona/default.aspx?id=25304&langtag=hr-hr hansen, m. j., clevenger, a. p. 2005: the influence of disturbance and habitat on the presence of non-native plant species along transport corridors. biological conservation, 125(2): 249-259. javorka, s., csapody, v. 1979: iconographia florae partis austro-orientalis europae centralis. g. fisher, third edition, stuttgart: 576 p. jovanović, s. 1993: pregled istraživanja ruderalne flore i vegetacije u svetu i na prostoru bivših jugoslovenskih republika. acta herbologica, 2: 3-23. lakušić, r. 1978: prodromus biljnih zajednica bosne i hercegovine, godišnjak biološkog instituta univerziteta u sarajevu, sarajevo. 87 p. lakušić, r., dizdarević, m., grgić, p., pavlović, b., redžić, s. 1991: ecological differentiation of the area of the una river system and its value. bilten društva ekologa bosne i hercegovine, 6: 15-23. leru, p. m., matei, d., ianovici, n. 2015: health impact of ambrosia artemisiifolia reflected by allergists practice in romania. a questionnaire-based survey. annals of west university of timisoara, ser. biology, 18 (1): 43-54. maslo, s. 2016: preliminary list of invasive alien plat species (ias) in bosnia and herzegovina. herbologia, 16 (1): 1-14. meunier, g., lavoie, c. 2012: roads as corridors for invasive plant species: new evidence from smooth bedstraw (galium molugo). invasive plant science and management, 5(1): 92-100. murrell, c., gerber, e., krebs, c., parepa, m., schaffner, u., bossdorf, o. 2011: invasive knotweed affects native plants through allelopathy. american journal of botany, 98 (1): 38-43. naiman, r.j., décamps, h., mclain, n. 2005: riparia: ecology, conservation, and management of streamside communities. elsevier academic press, burlington, mass: 448 p. nikolić, t., mitić, b., boršić, i. 2014: flora hrvatske: invazivne biljke. udžbenici sveučilišta u zagrebu, zagreb: 295 p. pyšek, p. 1998: is there a taxonomic pattern to plant invasions? oikos 82: 282-294. raunkiaer, c. 1934: the life form of plants and statisti-cal plant geography. clarendon press, oxford: 729 p. biologica nyssana ● 11 (1) september 2020: 65-69 hasanović et al. ● contribution to the knowledge of invasive flora in kozara national park biologica nyssana ● 11 (1) september 2020: 65-69 hasanović et al. ● contribution to the knowledge of invasive flora in kozara national park slavnić, ž. 1960: o useljavanju, širenju i odomaćivanju nekih adventivnih biljaka u bosni i hercegovini. godišnjak biološkog instituta univerziteta u sarajevu, 8 (1-2): 117-146. sarajevo. slavnić, ž. 1964: rod bidens l. u flori bosne i hercegovine. radovi naučnog društva sr bih, 25 (7): 155-162. šarić, t. 1991: atlas korova: 100 najvažnijih vrsta korovskih biljaka u jugoslaviji. svjetlost, sarajevo: 221 p. šilić, č. 2005: atlas dendroflore (drveće i grmlje). matica hrvatska, zagreb: 575 p. the catalogue of life partnership 2017: iopi global plant checklist in the catalogue of life. checklist dataset. https://doi.org/10.15468/ topalić-trivunović, lj. 2006: ruderalna flora i vegetacija područja banje luke, phd thesis, prirodno-matematički fakultet univerziteta u banjoj luci weber, h.e., moravec, j., theurillat, j.-p. 2000: international code of phytosociological nomenclature. 3rd edition. journal of vegetation science, 11: 739-768. 69 stojanović-radić et al. 2021, biologica nyssana 12(1) 12 (1) september 2021: 33-45 doi: 10.5281/zenodo.5522981 antistaphylococcal activity of thymus vulgaris and origanum vulgare essential oils: timelapse kinetics, antibiofilm activity and synergistic potential original article zorica stojanović-radić department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, niš, serbia zorica.stojanovic-radic@pmf.edu.rs (corresponding author) marina dimitrijević department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, niš, serbia dimitrijevicmarina92@yahoo.com ana aleksić department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, niš, serbia 90anna.aleksic@gmail.com nikola stanković department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, niš, serbia nikola.stankovic@pmf.edu.rs received: may 21, 2021 revised: june 23, 2021 accepted: september 01, 2021 abstract: staphylococcus aureus is one of the most frequent human pathogens, whose high virulence and severity of chronic infections are related to their ability to produce biofilms. in the present study, antimicrobial potential of oregano and thyme essential oils on the planktonic growth of s. aureus clinical strains (isolated from nose, throat and wound), as well as the effect of the oils on the biofilm production, compared with conventional antibiotic streptomycin were investigated. in addition, time-lapse kinetics and combinations of individual oils with streptomycin were investigated for synergism against the selected staphylococcal strains. the results showed high potential of both oils where minimal inhibitory values ranged between 0.078 and 2.50 mg/ml, while biofilms were reduced up to 96%. in the case of biofilms, the reverse concentration dependency has been observed. time-lapse kinetics showed recovery of some strains after 4 h of contact with the oils, but these strains demonstrated significantly reduced growth in comparison to the control. all mentioned assays showed slightly higher efficacy of oregano essential oil. calculated fics showed either synergistic or additive effect of all tested combinations (thyme-oregano, thyme-streptomycin, oregano-streptomycin), with the thyme-streptomycin as the most efficient one. all mentioned results point to a very high potential of both oils to be used as an adjuvant agent for control of human staphylococcal infections. key words: thyme, oregano, staphylococcus aureus, antibiofilm activity, synergism, clinical isolates apstract: antistafilokokna aktivnost etarskih ulja thymus vulgaris i origanum vulgare: vremenska kinetika, antibiofilm aktivnost i sinergistički potencijal staphylococcus aureus is one of the most frequent human pathogens, whose hstaphylococcus aureus je jedan od najčešćih humanih patogena, čija se visoka virulencija i ozbiljnost hroničnih infekcija povezuju sa sposobnošću produkcije biofilmova. u ovom istraživanju, ispitivani su antimikrobni potencijal etarskih ulja origana i timijana na planktonski rast kliničkih sojeva s. aureus (izolovanih iz nosa, grla i rane), kao i efekat ulja na produkciju biofilma u poređenju sa konvencionalnim antibiotikom streptomicinom. u dodatku, u odnosu na odabrane sojeve stafilokoka istraživani su vremenska kinetika i kombinovan efekat etarskih ulja sa streptomicinom u smislu sinergizma. rezultati su pokazali visok potencijal oba ulja gde je minimalna inhibitorna aktivnost bila u opsegu između 0,078 i 2,50 mg/ml, dok su biofilmovi bili smanjeni za do 96%. u slučaju biofilmova, uočena je obrnuta koncentraciona zavisnost. vremenska kinetika je ukazala na oporavak nekih sojeva nakon 4 h kontakta sa uljem, ali su ovi sojevi imali značajno umanjen rast u odnosu na kontrolu. svi pomenuti testovi pokazali su nešto značajniji efekat ulja origana. preračunati fik-ovi pokazali su ili sinergistički ili aditivni efekat svih testiranih kombinacija (timijan-origano, timijan-streptomicin, origanostreptomicin), dok je najefikasnija bila timijan-streptomicin. svi pomenuti rezultati ukazuju na veoma visok potencijal oba ulja za primenu u vidu dopunskih agenasa za kontrolu humanih infekcija izazvanih stafilokokama. ključne reči: timijan, origano, staphylococcus aureus, antibiofilm aktivnost, sinergizam, klinički izolati © 2021 stojanović-radić et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work noncommercially under the same license as the original. 33 introduction in the past few decades, increased number of bacterial infections, reduced antimicrobial efficacy and newly emerged microbial resistance have become global public concern, leading to improving interest in the examination of the antimicrobial activity of plantbased therapeutics (xiao et al., 2019; zhang et al., 2020). among all alternative natural antimicrobial agents, essential oils display the most significant antimicrobial potential. it is important to note that no tolerance or resistance to essential oils has been discovered yet, which can be explained by the great complexity of their structure and action on several target places at the same time, rather than conventional antibiotics (man et al., 2019). in the contemporary experimental research, an increasing interest is directed to the modulation of virulence factors of microorganisms, in terms of avoiding resistance development (zhang et al., 2020). biofilm is considered to be a major virulence factor that contributed to >80% of all human infections. biofilms are highly organized multicellular bacterial communities consisting of a complex matrix composed of polysaccharide, proteins, lipids and extracellular dna. the best way of controlling biofilms is to prevent their development (abdallah et al., 2020). staphylococcus aureus (s. aureus) is one of the most dominant human pathogen, responsible for a variety of chronic and severe infections including simple soft skin infections, endocarditis, bacteremia, and severe pneumonia (xiao et al., 2019). this bacteria is the member of the upper respiratory microbiome in more than 30% of healthy individuals (prince, 2013). once hosts defense mechanisms are disrupted, s. aureus can proliferate and the host begin to be a nasal carrier (najem, 2020). there are substantial epidemiological data indicating an association between nasal (or other mucosal site) colonization and subsequent s. aureus infection. surgical wounds contamination with skin staphylococci is not a rare case, and the results of the one study confirmed the molecular similarity of strains isolated from wounds and those found in the nose of the same patients (prince, 2013; najem, 2020). since 1960, with the emergence of mrsa-strains (methicillin-resistant s. aureus), s. aureus has become a key pathogen associated with many health-care infections, that are usually difficult to treat (uzair et al., 2017). staphylococcus aureus possesses a variety of virulence factors and their ability to form biofilm plays a critical role in chronic infections. a negative modulation or complete inhibition of biofilm formation may represent an important strategy for infection control and is considered as a major target for the development of novel therapeutic agents (papa et al., 2020). aromatic plants from the lamiaceae family present great sources of biologically active substances with wide use and proven antimicrobial and antioxidant properties (pecarski et al., 2016; uzair et al., 2017; man et al., 2019; zhang et al., 2020). anti-virulence activity of these essential oils have been well investigated and some of them such as cinnamon, oregano, clove, and ginger oil (zhang et al., 2020) are classified as gras (generally recognized as safe) by the u.s. food and drug administration (fda). anti-biofilm activity of these oils and its main compounds (thymol and carvacrol) against food-born (bilge et al., 2010; vázquez-sánchez et al., 2014) and ocular infections isolates (nostro et al., 2007) or mrsa strains (abdallah et al., 2020) has been investigated in several research papers. in the recently published study, the effectiveness of the essential oils (eucalyptus, cinnamon, clove, and tea tree) on the biofilm-producing ability of s. aureus nasal isolates was demonstrated (najem, 2020). in the present study we investigated antimicrobial potential of oregano and thyme essential oils on the planktonic growth of s. aureus clinical strains (isolated from nose, throat and wound), as well as the effect of the oils on the biofilm production, compared with conventional antibiotic streptomycin. in addition, time-lapse kinetics analysis was performed to determine the time required for exhibiting antimicrobial activity and the time necessary for recovery of the cells at sub/inhibitory concentrations. finally, to explore the potential of these two oils in enhancing the effect of other antimicrobials, the combined oils and also combinations of individual oils with streptomycin were tested for synergism against the selected staphylococcal strains. materials and methods isolation of essential oils plant material of origanum vulgare l. and thymus vulgaris l. in pulverized form (aerial parts) was purchased at the local herb market and used for extraction of the essential oils. isolation of the essential oils was performed by using the original clevenger-type apparatus and the obtained oils were separated by extraction with diethyl ether, dried over anhydrous mgso4 and stored at -20 °c before use. test microorganisms the essential oils were tested against a total of 12 strains of staphylococcus aureus clinical isolates of different origin (nose, throat, wound and atcc – american type culture collection strain 6538). 34 biologica nyssana ● 12 (1) september 2021: 33-45 stojanović-radić et al. ● antistaphylococcal activity of thymus vulgaris and origanum vulgare essential oils: time-lapse kinetics... 35 after isolation on blood agar and identification (selective media-msa agar and coagulase test), the cultures were maintained on nutrient agar until used for testing. determination of minimal inhibitory concentration (mic) sensitivity of the staphylococcal strains to the action of oregano and thyme essential oils was determined by a broth microdilution method as previously described (stojanović-radić et al., 2020). overnight cultures (18 h, exponential phase) were used for making suspensions of bacteria in sterile physiological saline (0.85% nacl). the obtained suspensions were adjusted to 0.5 mcfarland turbidity (den-1, biosan densitometer) and further used to inoculate the wells of a 96-well microtiter plates. stock solutions of the essential oils made in 100% dmso were serially diluted in the microtiter plate in the concentration range from 0.01-5.00 mg/ ml and then inoculum was added to each well. the final density of bacterial inoculum was 1-5 x 106 cfu (colony forming unit)/ml in each well. the plates included growth control (mueller hinton broth + 10% dmso + inoculum), sterility control (mhb + 10% dmso + test oil) as well as positive control (mhb + inoculum + serial double dilutions of streptomycin). after incubation of the plates at 37 °c for 24 h, the bacterial growth was determined by adding 20 µl of 0.5% (w/w) 2,3,5-triphenyltetrazolium chloride aqueous solution. minimal inhibitory concentration of the oil was defined as the lowest concentration of the tested samples where visible growth was not detected after the addition of the growth indicator. anti-biofilm assay to investigate the potential of the isolated strains to form biofilm, the tested isolates were first subjected to biofilm forming ability crystal violet (cv) assay as previously described (pejčić et al., 2020). the wells of the microtiter plates containing 200 μl of tryptone soy broth containing 0.5% (w/v) glucose were inoculated with suspensions prepared as described for microdilution method, to achieve the final concentration of ~106 cfu/ml. the plates were incubated 24 h at 37 °c afterwards the well content was aspirated, and the wells were washed twice with phosphate saline buffer (ph 7.4). then the plates were dried, stained with 0.5% (w/v) solution of cv for 20 min, washed and distained by addition of 250 μl of ethanol (96%, v/v). following 45 min of distaining procedure, the obtained solutions were transferred into a new microtiter plate and the absorbance of each well content was measured at 595 nm using an elisa reader (multiscan ascent, labsystems, finland). according to their biofilmproducing ability, the strains were classified into the following groups: none, weak, moderate, and strong biofilm producers (stepanović et al., 2007). the potential of oregano and thyme essential oils to inhibit biofilm production of the investigated s. aureus strains was done by using the same procedure. the only difference was that the wells contained serial doubling dilutions of the essential oils in the desired concentration range (0.01-5.00 mg/ml) together with inoculum and cultivation media. the percentage inhibition of biofilm formation caused by oregano and thyme essential oils was calculated according to the following equation: percentage inhibition= 100[(a595 of the well containing the essential oil / a595 of the well without essential oil) * 100]. the results are presented on the graph as absorbances of the control and experimental wells, where absorbance of the control represents 100%. time-lapse kinetics of inhibitory action to investigate whether the oils achieve long term inhibitory action and the amount of obtained inhibition against each strain, the modified timekill assay has been performed in microtiter plates. the assay was done by inoculation of the media (final cell density was ~106 cfu/ml) containing the essential oils in mic and mic/2 concentrations and subsequent incubation of the plates for total of 48 h at 37 °c. after inoculation, the absorbance of each well was measured at 600 nm using an elisa reader (multiscan ascent, labsystems, finland) at the following incubation periods: 0, 2, 3, 4, 22, 24, 46 and 48 h. the control wells contained only inoculated medium without essential oils. checkerboard assay the type of interaction between the two essential oils and also between the single oil and antibiotic streptomycin was tested using a microdilution checkerboard assay (stojanović-radić et al., 2020). the testing was performed against the three strains, two isolates and one atcc strain. briefly, the medium containing various combinations of antibacterial agent in question (essential oil or antibiotic), obtained by two-fold serial dilutions, were inoculated to achieve the final concentration of ~106 cfu/ml per well. the plates were incubated for 24 h at 37 °c and the combined effects of essential oils with antibiotic or with other essential oil were calculated. the obtained results are expressed as fractional inhibitory concentrations (fics) and the calculation was done using the following equations: ficeo= miceo-s/miceo fics=mics-eo/mics biologica nyssana ● 12 (1) september 2021: 33-45 stojanović-radić et al. ● antistaphylococcal activity of thymus vulgaris and origanum vulgare essential oils: time-lapse kinetics... 36 fici= ficeo + fics ficeo1=miceo1-eo2/miceo1 ficeo2=miceo2-eo1/miceo2 fici=ficeo1 + ficeo2 where ficeo is the fractional inhibitory concentration of essential oil (oregano or thyme), miceo-s is mic of the oil in combination with streptomycin, miceo is the mic of the oil alone; fics is the fractional inhibitory concentration of streptomycin, mics-eo is mic of the streptomycin in combination with the oil, mics is the mic of the streptomycin alone; ficeo 1 is the fractional inhibitory concentration of oregano, miceo1-eo2 is mic of the oregano oil in combination with thyme oil, miceo1 and miceo2 is the mic of the oregano and thyme oil alone, respectively; ficeo2 is the fractional inhibitory concentration of thyme, and miceo2-eo1 is mic of the thyme oil in combination with oregano oil. fici was interpreted as synergistic when the value was ≤0.5, as an additive or indifferent when fici was >0.5 and ≤2, and as antagonistic interaction when it was ≥2. the antimicrobial activity of o. vulgare and t. vulgaris essential oils was exhibited against all tested strains and minimal inhibitory values ranged between 0.078 and 2.50 mg/ml (tab. 1). the oregano essential oil showed higher antistaphylococcal potential by inhibiting the growth of the tested panel of staphylococci in the range 0.156-0.625, with the average mic of 0.45 mg/ml. on the other hand, thyme essential oil exhibited inhibition in the broader mic range (0.078 2.50 mg/ml, micavrg=1.1 mg/ ml) and mostly at much higher concentrations in comparison to oregano essential oil. among the strains of staphylococci, the strain no. 7 showed the highest resistance, while the most sensitive one has been the strain no. 4. these data are not corresponding to those for antibiotic, since the two strains showed the same sensitivity to streptomycin. this is one more proof that the oils possess different mode of action then the antibiotic, used as a control antibacterial agent. previous studies on the t. vulgaris essential oils antimicrobial efficacy reported variable active concentrations against s. aureus, which were highly dependent on the essential oil composition and ranged from ˂0.2 µl/ml to 4 mg/ml (rota et al., 2008; kazemi et al., 2012; kon & rai, 2012; de carvalho et al., 2015; pesavento et al., 2015; benabed et al., 2016; valizadeh et al., 2016; fani & kohanteb, 2017; gedikoğlu et al., 2019; pourazar et al., 2018). studies on the antimicrobial activity of oregano essential oil showed very high efficacy against many bacterial species due to high content of a well-known antibacterial compounds, carvacrol and thymol, also present in the thyme essential oil (sakkas & papadopoulou, 2017). it has been reported that oregano essential oil inhibited the growth of mrsa (methicillin resistant s. aureus strains) at concentrations of 0.16 and 0.32 mg/ml (boskovic et al., 2015; lu et al., 2018). in other studies where efficacy of oregano has been investigated against s. aureus strains by microdilution method, inhibitory concentrations ranged from 0.15 µg/ml to 0.8 mg/ml (nostro et al., 2007; özkalp et al., 2010; boskovic et al., 2015; marques et al., 2015; pesavento et al., biologica nyssana ● 12 (1) september 2021: 33-45 stojanović-radić et al. ● antistaphylococcal activity of thymus vulgaris and origanum vulgare essential oils: time-lapse kinetics... no. strains thymus vulgarismic *mg/ml origanum vulgare mic *mg/ml streptomycin mic *mg/ml origin 1 staphylococcus aureus 1.250 0.625 0.00625 nose 2 staphylococcus aureus 1.250 0.312 0.00156 nose 3 staphylococcus aureus 1.250 0.625 0.00078 throat 4 staphylococcus aureus 0.078 0.312 0.00078 nose 5 staphylococcus aureus 1.250 0.312 0.0001 wound 6 staphylococcus aureus 0.625 0.312 0.00039 nose 7 staphylococcus aureus 2.500 0.625 0.00078 nose 8 staphylococcus aureus 1.250 0.625 0.00078 atcc 9 staphylococcus aureus 0.625 0.625 0.0001 nose 10 staphylococcus aureus 1.250 0.625 0.00312 nose 11 staphylococcus aureus 1.250 0.312 0.00078 nose 12 staphylococcus aureus 0.625 0.156 0.00078 nose table 1. antistaphylococcal activity of the thymus vulgaris and origanum vulgare essential oils (mic: minimum inhibitory concentration; atcc: american type culture collection) 37 biologica nyssana ● 12 (1) september 2021: 33-45 stojanović-radić et al. ● antistaphylococcal activity of thymus vulgaris and origanum vulgare essential oils: time-lapse kinetics... fig. 1. antistaphylococcal activity of the thymus vulgaris and origanum vulgare essential oils *the results are presented as the percent of formed biofilms, where the control biofilm absorbance represents 100%. ovabsorbance of the wells containing origanum vulgare essential oil; tv absorbance of the wells containing thymus vulgaris essential oil; str streptomycin; c control biofilm. 38 biologica nyssana ● 12 (1) september 2021: 33-45 stojanović-radić et al. ● antistaphylococcal activity of thymus vulgaris and origanum vulgare essential oils: time-lapse kinetics... fig. 2. antibiofilm activity of oregano and thyme essentail oils against the isolates of staphylococcus aureus no. 7-12 *the results are presented as the percent of formed biofilms, where the control biofilm absorbance represents 100%. ovabsorbance of the wells containing origanum vulgare essential oil; tv absorbance of the wells containing thymus vulgaris essential oil; str streptomycin; c control biofilm. 2015; araujo & longo, 2016; evangelista-martínez et al., 2018; simirgiotis et al., 2020; xiao et al., 2020). in comparison to these results, the oregano essential oil investigated in this paper fits into the previous range of active concentrations. biofilm producing ability and antibiofilm activity the results on the biofilm producing ability of investigated s. aureus strains demonstrated that only one strain was a weak biofilm producer (no. 5), three strains were moderate producers (no. 7, 9, 11), while the remaining strains showed strong biofilm producing ability. the testing of the two essential oils showed that oregano oil had higher antibiofilm activity and that this activity was the most efficient at half-mics (fig. 1 and fig. 2). the double and four-time increment of the oils concentration (mic, 2 x mic) did not cause higher reduction. in contrast, reduction was lower with higher oil concentrations. the only exception was the action of this oil against isolate no. 5, a weak biofilm producer. the observed reductions were higher than 90% of the control biofilm for strains no. 1, 2, 4, 6 and 8 and more than 80% in strains no. 3, 7, 9 and 10. in contrast to these strains, the strain no. 5 demonstrated direct concentration-dependency where the reduction ranged from 39% (half mic) to 48% (2 x mic). in strain no. 11, the reductions were concentration dependent and ranged from 55 to 60%. finally, the strain no. 12 demonstrated very unusual effect of oregano essential oil, where half mic reduced biofilm by 72%, but higher concentrations promoted biofilm formation. similar results were obtained when these three concentrations were tested against p. aeruginosa in the study of pejčić et al. (2020) where in many cases, higher concentrations exhibited a lower reduction of biofilm formation. this was explained by an enhanced production of exopolysaccharides (eps) as a response of the cells to stress caused by high concentrations of antimicrobials. by this production, eps interfere with diffusion and reduce their toxicity. also, these high concentrations can cause decreased interaction with cells and/or activation of some resistance mechanisms in the biofilm cells (pejčić et al., 2020). therefore, herein obtained results suggest that similar processes occur in s. aureus biofilms as well. in the study of nostro et al. (2007), oregano essential oil antibiofilm efficacy was tested at three sub-inhibitory concentrations (mic/2, mic/4 and mic/8). their results showed increased reduction with higher concentrations, which is opposite to our results. the explanation might be that in present work, we tested only one subinhibitory concentration and it obtained very high efficiency. therefore, only subinhibitory concentrations should be tested in the future. lu et al. (2017) also confirmed high efficacy of oregano essential oil against 24-h-old multidrugresistant (mrsa) staphylococcal biofilms at concentration of 0.4 mg/ml. in the case of the thyme essential oil, the mentioned reverse concentration dependence has also been observed. here, in almost half of the strains (no. 1, 7, 8, 9, 11) the mentioned concentration dependency was observed, while in others the reductions exhibited by all three concentrations were statistically insignificant (no. 2, 3, 4 and 10). only in strain no. 12, the twice mic demonstrated the highest efficiency. up to now, several studies investigated potential of the thyme essential oil against staphylococcal biofilms (kavanaugh & ribbeck, 2012; vázquez-sánchez et al., 2014; ben abdallah et al., 2020). when the thyme essential oil was tested against sc-01, which is a biofilm-forming, oxacillinand methicillin-resistant clinical isolate, it has been efficient at ∼ 0.8% (v/v) concentration against its biofilm (kavanaugh and ribbeck, 2012). in the study of vazquez-sanchez et al. (2014), the thyme oil showed the highest efficacy with mic of 0.04% (v/v) among several tested essential oils and the most efficiently eradicated 48-h-old biofilms formed on stainless steel. finally, the recent research od abdallah et al. (2020) showed that the essential oil of thymus zygis reduced the biofilms of mrsa isolates up to 91%. in the present study, the reductions exhibited by the thyme essential oil ranged from 31% to 96%. time-lapse kinetics of inhibitory action the present study also dealt with kinetics of inhibitory activity in order to investigate the efficiency of the two oils during a time period of 48 h and the results are presented in fig. 3 and 4. in these experiments, two concentrations were tested, mics and halfmics. the strains 7, 8 and 9 were completely inhibited throughout the entire incubation period by both concentrations of both essential oils. in the case of oregano oil, it has been observed that in all strains the inhibition of growth was complete during the first four hours, afterwards the cultures started recovery and exponential growth in strains 1, 2, 3, 5, 6, 10 and 12. however, this growth was significantly reduced in comparison to the control and also the observed growth was noticed only at half mics (no. 1, 2, 3, 10, 11 and 12) or were concentration dependent (no. 5 and 6). also, in isolates no. 11 and 12, it is notable that the recovery has been prolonged, and the growth started after 24 h for both oils. the thyme essential oil exhibited similar action, where complete inhibition during the entire period of incubation has been observed in strains no. 1, 7, 8, 9 and 12, while some strains recovered 39 biologica nyssana ● 12 (1) september 2021: 33-45 stojanović-radić et al. ● antistaphylococcal activity of thymus vulgaris and origanum vulgare essential oils: time-lapse kinetics... but only at the lower tested concentration (no. 2, 3, 10 and 11). finally, in strains no. 4, 5 and 6, the recovery was observed at both mic and mic/2, but the final growth of bacterial populations has been significantly reduced in comparison to the control values. according to this, the efficiency against planktonic cells is the highest during the first day of incubation and this information might be used for application of some synergistic antimicrobials during this period to achieve the complete killing effect at lower oil/antimicrobial agent concentrations. biologica nyssana ● 12 (1) september 2021: 33-45 40 stojanović-radić et al. ● antistaphylococcal activity of thymus vulgaris and origanum vulgare essential oils: time-lapse kinetics... fig. 3. time-lapse kinetics of oregano and thyme essential oils inhibitory action against staphylococcus aureus clinical isolates (strains no. 1-6) 41 biologica nyssana ● 12 (1) september 2021: 33-45 stojanović-radić et al. ● antistaphylococcal activity of thymus vulgaris and origanum vulgare essential oils: time-lapse kinetics... fig. 4. time-lapse kinetics of oregano and thyme essential oils inhibitory action against staphylococcus aureus clinical isolates (strains no. 7-12) checkerboard assay to find out which is mutual relationship between the oils and also of the oils alone with the reference antimicrobial drug, the testing of sinergism was done by checkerboard assay. calculated fics showed either sinergistic or additive effect of all tested combinations (tab. 2). when the essential oils were combined together, mics of both oils reduced from two to eight times. in combination with streptomycin, sinergistic and additive effect was observed for both oils, with reductions of mics up to eight times for both participants in the combination. biologica nyssana ● 12 (1) september 2021: 33-45 42 stojanović-radić et al. ● antistaphylococcal activity of thymus vulgaris and origanum vulgare essential oils: time-lapse kinetics... c om bi na tio ns st ap hy lo co cc us a ur eu s a t c c 6 53 8 st ap hy lo co cc us a ur eu s * n o. 1 st ap hy lo co cc us a ur eu s * n o. 1 2 m ic al on e m ic co m bi ne d fi c m ic al on e m ic co m bi ne d fi c m ic al on e m ic co m bi ne d fi c 1 th ym e + or eg an o th ym e 1. 25 0. 15 6 0. 12 5 1. 25 0. 62 5 0. 5 0. 62 5 0. 31 2 0. 5 or eg an o 0. 62 5 0. 07 8 0. 12 5 0. 62 5 0. 31 2 0. 5 0. 15 6 0. 07 8 0. 5 *f ic i in te ra ct io n 0. 25 sy n 1 a dd 1 a dd 2 th ym e + st re pt om yc in th ym e 1. 25 0. 15 6 0. 12 5 1. 25 0. 15 6 0. 12 5 0. 62 5 0. 15 6 0. 25 st re pt om yc in 0. 00 07 8 0. 00 02 4 0. 31 3 0. 00 62 5 0. 00 09 0. 15 6 0. 00 07 8 0. 00 04 8 0. 62 6 *f ic i in te ra ct io n 0. 43 8 sy n 0. 28 1 sy n 0. 87 6 a dd 3 or eg an o + st re pt om yc in or eg an o 0. 62 5 0. 07 8 0. 12 5 0. 62 5 0. 15 6 0. 25 0. 15 6 0. 07 8 0. 5 st re pt om yc in 0. 00 07 8 0. 00 04 8 0. 62 5 0. 00 62 5 0. 00 09 0. 15 6 0. 00 07 8 0. 00 09 7 1. 25 *f ic i in te ra ct io n 0. 75 a dd 0. 40 6 sy n 1. 65 a dd ta bl e 2. m in im um in hi bi to ry c on ce nt ra tio ns (m g/ m l) an d fra ct io na l i nh ib ito ry c on ce nt ra tio n va lu es o f t he m ut ua l c om bi na tio ns o f e o s an d w ith s tre pt om yc in fi c : fr ac tio na l i nh ib ito ry c on ce nt ra tio n; f ic i: fr ac tio na l i nh ib ito ry c on ce nt ra tio n in de x; m ic : m in im um in hi bi to ry c on ce nt ra tio n; a tc c : a m er ic an t yp e cu ltu re c ol le ct io n; s yn : s yn er gi sm ; a dd : a dd iti ve the results pointed to the thyme-streptomycin as the most efficient combination, where sinergism was demonstrated against two out of the three tested strains of staphylococci. also, important to mention is the fact that these combinations were sinergistic for the strains with higher mics of oils. in the third strain, which was more susceptible, additive effect has been observed for all tested combinations. up to now, it has been reported that the thyme oil exhibits sinergistic potential against staphylococci when combined with garlic (garcía-díez et al., 2017), myrtus communis essential oil (sadiki et al., 2014) and with mustard (reyes-jurado et al., 2016). on the other hand, van vuuren et al. (2009) demonstrated the predominance of antagonism against staphylococci (and some other microbial species) when the thyme essential oil was combined with cyprofloxacin. in the case of oregano, it has been reported that it possesses an additive effect in combination with acetic acid (de souza et al., 2009), basil or bergamot essential oils (lv et al., 2011), silver nanoparticles (scandorieiro et al., 2016), as well as with mustard (reyes-jurado et al., 2016). the study of rejes-jurado et al. (2016) demonstrated that in combination, thyme and mexican oregano exhibited additive effect only. our study partially confirms these results since additive effect has been observed for one of the three tested strains. together with this, a recent study of xiao et al. (2019) revealed that oregano when combined with quinolone drugs (tosufloxacin, levofloxacin, ciprofloxacin) and rifampin completely eradicated all stationary phase s. aureus cells. based on the herein presented results, we can suggest that both oils might be used in combination or for an effective enhancement of the streptomycin efficacy against human infections caused by staphylococci. conclusion the present study tested the antimicrobial potential of the thyme and oregano essential oils against clinical isolates of s. aureus. the results showed moderate to high action against planktonic cells and significant antibiofilm potential of both oils. oregano exhibited higher activity in all tests in comparison to the thyme essential oil. time-lapse kinetics study revealed that the action of both oils is strain-dependent and that in most cases, the mics very significantly reduced or even completely eradicated the growth of the cells. also, inhibitory effect in recovering cultures is the most prominent during the first four up to 24 h of cultivation, so the effect of some compound that possess sinergistic relationship with the essential oils will be the highest if applied during the first 24 h. finally, the sinergy has been observed when the oils were applied in combination, but also when individually combined with streptomycin. all mentioned results point to a very high potential of both oils to be used as an adjuvant agent for control of human staphylococcal infections. references abdallah, f. ben, lagha, r., & gaber, a. 2020: biofilm inhibition and eradication properties of medicinal plant essential oils against methicillinresistant staphylococcus aureus clinical isolates. pharmaceuticals, 13: 369. araujo, m. m. de, & longo, p. l. 2016: teste da ação antibacteriana in vitro de óleo essencial comercial de origanum vulgare (orégano) diante das cepas de escherichia coli e staphylococcus aureus. arquivos do instituto biológico, 83: 1–7. ben abdallah, f., lagha, r., & gaber, a. 2020: biofilm inhibition and eradication properties of medicinal plant essential oils against methicillinresistant staphylococcus aureus clinical isolates. pharmaceuticals, 13(11): 1–15. benabed, k. h., gourine, n., ouinten, m., yousfi, m., bombarda, i., & yousfi, m. 2016: chemical composition, antioxidant and antimicrobial activities of the essential oils of three algerian lamiaceae species. current nutrition and food science, 13(2): 97–109. bilge, n., vatansever, l., & duman, b. 2010: effect of oregano essential oil on biofilms formed by staphylococci and escherichia coli. kafkas üniversitesi veteriner fakültesi dergisi, 16 (suppl-a): 22–29. boskovic, m., zdravkovic, n., ivanovic, j., janjic, j., djordjevic, j., starcevic, m., & baltic, m. z. 2015: antimicrobial activity of thyme (tymus vulgaris) and oregano (origanum vulgare) essential oils against some food-borne microorganisms. procedia food science, 5: 18–21. de carvalho, r. j., de souza, g. t., honório, v. g., de sousa, j. p., da conceição, m. l., maganani, m., de souza, e. l. 2015: comparative inhibitory effects of thymus vulgaris l. essential oil against staphylococcus aureus, listeria monocytogenes and mesophilic starter co-culture in cheese-mimicking models. food microbiology, 52: 59–65. de souza, e. l., de barros, j. c., da conceição, m. l., neto, n. j. g., & da costa, a. c. v. 2009: combined application of origanum vulgare l. essential oil and acetic acid for controlling the growth of staphylococcus aureus in foods. brazilian biologica nyssana ● 12 (1) september 2021: 33-45 stojanović-radić et al. ● antistaphylococcal activity of thymus vulgaris and origanum vulgare essential oils: time-lapse kinetics... 43 journal of microbiology, 40(2): 387–393. evangelista-martínez, z., reyes-vázquez, n., & rodríguez-buenfil, i. 2018: antimicrobial evaluation of plant essential oils against pathogenic microorganisms: in vitro study of oregano oil combined with conventional food preservatives. acta universitaria, 28(4): 10–18. fani, m., & kohanteb, j. 2017: in vitro antimicrobial activity of thymus vulgaris essential oil against major oral pathogens. journal of evidence-based complementary and alternative medicine, 22(4): 660–666. garcía-díez, j., alheiro, j., pinto, a. l., falco, v., fraqueza, m. j., & patarata, l. 2017: synergistic activity of essential oils from herbs and spices used on meat products against food borne pathogens. natural product communications, 12(2): 281–286. gedikoğlu, a., sökmen, m., & çivit, a. 2019: evaluation of thymus vulgaris and thymbra spicata essential oils and plant extracts for chemical composition, antioxidant, and antimicrobial properties. food science and nutrition, 7(5): 1704– 1714. kavanaugh, n. l., & ribbeck, k. 2012: selected antimicrobial essential oils eradicate pseudomonas spp. and staphylococcus aureus biofilms. applied and environmental microbiology, 78(11): 4057– 4061. kazemi, m., mousavi, e., & bandrez, n. 2012: chemical compositions and antibacterial activity of the essential oils of thymus vulgaris and tanacetum perthenium. research journal of soil biology, 4(2): 21–31. kon, k., & rai, m. 2012: antibacterial activity of thymus vulgaris essential oil alone and in combination with other essential oils. nusantara bioscience, 4(2): 50–56. lu, m., dai, t., murray, c. k., wu m. x. 2018. bactericidal property of oregano oil against multidrug-resistant clinical isolates. frontiers in microbiology, 9: 2329. lv, f., h. liang, q. yuan, and c. li. 2011. in vitro antimicrobial effects and mechanism of action of selected plant essential oil combinations against four food-related microorganisms. food research international, 44: 3057–3064. man, a., santacroce, l., jacob, r., mare, a., & man, l. 2019: antimicrobial activity of six essential oils against a group of human pathogens: a comparative study. pathogens, 8(15): 1–11. marques, j. de l., volcão, l. m., funck, g. d., kroning, i. s., da silva, w. p., fiorentini, â. m., & ribeiro, g. a. 2015: antimicrobial activity of essential oils of origanum vulgare l. and origanum majorana l. against staphylococcus aureus isolated from poultry meat. industrial crops and products, 77: 444–450. najem, a.h. 2020: in vitro antimicrobial and antibiofilm activity of some plant essential oils against nasal staphylococcus aureus. journal of critical reviews, 7(5): 892–898. nostro, a., roccaro, a. s., bisignano, g., marino, a., cannatelli, m. a., pizzimenti, f. c., cioni, p. l., procopio, f., & blanco, a. r. 2007: effects of oregano, carvacrol and thymol on staphylococcus aureus and staphylococcus epidermidis biofilms. journal of medical microbiology, 56: 519–523. özkalp, b., sevgi, f., özcan, m., & özcan, m. m. 2010: the antibacterial activity of essential oil of oregano (origanum vulgare l.). journal of food, agriculture and environment, 8(2): 272–274. papa, r., garzoli, s., vrenna, g., sabatino, m., sapienza, f., relucenti, m., donfrancesco, o., fiscarelli, e. v., artini, m., selan, l., & ragno, r. 2020: essential oils biofilm modulation activity, chemical and machine learning analysis. application on staphylococcus aureus isolates from cystic fibrosis patients. international journal of molecular sciences, 21(23): 1–20. pecarski, d., ketin, s., omerovic, i., mirkovic, m., jugovic, z., & biocanin, r. 2016: chemical compositions and antimicrobial activities of oregano and thyme essential oils. bulgarian chemical communications, 48(4): 678 – 683. pejčić, m., stojanović-radić, z., genčić, m., dimitrijević, m., & radulović, n. 2020: antivirulence potential of basil and sage essential oils: inhibition of biofilm formation, motility and pyocyanin production of pseudomonas aeruginosa isolates. food and chemical toxicology, 141: 111431. pesavento, g., calonico, c., bilia, a. r., barnabei, m., calesini, f., addona, r., mencarelli, l., carmagnini, l., di martino, m. c., & lo nostro, a. 2015: antibacterial activity of oregano, rosmarinus and thymus essential oils against staphylococcus aureus and listeria monocytogenes in beef meatballs. food control, 54: 188–199. pourazar dizaji‎, s., soleimani, n., afrugh, p., & saedi‎, s. 2018: in vitro antibacterial activity of thymus vulgaris essential oil mycobacterium tuberculosis. infection epidemiology and microbiology, 4(2): 47–51. biologica nyssana ● 12 (1) september 2021: 33-45 stojanović-radić et al. ● antistaphylococcal activity of thymus vulgaris and origanum vulgare essential oils: time-lapse kinetics... 44 biologica nyssana ● 12 (1) september 2021: 33-45 stojanović-radić et al. ● antistaphylococcal activity of thymus vulgaris and origanum vulgare essential oils: time-lapse kinetics... prince, a. (2013). staphylococcus aureus infection in the respiratory tract. in mucosal immunology of acute bacterial pneumonia. springer-verlag, pp. 239. reyes-jurado, f., lópez-malo, a., & palou, e. 2016: antimicrobial activity of individual and combined essential oils against foodborne pathogenic bacteria. journal of food protection, 79(2): 309–315. rota, m. c., herrera, a., martínez, r. m., sotomayor, j. a., & jordán, m. j. 2008. antimicrobial activity and chemical composition of thymus vulgaris, thymus zygis and thymus hyemalis essential oils. food control, 19(7): 681–687. sadiki, m., balouiri, m., barkai, h., maataoui, h., koraichi, s. i., & elabed, s. 2014: synergistic antibacterial effect of myrtus communis and thymus vulgaris essential oils fractional inhibitory concentration index. international journal of pharmacy and pharmaceutical sciences, 6(6): 121– 124. sakkas, h., & papadopoulou, c. 2017: antimicrobial activity of basil, oregano, and thyme essential oils. journal of microbiology and biotechnology, 27(3): 429–438. scandorieiro, s., de camargo, l. c., lancheros, c. a. c., yamada-ogatta, s. f., nakamura, c. v., de oliveira, a. g., andrade, c. g. t. j., duran, n., nakazato, g., & kobayashi, r. k. t. 2016: synergistic and additive effect of oregano essential oil and biological silver nanoparticles against multidrug-resistant bacterial strains. frontiers in microbiology, 7: 1–14. simirgiotis, m. j., burton, d., parra, f., lópez, j., muñoz, p., escobar, h., & parra, c. 2020: antioxidant and antibacterial capacities of origanum vulgare l. essential oil from the arid andean region of chile and its chemical characterization by gcms. metabolites, 10(10): 1–12. stepanović, s., vuković, d., hola, v., bonaventura, g. di, djukić, s., ćircović, i., & ruzicka, f. 2007: quantification of biofilm in microtiter plates. apmis, 115(8): 891–899. stojanović-radić, z., dimitrijević, m., genčić, m., pejčić, m., & radulović, n. 2020: anticandidal activity of inula helenium root essential oil: synergistic potential, anti-virulence efficacy and mechanism of action. industrial crops and products, 149: 112373. uzair, b., niaz, n., bano, a., khan, b. a., zafar, n., iqbal, m., tahira, r., & fasim, f. 2017: essential oils showing in vitro anti mrsa and synergistic activity with penicillin group of antibiotics. pakistan journal of pharmaceutical sciences, 30(5): 1997– 2002. valizadeh, s., mahmodi, r., fakheri, t., katiraie, f., & rahmani, v. 2016: investigating the phytochemical, antibacterial and antifungal effects of thymus vulgaris and cuminum cyminum essential oils. medical laboratory journal, 10(1): 36–43. van vuuren, s. f., suliman, s., & viljoen, a. m. 2009: the antimicrobial activity of four commercial essential oils in combination with conventional antimicrobials. letters in applied microbiology, 48(4): 440–446. vázquez-sánchez, d., cabo, m. l., & rodríguezherrera, j. j. 2015: antimicrobial activity of essential oils against staphylococcus aureus biofilms. food science and technology international, 21(8): 559–570. xiao, s., cui, p., shi, w., & zhang, y. 2019: identification of essential oils with strong activity against stationary phase staphylococcus aureus. biorxiv, 4: 1–10. zhang, d., li, h., gan, r., zhu, f., zhang, j., wang, x., farha, a. k., & corke, h. 2020: antivirulence properties and related mechanisms of spice essential oils: a comprehensive review. comprehensive reviews in food science and food safety, 2020: 1–39. 45 anticancer compounds from medicinal plants biologica nyssana 5 (1)  september 2014: 63-69 zlatković, b. et al.  report on the new and insufficiently studied taxa… 63 original article received: 3 september 2014 revised: 6 september 2014 accepted: 10 september 2014 report on the new and insufficiently studied taxa in the flora of serbia bojan zlatković 1 *, stefan bogosavljević 2 , nenad smiljković 3 , vladimir ranđelović 1 1 university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia 2 the pharmaceutical chamber of serbia, dečanska 8a, belgrade, serbia 3 serbian herpetological society "milutin radovanovic", despota stefana 142, belgrade, serbia *e-mail: bojanzlat@yahoo.com abstract: zlatković, b., bogosavljević, s., smiljković, n., ranđelović, v.: report on the new and insufficiently studied taxa in the flora of serbia. biologica nyssana, 5 (1), septemeber 2014: 63-69. several important floristic records were established during our recent floristic investigations of southeastern serbia and province kosovo-metohija. minuartia mediterranea (caryophyllaceae) and arabis glabra subsp. pseudoturritis (brassicaceae) are reported for the first time from the territory of serbia, while presence of neglected species symphytum bulbosum (boraginaceae) and valerianella microcarpa (valerianaceae) is reevaluated, as they were reported from new localities. key words: vascular flora, new and neglected taxa, distribution, serbia, balkans. apstrakt: zlatković, b., bogosavljević, s., smiljković, n., ranđelović, v.: izveštaj o novim i manje poznatim taksonima za floru srbije. biologica nyssana, 5 (1), septemeber 2014: 63-69. tokom florističkih istraživanja jugoistočne srbije i kosova i metohije, zabeležen je veći broj značajnih podataka za floru srbije. minuartia mediterranea (caryophyllaceae) i arabis glabra subsp. pseudoturritis (brassicaceae) su po prvi put ustanovljeni na teritoriji srbije. rasprostranjenje symphytum bulbosum (boraginaceae) i valerianella microcarpa (valerianaceae) dopunjeno je novim lokalitetima, čime je potvrđeno prisustvo ovih, u flori srbije, zanemarenih taksona. ključne reči: vaskularna flora, novi i manje poznati taksoni, distribucija, srbija, balkan introduction the territory of serbia, in common with the other regions of balkan peninsula, is characterized by relatively high floristic richness compared to the rest of europe (t u r r i l l , 1929, s t e v a n o v i ć et al. 1995; 1999). flora of serbia has been studied since g r i s e b a c h (1843) and p a n č i ć (1874, 1884), and this long period of continuous research has significantly increased the number of reported floristic data from this country. the largest amount of data from the recent period is included in the 8volume publication flora of sr serbia (j o s i f o v i ć , ed., 1970-1976), summarizing the 5 (1) • september 2014: 63-69 biologica nyssana 5 (1)  september 2014: 63-69 zlatković, b. et al.  report on the new and insufficiently studied taxa… 64 overall knowledge collected by the date of publication of each volume. however, as a large amount of new floristic data was reported for the region during the last decades, additional papers were published either simultaneously or after the publication date of the volume dealing with those taxa (d i k l i ć 1977; n i k o l i ć et al. 1986). even though the flora of serbia presently comprises accounts on more than 3600 species and subspecies, including data in two more recent volumes (s t e v a n o v i ć 1992, 2012), the number of newly reported taxa still fluctuates in the positive direction. during the most recent period of floristic research of serbia (and particularly its southern provinces) many taxa new or poorly known for this area have been registered and reassessed on the topic of their distribution, ecology and conservation (z l a t k o v i ć et al. 2009; 2011). the aim of this paper is to report findings of two recently discovered plant taxa in the flora of serbia, and to emphasize presence of two other neglected species recently established at new localities in serbia. resuming their distribution at the local scale would be useful as an addition to the forthcoming volumes of “the flora of serbia (i-x)” (j o s i f o v i ć , ed., 1970-1977; s a r i ć & d i k l i ć 1986), offering newly obtained floristic data. material and methods distribution of the studied taxa within the territory of serbia was determined and mapped in utm grid system (10 x 10 sq. km., utm zone 34t). in addition to the field survey, herbarium collections and literature sources were also checked for supplementation of distribution data. recent volumes of atlas florae europae (j a l a s & s u o m i n e n 1986; j a l a s et al. 1999) and comparative chorology of the central european flora for selected taxa (m e u s e l et al. 1965; 1978; m e u s e l & j ä g e r 1992) were used in order to discuss and compare recent and previously published data on distribution. in order to align plant distribution with geology substrate, adequate sections of geology map of yugoslavia (k 34-56, 34-57, 34-68) were used at scale 1:100,000 (b a b o v i ć & c v e t k o v i ć 1968; k a r a j o v a n o v i ć & h r i s t o v 1972; t e r z i n et al. 1970). descriptions of taxa match those in the selected volumes of the flora of europe (h a l l i d a y 1993; j o n e s 1964; p a w l o w s k i 1972; e r n e t et al. 1976). the main taxonomic characters of enumerated taxa were briefly compared with other similar representatives of their genera from serbian flora. in order to provide explanation of the ecological conditions in relation to the new parts of their range, short notes on ecological conditions of their habitats are added. nomenclature and synonyms are aligned with the euro+med (http://ww2.bgbm.org/europlusmed/) databases and adequate volumes of the flora of europe (e r n e t et al. 1976). voucher specimens are deposited in the herbarium of the institute of botany and botanical garden “jevremovac”, university of belgrade (beou), with acquisition numbers given in the brackets. results and discussion minuartia mediterranea (link) k. malý general distribution: algeria, albania, bosnia and herzegovina, bulgaria, croatia, cyprus, egypt, france [incl. corse], greece [incl. crete & aegean islands], spain [incl. balearic islands], israel, jordan, italy [incl. sardinia & sicily], libya, lebanon, portugal, syria, malta, macedonia, montenegro, morocco, slovenia, tunisia, turkey. new distribution data: prizrenska bistrica gorge (rečane), xeric pastures (thero-brachypodietea) on shallow soil, limestone, 430 m, 26-05-1997, dm77, coll./det. b. zlatković (beou 16853); prizrenska bistrica gorge (st. arhangeli monastery), rocky ground and screes, limestone, 390 m, 26-05-1997, dm87, coll./det. b. zlatković (fig. 1). figure 1. distribution of minuartia mediterranea in serbia. biologica nyssana 5 (1)  september 2014: 63-69 zlatković, b. et al.  report on the new and insufficiently studied taxa… 65 description: annual plant, with erect stems branching from the base or growing upward. stem 3-15 cm tall, glabrous or subglabrous. leaves linear, 10-12 mm long. inflorescence dense, almost crowded. pedicels usually shorter than sepals. sepal segments 3-5 mm long and 0.5-0.7 mm wide, 3veined, linear-lanceolate. petals shorter than sepals or absent. capsule usually shorter than sepals, with numerous almost smooth seeds, 0.4 mm in diameter (h a l l i d a y 1993). in the flora of serbia this is the fourth reported species from sect. sabulina (rchb.) graebner of genus minuartia, after m. verna, m. hybrida and m. mesogitana (diklić & stevanović 2012). among the enumerated group of species, the newly reported m. mediterranea is most similar to m. hybrida. main differences are in inflorescences, as cymes of m. mediterranea are denser and more crowded, while pedicels are shorter than sepals in the second species. this species inhabits xeric and rocky pastures on shallow limestone soils, mainly occupied by annual plants. it was also recorded in warm positions of limestone screes in the gorges of upland region. it grows mainly as a constituent of communities in thero-brachypodietea alliance, together with phleum exaratum, trachynia distachya, helianthemum salicifolium, valerianella coronata, pterocephalus plumosus etc. arabis glabra (l.) bernh. subsp. pseudoturritis (boiss. & heldr.) maire general distribution: algeria, italy [c & s, incl. sicily], albania, bulgaria, greece [incl. aegean islands], macedonia, malta, morocco. new distribution data: kozjak mt. (delinovački rid), querco-carpinetum orientalis, granite and migmatites, 560 m, 16-05-2004, em78, coll./det. b. zlatković (beou 16883), subnom. turritis pseudoturritis; starac mt. (gornji starac), ostryocarpinion aegeicum, migmatites, 800 m, 02-052005, em78, coll./det. b. zlatković (beou 16882), subnom. turritis pseudoturritis; pčinja river gorge (village barbace), querco-carpinetum orientalis, fine-grained biotite and biotite-muscovite gneiss, 530 m, 23-04-2003, em79, coll./det. b. zlatković; rujan mt. (orljak), querco-fagetea, silicate, 742 m, 26-06-2009, em67, coll./det. b. zlatković & n. smiljković (beou 16884), subnom. turritis pseudoturritis (fig. 2). description: biennial to perennial plant with usually erect and non-branching stems (40-70 cm), pubescent on ventral side. basal leaves lanceolate, sinuate-dentate to lyrate, with stellate hairs. cauline leaves usually ovate, sagittate in the basal part, figure 2. distribution of arabis glabra subsp. pseudoturritis in serbia. glabrous and glaucous. petals 4-8 mm, cream or whitish, compound in lax, elongate inflorescence. siliqua 50-80 (100) x 0.8-1.2 mm, erect, somewhat compressed and with prominent median veins. seeds in a single row in each locus (j o n e s , 1964). the taxonomic position of this taxon is still uncertain as various authors have different opinions. in the earlier floristic literature it was either regarded as a well-defined species: turritis pseudoturritis (j o n e s , 1964, a s e n o v , 1970, t r i n a j s t i ć , 1976) or a. pseudoturritis (m i c e v s k i , 1995), or in the later period as a variety or synonym for a. glabra (j o n e s & a k e r o y d , 1993; a n č e v , 2007). according to several morphological traits, subsp. pseudoturritis is distinguishable from the typical subspecies of a. glabra, in our opinion indicating subspecies level. the most prominent difference from subsp. glabra is uniseriate seed alignment within the fruit and generally longer siliquae (up to 110 mm). some of the authors considered both shape and venal prominence of the fruit wall as valuable additional characters for making difference of two taxa more clear. the valves of siliquae are more compressed and prominently three-veined in subsp. pseudoturritis, compared to the single central vein in subsp. glabra. the typical subspecies is distributed throughout europe except for extreme north, while subsp. pseudoturritis is confined to southern europe and northern africa, overlapping biologica nyssana 5 (1)  september 2014: 63-69 zlatković, b. et al.  report on the new and insufficiently studied taxa… 66 the southernmost part of the species range. a. glabra subsp. pseudoturritis may be confused with a. laxa which inhabits southern balkans and transcaucasia. this last species is distinguished from a. glabra subsp. pseudoturritis by oblong, sparsely ciliate, cauline leaves cordate in the base, and petals that are rather white or lilac in color. it is also similar with a. pauciflora, which is possibly erroneously reported for southern serbia (a d a m o v i ć , 1899). a. glabra subsp. pseudoturritis inhabits forest edges and stony places on silicate soils in the mountains of study region. records of this taxon in the neighboring countries also show its sporadic distribution in mountains, but usually at higher altitudes. it was recorded in bulgaria between 1400 and 2200 m above sea level (a s e n o v , 1970), at higher altitude than records from serbia. on the other hand, in macedonia it was reported from a much wider altitudinal range, from 800 to 2300 m above sea level (m i c e v s k i , 1995), similar to the recorded positions in serbia. this subspecies has probably wider distribution in other mountain regions of serbia that should be investigated in the future. symphytum bulbosum k. f. schimp. general distribution: algeria, albania, bulgaria, france [corse], croatia, germany, greece, switzerland, italy [incl. sardegna and sicily], slovenia, turkey. new records: pčinja river gorge (village jablanica), humid and shady places in the woods (quercofagetea), fine-grained biotite and biotite-muscovite gneiss, 560 m, 15-05-2005, em78, coll./det. b. zlatković (beou 16856); literature data for serbia: bosilegrad (village gornja ljubata) – fn00, bosilegrad (village musulj) – fn00 (u r u m o f f 1937), (fig. 3). description: perennial plant with slender and creeping stock, producing subglobose underground tubers. stems up to 50 cm, poorly branched in the upper part or simple, covered with short hooked hairs and (about 1.5 mm long) setae like the remaining part of the plant. basal leaves large, with ovate to elliptical-lanceolate lamina, tapered to a distinct, long petiole. the cauline leaves elliptical to lanceolate with short petioles, the upper sessile and narrowly decurrent. corolla about 8 – 11 mm long, pale yellow, with short and erect lobes. tubular scales acute, lanceolate-subulate, papilose and exerted about 1-4 (5) mm from the tube. stamens small and included, with minutely apiculate 2-2.5 mm long anthers (p a w l o w s k i , 1972). according to the phenotype, this plant is similar to s. tuberosum, and may be confused because both species have swollen subterranean parts, especially in the vegetative stage. however, s. bulbosum is well distinguished according to several flower traits, first of all longer and exerted scales. corolla is generally smaller, with terminal lobes that are not deflexed like in s. tuberosum. figure 3. distribution of symphytum bulbosum in serbia. the reconstruction of its occurrence in serbia is based at the data by u r u m o f f (1937), who reported this species for two localities in the serbian part of krajište region, formerly regarded as bulgarian territory. the species inhabits different types of deciduous, termophilous forests mainly at lower altitudes of the montane region, in the zone of oak forests. it is sporadically recorded in the lower part of pčinja valley, close to the river at somewhat damp and shady habitats in the oak woods, together with lathyrus laxiflorus, primula vulgaris, arum orientale, cardamine bulbifera, helleborus odorus, galanthus nivalis, corydalis solida and other species of the herb layer. valerianella microcarpa loisel. general distribution: bulgaria, france [incl. corse], greece [incl. crete & aegean islands], spain [incl. balearic islands], italy [incl. sardinia & sicily], portugal, turkey [european part], northern africa. biologica nyssana 5 (1)  september 2014: 63-69 zlatković, b. et al.  report on the new and insufficiently studied taxa… 67 new records in serbia: prizren (village našec), thero-brachypodietea, xerophilous pastures on shallow soil, limestone, 390 m, 26-05-1997, dm77, coll./det. b. zlatković (beou 16852); prizrenska bistrica gorge (st. arhangeli monastery), therobrachypodietea, thermophilic pastures on shallow surface, limestone, 390 m, 26-05-1997, dm87, coll./det. b. zlatković (beou 16851); literature data for serbia: bosilegrad – fn20 (u r u m o f f 1937); (fig. 4). figure 4. distribution of valerianella microcarpa in serbia. description: dwarf plant with stems 5-15 (35) cm long, in upper part dichotomously branched. lower cauline and basal leaves ovate-spatulate, obtusate, entire to sinuate. inflorescence dichotomous, dense, crowded. bracts more or less auriculate, green, with a narrow scarious margin, the lower linear to spatulate, obtusate, the upper narrowly triangular acuminate. fruits ovate to connate, densely hairy or glabrous, c. 1-1.5 mm long, numerous, arranged in fasciculate terminal clusters. sterile loculi of the fruit reduced to tiny ribs, separated by ovate flat area. calyx reduced to an indistinctly dentate, narrow rim (e r n e t & r i c h a r d s o n , 1976). as one of the representatives with smallest fruit size, this species is easily recognized among the species of genus valerianella in serbian flora. according to the shape of the fruit it might be confused with v. dentata and v. muricata, but both have larger fruits. after v. costata, this is the second report of the presence of neglected species of this genus in serbia (z l a t k o v i ć et al., 2011) after the reports by k o j i ć (1973) and r a n đ e l o v i ć (1975). occurrence of v. microcarpa was first reported by u r u m o f f (1937) from the former territory of bulgaria close to bosilegrad in se serbia. remarkable flora of this area was consequently investigated in the later period (r a n d ž e l o v i č & s t a m e n k o v i č , 1986; 1987; r a n d ž e l o v i č et al., 1988), but presence of v. microcarpa has not been confirmed there yet. as in the case of v. costata, existence of this mediterranean species may be expected from several other new sites influenced by mild climate in southern serbia. the species is recorded in dense plant communities at hill slopes and along the roadsides, formed at slightly eutrophic soils, consisting mainly of grasses and annuals, e.g. poa bulbosa, vulpia cilliata, trifolium dalmaticum, medicago minima, parentucellia latifolia. conclusion according to the recent floristic studies, presence of two completely new and two neglected taxa for the flora of serbia is reported from its southern regions and the province of kosovometohija. to the best of our knowledge, presence of minuartia mediterranea and arabis glabra subsp. pseudoturritis has not been reported from this region before. reports on their occurrence from the new, remote sites in the central part of balkan peninsula are especially thought-provoking. the new records of these two taxa extend their ranges in northward direction, out of their known distribution in southern areas. the presence of neglected species symphytum bulbosum and valerianella microcarpa in the flora of serbia is confirmed by adding new floristic data on their distribution. all new records contributed to knowledge of their general distribution and are important for the comprehensive publications that consider diversity of vascular plants of serbia. details on habitat preferences for the reported taxa offer additional information on their ecology in the newly established parts of their range in balkan peninsula. acknowledgements. the ministry of education, science and technological development of the republic of serbia (grant 173030) supported this research. we are grateful to dr marjan niketić for his valuable suggestions considering arabis glabra subsp. pseudoturritis. biologica nyssana 5 (1)  september 2014: 63-69 zlatković, b. et al.  report on the new and insufficiently studied taxa… 68 references adamović, l., 1899: neue beiträge zur flora von serbien. botanisches centralblatt, 78: 1-8. ančev, m., 2007: catalogue of the family brassicaceae (cruciferae) in the flora of bulgaria. phytologia balcanica, 13(2): 153-178. asenov, i., 1970: turritis l. in: jordanov, d. (ed.), flora na narodna republika b’lgarija. 4: 448452. b’lgarskata akademija na nauki. sofia. babović, m., cvetković, d., 1968: trgovište sa radomirom, 34-57. in: dimitrijević, m. et al. (eds.): osnovna geološka karta 1:100 000, socijalistička federativna republlika jugoslavija. savezni geološki zavod, beograd. diklić, n., 1977: dopuna flori sr srbije novim podacima o rasprostranjenju biljnih vrsta in: josifović, m. (ed.). flora sr srbije. 9: 205-210. srpska akademija nauka i umetnosti. beograd. diklić, n., stevanović, v., 2012: minuartia l. in: stevanović, v. (ed.). flora sr srbije. 2: 209235. srpska akademija nauka i umetnosti. beograd. ernet, d. m., richardson, i. a. k., 1976: valerianella miller. in: tutin, t. g. et al. (eds.), flora europaea 4: 48-52, university press, cambridge. grisebach, a., 1843: spicilegium florae rumelicae et bithynicae exhibens synopsin plantarum quas in aest. 1839 legit auctor a. grisebach 1. fridericus vieweg et filius, brunsvigae (braunschweig). halliday, g., 1993: minuartia l. in: tutin, t. g. et al. (eds.), flora europaea ed. 2, 1: 152-160, university press, cambridge. jalas j., suominen j., (eds.), 1986: atlas florae europaeae. distribution of vascular plants in europe. 7. the committee for mapping the flora of europe & societas biologica fennica vanamo, helsinki. jalas j., suominen j., lampinen r., kurtto a., (eds.), 1999: atlas florae europaeae. distribution of vascular plants in europe. 12. the committee for mapping the flora of europe & societas biologica fennica vanamo, helsinki. jones, b. m. g., 1964: arabis l. in: tutin, t. g. et al. (eds.), flora europaea 1: 290-294, university press, cambridge. jones, b. m. g., akeroyd, j.r., 1993: arabis l. in: tutin, t. g. et al. (eds.), flora europaea ed. 2, 1: 352-356, university press, cambridge. josifović, m. (ed.), 1970-1977: flora sr srbije 1-9. – srpska akademija nauka i umetnosti, beograd. karajovanović, m., hristov, s., 1972: kumanovo, 34-68.in: dimitrijević, m. et al (eds.): osnovna geološka karta 1:100 000, socijalistička federativna republlika jugoslavija. savezni geološki zavod, beograd. kojić, m., 1973: valerianella moench. in: josifović, m. (ed.), flora sr srbije ed. 5: 522-528, srpska akademija nauka i umetnosti, beograd. meusel, h., jäger, e., weinert, e., 1965: vergleichende chorologie der zentraleuropäischen flora «1». karten. gustav fischer, jena. meusel, h., jäger, e., weinert, e., 1978: vergleichende chorologie der zentraleuropäischen flora «2». karten. gustav fischer, jena. meusel, h., jäger, e., 1992: vergleichende chorologie der zentraleuropäischen flora 3. karten, literatur, register. gustav fischer, jena, stuttgart, new york. micevski, k., 1995: flora na republika makedonija 1(3). makedonska akademija na naukite i umetnostite, skopje. nikolić, v., sigunov, a., diklić, n., 1986: dopuna flori sr srbije novim podacima o rasprostranjenju biljnih vrsta in: sarić, m., diklić, n. (eds.), flora sr srbije. 10: 259-336. srpska akademija nauka i umetnosti. beograd. pančić, j., 1874: flora kneževine srbije. državna štamparija. beograd xxxiv + 802 pp. pančić, j., 1884: dodatak flori kneževine srbije. kraljevsko-srpska državna štamparija. beograd 253 pp. pawlowski, b., 1972: symphytum l. in: tutin, t. g. et al. (eds.), flora europaea ed. 3: 103-105, university press, cambridge. randželovič, n., stamenkovič, v., 1986: priloženie kom florata v okolnostta na bosilegrad. most. 100: 80-86. randželovič, n., stamenkovič, v., 1987: florata v okolnostta na bosilegrad ii. most. 105: 50-54. randželovič, n., stamenkovič, v., sotirov, s., randželovič, v., 1988: florata v okolnostta na bosilegrad iii. most. 112: 47-53. sarić, m., diklić, n., (eds.), 1986: flora sr srbije 10. srpska akademija nauka i umetnosti, beograd. stevanović, v., (ed.), 1992: flora sr srbije 1 (second edition). – srpska akademija nauka i umetnosti, beograd. stevanović, v., (ed.), 2012: flora sr srbije 2 (second edition). – srpska akademija nauka i umetnosti, beograd. stevanović, v., jovanović, s., lakušić, d., 1995: biogegrafska podela teritorije jugoslavije. in: stevanović, v., vasić, v., (eds.): biodiverzitet jugoslavije sa pregledom vrsta od međunarodnog značaja. ecolibri, beograd, biološki fakultet, beograd. 117-127. biologica nyssana 5 (1)  september 2014: 63-69 zlatković, b. et al.  report on the new and insufficiently studied taxa… 69 stevanović, v., jovanović, s., lakušić, d., niketić, m., 1999: karakteristike i osobenosti flore srbije i njen fitogeografski položaj na balkanskom poluostrvu i u evropi. in: stevanović, v. (ed.), crvena knjiga flore srbije 1. iščezli i krajnje ugroženi taksoni. 9-18. ministarstvo za životnu sredinu republike srbije, biološki fakultet univerziteta u beogradu, zavod za zaštitu prirode republike srbije. beograd. terzin, v. et al., 1970: vranje, 34-54. in: dimitrijević, m. et al. (eds.): osnovna geološka karta 1:100 000, socijalistička federativna republlika jugoslavija. savezni geološki zavod, beograd. the euro+med plantbase the information resource for euro-mediterranean plant diversity http://ww2.bgbm.org/europlusmed/ trinajstić, i., 1976: turritis l. in: trinajstić, i. (ed.). analitička flora jugoslavije 2(2). 237-240. institut za botaniku sveučilišta u zagrebu, zagreb. turrill, w.b., 1929: the plant-life of the balkan peninsula. a phytogeographical study. clarendon, oxford. 490 p. urumoff, i. k., 1935: florata na kjustendilskij okržja (flora des kustendiler kreises). sbornik na b’lgarskata akademija na nauki. 30: 1-235. zlatković, b., tomović. g., ranđelović, v., vukojičić, s., niketić, m. 2009: distribution and conservation status of several new and neglected vascular plants in serbia. phytologia balcanica, 15(1): 95-105. zlatković, b., ranđelović, v., lakušić, d., stevanović, v., 2011: novelties for the vascular flora of serbia. botanica serbica, 35(2):103-111. http://ww2.bgbm.org/europlusmed/ biologica nyssana 5 (1)  september 2014: 63-69 zlatković, b. et al.  report on the new and insufficiently studied taxa… 70 microsoft word 0105_matovic_et_al biologica nyssana 1 (1-2) december 2010: 43-47 matović, m. et al. natural potentials of the medicinal plants… 43 original article ! natural potentials of the medicinal plants from the orchidaceae family with mucus as the main ingredients from zlatar mountain milić matović1, biljana nikolić2*, gorica đelić3, marija marković1 1 university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia 2 institute of forestry, kneza višeslava 3, 11030 belgrade, serbia 3faculty of sciences, university of kragujevac, radoja domanovića 12, 34000 kragujevac, serbia * e-mail: smikitis@gmail.com abstract: matović, m., nikolić, b., đelić, g., marković, m.: natural potentials of the medicinal plants from the orchidaceae family with mucus as the main ingredients from zlatar mountain. biologica nyssana, 1 (12), december 2010: 43-47. the spontaneous medicinal flora of zlatar mountain was studied in the aim of realizing the possibilities of its sustainable use for the needs of the pharmaceutical industry. the special attention was paid to genera orchis, ophrys, plathanthera, gimnadenia, etc. from the orchid family (orchidaceae) of which salep is made (tuber salep). salep is a typical mucous drug (contains over 50% of mucus), which is very beneficial and useful. the primary role of salep is to heal and strengthen the organism and urge the sexual and every other biological ability. orchids of which salep is made (orchis coriophora, orchis laxiflora, orchis morio, orchis mascula, orchis pallens, orchis purpurea, orchis simia, orchis tridentata and orchis ustulata) are to be found on numerous habitats of zlatar (in the bright forests, clearing areas and on forest meadows). key words: tuber salep, orchis, drug, pharmacopoeia, production of the orchids introduction ! species from orchidaceae family (order asparagales) are spread over the whole planet. there are about 22000 to 26000 species, classified in about 880 genera (s t e v e n s , 2001). the main development orchids reached in the tropic areas (particularly in the tropic forests), where they live as epiphytes or lianas. in the mild and cold zones they are quite sparse, and they usually grow on the lime base (p a v i ć e v i ć et al., 1968; s a r i ć , ed., 1986; o b r a t o v & m a t o v i ć 1993a; m a t o v i ć et al., 2005). because of the unusually beautiful, impressive and fragrant flowers, numerous species are grown as decorative plants in the form of the numerous strains and hybrids. as economically important plants, orchids has proportional small role. the most important of them is vanilla planifolia jacks., originating from mexico. some species from the orchid family (orchidaceae), are used in medicine because of the abundant content of mucus in the tuber (genus orchis, ophrys, plathantera, gimnadenia, etc). genus orchis consists of about 75 species which grow in europe, mild part of asia, in north africa, on the canary islands and in north america. endangerment of many plant species, their ecology as well as measures for their protection have already proposed (m a t o v i ć , 1985, 1986, 1993; m a t o v i ć et al., 1994, 2005; m a t o v i ć & t a t i ć , 1999; n i k o l i ć et al., 2006). 10th sfses • 17-20 june 2010, vlasina lake1 (1-2) • december 2010: 43-47 biologica nyssana 1 (1-2) december 2010: 43-47 matović, m. et al. natural potentials of the medicinal plants… 44 material and methods ! the greatest part of the data was collected by personal investigation in zlatar, starting from the very foot at the banks of lim, mileševka and uvac, to the greatest peaks of the mountain. this research has been performed since 2006. the spontaneous medicinal flora of zlatar as the natural healing factors in the forest ecosystems was studied in the aim of perceiving the possibilities of its sustainable use for the needs of the health tourism. the orchids of which salep is made are to be found on the numerous forest habitats of zlatar. the optimal conditions the orchids have in the bright forests, clearing areas and forest meadows. the plant species from the orchid family (orchidaceae) which are the subject of the research in this paper were determined by the keys of the modern flora: “pharmacopoea serbica“ (1881-1926), “lekovito bilje u srbiji“ (p e t r o v i ć , 1883), “parmacopoea jugoslavica“ (1933-1984), “flora europaea“ (t u t i n at al., 1964–1980), “flora sr srbije“ (j o s i f o v i ć et al., 1970–1977), “flora of serbia“ (s a r i ć 1986),“lekovite biljke srbije“ (s a r i ć , ed., 1989), o b r a t o v (1992). results and disscusion there are 13 species of orchids in flora of serbia (s a r i ć , 1989), and 9 of them from the genus orchis are present in the zlatar mountain: 1. orchis coriophora l. 1753, 2. orchis laxiflora lam. 1779, 3. orchis morio l. 1753, 4. orchis mascula l. 1755, orchis pallens l. 1771, 6. orchis purpurea hudson 1762, 7. orchis simia lam. 1779, 8. orchis tridentata scop. 1772, and 9. orchis ustulata l. 1753 (diklić in j o s i f o v i ć , 1976; o b r a t o v & m a t o v i ć 1992, 1993b,c; m a t o v i ć & m i h a j l o v , 1993; m a t o v i ć et al., 1993). orchids from zlatar mountain are extremely beautiful, colourful, small, but long-lasting herbaceous plants. their leaves are green, succulent, whole across the rib and have parallel nerves. flower stalk is straight and in the upper part it carries a bunch of unusually beautiful, irregular flowers, which vary in colours, and to the greater extent they vary in form and size. the orchids blossom from spring to the mid summer, which depends upon the altitude. the orchids sometimes have several rootlets and two tubers which are most often egg-shaped, after which the whole genus was named, which was derived from the greek word orchis = egg, because of the appearance of the tuberlets. the egg out of which the overground stem grows in the spring is bigger, but rugged, soft and dark. the egg which is smaller, but succulent, solid, firm, whole, young and bright is placed next to it. as summer goes by, the young egg becomes bigger, and the old one deteriorates and disappears. the healing power of the orchid is placed in the egg-shaped tubers of which salep is made (tuber salep). fig. 1. orchis morio , zlatar mt. (author: m. matović, 2010) the tubers are of different form and size. some of them are egg-shaped, whereas the others are paw and fork shaped. round tubers are of the smaller walnuts' size. the demand for them and their prices are higher than of the fork sized ones, although their healing value is the same. round tubers have the following orchid species from zlatar mountain: orchis mascula, orchis morio, orchis tridentata and orchis purpurea. they all have red or purple flowers. according to round tubers they are similar to some other orchids: orchis militaris and orchis mascula ssp. signifera. this group of the orchids is accompanied with the other similar species with the egg-shaped tubers from which salep is made. there are following species of such orchids: platanthera bifolia which have the white, fragrant flowers and oval leaves and ophrys sphegodes with the vertically eliptic to wide lancet-shaped leaves, the widest in the middle, and ponted on the top). finger-shaped tubers have the following orchids: orchis maculata, orchis latifolia, orchis palustris, orchis sambucina, as well as related species with the finger-shaped tubers (gymnadenia conopsea). while the orchids blossom only the young tuber is extracted. the trained collectors of medicinal plants also extract it after the blossom, as the yield is bigger at the time, and the young tuber is larger. however, one should be careful not to dig the tubers of the other non-medicinal or poisonous biologica nyssana 1 (1-2) december 2010: 43-47 matović, m. et al. natural potentials of the medicinal plants… 45 plants. the special attention should be paid to autumn saffron. extracted tubers are cleaned of soil, washed in cold water, stringed on the thread or place in the net, put in the boiling water, kept for a few minutes until the water boils again or scalded by hot water, then they are dried in the draft, in the sun or in the drier at temperature lower than 700c. by boiling water the tubers lose the unpleasant odour and bitterness which they have when they are fresh, they dry more quickly, starch grains are turned into the glue, and the dried tuber as a result becomes transparent and hornlike. the tubers which are not scalded dry slowly, darken and become moldy before they are completely dried. boling water destroy the enzymes and the salep obtained in such way can be preserve for a long time. the tubers prepared in such way are round, heart-shaped, whereas some resemble fist or are paw-fork-shaped, very hard, hornlike and very heavy (they look like pebble), little transparent on the surface, 1-4 cm long, and 0.5-3 cm wide. on the top they have a stamp of the stem bud. on the surface they are irregulary furrowlike, uneven, netlike rugged, whereas some are almost smooth or little rough. their colour is brownish or whiteyellow. their taste is very mucous and insipid. the name salep is derived from the persian sahlep i schalap= slimy, the name for the little tubers and for the hot drink which is made from them, either from the orchid species of the orchis genus or from the related genera gymnadenia, ophrys, platanthera, etc. the main ingredient of salep is mucus (about 50%), there are 25% of starch, about 5% of albumen, 1% of sugar. it also contains cellulose, fat substances, organic acid and other pharmacodynamic active substances. salep is a typical mucous drug, which is very beneficial and useful. by cooking in water the thick, sinewy, sticky, colloidal solutions are obtained. when used, it forms a noble membrane on the mucosa which alleviates pains and prevents inflammation. therefore, salep has been used in the scientific medicine for centuries as a mild, harmless, efficient healing remedy for treatment of the various diseases. the primary role of salep is to heal and strengthen the organism. salep is often used in the form of so-called "salep mucus". according to the regulations of our pharmacopoeia (pharmacopoeia jugoslavica) our pharmacies prepare this mucus when it is needed, with the doctor's prescription, in the following way: 1 g of salep in powder and 1 g of lactose (milky sugar) are mixed well and this mixture is put in the measured bottle. the mixture is drenched with 2 g of alcohol, mixed, filled up with 10-15 g of boiled destilled water the weight of which is up to 100g. the liquid is often shaked until it is completely cool and the even thich mucus is formed. the mucus prepared in this way is very useful and healing, but children do not like drinking it. therefore, prior to use it has to be mixed well in a glass with currant, orange, lemon, cherry juice, or some other refreshing drink. french and some other pharmacopoeias give this description for making the medicinesweet, pleasant for use, in contrast to a pure salep which is insipid and disagreeable, owing to which many persons, particularly children do not like drinking it: 30g of chocolate is dissolved in the mild heat, 1,000g of salep in the form of the noble powder is added, mixed well and poured in the forms which the children prefer. salep is drunk as a remedy for enjoyment, sweet, and nourishing drink. salep which is cooked in water or milk for a long time the makers sweetened with honey, spiced with aromatic herb powders and poured in order that it is drunk hot. salep is harmlees, nourishing and healing drink, and easily digested food, the use of which is yet to be affirmed. an important precondition for the preservation of the species diversity of orchids on their natural habitats is the large-scale orientation towards the plantation production (fig. 2 and 3). fig. 2. the production of the orchids from the culture, zlatar mt. (author: m. matović, 2010) conclusion relations between man and his search for the medicines in nature have a long tradition, about which different sources in the form of the written data, preserved monuments and original plant medicines testify. knowledge which these sources give to use is undoubtedly the result of a long human struggle with diseases, in which man learnt biologica nyssana 1 (1-2) december 2010: 43-47 matović, m. et al. natural potentials of the medicinal plants… 46 to find the medicines in plants, their bark, seedlings, fruits, leaves and other organs. for many medicines of plant origin, which were known in the oldest human civilizations as well and which have been used for millennia, the modern science confirmed the active effect and incorporated them in pharmacopoeia. fig. 3. experimental plots of orchids from the culture, zlatar mt. (author: m. matović, 2010) the orchids are widely spread in zlatar from the very foot to the top of the mountain. out of 75 species, which grow in europe, the mild part of asia, north africa, on the canary islands and in north america, 13 species are present in flora of serbia and some of them are rare and endangered, and we found 9 orchis species at zlatar mt. alongside with systematic and chemical diversity, zlatar orchids are very variable and morphological. this is particularly expressed in the variability of the shapes of the tubers ranging from round and egg-shaped to the paw-shaped with the numerous transitory forms. salep is obtained from little tubers is typical, mucous drug, which is very efficient and useful. by cooking in water the thick, sinewy, sticky and colloidal solutions are obtained. upon use, salep forms a noble membrane on the mucosa, which covers the mucus, alleviate and soothes the pains and inhibits the inflammation, because of which is has been used in medicine for centuries as a mild, harmless, efficient healing remedy for the treatment of the various diseases. thus, the primary role of salep is to heal and strengthen organism. a number of the medicinal plants which are in circulation have a limited spread in serbia, and owing to the use in folk and official medicine they become endangered, which points to the need of the limited collecting and possibility of the prohibition of the total use in serbia. primary protection of some representatives of orchid family (orchidaceae) comes from the 1973 cites (convention on international trade in endangered wild flora and fauna) and later, from iucn red list (orchid specialist group). for the individuals of these species, their parts and derivates, the trade is prohibited, except for seeds and pollen, seedlings or culture of tissues in vitro obtained individuals, as well as cut plants of flowers produced in the culture. alongside with the protection of these species, it is needed to protect the areas, such as protected natural properties, identify the centers of medicinal plants, as well as the parts of the network of the important plant areas. the protection is carried out in the habitat (in situ) and outside of it (ex situ). medicinal plants with mucous substances have a concrete ability as every other creatures or their groups to take part in the preservation of the accord and harmony in the biosphere. nature is perfectly rational and useful, so it left to a chance not even a particle of the biosphere which would endanger its sustainable development. at preservation and improvement of biodiversity of medicinal plants and other plant species the way of sustainable use of biological resources can be defined, if the economically important biological resources are concerned, or the revitalisation can be applied on the nature habitats, if we want to protect the economically important species. for both approaches it is needed to make the plan and programme of the protection, which contains the information about spread, ecology of the species, endangerment, measures for protection. biologica nyssana 1 (1-2) december 2010: 43-47 matović, m. et al. natural potentials of the medicinal plants… 47 references josifović, m., ed., 1970-1977: flora sr srbije. i-ix. srpska akademija nauka i umetnosti, beograd. matović, m. 1985: vegetacija kanjona mileševke. polimlje (prijepolje), 96 p. matović, m. 1986: biljni pokrivač okoline prijepolja. polimlje (prijepolje), 166 p. matović, m. 1993: reliktna vegetacija srednjeg polimlja. prirodno-matematički fakultet, kragujevac, 144 p. matović, m., mihajlov, m. 1993: contribution to the medical forests plants serbien pancicia and spruce (pancicio-piceetum abietis mat.) on mountain zlatar (serbia). archiv of farmacy, 56, belgrade, pp. 131-135. matović, m., mihajlov, m., svilkić, b., topuzović, m. 1993: the medical plants of the pastures and meadows in the valley of prijepolje (serbia), archiv of farmacy, 5-6, belgrade, pp. 141-144. matović, m., bukvić, s., jovičić, d. 1994: zaštita životne sredine i biljni svet, naučna knjiga, beograd. matović, m., tatić, b. 1999: endemične biljke. zavod za udžbenike i nastavna sredstva, beograd. matović, m., đelić, g., nikolić, b., braunović, s., brašanac, lj. 2005: istraživanje ugroženih retkih biljnih vrsta na području jugozapadne srbije. 8th symposium on flora of southeastern serbia and neighbouring regions. june 20 24, 2005, niš, abstracts, p. 45. nikolić, b., matović, m., rakonjac, lj. 2006: endemorelic tree species in southwest serbia. international scientific conference in occasion of 60 year of operation of institute of forestry, belgrade, serbia: sustainable use of forest ecosystems, the challenge of the 21th century, 8 10 th november 2006, donji milanovac, serbia, the book of abstracts, p. 145, proceeedings, pp. 575-582. obratov, d. 1992: flora i vegetacija planine zlatar. beograd: biološki fakultet, doktorska disertacija. obratov, d., matović, m. 1992: rare plant speces in the forest associations in the central polimlje. protection of nature, 45, belgrade, pp. 47-54. obratov, d., matović, m. 1993a: zastupljenost vrsta sa taninskim svojstvima u flori planine zlatar. iii simpozijum o flori jugoistočne srbije, leskovacpirot, zbornik radova, pp. 17-27. obratov, d., matović, m. 1993b: lekovite biljke smrčevih šuma planine zlatar. savetovanje o lekovitim i aromatičnim biljkama jugoslavije, zlatibor, 8-10. septembar 1993, izvodi radova, p. a-3. obratov, d., matović, m. 1993c: chorological ecomorphologie, taxonomie and phytocoenological characteristics of the species pancicia serbica vis., optima meeting, borovetz, bulgaria, pp. 8-9. pavićević, n. et al. 1968: zemljište starog vlaha i raške, institut za proučavanje zemljišta, beograd. petrović, s. 1883: lekovito bilje u srbiji. srpski arhiv za celokupno lekarstvo, 2, xvi. kraljevska srpska državna štamparija, beograd. ... ph. serb. i, 1881: pharmacopoea serbica, editio prima, typographia principetus serbe, belgradi. ... ph. serb. ii, 1908: srpska farmakopeja, drugo izdanje pharmakopoea serbica, edittio secunda, izdanje kralj. srb. ministarstva unutrašnjih dela, beograd. ... ph. serb. ii, 1926: srpska farmakopeja, drugo izdanje. pharmakopoea serbica, editio secunda, apotekarska komora kraljevine srba, hrvata i slovenaca, beograd. ... ph. jug. i, 1933: parmacopoea jugoslavica mcmxxxiii, jugoslovenska farmakopeja 1933, apotekarska komora, beograd. ... ph. jug. ii, 1951: farmakopeja fnrj, pharmacopoea jugoslavica, editio secunda, medicinska knjiga, beograd. ... ph. jug. iii, 1972: farmakopeja sfrj, pharmacopoea jugoslavica, editio quarta, vol. iii, savezni zavod za zdravstvenu zaštitu, beograd. ... ph. jug. iv, 1984: farmakopeja sfrj, pharmacopoea jugoslavica, edittio quarta, vol. iii, savezni zavod za zdravstvenu zaštitu, beograd. sarić, m. (ed.) 1989: lekovite biljke srbije, srpska akademija nauka i umetnosti, beograd. stevens, p.f. 2001: angiosperm phylogeny website, july 2010. http://www.mobot.org/mobot/research/apweb/ tutin, t.g., v.h. heywood, n.a. burges, d.m. moore, d.h. valentine, s.m. walters & d.a. webb (eds.), 1964-1980: flora europaea, i-v. cambridge university press. london nikolic et al. 2021, biologica nyssana 12(1) 12 (1) september 2021: 23-32 doi: 10.5281/zenodo.5522967 variability of headspace volatiles in native population of abies x borisii-regis from the central rhodopes original article jelena s. nikolić department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, niš, serbia jelenanikolic9311@gmail.com (corresponding author) snežana č. jovanović department of chemistry, faculty of sciences and mathematics, university of niš, višegradska 33, niš, serbia coka.sslagalica@gmail.com bojan zlatković department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, niš, serbia bojanzlat@yahoo.com gordana s. stojanović department of chemistry, faculty of sciences and mathematics, university of niš, višegradska 33, niš, serbia gordanastojanovic2411960@gmail.co zorica mitić department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, niš, serbia saraczorica@gmail.com received: march 31, 2021 revised: july 05, 2021 accepted: july 07, 2021 abstract: the present study investigates variability of headspace needle volatiles in native population of abies x boisii-regis (king boris fir) from the central rhodopes (southern bulgaria) as well as the relationships among this and seventeen previously studied fir populations from the presumed zones of natural hybridization in the balkan peninsula. according to multivariate statistical analyses, all four populations identified as a. x borisii-regis from southern bulgaria, northern and central greece included volatile profiles of both supposed parent species (a. alba and a. cephalonica), where profile frequencies changed clinally along the latitudinal gradient. considering that one of the indicators of the presence of a hybrid zone is the clinical variation of all or a larger number of characters located in the same area, the observed geographic distribution of volatile entities supports the hypothesis that studied a. x borisii-regis populations are of secondary origin due to hybridization between a. alba and a. cephalonica. key words: abies x borisii-regis, rhodopes, headspace, needle volatiles, chemodiversity apstract: varijabilnost hedspejs isparljivih jedinjenja u prirodnoj populaciji abies x borisii-regis sa centralnih rodopa ovo istraživanje ispituje varijabilnost hedspejs isparljivih jedninjenja četina prirodne populacije abies x borisii-regis (jela kralja borisa) sa centralnih rodopa (južna bugarska), kao i odnose između ove i 17 prethodno istraživanih populacija jela iz pretpostavljene zone prirodne hibridizacije na bakanskom poluostrvu. na osnovu multivarijantnih statističkih analiza, sve četiri populacije identifikovane kao a. x borisii-regis iz južne bugarske, severne i centralne grčke uključivale su ispaljive profile obe pretpostavljene roditeljske vrste (a. alba and a. cephalonica), gde su se učestalosti profila klinalno menjale po gradijentu georgrafske širine. uzimajući u obzir da je jedan od pokazatelja prisustva hibridne zone klinalno variranje svih ili većeg broja karaktera lociranih na istom području, dobijena geografska distribucija isparljivih entiteta podržava hipotezu da su ispitivane populacije a. x borisiiregis sekundarnog porekla nastale hibridizacijom a. alba i a. cephalonica. ključne reči: abies x borisii-regis, rodopi, hedspejs, isparljiva jedinjenja četina, hemodiverzitet introduction conifers of the genus abies mill. (pinaceae) play an important ecological role in forest ecosystems of the northern hemisphere (liepelt et al., 2010). most of the abies species are in the temperate zone, with a few of them on high mountains in subtropical areas (xiang et al., 2007). the mediterranean area is considered as one of the abies distribution centers with about eight species depending on particular classification scheme. mediterranean abies spp. are weakly reproductively isolated and relatively easily hybridize in natural and artificial conditions as shown by crossability studies (kormutak, 1985; 2004; moulalis, 1986). © 2021 nikolić et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 23 on the balkan peninsula, there are two native species of the section abies: a. alba mill. (silver fir), widely distributed across central europe and some parts of southern and eastern europe, and a. cephalonica loudon (greek fir), endemic to the mountain massifs of central and southern greece (panetsos, 1975). except them, a. x borisii-regis matff. (king boris fir) was described from the central rhodopes in bulgaria by mattfeld (1926) as a hybrid between a. alba and a. cephalonica. subsequently, turrill (1937) considered as equally possible that king boris fir is an ancient lineage from which a. alba diversified towards the north and a. cephalonica towards the south. in addition, there are a few more mutually exclusive hypotheses about its origin (krajmerová et al., 2015). nevertheless, in most previous classifications, a. x borisii-regis has the status of a hybrid between a. alba and a. cephalonica (chater, 1964; christensen, 1986; christensen, 1997). namely, it is considered that a. alba and a. cephalonica have experienced secondary contact in the refuges during periods of glacial expansion in pleistocene. these events probably led to the exchange of genetic material between these two species in the glacial refugia which contributed to appearance of a. x borisii-regis (liepelt et al., 2010). the reports of a. x borisii-regis distribution range 24 biologica nyssana ● 12 (1) september 2021: 23-32 nikolić et al. ● variability of headspace volatiles in native population of abies x borisii-regis from the central rhodopes fig. 1. geographical position of analyzed populations. for the description of the location and habitat conditions of the populations, cf. tab. 1. 25 are highly controversial: according to one concept a. x borisii-regis appears parapatrically along with the putative parent species, i.e. in its northern distribution with a. alba and in southern with a. cephalonica (jalas & suominen, 1973; christensen, 1997; andreev, 1992). thus, it occurs in southern albania, almost the whole greece (except the cephalonia island and the southern half of the peloponnese), the southernmost parts of northern macedonia and across southwestern and southern bulgaria (jalas & suominen, 1973). on the other hand, based on the atlas of the word’s conifers (farjon & filer, 2013), a. alba, a. x borisii-regis, and a. cephalonica are mostly allopatric, i.e., king boris fir is distributed in southern albania, northern and northeastern greece, and rhodope mt. in southern bulgaria. finally, fady (1993) considered only the populations from pindos mt. and central greece as a. x borisii-regis. the usefulness of terpenes as volatile chemophenetic markers has been addressed in detail by hanover (1992), who emphasized their importance in research of biodiversity, geographic variability, evolution, and systematics, particularly in studies of conifer order pinales. static headspace (hs) extraction is a common technique for analyzing volatile organic compounds, enabling the analyst to study a variety of sample matrices while avoiding the costly and time-consuming preparation involved with traditional gas chromatography (kolb & ettre, 2006). the advantage of combining of hs and gcms/fid (gas chromatography-mass spectrometry/ flame ionization detection) methods in rapid and effective extraction and identification of highly volatile compounds as well as for chemotyping conifers has already been reported (mitić et al., 2020; nikolić et al., 2021). in our previous study, seventeen native fir populations from the presumed zones of natural hybridization in the balkan peninsula were analyzed using hs needle volatiles as chemophenetic markers (nikolić et al., 2021). abies alba and a. cephalonica were characterized by distinct volatile profiles, while three populations from northern and central greece identified as a. x borisii-regis shared the volatile profiles with both of the supposed parent species (nikolić et al., 2021). although king boris fir was described from the central rhodopes in bulgaria (mattfeld, 1926), recent molecular study did not provide evidence on hybrid origin of any bulgarian fir populations including populations from this mountain massif (krajmerová et al., 2015). therefore, the aim of the present study was to shed more light on chemotaxonomic status of controversial bulgarian fir populations. hence, in this article we presented new results for one population of a. x borisii-regis from the southern bulgaria (the central rhodopes) regarding diversity of hs needle volatiles. for multivariate statistical analyses (canonical discriminate analysis and agglomerative hierarchical clustering), in addition to the results obtained in this study, we used our previously published data relating to the broader transition zone between a. alba and a. cephalonica (nikolić et al., 2021). in this way, hs needle volatiles of 18 populations of three abies taxa from the balkan peninsula have been analyzed for the first time (fig. 1). materials and methods plant material plant material of a. x borisii-regis mattf. was collected in the southern bulgaria (the central rhodopes, pamporovo). three-year-old needles were sampled during september 2021 from 14 individuals, which were randomly selected and distant at least 30 m. plant material, deposited in labeled polyethylene bags (sample plot, date of collection, and locality), was immediately transferred to a freezer and stored at -20 °c prior to further analysis. voucher specimen was deposited in the “herbarium moesiacum niš” (hmn) of the department of biology and ecology, faculty of sciences and mathematics, university of niš under the acquisition number 14389. locations of the presently studied population, as well as 17 populations previously analyzed by nikolić et al. (2021) are presented in fig. 1. the corresponding geographic and geologic data, date of collection, voucher information as well as the number of studied individuals for every population are listed in the tab. 1. headspace isolation three-year-old needles of every individual tree were cut into pieces of 2-3 mm length. five hundred milligrams of chopped plant material was placed into 20-ml headspace vials and 1 ml of distilled water was added in each. the prepared vials were placed into a tray for further automated procedure. each sample was heated at 80 °c for 20 minutes with the following program: shaking for 5 seconds, pausing for 2 seconds. after equilibration, the gas vapor of hs volatiles above the aliquot of volatiles was sampled with a gas syringe and subsequently injected into a gc analyzer. gc-ms/fid analyses qualitative analysis of the hs volatiles was performed by gas chromatography-mass spectrometry (gc-ms), while the quantitative data were obtained by gas chromatography-flame ionization detector (gc-fid) analysis. samples biologica nyssana ● 12 (1) september 2021: 23-32 nikolić et al. ● variability of headspace volatiles in native population of abies x borisii-regis from the central rhodopes 26 biologica nyssana ● 12 (1) september 2021: 23-32 nikolić et al. ● variability of headspace volatiles in native population of abies x borisii-regis from the central rhodopes ta bl e 1. l oc at io n an d ha bi ta t d es cr ip tio n of th e se le ct ed p op ul at io ns o f t hr ee a bi es ta xa ta xo n l oc al it y p op ul at io n co de l at it ud e (n ) l on gi tu de (e ) a lt it ud e (m .a .s .l. ) su bs tr at um n um be r of in di vi du al s an al yz ed d at e of co lle ct io n n um be r of va uc he rs a . a lb a r om an ia , s ou th er n c ar pa th ia ns , t ra ns fã gã rã şa n a r c 45 °2 3' 30 " 24 °3 7' 56 " 95 7 si lic at e 5 a ug us t, 20 18 13 95 0 h m n a . a lb a se rb ia , m t. ta ra , p re do v k rs t a st 40 °1 1' 47 " 22 °1 0' 48 " 10 96 l im es to ne 15 ju ne , 2 01 8 13 94 9 h m n a . a lb a se rb ia , m t. k op ao ni k, k ad ije va c a sk 43 °1 9' 43 " 20 °4 5' 41 " 13 50 si lic at e 15 ju ne , 2 01 7 13 94 6 h m n a . a lb a se rb ia , m t. st ar a pl an in a, a rb in je a ss 43 °1 8' 25 " 22 °4 7' 18 " 12 95 si lic at e 15 a ug us t, 20 18 13 94 4 h m n a . a lb a n or th m ac ed on ia , m t. ša rpl an in a, l eš ni ca a m s 42 ° 1' 17 " 20 °4 7' 04 " 15 05 l im es to ne 15 a ug us t, 20 17 13 94 5 h m n a . a lb a n or th m ac ed on ia , m t. n id že a m n 40 °5 8' 54 " 21 °4 7' 31 " 19 00 si lic at e 15 a ug us t, 20 18 13 94 8 h m n a . a lb a b ul ga ri a, m t. pi ri n, a bo ve b an sk o a b p 23 °2 8' 33 " 12 90 si lic at e 15 o ct ob er , 2 01 8 13 94 2 h m n a . a lb a b ul ga ri a, m t. r ila , a bo ve r ila m on as te ry a b r 42 °0 9' 17 " 23 °2 4' 49 " 15 36 si lic at e 15 o ct ob er , 2 01 8 13 94 7 h m n a . x b or is iire gi s b ul ga ri a, m t. r ho do pe s, pa m po ro vo b b r 41 °3 9' 01 " 24 °4 1' 40 " 16 20 si lic at e 14 se pt em be r, 20 20 14 38 9 h m n a . x b or is iire gi s g re ec e, m t. pi er ia , f te ri b g p 40 °1 1' 48 " 22 °1 1' 72 " 92 1 si lic at e 15 o ct ob er , 2 01 8 13 94 3 h m n a . x b or is iire gi s g re ec e, m t. o ly m pu s, a bo ve l ep to ka ry a b g o 40 ° 01 '5 4" 22 °3 0' 25 " 64 0 l im es to ne 15 o ct ob er , 2 01 8 13 95 1 h m n a . x b or is iire gi s g re ec e, m t. ty m fr is to s, s el lo s b g t 38 °5 4' 33 " 21 °5 4' 36 " 97 1 l im es to ne 15 se pt em be r, 20 19 13 99 7 h m n a . c ep ha lo ni ca g re ec e, m t. o iti c g o 38 °4 4' 15 " 22 °2 2' 54 " 89 5 l im es to ne 15 se pt em be r, 20 19 14 00 2 h m n a . c ep ha lo ni ca g re ec e, m t. pa rn as so s, pl ei ad es c g p 38 °3 3' 06 " 22 °3 4' 10 " 16 60 l im es to ne 15 se pt em be r, 20 19 14 00 1 h m n a . c ep ha lo ni ca g re ec e, p el op on ne se , m eg a sp ila io m on as te ry c g m 38 °0 5' 17 " 22 °1 0' 23 " 86 0 l im es to ne 15 se pt em be r, 20 19 14 00 0 h m n a . c ep ha lo ni ca g re ec e, p el op on ne se , m t. c he lm os , k al av ry ta c g k 38 °0 0' 41 " 22 °0 9' 52 " 13 68 l im es to ne 15 se pt em be r, 20 19 13 99 8 h m n a . c ep ha lo ni ca g re ec e, p el op on ne se , m t. c he lm os c g c 38 °0 0' 56 " 22 °1 0' 57 " 15 50 l im es to ne 15 se pt em be r, 20 19 13 99 9 h m n a . c ep ha lo ni ca g re ec e, c ep ha lo ni a, m t. a in os c g a 38 °0 9' 37 " 20 °3 7' 13 " 10 05 l im es to ne 15 ju ly , 2 01 9 13 99 6 h m n were analyzed on agilent technologies 7890b gc equipped with a fused silica capillary column (hp5ms, 250 μm×25 m, film thickness 0.25 μm, agilent technologies, santa clara, ca, usa) and coupled with a 7890a flame ionization detector (fid) and 7000b ms/ms spectrometer (operating in ms1 scan mode) from the same company. the gc was operated under the following conditions: injector temperature 250 °c; gc-ms interface temperature 300 °c; oven temperature programmed from 50 °c for 2.25 minutes, then to 200 °c at 4 °c/minute (carrier gas he, 1.0 ml/min, constant flow mode). ms conditions were as follows: ionization voltage of 70 ev; acquisition mass range 40-440; scan time 0.32 seconds. hs volatiles were identified from tic (total ion chromatogram) by comparison of their linear retention indices relative to c8-c20 n-alkanes recorded on the same column/temperature program with literature values (nist ms search 2.0; nist chemistry webbook srd69; adams, 2007) and their mass spectra with those of standards from databases (wiley 6, nist02, adams) by the application of the amdis software (the automated mass spectral deconvolution and identification system, ver. 2.7, distributed within the software package for 78907000 bgc-ms/ms triple quadrupole system). the percentage composition of the hs volatiles was computed from the gc-fid peak areas without any corrections. statistical analyses statistical data processing was carried out by statistica 8 software (statsoft, inc., tulsa, ok, usa). statistical matrices included hs volatiles that were detected in the content ≥0.5% in at least one of the analyzed taxa (12 components in total: santene, tricyclene, α-pinene, camphene, β-pinene, myrcene, α-phellandrene, limonene+βphellandrene, terpinolene, borneol, bornyl acetate, (e)-caryophyllene). multivariate analysis included canonical discriminant analysis (cda) and agglomerative hierarchical clustering (ahc). overall differences between the compared groups are presented by mahalanobis distances. the calculated matrix distance was used for agglomerative hierarchical clustering (ahc), using the ward’s method. results and discussion variability of hs needle volatiles gc-ms/fid analyses of hs needle volatiles in a. x borisii-regis population from the central rhodopes revealed the presence of 34 compounds (tab. 2). the experimental and literary retention indices, identification methods, and classes to which identified compounds belong are also presented in tab. 2. as the most volatile compounds, monoterpene hydrocarbons represented the major compound class (95.3%), while contents of oxygenated monoterpenes and sesquiterpene hydrocarbons were significantly lower (2.2 and 2.4%, respectively). further, the phytochemical profile of the main hs volatiles for studied population could be presented as follows: β-pinene >>> α-pinene > camphene > limonene+βphellandrene (where, based on petrakis et al. (2001), =, >, >>, and >>> represent difference of 0.1-1.0%, 1.15.0%, 5.1-15.0%, and more than 15.1%, resp.). according to nikolić et al. (2021), the phytochemical profiles of the dominant hs volatiles of a. alba, a. x borisii-regis, and a. cephalonica were: a. alba: β-pinene >> limonene+β-phellandrene > camphene > α-pinene a. x borisii-regis: β-pinene >> α-pinene >> camphene > limonene+β-phellandrene a. cephalonica: β-pinene > α-pinene >>> camphene > limonene+β-phellandrene thus, presently reported volatile profile of bulgarian fir population from the central rhodopes fits well with described profile of greek a. x borisiiregis populations. multivariate analyses (cda and ahc) of hs needle volatiles at the population level the cda based on 259 individuals from 18 populations of a. alba, a. x borisii-regis, and a. cephalonica showed that the first two canonical axes participated in 62.9% of the total discrimination, of which the first axis (ca1) accounted for 44.5% (fig. 2 and tab. 3). four compounds, i.e. β-pinene, limonene+β-phellandrene, camphene, and α-pinene had significant impact on both axes, while bornyl acetate significantly affected only the ca2 (tab. 3). based on the obtained scatterplot (fig. 2), all populations of a. alba showed positive values for the ca1 (yellow ellipse), while a. cephalonica populations formed a group at the negative part of ca1 (blue ellipse). according to the results of the post-hoc test that was done in the study of nikolić et al. (2021), the “alba” profile was characterized by higher content of limonene+β-phellandrene and camphene and lower content of α-pinene compared to the second profile; vice versa the “cephalonica” profile was characterized by lower amount of limonene+β-phellandrene and camphene and higher 27 biologica nyssana ● 12 (1) september 2021: 23-32 nikolić et al. ● variability of headspace volatiles in native population of abies x borisii-regis from the central rhodopes 28 biologica nyssana ● 12 (1) september 2021: 23-32 nikolić et al. ● variability of headspace volatiles in native population of abies x borisii-regis from the central rhodopes table 2. relative percentage composition of hs needle volatiles of bulgarian a. x borisii-regis population from the central rhodopes entry compounds ri1 li2 content (%)3 n=14 class of compounds identification method 1. hexanal 801 801 tr o ri, ms 2. (2e)-hexenal 849 846 0.1±0.2 o ri, ms 3. (3z)-hexenol 850 850 tr o ri, ms 4. hexanol 866 863 tr o ri, ms 5. santene 884 884 0.5±0.2 mh ri, ms 6. tricyclene 921 921 3.3±1.2 mh ri, ms 7. α-pinene 934 932 19.7±2.5 mh ri, ms 8. camphene 949 946 16.3±4.2 mh ri, ms 9. β-pinene 980 974 40.3±5.5 mh ri, ms 10. myrcene 990 988 1.2±0.3 mh ri, ms 11. α-phellandrene 1004 1002 0.3±0.2 mh ri, ms 12. limonene+βphellandrene 1029 1024/1025 13.3±2.1 mh ri, ms 13. 2-heptyl acetate 1040 1038 tr o ri, ms 14. γ-terpinene 1057 1054 tr mh ri, ms 15. terpinolene 1087 1086 0.4±0.1 mh ri, ms 16. 2-nonanone 1089 1087 tr o ri, ms 17. borneol 1166 1165 1.0±0.9 mo ri, ms 18. linalyl acetate 1254 1254 tr mo ri, ms 19. bornyl acetate 1287 1287 1.2±1.0 mo ri, ms 20. α-longipinene 1353 1350 0.1±0.1 sh ri, ms 21. α-ylangene 1373 1373 tr sh ri, ms 22. α-copaene 1377 1374 tr sh ri, ms 23. sibirene 1404 1400 tr sh ri, ms 24. longifolene 1409 1407 tr sh ri, ms 25. (e)-caryophyllene 1420 1417 1.6±1.2 sh ri, ms 26. himachala-2,4-diene 1429 14292a 0.1±0.2 sh ri, ms 27. humulene 1456 1452 0.4±0.3 sh ri, ms 28. β-selinene 1490 1489 tr sh ri, ms 29. δ-selinene 1494 1492 0.1±0.1 sh ri, ms 30. α-selinene 1498 1498 tr sh ri, ms 31. β-himachalene 1503 1500 tr sh ri, ms 32. δ-amorphene 1509 1511 tr sh ri, ms 33. γ-cadinene 1516 1513 tr sh ri, ms 34. δ-cadinene 1525 1522 tr sh ri, ms total 100.0±0.2 monoterpene hydrocarbons (mh) 95.3±1.9 oxygenated monoterpenes (mo) 2.2±1.8 sesquiterpene hydrocarbons (sh) 2.4±1.8 others (o) 0.2±0.2 1ri: experimental linear retention indices relative to c8-c20 alkanes. 2li:literature indices-adams’ retention indices and 2aaccording to nist data base. 3contents are given as percentages (mean±standard deviation) of the total compounds content; n: the number of analyzed individuals; tr trace<0.05%; (-) not detected compounds; the most abundant hs volatiles are in boldface. amount of α-pinene compared to the “alba” profile. accordingly, eastern a. alba lineage and a. cephalonica may be undoubtedly characterized by volatile markers, while populations of a. x borisiiregis were positioned in the intermediate position. specifically, all four populations identified as a. x borisii-regis included volatile profiles of both supposed parent species, whereby profile frequencies changed clinally along the latitudinal gradient (fig. 2). in the northernmost population of a. x borisiiregis from southern bulgaria (rhodopes mt. bbr) most individuals belonged to the “alba” profile (the largest number of green diamonds is situated within the yellow ellipse), while individuals from two northern greece populations were more evenly distributed on both sides of ca1. nevertheless, in a slightly more northern population (pieria mt. bgp) a larger number of individuals belonged to the “alba” profile (green squares more abundant within the yellow ellipse), while individuals from a somewhat southern population (olympus mt. bgo) were evenly distributed on both sides of ca1, indicating approximately equal frequencies of both profiles (green circles with approximately equal representation within both ellipses). finally, in the southernmost a. x borisii-regis population from the central greece (tymfristos mt. bgt) most individuals were located in the negative part of ca1, closer to the “cephalonica” profile (the largest number of green triangles is situated within the blue ellipse). therefore, all four studied a. x borisii-regis populations included both the a. alba and a. cephalonica volatile profiles, but distributed along a latitudinal gradient connecting the ranges of presumed parent species, which confirms the hypothesis proposed in our previous study (nikolić et al., 2021). considering that one of the indicators of the presence of a hybrid zone is the clinical variation of all or a larger number of characters located in the same area, the observed geographic distribution of volatile entities supports the hypothesis that studied a. x borisii-regis populations are of secondary origin due to hybridization between a. alba and a. cephalonica. in addition, clinal (north-south) pattern in the distribution of morpho-anatomical (mitsopoulos & panetsos, 1987) and genetic traits (krajmerová et al., 2015) over the a. x borisii-regis range was previously reported. the dendrogram obtained by ahc analysis has also shown a tendency to divide analyzed fir populations in similar way (fig. 3). namely, populations of a. alba and a. cephalonica were divided in two separate clusters, while populations of a. x borisii-regis appeared heterogeneous. actually, the northernmost population of a. x borisii29 biologica nyssana ● 12 (1) september 2021: 23-32 nikolić et al. ● variability of headspace volatiles in native population of abies x borisii-regis from the central rhodopes variables ca1 ca2 santene -0.105 -0.164 tricyclene 0.827 -0.905 α-pinene 1.123 1.526 camphene 2.193 2.934 β-pinene 4.207 2.859 myrcene 0.063 0.056 α-phellandrene -0.089 0.203 limonene+β-phellandrene 3.690 2.987 terpinolene 0.066 -0.177 borneol 0.468 -0.712 bornyl acetate 0.610 1.239 (e)-caryophyllene 0.730 0.436 eigenvalue 4.197 1.729 % explained variation 0.445 0.629 table 3. standardized coefficients for the first two canonical axes (ca) of variation in 12 needle volatiles from the discriminant functional analysis of 18 a priori groups. the most significant coefficients are in boldface fig. 2. canonical discriminant analysis (cda) based on contents of 12 hs needle volatiles isolated from 259 individuals of 18 populations. abbreviations and symbols refer to populations as indicated in tab. 1. and fig 1. abbreviation colors: yellow – a. alba, green – a. x borisii-regis, and blue – a. cephalonica. the yellow and blue ellipses encompass all individuals of supposed parent species. regis from southern bulgaria (bbr) fitted within the “alba” and all greek populations of a. x borisii-regis (bgp, bgo, and bgt) within the “cephalonica” cluster. furthermore, the northernmost (bbr) and southernmost (bgt) population of a. x borisiiregis showed the greatest resemblance to the geographically closest populations of the parent species: southwestern bulgarian populations of a. alba (abr and abp) and population of a. cephalonica from central greece (cgo), respectively (fig. 3). the history of fir populations from rhodope mt. is largely unknown; fluctuating presence of fir pollen was recorded in rhodope sites since the end of the pleniglacial until the early holocene and an expansion occurred only after 8500 bp (terhürneberson et al., 2004). although king boris fir was described from the central rhodopes (mattfeld, 1926), recent molecular study did not provide evidence on hybrid origin of any of 10 analyzed populations from this mountain massif, based on maternally inherited mitochondrial (mtdna) markers (krajmerová et al., 2015). moreover, these authors found exclusively a. alba mitotype in all fir populations from bulgaria, north macedonia, and northern greece, while only two populations from central greece (tymfrostos and rentina mts.) were mixed and contained predominantly the a. cephalonica mitotype, with a lower proportion of the a. alba mitotype. even though bella et al. (2014) analyzed the same mtdna marker (nad5-4) as in the study of (krajmerová et al., 2015), they found a mixture of maternal lineages (mitotypes of both parent species) in two northern greek populations (pieria and pindos mts.). nevertheless, the second study did not include any of fir populations from central greece, bulgaria, or north macedonia. according to phytochemical markers selected in the present study, presence of both volatile profiles was recorded in a somewhat wider geographical area. i.e. four populations from the southern bulgaria, northern and central greece, where the northernmost population contained predominantly a. alba and the southernmost a. cephalonica profile (fig. 2). thus, based on this concept, a. alba and a. cephalonica were co-occurring at least in the southern bulgaria, northern and central greece, suggesting that this region may have served as a refugium for a. alba during the pleistocene glacial cycles. bearing in mind several proposed distribution concepts of king boris fir, our results are mostly in agreement with the concept given in the atlas of the word’s conifers (farjon & filer, 2013) with the addition of central greece. conclusions gc-ms/fid analyses of hs needle volatiles in population a. x borisii-regis from the southern bulgaria (the central rhodopes) revealed presence biologica nyssana ● 12 (1) september 2021: 23-32 30 nikolić et al. ● variability of headspace volatiles in native population of abies x borisii-regis from the central rhodopes fig. 3. dendrogram obtained by agglomerative hierarchical clustering (ahc) of 18 populations based on 12 hs needle volatiles. abbreviations and symbols refer to populations as indicated in tab. 1. and fig 1. abbreviation colors: yellow a. alba, green a. x borisii-regis, and blue a. cephalonica of 34 compounds. the major hs volatiles were β-pinene (40.3%), α-pinene (19.7%), camphene (16.3%) and limonene+β-phellandrene (13.3%). the presently reported phytochemical profile of the main hs volatiles fits well with previously described profile of greek a. x borisii-regis populations. based on multivariate statistical analyses, all four populations identified as a. x borisii-regis from southern bulgaria, northern and central greece included volatile profiles of both supposed parent species, where profile frequencies changed clinally along the latitudinal gradient connecting the ranges of parent species. thus, based on hs needle volatiles, a. alba and a. cephalonica were co-occurring at least in the southern bulgaria, northern and central greece, suggesting that this region may have served as a refugium for a. alba during the pleistocene glacial cycles. as a natural hybrid, populations of king boris fir can be interesting from the point of view of commercial forestry for the reason that offer the possibility to cope with the predicted forest decline in southern europe as a result of climate change and can be used for reforestation. acknowledgements. this research was supported by grants no 451-03-9/2021-14/200124 by the ministry of education, science and technological development of the republic of serbia. references andreev, n. 1992: pinophyta, in: kozuharov, s. (ed.), field guide to the vascular plants in bulgaria, 81-85, naouka & izkoustvo, sofia (in bulgarian). bella, e., liepelt, s., parducci, l., drouzas, a. 2014: genetic insights into the hybrid origin of abies x borisii-regis mattf. plant systematics and evolution, 301: 749-759. chater, a.o. 1964: abies miller. in: tutin, t.g., heywood, v.h., burges, n.a., valentine, d.h., walters, s.m., webb, d.a. (eds.), flora europaea, 1: 37-38, cambridge university press, cambridge. christensen, k.i. 1986: abies miller. in: strid, a. (ed.), mountain flora of greece, 1: 38-41, cambridge university press, cambridge. christensen, k.i. 1997: abies miller. in: strid, a., tan, k. (eds.), flora hellenica, 1: 1-3, koeltz scientific books, königstein. fady, b. 1993: caractéristiques écologiques et sylvicoles des sapins de grèce dans leur aire naturelle et en plantation dans le sud de la france. perspectives pour le reboisement en règion méditerrané enne. revue forestière française, 45: 119-133. farjon, a., filer, d. 2013: an atlas of the world’s conifers: an analysis of their distribution, biogeography, diversity and conservation status. koninklijke brill nv, leiden. 512 p. hanover, j.w. 1992: applications of terpene analysis in forest genetics. new forests, 6: 159-178. jalas, j., suominen, j. 1973: atlas florae europaeae. 2 gymnospermae (pinaceae to ephedraceae). the committee for mapping the flora of europe & societas biologica fennica vanamo, helsinki, 40 p. kolb, b., ettre, l.s. 2006: static headspace-gas chromatography: theory and practice. wileyinterscience, new york. 349 p. kormutak, a. 1985: study on species hybridization within the genus abies. acta dendrobiologica. veda, bratislava. 127 p. kormutak, a. 2004: crossability relationships between some representatives of the mediterranean, northamerican and asian firs (abies sp.).veda, bratislava. krajmerová, d., paule, l., zhelev, p., voleková, m., evtimov, i., gagov, v., gömöry, d. 2015: natural hybridization in eastern-mediterranean firs: the case of abies borisii-regis. plant biosystematics, 150: 1189-1199. liepelt, s., mayland-quellhorst, e., lahme, m., ziegenhagen, b. 2010: contrasting geographical patterns of ancient and modern genetic lineages in mediterranean abies species. plant systematics and evolution, 284: 141-151. mattfeld, j. 1926: die europäischen und mediterranen abies arten. die pflanzenareale, 1: 22-29 (in german). mitić, z.s., jovanović, s.č., zlatković, b.k., milanovici, s.j., nikolić, b.m., petrović, g.m., stojanović g.s., marin, p.d. 2020: variation of needle volatiles in native populations of pinus mugo evidence from multivariate statistical analysis. plant biosystems-an international journal dealing with all aspects of plant biology, 155(4): 700-710. mitsopoulos, d.j., panetsos, c.p. 1987: origin of variation in fir forests of greece. silvae genetica, 36: 1-15. moulalis, d. 1986: diagnosis and characterization of fir hybrids. scientific annals of the department of forestry and natural environment, aristotle university of thessaloniki, 29: 371-404. nikolić, j.s., zlatković, b.k., jovanović, s.č., stojanović, g.s., marin, p.d., mitić, z.s. 2021: needle volatiles as chemophenetic markers in 31 biologica nyssana ● 12 (1) september 2021: 23-32 nikolić et al. ● variability of headspace volatiles in native population of abies x borisii-regis from the central rhodopes biologica nyssana ● 12 (1) september 2021: 23-32 32 nikolić et al. ● variability of headspace volatiles in native population of abies x borisii-regis from the central rhodopes differentiation of natural populations of abies alba, a. x borisii-regis, and a. cephalonica. phytochemistry, 183: 112612. panetsos, c.p. 1975: monograph of abies cephalonica loudon. annals of forest science, 7: 1-18. petrakis, p.v., tsitsimpikou, c., tzakou, o., couladis, m., vagias, c., roussis, v. 2001: needle volatiles from five pinus species growing in greece. flavour and fragrance journal, 16: 249-252. terhürne-berson, r., litt, t., cheddadi, r. 2004: the spread of abies throughout europe since the last glacial period: combined macrofossil and pollen data. vegetation history and archaeobotany, 13: 257-268. turrill, w.b. 1937: xi-on the flora of the near east: xviii. new species, new records and notes. kew bulletin, 2: 79-86. xiang, x.g., cao, m., zhou, z.k. 2007: fossil history and modern distribution of the genus abies (pinaceae). frontiers of forestry in china, 2: 355365. microsoft word 0304_nesic_et_al biologica nyssana 1 (1-2) december 2010: 65-69 nešić, m. et al. total content of organic acid in plants… 65 original article ! total content of organic acids in plants from fire affected forest marija nešić1*, marija marković1, radmila trajković2, dragana pavlović3, marija ilić4, violeta mitić4, vesna stankov-jovanović4 1 university of niš, faculty of science and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia 2 faculty of science and mathematics, university of kosovska mitrovica, serbia 3 faculty of science and mathematics, university of kragujevac, department of biology and ecology, radoja domanovića 12, 34000 kragujevac, serbia 4 university of niš, faculty of science and mathematics, department of chemistry, ćirila i metodija 1, 18000 niš, serbia *e-mail: nesic_marija@yahoo.com abstract: nešić, m., marković, m., trajković, r., pavlović, d., ilić, m., mitić, v., stankov-jovanović, v.: total content of organic acids in plants from fire affected forest. biologica nyssana, 1 (1-2), december 2010: 65-69. in 2007 catastrophic fire on vidlič mountain had been occurred. it had been burned down nearly 1000 hectares of forest. that year vegetation was totally destroyed. ecosystems affected by fire are those with great changes in variety of ecological parameters and they can recover by natural succession. post fire areas are being occupied by pioneer plants which start one natural cycle. total organic acids content in plants from fire affected forest was studied and the same plant species from forest which had not been affected by fire were taken as a control. total organic acids content for all plants from forest affected by fire was higher than for plants from fire non affected forest except one plant (aegopodium podagraria). key words: organic acids, forest affected by fire, pioneer plants, vidlič mountain introduction ! fire is one of the basic natural forces that influence plant communities over evolutionary period of time. certain plant communities require periodic fires to maintain their position in the ecosystem (m u t c h 1970). according to hypothesis of m u t c h (1970) fire-dependent plant communities burn more readily than fire-nondependent communities, because natural selection has favored development of characteristics that make them more flammable. but, not every forest fire is consequence of natural disturbance. some of them are appeared after human activity and have more sever consequences on landscape and ecosystems. one of such had been appeared on vidlič mountain near pirot town in june 2007 and had last for 10 days and almost 1000 hectares of forest were burnt down. after expiration of fire only grate amount of dust was remained. forest fires create the conditions which are favorable for certain plant species that were not present in that ecosystem earlier or which were presented but with very few number, and they are known as pioneer plants. these plants are specific because they face with many unfavorable ecological factors such as: high illumination, high temperature, low moisture, increased evaporation and finally significant changes in soil composition. fire affected landscapes are very specific according their ecological factors and soil compositions. so, it is very interesting to examine 10th sfses • 17-20 june 2010, vlasina lake1 (1-2) • december 2010: 65-69 biologica nyssana 1 (1-2) december 2010: 65-69 nešić, m. et al. total content of organic acid in plants… 66 biochemical and physiological parameters in plants which inhabit that kind of landscapes. the total amount of organic acids in pioneer plants from fire affected forest has been investigated, taking into consideration their importance as indicators of physiological status of plants and effects of ecological factors on them. material and methods the plant material had been collected one year after forest fire. four plant species have been chosen. control group of plants was collected in the same time but from forest which hadn’t been affected by fire. tested plants were: fagus moesiaca (k.malỳ) czeczott, tussilago farfara l., doronicum columnae ten. and aegopodium podagraria l.. the method by p l e s h k o v (1985) was applied for determination of total organic acids content. accurately weighted mass (20.00 g) of fresh plant material was extracted in blender with 150 ml of distillated water, transferred into erlenmeyer flask and heated in water bath at 70ºc for 30 minutes. after that extract was filtered and total volume of filtrate was adjusted to 200 ml. the filtrate aliquot of 50 ml was transferred into erlenmeyer flask and titrated with 0.1 mol/l standard solution of naoh. the titration endpoint was registered potentiometrically. acidity was calculated by formula: x=axtx200x10/nx50 where: x=content of acids in analyzed solution in mekv/g of fresh plant material, a = volume of 0.1 mol/l standard solution of naoh used for titration in ml, t = factor of 0.1 mol/l standard solution of naoh, 200 = total filtrate volume in ml, 10 = factor for calculating mekv of acids n = mass of plant material used for extract preparation, 50 = volume of filtrate aliquote results and discussion organic acids are secondary metabolites, produced by degradation of carbohydrates, fats and proteins and mostly include: oxalic, formic, citric, fumaric, malic, succinic, acetic and phosphoric acids as well as many others which arise in lower amount (r i v a s s e a u et al., 2006). accumulation of some specific organic acids in plants is in relationship with their enzymatic reactions, growing and development. also, their abundance is under effect of different ecological factors. biochemical function of organic acids is dependent on different ecological conditions. they are donors of protons in some oxido-reduction reactions, for example -conversion of malic to oxalic acid. some earlier studies showed that content of organic acids in plants is dependent on respiration, transpiration, various biochemical processes in plants, plant’s phenophase (g a š i ć , 1992). fig. 1. doronicum columnae ten. fig. 2. aegopodium podagraria l. biologica nyssana 1 (1-2) december 2010: 65-69 nešić, m. et al. total content of organic acid in plants… 67 fig. 3. total content of organic acids in some plant species (ap c= aegopodium podagraria control; ap t= aegopodium podagraria test plant; tf c= tussilago farfara control; tf t= tussilago farfara test plant; fm c= fagus moesiaca control; fm t= fagus moesiaca test plant; dc c= doronicum columnae control; dc t= doronicum columnae test plant) obtained results indicate that the content of organic acids in plants are different in analyzed plant species but, in general, they are higher for plants from fire affected areas than in control plants. the only exception is aegopodium podagraria which has lower total content of organic acids compared with control plant and it is 0.05 mekv/g of fresh weigh in test plant and 0.162 mekv/g of fresh weigh (tab. 1). the highest total content of organic acids in tested plants compared with control had been found in f. moesiaca and it is 115.38% in comparison to the control. the content total content of organic acids in plants from post fire area was 0.270 mekv/g, while in control plants that amount was 0.234 mekv/g (tab. 1) common beech is perennial plant and seedlings which were examined were one year old shoots. acorns had been produced in the same year when forest fire had occurred or one year before that. they had been exposed to heat, fire, fume, dust, high illumination and low humidity which are unfavorable conditions for germination and development of seedlings and so they can be considered as stressed. in those situations plants activate defense mechanisms which are numerous. some of them are species specific, while the others are common for huge number of species (trajković et al., 2007). plant’s organic acids have very important role in plant detoxification caused by heavy metals (j o n e s , 1998; m a , 2000; m a et al., 2001). the lowest level of total organic acids had been found in a. podagraria, and it was supposed that it has different defense mechanisms as well as specific morphological characteristics that are very important for adaptations and survivor in stress conditions (t r a j k o v i ć , 1995; t r a j k o v i ć et al., 2007). in d. columnae the total content of organic acids was 0.202 mekv/g in tested plants and 0.189 mekv/g in control plants. in t. farfara the content in tested plants is 0.108 mekv/g and 0.103 mekv/g in control plants. it had been established that content of organic acids can vary in underground and up ground plant organs in the same species (t r a j k o v i ć , 1995; t r a j k o v i ć et al., 2007). they are of different anatomy, morphology and defenses mechanisms which are genetically caused. it was supposed that plant organs have different activities, different needs and different active processes. analysis of soil indicates that soil which was affected by fire was mineralized and enriched with mineral elements (tab. 2) and some of them are not favorable for plant grow and development (j o n e s , 1998). organic acids are needed for detoxification from heavy metals. they make helate complexes with them and enable their circulation in plant. in the same time that complexes are accumulated in plant organs which later can be excreted (j o n e s , 1998; m a , 2003; a r n e t o l i et al., 2008.). on the other hand it had been demonstrated that iron deficiency causes a substantial accumulation of organic acids in root tissues and also includes a large increase in h+ and organic acids excretion (d e v o s et al., 1986; g u e r i n o t & y i , 1994; o h w a k i & s u g a h a r a , 1997). except that, deficiency of phosphorus (h o f f l a n d et al., 1989; j o h n s o n et al., 1996; d a k o r a & p h i l l i p s , 2002) or elevated concentrations of al3+ ions lead to increasing exudation of organic acid anion in some species (l i et al., 2002; p i r e n o s et al., 2002). it is reported that higher content of organic acids have plants which are tolerant to particular metal (d e l h a i z e et al., 1993; b a s u et al., 1994). conclusion according all this facts and results which were presented one conclusion is irrevocably: synthesis of organic acids is one of the major defense mechanism in plant reaction on stress conditions. it is known that high concentrations of heavy metals cause increase content of organic acids biologica nyssana 1 (1-2) december 2010: 65-69 nešić, m. et al. total content of organic acid in plants… 68 table 1. comparison of total content of organic acids in some plants from post fire region from vidlic mountain with control plant group (mekv/g fresh weigh) plant species control test plant % compared with control aegopodium podagraria l. 0.162 0.050 30.86 tussilago farfara l. 0.103 0.108 104.85 doronicum columnae ten. 0.189 0.202 106.88 fagus moesiaca (k. malý) czeczott 0.234 0.270 115.38 table 2. total content of heavy metals in soil from fire affected and fire non affected forest areas metal ( ppm) plant species pb cd cu zn fe fr om fi re af fe ct ed so il a. podagraria 113.12 0.21 15.78 50.93 9893.64 f. moesiaca 1.94 0 3.92 0 7367.97 t. farfara 202.79 3.545 35.25 130.14 12840.17 d. columnae 146.23 1.89 25.91 56.22 5236.11 fr om no n fi re af fe ct ed so il a. podagraria 30.92 0 13.60 18.10 10764.98 f. moesiaca 14.18 0 18.94 0 16813.46 t. farfara 10.14 2.29 12.32 0 10687.54 d. columnae 34.33 3.50 16.07 37.01 14401.91 in plants (v e r g a n a & g a b r i e l l i , 1987; t r a j k o v i ć , 1995; t r a j k o v i ć et al., 2007). plants on fire affected forest region are high adaptive plants and have mechanism for survival. they can change environment and make favorable conditions for natural succession and invasion of the other plant species. references arnetoli, m., montegrossi, g., buccianti, a., gonnelli, c. 2008: determination of organic acids in plants of silene paradoxa l. by hplc. journal of agricultural and food chemistry 56: 789-795. basu, u., godbold, d., taylor, g.j. 1994: aluminum resistance in triticum aestivum associated with enhanced exudation of malate. journal of plant physiology 144: 747-753. dakora, f.d. and phillips, d.a. 2002: root exudates as mediators of mineral acquisition in law-nutrient environments. plant soil 245: 3547. delhaize, e., ryan, p.r., randall, p.j. 1993: aluminum toxicity and tolerance in plants. plant physiology 103: 695-702. de vos, c.r., lubberding, h.j., bienfait, h.f. 1986: risosphere acidification as a response to iron-deficiency in bean plants. plant physiology 81: 842-846. gašić, olga 1992: biohemija biljaka, naučna knjiga, beograd guerinot, m.l. and yi y. 1994: iron: nutritious, noxious, and not readily available. plant physiology 104: 815-820. hoffland, e., van den boogaard, r., nelemans, j., findenegg, g. 1992: biosynthesis and root exudation of citric and alic acids in phosphatestarved rape plants. new phytology 122: 675680. johnson, j.f., allan, d.l., vance, c.p., weiblen, g. 1996: root carbon dioxide fixation by phosphorous-deficient lupus albus. contribution to organic acid exudation by proteoid roots. plant physiology 112: 19-30. jones, david l. 1998: organic acids in rhizosphere – a critical review. plant and soil 205: 25-44. li, x.f., ma, j.f., matsumoto, h. 2002: aluminium-induced secretion of both citrate and malate in rye. plant soil 242: 235-243. ma, jian feng 2000 role of organic acids in detoxification of aluminium i nhigher plants. plant cell physiology, 41(4): 383-390. ma, jian feng, ryan, peter r. delhaize emmanuel 2001: aluminium tolerance in plants and the complexing role of organic acids. trends in plant science, 6 (6): 273-278. ma, j.f. and furukawa, j. 2003: recent progress in the research of external al detoxification in higher plants: a minir eview. journal of inorganic biochemistry 97: 46-51. mutch, robert, w. 1970: wildland fires and ecosystems – a hypothesis. ecology, 51 (6): 1046-1051. ohwaki, y. and sugahara, k. 1997: active extrusion of protons and exudation of carboxylic acids in response to iron deficiency by roots of chickpea (cicer arietinum l.). plant soil 189: 49-55. biologica nyssana 1 (1-2) december 2010: 65-69 nešić, m. et al. total content of organic acid in plants… 69 pirenos, m.a., magalhaes, j.v., alves, v.m.c., kochian l.v. 2002: the physiology and biophysics of an aluminium tolerance mechanism based on root citrate exudation in maze. plant physiology 129: 1194-1206. плешков, б.п., 1985: практикум по биохемии растений. издателство „колос“, москва. rivasseau, corinne; boisson, anne-marie; mongélard, gaëlle; couram, georgy; bastien, oliver; bligny, richard 2006: rapid analysis of organic acids in plant estracts by capillary electrophoresis with indirect uv detection directed metabolic analyses during metal stress. journal of chromatography a, 1129: 283-290. trajković, radmila 1991: some biochemical indications of effects of air pollutants on plants, master thesis, skopje. trajković, radmila 1995: air pollutants effects on some biochemical and physiological parameters in plants from industrial areas in kosovska mitrovica and obilic, phd disseratation, faculty of science and mathematic, priština. trajković, r., nesić, m., blagojević, n. 2007: content of totally organic acids in plant from industrial area of bor town. 9th symposium of flora and vegetation of southeaster serbia and neighboring areas, nis, 145-151. vergano, o. and gabrielli, r. 1987: the response of plants to heavy metals, organic acids production. g.bot “ital.” 121, n 3-4: 209-212. stojanović et al., 2019, biologica nyssana 10(2) 10 (2) december 2019: 125-133 doi: 10.5281/zenodo.3600189 comparative morphoanatomical analysis of gagea pratensis (pers.) dumort. (liliaceae) from serbia and montenegro original article jovana stojanović university of niš, faculty of sciences and mathematics, department of biology and ecology, niš, serbia jovanagreen6@gmail.com (corresponding author) irena raca university of niš, faculty of sciences and mathematics, department of biology and ecology, niš, serbia racairena@gmail.com jelena jevtić university of niš, faculty of sciences and mathematics, department of biology and ecology, niš, serbia j.jevtic93@gmail.com marina jušković university of niš, faculty of sciences and mathematics, department of biology and ecology, niš, serbia marinaju@pmf.ni.ac.rs vladimir ranđelović university of niš, faculty of sciences and mathematics, department of biology and ecology, niš, serbia vladar@pmf.ni.ac.rs received: november 12, 2019 revised: december 23, 2019 accepted: december 24, 2019 abstract: in this study, morphological and anatomical properties of species gagea pratensis (pers.) dumort. were investigated and described. the comparative analysis included six populations from serbia and montenegro. this study aimed to examine potential morpho-anatomical differentiation and the characters that contribute most to the differences between populations. statistical analyses were carried out for 19 morphological and 5 anatomical characters of 122 specimens using program statistica 7, including the analysis of variance (anova), principal component (pca) and discriminant analysis (cda). analysis of variance showed that almost all investigated characters are statistically significant for differentiation of analysed populations. according to the principal component analysis characters that contributed the most to the separation of populations were height of the whole plant, bulb width, width and length of basal leaf, width and indumentum type of peduncle, length of first cauline leaf, width and length of second cauline leaf, and width of outer and inner segments of perigone. the results of thr discriminant analysis showed the existence of morphoanatomical differentiation between analysed populations, separating the population from locality gamzigradska banja from all other populations. key words: gagea pratensis, morphology, anatomy, variability, differentiation apstract: komparativna morfo-anatomska analiza vrste gagea pratensis (pers.) dumort. (liliaceae) iz srbije i crne gore u ovoj studiji ispitivane su i opisivane morfološke i anatomske karakteristike vrste gagea pratensis (pers.) dumort. komparativnom analizom obuhvaćeno je šest populacija sa prostora srbije i crne gore. cilj istraživanja bio je određivanje potencijalne morfo-anatomske diferencijacije, kao i definisanje onih karaktera koji najviše doprinose postojanju razlika između populacija. sprovedene su statističke analize za 19 morfoloških i 5 anatomskih karaktera na 122 uzorka primenom programa statistica 7, uključujući analizu varijanse (anova), analizu glavnih komponenti (pca) i diskriminantnu analizu (cda). analiza varijanse je pokazala da gotovo svi istraživani karakteri imaju statistički značaj u diferencijaciji analiziranih populacija. na osnovu analize glavnih komponenti izdvojeni su karakteri koji su imali najveći uticaj na razdvajanje populacija i to su visina cele biljke, širina lukovice, širina i dužina bazalnog lista, širina i tip indumentuma stabla, dužina prvog pricvetnog lista, širina i dužina drugog pricvetnog lista i širina unutrašnjih i spoljašnjih segmenata perigona. rezultati diskriminantne analize ukazali su na postojanje morfo-anatomske diferencijacije, pri čemu je populacija sa gamzigradske banje bila jasno izdvojena od svih ostalih populacija. ključne reči: gagea pratensis, morfologija, anatomija, varijabilnost, diferencijacija introduction the genus gagea salisb. (liliaceae) is a taxonomically complicated group, comprising more than 320 species (levichev, 2013; peterson et al., 2016), distributed in temperate and subtropical regions of europe and asia (levichev, 1999a). the primary cause of the taxonomic problems in the genus gagea is the superficial similarity of most of the species (rix & woods, 1981). in the last few decades several new species of the genus gagea salisb. were described based on analyses of © 2019 stojanović et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 125 13th symposium on the flora of southeastern serbia and neighboring regions morphological and anatomical characters (zarrei and zarre, 2003; peruzzi et al., 2007; hamzaoğlu et al., 2008, zarrei et al., 2010a,b; levichev et al., 2019). according to levichev (in peterson et al., 2008) gagea pratensis (pers.) dumort. belongs to section gagea davlianidze, 1972, not. syst. geogr. inst. bot. thbilissiensis. 29: 73., genus gagea salisb., tribe tulipeae and family liliaceae (peruzzi, 2011). section gagea forms a well-defined monophyletic clade which is marked by four morphological synapomorphies angular peduncles and pedicels, second leaf concrescent with peduncle and seeds with distinct arillode (peruzzi et al., 2008a). gagea pratensis is distributed in most parts of europe and asia minor (diklić in josifović, 1975). it is characterized by the bulb and bulblet being exserted (peruzzi et al., 2007). basal leaves have nine or more vascular bundles, first cauline leaves have five barely pronounced keels, pedicels are triangular with six to eight vascular bundles and peduncles are polyedric with a ‘channel-like’ structure inside (peruzzi et al., 2007). gagea pratensis is a highly variable, complex and critical taxon, which is poorly investigated in the balkan peninsula. therefore, in this paper, the morphology and anatomy of g. pratensis from serbia and montenegro were studied in detail. the aim of the study was to examine potential morpho-anatomical differentiation of the studied populations, and to determine the characters that contribute most to the differences between populations. material and methods plant material plant samples were collected from 6 populations of g. pratensis from serbia and montenegro during the flowering time. the sampling localities with associated information are given in the tab. 1. one part of the collected plant material was deposited in herbarium moesiacum niš (hmn). the other part was fixed in 50% alcohol for anatomical study, which was done in the laboratory for plant systematics and ecology (department of biology and ecology, faculty of sciences and mathematics, university of niš, serbia). 126 biologica nyssana ● 10 (2) december 2019: 125-133 stojanović et al. ● comparative morpho-anatomical analysis of gagea pratensis (pers.) dumort. (liliaceae) from serbia and montenegro fig. 1. gagea pratensis: a – habitus, b – underground organs, c – floral details table 1. the sampling localities with associated information (v.r. v. ranđelović, i.r. i. raca, j.j. j. jevtić, d.h. d. harpke) locality n e altitude (m) habitat date collectors voucher gamzigradska banja 43° 92’38” 22° 17’ 03” 164 clearing in an oak forest 18.03.2017. v.r. hmn13289 the jelašnička gorge 43° 28’ 41” 22° 06’ 61” 460 arid subcontinental steppic grassland 23.03.2017. v.r, i.r., j.j. hmn13284 the hill rgotski kamen 44° 02’ 62” 22° 23’ 32” 225 arid subcontinental steppic grassland 18.03.2017. v.r. hmn13287 mt. seličevica 43° 18’ 38” 21° 83’ 09” 802 oak forest 23.03.2017.v.r, i.r., j.j. hmn13285 mt. lovćen 42° 24’ 02” 18º 49’ 33” 1749 subalpine pasture 07.04.2018.v.r, i.r., d.h. hmn13666 mt. vjetarnik 42° 36’ 30” 19º 25’ 49” 1075 10.04.2018.v.r, i.r., d.h. hmn13667 tissue was present below both adaxial and abaxial epidermis in all populations, except that from mt. vjetarnik, where some individuals had palisade tissue only below their abaxial epidermis. big and small vascular bundles were alternately lined in leaf mesophyll. every vascular bundle was surrounded by parenchymatous tissue organised in one or two layers. indumentum was absent from basal leaves. the anatomical characteristics of the first cauline leaf are presented in fig. 3. cross-sections of first cauline leaves of the species g. pratensis had a crescent shape. the anatomy of first cauline leaves is similar to the anatomy of basal leaves. the epidermis was made of square, rectangular to oval-shaped cells all arranged in one layer. leaves were amphystomatic. mesophyll was usually made of spongy parenchyma and one or two layers of palisade parenchyma. palisade tissue can be located only below abaxial (populations from mt. seličevica, mt. vjetarnik and mt. lovćen, and some individuals from the hill rgotski kamen and the jelašnička gorge), below both abaxial and adaxial epidermis (population from gamzigradska banja and some individuals from the hill rgotski kamen) or it can be absent (some individuals from the jelašnička gorge had mesophyll made only of spongy tissue). big and small vascular bundles were alternately lined in leaf morpho-anatomical analysis 19 morphological characters were defined from the fresh plant material. manual microtome (gligorijević & pejčinović, 1983) was utilized in order to make cross-sections of the basal leaves, first cauline leaves, peduncles and pedicels at their widest part. cross-sections were stained with safranin alcian blue, examined by leica dm 2500 microscope and photographed with camera leica dfc490. 5 anatomical characters were analysed. all investigated characters are represented in the tab. 3. statistical analysis basic descriptive statistics was performed for each quantitative character. the significance of differences between the populations studied was established by analysis of variance (anova). principal component analysis (pca) was conducted to determine which characters contributed most to the delimitation of populations, while discriminant analysis (cda) was used to determine the morpho-anatomical differentiation of investigated populations. all statistical analyses were performed using statistica 7.0 software (statsoft. inc., 2004). results the general habitus is represented in fig. 1a, including the details of the underground organs (fig. 1b) and flower (fig. 1c) (photo by j. stojanović). tunic’s colour in all flowering plants was either light or dark brown. in all investigated populations bulbils on peduncle and in the base of cauline leaves were absent. middle stem leaves and indumentum of basal leaves were lacking in all investigated populations. first cauline leaves were sparse ciliate, except some individuals from population from the hill rgotski kamen. peduncles were hairless in all populations, excluding some specimens from the locality of gamzigradska banja and mt. seličevica with sparse hairy peduncles. pedicels, outer perigone and inner perigone segments were glabrous in all populations. perigone segments were yellow on the inside (white colour was only present in the population from the hill rgotski kamen) and green on the outside. the anatomical characteristics of the basal leaf are presented in fig. 2. cross-sections of basal leaves of the species g. pratensis had recognisable v-shape in all investigated populations. both adaxial and abaxial epidermis were made of square, rectangular to oval-shaped cells all arranged in one layer. leaves were amphystomatic and stomata were at the same level as epidermal cells. some indications of mesophyll differentiation were noticed in all investigated populations. palisade biologica nyssana ● 10 (2) december 2019: 125-133 127 figure 2. a – v-shaped cross-section of basal leaf of g. pratensis; b – central part of basal leaf cross-section; c – central vacular bundle (ade – adaxial epidermis, abe – abaxial epidermis, ph – phloem, pp – palisade parenchyma, sp – spongy parenchyma, st – stomata, vb – vascular bundle, vs – vascular sheath, xy – xylem) stojanović et al. ● comparative morpho-anatomical analysis of gagea pratensis (pers.) dumort. (liliaceae) from serbia and montenegro mesophyll. every vascular bundle was surrounded by parenchymatous tissue organized in one or two layers. indumentum was present on first cauline leaves in all investigated populations. the anatomical characteristics of pedicels are presented in fig. 4. crosssections of pedicels of the species g. pratensis had recognizable triangular shape. the epidermis was made of square to oval cells organised in one layer. stomata were at the same level as epidermal cells. both cortex and pith, were made of parenchymatous cells with thin walls. the cortex was made of 3-5 layers of cells. vascular bundles were arranged in a circle, with xylem oriented toward the pith and surrounded by its cells. each vascular bundle was surrounded by a parenchymatous vascular sheath. sclerenchymatous 128 tissue was also present in the form of a ring, made of 2-3 layers of cells, located beneath the cortex and covering only the phloem. indumentum was absent from pedicels. microphotographs of peduncle cross-sections for all investigated populations are presented in fig. 5. this organ was very variable in shape. peduncles with square shape without channel-like structures were noticed in populations from the hill rgotski kamen, mt. lovćen and mt. vjetarnik; deltoid shape without channel-like structures from mt. lovćen and fig. 3. a – cross-section of first cauline leaf of g. pratensis; b – central part of cross-section; c – central vascular bundle; d – leaf edge (ade – adaxial epidermis, abe – abaxial epidermis, ph – phloem, pp – palisade parenchyma, sp – spongy parenchyma, st – stomata, t – trichome, vb – vascular bundle, vs – vascular sheath, xy – xylem) fig. 4. a – general outlook and b – anatomical features of triangular cross-section of pedicel of g. pratensis (cr – cortex, e epidermis, ph – phloem, pi – pith, sc – sclerenchyma, st – stomata, vb – vascular bundle, vs – vascular sheath, xy xylem) fig. 5. comparative overview of peduncle cross-sections of g. pratensis from localities: a – gamzigradska banja, b – seličevica, c – jelašnička klisura, d – rgotski kamen, e – lovćen, f – komovi. biologica nyssana ● 10 (2) december 2019: 125-133 stojanović et al. ● comparative morpho-anatomical analysis of gagea pratensis (pers.) dumort. (liliaceae) from serbia and montenegro 129 mt. komovi; deltoid shape with one channel-like structure from all populations except the populaton from locality gamzigradska banja. deltoid shape with an arm and two channel-like structures was present only in populations from gamzigradska banja and mt. seličevica. deltoid shape without arms and with 2 channel-like structures, as well as, irregular shape with two arms and 2 or 3 channellike structures was noticed only in population from gamzigradska banja. table 2. descriptive statistics of quantitative morphological and anatomical characters of analysed populations of the species g. pratensis. quantitative numerical values are expressed in mm, as mean±standard deviation locality gamzigradska banja jelašnička gorge rgotski kamen seličevica lovćen vjetarnik morphological characters mean±std.dev. height of the whole plant 162.16±26.63 77.85±15.12 113.19±24.54 124.44±14.79 95.98±21.22 85.38±23.95 number of bulbs in common tunic 3.00±0.00 1.00±0.00 1.00±0.00 3.00±1.00 1.00±0.00 1.00±0.00 width of bulb 20.17±4.41 7.03±1.17 9.55±1.83 15.95±3.59 4.74±1.46 7.07±1.55 width of basal leaf 4.51±0.74 2.30±0.63 2.71±0.53 3.54±0.48 2.19±0.63 2.97±0.74 length of basal leaf 217.61±40.80 113.99±30.33 147.28±34.12 159.38±19.89 140.36±38.16 162.93±31.39 width of peduncle 1.84±0.38 1.19±0.21 1.52±0.35 1.40±0.19 0.84±0.22 1.18±0.33 heigh of peduncle from bulb to first cauline leaf 91.23±20.35 46.38±17.10 65.48±22.72 67.62±16.12 56.53±18.27 38.70±18.29 width of first cauline leaf 6.79±1.30 4.73±6.19 5.68±1.20 5.07±0.91 5.70±1.34 6.48±1.72 length of first cauline leaf 60.63±16.64 23.73±6.25 42.59±14.51 42.65±9.24 40.02±9.22 48.03±15.73 width of second cauline leaf 5.07±1.12 1.64±0.57 2.60±0.61 2.88±0.63 2.67±0.59 2.84±0.85 length of second cauline leaf 41.90±9.90 13.13±4.15 24.48±7.90 25.96±5.56 21.69±5.04 26.17±10.41 width of pedicels 1.13±0.16 0.96±0.17 0.88±0.16 1.01±0.10 0.76±0.20 0.75±0.12 number of flowers 3.00±1.00 1.00±1.00 4.00±2.00 2.00±1.00 2.00±1.00 3.00±2.00 length of outer perigone segments 16.65±1.69 12.74±2.93 12.76±2.47 14.57±1.47 12.95±1.62 15.11±2.01 length of inner perigone segments 15.21±1.59 11.63±2.06 11.37±2.25 13.55±1.39 12.21±1.66 14.26±1.95 width of outer perigone segments 4.24±0.72 2.59±0.57 2.52±0.49 3.02±0.32 2.72±0.46 3.42±0.61 width of inner perigone segments 3.23±0.56 2.00±0.49 2.10±0.43 2.47±0.21 2.41±0.34 2.72±0.36 anatomical characters no. of vascular bundles in basal leaf 9.00±0.00 8.00±1.00 8.00±1.00 7.00±1.00 8.00±1.00 8.00±2.00 no. of vascular bundles in first cauline leaf 12.00±3.00 10.00±2.00 12.00±2.00 9.00±2.00 14.00±2.00 13.00±3.00 no. of vascular bundles in pedicel 6.00±1.00 6.00±0.00 6.00±0.00 6.00±0.00 6.00±0.00 6.00±0.00 no. of vascular bundles in peduncle 13.00±1.00 8.00±1.00 9.00±1.00 9.00±1.00 9.00±2.00 11.00±2.00 no. of channel-like structures in peduncle 2.00±0.00 1.00±0.00 0.00±1.00 1.00±1.00 0.00±1.00 1.00±0.00 biologica nyssana ● 10 (2) december 2019: 15-133 stojanović et al. ● comparative morpho-anatomical analysis of gagea pratensis (pers.) dumort. (liliaceae) from serbia and montenegro the anatomical structure was the same regardless the shape of peduncle (fig. 6). the epidermis was made of cubed to oval-shaped cells arranged in one layer. channel-like structures were limited by the cells which are similar to epidermal cells regarding their shape and size. stomata were present in epidermis, as well as in the layer of cells surrounding channel-like structures. cortex was made of 4-5 layers of oval-shaped parenchymal cells with thin walls. sclerenchymatous tissue was present in the form of a ring, made of 2-3 layers of cells, located beneath the cortex and covering only the phloem. vascular bundles were also arranged in the form of a ring. in the case of individuals with channel-like structures, there were additional vascular bundles (1-3) in the area surrounding this structure. it was common that two vascular bundles fuse in the region of xylem, while the phloem stays separated. pith was surrounding xylem, and it was made of parenchymal cells. indumentum was absent from the peduncle, except populations from localities gamzigradska banja and mt. seličevica, where some plants with hairy peduncles were recorded. descriptive statistics is represented in the tab. 2. the results of one-way analysis of variance (tab. 3) showed that all analysed characters had statistical 130 fig. 6. a – cross-section of peduncle of g. pratensis, b – channel-like structure, c – fusion of two vascular bundles in the region of xylem (ch – channel-like structure, cr – cortex, e epidermis, ph – phloem, pi – pith, sc – sclerenchyma, st – stomata, vb – vascular bundle, vs – vascular sheath, xy xylem) table 3. results of one-way analysis of variance (* p<0.05) and principal component analysis (pca) of morphological and anatomical characters of six populations of the species g. pratensis. the first three principal components accounted for 62.69% of the variance characters anovap-values pca 1 pca 2 pca 3 height of the whole plant 0.00* -0.81 -0.04 -0.31 number of bulbs in common tunic 0.00* -0.69 -0.43 0.15 width of bulb 0.00* -0.85 -0.35 -0.01 width of basal leaf 0.00* -0.89 -0.02 -0.06 length of basal leaf 0.00* -0.79 0.22 -0.18 width of peduncle 0.00* -0.72 -0.11 -0.39 height of peduncle from bulb to first cauline leaf 0.00* -0.61 -0.10 -0.36 indumentum of peduncle 0.00* -0.82 -0.18 0.11 first cauline leaf width 0.28 -0.31 0.36 -0.09 length of first cauline leaf 0.00* -0.78 0.41 -0.15 width of second cauline leaf 0.00* -0.90 0.21 -0.05 length of second cauline leaf 0.00* -0.88 0.26 -0.12 width of pedicel 0.00* -0.55 -0.42 -0.06 number of flowers 0.00* -0.36 0.45 -0.58 length of outer perigone segments 0.00* -0.64 0.13 0.35 length of inner perigone segments 0.00* -0.64 0.16 0.36 width of outer perigone segments 0.00* -0.80 0.18 0.30 width of inner perigone segments 0.00* -0.73 0.27 0.23 color of the inner side of perigone segments 0.00* -0.07 -0.01 0.53 no. of vascular bundles in basal leaf 0.00* -0.30 0.18 0.18 no. of vascular bundles in first cauline leaf 0.00* 0.00 0.68 0.16 no. of vascular bundles in pedicel 0.00* -0.27 -0.22 0.43 no. of vascular bundles in peduncle 0.00* -0.66 0.25 0.32 number of channel-like structures in peduncle 0.00* -0.68 -0.46 0.17 biologica nyssana ● 10 (2) december 2019: 125-133 stojanović et al. ● comparative morpho-anatomical analysis of gagea pratensis (pers.) dumort. (liliaceae) from serbia and montenegro significance for the formation of differences between analysed populations of species g. pratensis, except the width of the first cauline leaf. the results of principal component analysis (pca) showed that the three principal components contained 62.69% of the total variability. the most significant contribution to the formation of variability was provided by the first principal component with 44.77%, followed by the second one with 9.96% and the third one with 7.96% of variation. according to pca analysis (tab. 3) characters which contributed most to the differentiation of populations (factor loadings > 0.7) were height of the whole plant, width of bulb, width and length of basal leaf, width and type of indumentum of peduncle, length of first cauline leaf, width and length of second cauline leaf and width of outer and inner perigone segments. the results of discriminant analysis (cda) showed the existence of morpho-anatomical differentiation of analysed populations. populations from the jelašnička gorge, the hill rgotski kamen, mt. lovćen and mt. komovi are on the positive side of the first canonical root, while populations from mt. seličevica and gamzigradska banja are on the negative side. separation is also present on the second canonical root, where populations from the jelašnička gorge and mt. seličevica are on the positive, while populations from mt. lovćen and mt. vjetarnik are on the negative side (fig. 7). discussion in this study, the detailed morpho-anatomical comparison of six populations of g. pratensis from serbia and montenegro was conducted. the primary goal was to examine potential morpho-anatomical differentiation and to determine the characters that contribute most to the differences between populations. as previous studies showed, the morphological characters of significance for taxonomy within the genus gagea salisb. are number and dimensions of underground organs (levichev, 1999b; peruzzi & caparelli, 2007; zarrei et al., 2007; hamzaoğlu et al., 2008; peruzzi et al., 2008b; peruzzi et al., 2011), presence/absence of bulbils (levichev, 1999b; peruzzi et al., 2011; zarrei et al., 2011) and indumentum (hamzaoğlu et al., 2008; wörz et al., 2012), number and dimensions of basal and cauline leaves (rix & woods, 1981; peruzzi & caparelli, 2007; peruzzi et al., 2011; zarrei et al., 2011; wörz et al., 2012), and number, dimensions and colour of flowers (peruzzi & caparelli, 2007; zarrei et al., 2007; hamzaoğlu et al., 2008; peruzzi et al., 2008b; peruzzi et al., 2011; zarrei et al., 2011; levichev et al., 2019). our research confirmed the significance of previously mentioned morphological traits. gagea pratensis is highly variable taxon, therefore wide ranges of values were obtained for almost all investigated characteristics. maximal values of the morphological characters studied were mostly found in the population from gamzigradska banja, which makes it easily distinguishable from all the other populations. also, significant role in the taxonomy of this genus plays the anatomy of basal leaves (zarrei and zarre, 2003; peruzzi et al., 2007; peruzzi et al., 2008a; zarrei et al., 2010b; zarrei et al., 2010c), first cauline leaves (peruzzi et al., 2007), pedicels (peruzzi et al., 2007; peruzzi et al., 2008a, zarrei et al., 2010b; zarrei et al., 2010c) and peduncles (peruzzi et al., 2007; peruzzi et al., 2008a; ajani et al., 2010; zarrei et al., 2010b; zarrei et al., 2010c). relevant anatomical markers are the shape of cross-sections (grossheim, 1935; zarrei et al., 2010c) and the number of vascular bundles (heyn and dafni, 1977; zarrei et al., 2010c). investigated populations of g. pratensis had recognisable v-shaped basal leaves with inner part that was not fistulose, and rather, it was made up of mesophyll, which was in accordance to the study conducted by peruzzi et al. (2008a). mesophyll was not differentiated into palisade and spongy tissue, which was also confirmed by akyol et al. (2015), but some indications of differentiation were noticed in all investigated populations. according to grossheim (1935) presence/absence 131 fig. 7. canonical discriminant analysis of the six g. pratensis populations from serbia and montenegro biologica nyssana ● 10 (2) december 2019: 15-133 stojanović et al. ● comparative morpho-anatomical analysis of gagea pratensis (pers.) dumort. (liliaceae) from serbia and montenegro of central parenchyma in basal leaves is one of the notable characters in terms of taxonomy. this tissue was absent from basal leaves in all investigated populations. the number of vascular bundles in basal leaves was less than nine in all populations, except in population from gamzigradska banja. these results differ from the results obtained by peruzzi et al. (2007). first cauline leaves had similar anatomical structure as basal leaves and presence of keels was noticed, as in the study carried out by peruzzi et al. (2007). cross-sections of pedicels of g. pratensis are triangular (peruzzi et al., 2008a) and number of vascular bundles ranges from 6-8 (peruzzi et al., 2007), which is also confirmed by our study. of all the organs analysed, peduncles showed the highest variability in shape. according to levichev (in ajani et al., 2010) the form of a peduncle is particular, because the second basal leaf accretes with a peduncle from the basis up to an inflorescence and functions as the lower floral leaf. this accretion of basal leaf with a peduncle leads to a dissymmetric and the specific configuration of the transverse section of the peduncle. peduncles are often polyhedric with a ‘channel-like’ structure inside (peruzzi et al., 2007) and two circles of vascular bundles (levichev, 2001). in our research, number of channel-like structures was one in all investigated populations except the populations from seličevica (some plants had one, while others had two channel-like structures) and gamzigradska banja (some plants had two, while others had three channel-like structures). these two populations are also distinguished by the presence of arms and hairs on their peduncles, which has not been recorded in the previous studies. we assume that the largest channel-like structure often followed by the presence of arms is a result of an incomplete concrescence between the first cauline leaf and peduncle. conclusion populations of g. pratensis analysed in this study are morpho-anatomically differentiated. population from gamzigradska banja is separated from all the other populations based on height of the whole plant, width of bulb, width and length of basal leaf, width and indumentum type of peduncle, length of first cauline leaf, width and length of second cauline leaf, and width of outer and inner segments of perigone. the values of the characters analysed in the population from seličevica tend to be transit in between population from gamzigradska banja and all the others populations. in order to determine the basis of morphological and anatomical differences, it is necessary to carry out analysis of the influence of ecological factors on investigated characters, perform experiments with cultivated plants, and conduct a molecular analysis. acknowledgements. the work was funded by the ministry of education, science and technological development of republic of serbia (project no. 173030). references ajani, y., noroozi, j., levichev, i. g. 2010: gagea alexii (liliaceae), a new record from subnival zone of southern iran with key and notes on sect. incrustatae. pakistan journal of botany, 42: 67-77. akyol, y., kocabas, o., yetisen, k. 2015: morphological and anatomical investigations of some gagea salisb. species (liliaceae) in turkey. journal of biological research and development, 5(1): 1-10. diklić, n. 1975: gagea salisb. in: josifović, m. (ed.), flora srbije, 7: 515–522, srpska akademija nauka i umetnosti, beograd. gligorijević, s., pejčinović, d. 1983: contribution to the methodology of anatomical sections and preparation. acta biologiae et medicinae experimentalis, 8: 43-45. grossheim, a. a. 1935: gagea. in: komarov, v. l. (ed.), flora ussr, 4: 61–112, botanical institute, leningrad. hamzaoğlu, e., budak, ü., aksoy, a. 2008: a new species of gagea salisb.(liliaceae) from sivas (central anatolia, turkey). turkish journal of botany, 32(4): 261-264. heyn, c.h., dafni, a. 1977: studies in the genus gagea (liliaceae) ii. the non-latyspermous species from the galilee, the golan heights and mt hermon. israel journal of botany 26: 11– 22. jakab, g., molnár, a. 2011: first record of gagea szovitsii in central europe. biologia, 66(3): 433438. levichev, i. g. 1999a: phytogeographical analysis of the genus gagea salisb. (liliaceae). komarovia, 1: 45-57. levichev, i. g. 1999b: the morphology of gagea salisb.(liliaceae) i. subterranean organs. flora, 194(4): 379-392. levichev, i.g. 2001: new species of the genus gagea salisb. (liliaceae) from western regions of asia. turczaninowia, 4(1-2): 5-35. levichev, i. g. 2006: four new species of the genus gagea salisb.(liliaceae) from western himalayas and the adjoining regions. pakistan journal of botany, 38(1): 47. 132 biologica nyssana ● 10 (2) december 2019: 125-133 stojanović et al. ● comparative morpho-anatomical analysis of gagea pratensis (pers.) dumort. (liliaceae) from serbia and montenegro levichev, i. g. 2013: structural features of shoots in lloydia, gagea, kharkevichia (liliaceae) as evolutionary variability of the modules of mesome nature in monocotyledons. botanicheskii zhurnal, 98(4): 409-452. levichev, i. g., jang, c. g., oh, s. h., lazkov, g. a. 2019: a new species of genus gagea salisb. (liliaceae) from kyrgyz republic (western tian shan, chatkal range, sary-chelek nature reserve). journal of asia-pacific biodiversity, 12(2): 341-343. peruzzi, l. 2011: nomenclatural novelties at sectional level in gagea (liliaceae). atti della società toscana di scienze naturali, memorie, serie b, 118: 23–24. peruzzi, l., bartolucci, f., frignani, f., minutillo, f. 2007: gagea tisoniana, a new species of gagea salisb. sect. gagea (liliaceae) from central italy. botanical journal of the linnean society, 155(3): 337-347. peruzzi, l., caparelli, k. f. 2007: gagea peduncularis (j. & c. presl) pascher (liliaceae) new for the italian flora. webbia, 62(2): 261-268. peruzzi, l., peterson, a., tison, j. m., peterson, j. 2008a: phylogenetic relationships of gagea salisb.(liliaceae) in italy, inferred from molecular and morphological data matrices. plant systematics and evolution, 276(3-4): 219-234. peruzzi, l., tison, j. m., peterson, a., peterson, j. 2008b: on the phylogenetic position and taxonomic value of gagea trinervia (viv.) greuter and gagea sect. anthericoides a. terracc.(liliaceae). taxon, 57(4): 1201-1214. peruzzi, l., peterson, a., tison, j. m., harpke, d. 2011: new light on phylogeny and taxonomy of the eurasian gagea villosa–g. fragifera complex (liliaceae). nordic journal of botany, 29(6): 722733. peterson, a., levichev, i. g., peterson, j. 2008: systematics of gagea and lloydia (liliaceae) and infrageneric classification of gagea based on molecular and morphological data. molecular phylogenetics and evolution, 46(2): 446-465. peterson, a., harpke, d., levichev, i. g., beisenova, s., schnittler, m., peterson, j. 2016: morphological and molecular investigations of gagea (liliaceae) in southeastern kazakhstan with special reference to putative altitudinal hybrid zones. plant systematics and evolution, 302(8): 985-1007. rix, e. m., woods, r. g. 1981: gagea bohemica (zauschner) ja & jh schultes in the british isles, and a general review of the g. bohemica species complex. watsonia, 13: 265-270. wörz, a., hohmann, n., thiv, m. 2012: morphological and molecular diversity of some populations of gagea (liliaceae) in southwest germany. stuttgarter beitr naturk a, neue serie, 5: 1-11. zarrei, m., zarre, s. 2003: a new species of gagea (liliaceae) from iran. nordic journal of botany, 23(3): 269-274. zarrei, m., zarre, s., wilkin, p., rix, m. 2007: systematic revision of the genus gagea salisb. (liliaceae) in iran. botanical journal of the linnean society, 154(4): 559-588. zarrei, m., wilkin, p., ingrouille, m., chase, m. w. 2010a: gagea calcicola (liliaceae), a new species from southwestern iran. kew bulletin, 65(1): 89-96. zarrei, m., wilkin, p., ingrouille, m. j., chase, m. w. 2010b: gagea robusta (liliaceae), a new species from flora iranica area. kew bulletin, 65(2): 327-336. zarrei, m., wilkin, p., ingrouille, m. j., zarre, s., chase, m. w. 2010c: the systematic importance of anatomical data in gagea (liliaceae) from the flora iranica area. botanical journal of the linnean society, 164(2): 155-177. zarrei, m., wilkin, p., noltie, h. j., ingrouille, m. j., chase, m. w. 2011: clarifying the nomenclature and taxonomy of gagea kunawurensis (royle) greuter (liliaceae) and allied taxa. edinburgh journal of botany, 68(1): 43-59. 133 biologica nyssana ● 10 (2) december 2019: 15-133 stojanović et al. ● comparative morpho-anatomical analysis of gagea pratensis (pers.) dumort. (liliaceae) from serbia and montenegro saliaj et al. 2022, biologica nyssana 13(1) 13 (1) september 2022: 11-25 doi: 10.5281/zenodo.7117406 critical revision of lamiaceae family in albanian flora original article oresta saliaj research center of flora and fauna, faculty of natural sciences, university of tirana, albania rr. petro nini luarasi, nr. 80, 1010, tirana, albania oresta.saliaj@fshn.edu.al (corresponding author) alfred mullaj research center of flora and fauna, faculty of natural sciences, university of tirana, albania rr. petro nini luarasi, nr. 80, 1010, tirana, albania julian shehu research center of flora and fauna, faculty of natural sciences, university of tirana, albania rr. petro nini luarasi, nr. 80, 1010, tirana, albania received: may 04, 2022 revised: june 29, 2022 accepted: july 11, 2022 abstract: the territory of albania includes a wide range of habitats and biodiversity. in the 3rd published volume of “flora of albania” lamiaceae family is recognized as labiatae family and includes 32 genera. the aim of this study is to bring an update of data on one of the large families of “flora of albania”, lamiaceae family. first, this study refers to “flora of albania” volumes, as the only complete publication for the albanian flora. most of the data for the critical revision has been extracted from literature, such as the four volumes of “flora of albania”, the five volumes of the “flora europaea”, the medchecklist and the euro+med plantbase. two genera: acinos miller and calamintha miller did not exist so far, and now this two genera are part of clinopodium l. genus. from 276 taxa of this family only 143 remain unchanged like in the 3rd edition of “flora of albania”, whereas 133 taxa are new for albania (with changed species/subspecies names or not even found in any of “flora of albania” published volumes). key words: lamiaceae, new taxa, albania apstrakt: kritički pregled porodice lamiaceae u flori albanije teritorija albanije uključuje širok spektar staništa i biodiverziteta. u trećem publikovanom volumenu “flora albanije” porodica lamiaceae je prepoznata kao porodica labiatae i sadrži 32 roda. cilj ovog istraživanja je da ažurira podatke o jednoj vrstama bogatijih porodica u publikaciji “flora albanije”, porodici lamiaceae. prvo, ovo istraživanje se odnosi na volumene “flora albanije”, kao jedinoj kompletnoj publikaciji flore albanije. većina podataka za kritičnu reviziju je preuzet iz literature, poput četiri volumena “flora albanije”, pet volumena “flora europaea”, medchecklist i euro+med plantbase. dva roda: acinos miller i calamintha miller do sada nisu postojali, a sada su oni deo roda clinopodium l. od 276 taksona ove porodice, svega 143 ostalo je nepromenjeno u odnosu na treće izdanje “flora albanije”, dok je 133 taksona novo za albaniju (sa promenjenim imenima vrsta/podvrsta ili uopšte nisu postojali u publikovanim volumenima “flora albanije”). ključne reči: lamiaceae, novi taksoni, albanija introduction many scientific studies and a lot of fieldwork have been carried out for the “flora of albania”. “flora of albania” represents the most important work in the field of floristic studies, which analyzes 3,235 species and 689 subspecies, grouped in 165 families and 300 genera. it has been published in 4 volumes (paparisto et al., 1988; qosja et al., 1992; qosja et al., 1996; vangjeli etal., 2000) from the institute of biological research, academy of sciences, albania. the material for “flora of albania” is taken mainly by the national herbarium and includes over 150,000 specimens. many scientific publications of foreign and albanian botanists, who have carried out expeditions throughout albania, have been used. the territory of albania includes a wide range of coastal habitats from maritime limestone and sandstone cliffs to sand dunes, saline grasslands and brackish waters to mountains above 2000 m (barina et al., 2017). from year 2000 when the publication of the 4th and last volume of “flora of albania” was completed and onwards, a considerable number of taxa have been reported by many national and foreign authors. from the general analysis of all these studies it results that the albanian flora already consists of about 3,629 species which belong to 960 genera and 170 families (meço & mullaj, 2017). © 2022 saliaj et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 11 lamiaceae (mint family) has a cosmopolitan distribution (heywood et al., 2007). the enlarged lamiaceae contain about 236 genera (raymond et al., 2004). the largest genera are salvia (900), scutellaria (360), stachys (300), plectranthus (300), hyptis (280), teucrium (250), vitex (250), thymus (220), and nepeta (200) (raymond et al., 2004) (https://en.wikipedia.org/wiki/lamiaceae). the stems usually, but not exclusively, have a square cross-sectional. its flowers are bilaterally symmetrical with 5 petals and 5 sepals. lamiaceae are commonly bisexual and they appear to have one whorl of flowers even though they are comprised of two clusters. the leaves are either decussated (crossing one another) or whorled. these plants are most frequently shrubs or trees but could also be vines. this family includes the characteristically aromatic herbs used in cooking such as rosemary, oregano, thyme, basil, mint, marjoram, sage, and lavender (patwardhan & mashelkar, 2009). in the 3rd published volume of “flora of albania” lamiaceae family is recognized as labiatae family and includes 32 genera. the aim of this study is to bring an update of data on one of the large families of “flora of albania”, lamiaceae family, obtained from the use of modern methods of molecular analysis, research expeditions for the recognition and discovery of new species by albanian botanists or foreigners, by completing the lists of families or genera with newly discovered species, establishing current names if they have changed starting from family, genus and species, etc. materials and methods most of the data for the critical revision of lamiaceae family in albanian flora has been extracted from literature, such as the four volumes of “flora of albania” (paparisto et al., 1988; qosja et al., 1992; qosja et al., 1996; vangjeli et al., 2000), the five volumes of the “flora europaea” (tutin et al., 19641980), the medchecklist (greuter et al., 1984-1989) and the euro+med plantbase (http://ww2.bgbm. org/europlusmed/query.asp). euro+med plantbase has been selected as a reference for the taxa names. we have also referred, as an important study on the flora of albania, to the monograph “checklist of vascular plants of albania” (barina et al., 2018). also, to complete the study with the discoveries of recent years, all new species found in the territory of albania (year 2018-2022) and published in international scientific journals have been taken into consideration. additionally, the rest of the information is gathered through visiting the national herbarium of albania at the research center of flora and fauna. results and discussion after revising the family lamiaceae using the above methodology, it results that in albania from this family already exist 32 genera, but most of them with lot of changes that needs to be updated for the albanian flora. all the genera that have undergone major changes have been analyzed one by one. genus ajuga l. ajuga genus is represented by 11 taxa (tab. 1), of which 3 (2 types and 1 species) are not published in any of the volumes of “flora of albania”: 1. ajuga chamaepitys (l.) schreb. subsp. chamaepitys (barina et al., 2018) 2. ajuga pyramidalis l. subsp. pyramidalis (the euro+med plantbase, 2010) 3. ajuga laxmannii (l.) benth. (barina et al., 2018) genus ballota l. ballota genus is represented by 7 taxa (tab. 1), of which 2 have new accepted names by “the euro+med plantbase”. 1. ballota hispanica (l.) benth. in the 3rd volume of “flora of albania” this species is known as ballota rupestris (biv.) vis. 2. ballota nigra subsp. ruderalis (sw.) briq. this subspecies in the same volume of “flora of albania” is known as subspecies unicata. genus clinopodium l. this genus for albania brings many changes. first, according to the euro+med plantbase (http://ww2. bgbm.org/europlusmed/query.asp), the genera calamintha and acinos, which are present in the 3rd published volume of “flora of albania”, have already changed the name of genus and are part of the genus clinopodium. from the genus clinopodium in “flora of albania”, only two taxa are mentioned: clinopodium vulgare l., and clinopodium vulgare l. subsp. vulgare. meanwhile, with all the analysis done, already, 23 taxa exist for the genus clinopodium (tab. 1). 1. clinopodium acinos (l.) kuntze. known as acinos arvensis (lam.) dandyin “flora of albania” 3rd edition. 2. clinopodium alpinum (l.) kuntze. known as acinos alpinus (l.) moench in “flora of albania” 3rd edition. 3. clinopodium alpinum subsp. albanicum (kümmerle & jáv.) govaerts. new subspecies according to the euro+med plantbase 2010, not published in any of the volumes of “flora of albania”. 12 biologica nyssana ● 13 (1) september 2022: 11-25 saliaj et al. ● critical revision of lamiaceae family in albanian flora table 1. lamiaceae family in albanian flora genera species ajuga l. ajuga chamaepitys (l.) schreb. ajuga chamaepitys subsp. chia (schreb.) ajuga chamaepitys (l.) schreb. subsp. chamaepitys ajuga genevensis l. ajuga orientalis l. ajuga piskoi degen & bald. ajuga pyramidalis l. ajuga pyramidalis l. subsp. pyramidalis ajuga reptans l. ajuga laxmannii (l.) benth. ajuga iva (l.) schreb. ballota l. ballota hispanica (l.) benth. ballota macedonica vandas ballota nigra l. ballota nigra subsp. foetida hayek ballota nigra l. subsp. nigra. ballota nigra subsp. sericea (vandas) patzak. ballota nigra subsp. ruderalis (sw.) briq. clinopodium l. clinopodium acinos (l.) kuntze clinopodium alpinum (l.) kuntze clinopodium alpinum subsp. alpinum clinopodium alpinum subsp. albanicum (kümmerle & jáv.) govaerts clinopodium alpinum subsp. hungaricum (simonk.) govaerts clinopodium alpinum subsp. meridionale (nyman) govaerts clinopodium grandiflorum (l.) kuntze clinopodium grandiflorum (l.) kuntze subsp. grandiflorum clinopodium menthifolium (host) stace clinopodium menthifolium subsp. ascendens (jord.) govaerts clinopodium menthifolium (host) stace subsp. menthifolium clinopodium nepeta (l.) kuntze clinopodium nepeta (l.) savi subsp. nepeta clinopodium nepeta subsp. glandulosum (req.) govaerts clinopodium suaveolens (sm.) kuntze clinopodium serpyllifolium (m. bieb.) kuntze clinopodium thymifolium (scop.) kuntze clinopodium vardarense (šilic) govaerts clinopodium vulgare l. clinopodium vulgare subsp. orientale bothmer clinopodium vulgare l. subsp. vulgare biologica nyssana ● 13 (1) september 2022: 11-25 saliaj et al. ● critical revision of lamiaceae family in albanian flora 13 14 biologica nyssana ● 13 (1) september 2022: 11-25 saliaj et al. ● critical revision of lamiaceae family in albanian flora genera species clinopodium l. clinopodium graveolens subsp. rotundifolium (pers.) govaerts clinopodium suaveolens (sibth. & sm.) g. don. galeopsis l. galeopsis bifida boenn. galeopsis ladanum l. galeopsis pubescens besser galeopsis speciosa mill. galeopsis tetrahit l. glechoma l. glechoma hederacea l. glechoma hirsuta waldst. & kit. hyssopus l. hyssopus officinalis l. hyssopus officinalis subsp. aristatus (godr.) nyman. hyssopus officinalis l. subsp. officinalis lamium l. lamium amplexicaule l. lamium amplexicaule l. var. amplexicaule lamium galeobdolon (l.) crantz. lamium galeobdolon (l.) crantz subsp. galeobdolon lamium galeobdolon subsp. montanum (pers.) hayek lamium garganicum l. lamium garganicum l. subsp. garganicum lamium garganicum subsp. striatum (sm.) hayek lamium maculatum (l.) l. lamium purpureum l. lamium purpureum l. var. purpureum lamium bifidum cirillo lamium flexuosum ten. lamium orvala l. leonurus l. leonurus cardiaca l. lavandula l. lavandula angustifolia mill. lycopus l. lycopus europaeus l. lycopus exaltatus l. f. marrubium l. marrubium anisodon k. koch. marrubium cylleneum boiss. & heldr. marrubium incanum desr. marrubium peregrinum l. marrubium thessalum boiss. & heldr. marrubium vulgare l. marrubium × paniculatum desr. melissa l. melissa officinalis l. melissa officinalis subsp. altissima (sm.) arcang. melissa officinalis subsp. officinalis 15 biologica nyssana ● 13 (1) september 2022: 11-25 saliaj et al. ● critical revision of lamiaceae family in albanian flora genera species melittis l. melittis melissophyllum l. melittis melissophyllum subsp. albida (guss.) p. w. ball mentha l. mentha aquatica l. mentha longifolia (l.) l. mentha longifolia (l.) l. subsp. longifolia mentha pulegium l. mentha spicata l. mentha spicata subsp. condensata (briq.) greuter & burdet. mentha spicata l. subsp. spicata mentha × dumetorum schult. mentha × piperita l. mentha × rotundifolia (l.) huds. mentha suaveolens ehrh. micromeria benth. micromeria albanica (k. malý) šilic micromeria cremnophila boiss. & heldr. micromeria cremnophila boiss. & heldr. subsp. cremnophila micromeria cristata (hampe) griseb. micromeria cristata (hampe) griseb. subsp. cristata micromeria cristata subsp. kosaninii (šilic) ined. micromeria croatica (pers.) schott. micromeria graeca (l.) benth. micromeria graeca subsp. fruticulosa (bertol.) guinea micromeria juliana (l.) benth. ex rchb. micromeria parviflora rchb. micromeria microphylla (d'urv.) benth. micromeria myrtifolia boiss. & hohen. micromeria nervosa (desf.) benth. nepeta l. nepeta cataria l. nepeta nuda l. nepeta nuda l. subsp. nuda nepeta parnassica heldr. & sart. nepeta spruneri boiss. nepeta argolica baden. nepeta argolica baden subsp. malacotrichos (baden) a.strid & kit. tan. ocimum l. ocimum basilicum l. origanum l. origanum vulgare l. origanum vulgare subsp. hirtum (link) ietsw. origanum vulgare l. subsp. vulgare origanum vulgare l. subsp. viridulum (martrindonos) nyman. origanum majorana l. 16 biologica nyssana ● 13 (1) september 2022: 11-25 saliaj et al. ● critical revision of lamiaceae family in albanian flora genera species phlomis l. phlomis fruticosa l. phlomis herba-venti l. phlomis herba-venti l. subsp. herba-venti phlomis tuberosa l. prasium l. prasium majus l. prunella l. prunella albanica pénzes. prunella grandiflora (l.) scholler. prunella grandiflora (l.) scholler subsp. grandiflora prunella laciniata (l.) l. prunella vulgaris l. prunella vulgaris l. subsp. vulgaris prunella × intermedia link. prunella × surrecta dumort. rosmarinus l. rosmarinus officinalis l. rosmarinus officinalis l. subsp. officinalis salvia l. salvia amplexicaulis lam. salvia argentea l. salvia candidissima vahl. salvia fruticosa mill. salvia glutinosa l. salvia nemorosa l. salvia nemorosa l. subsp. nemorosa salvia officinalis l. salvia officinalis l. subsp. officinalis salvia pratensis l. salvia pratensis l. subsp. pratensis salvia ringens sm. salvia sclarea l. salvia tomentosa mill. salvia verbenaca l. salvia verticillata l. salvia verticillata l. subsp. verticillata salvia virgata jacq. salvia viridis l. salvia aethiopis l. salvia microphylla kunth. salvia splendens sellow ex schult. satureja l. satureja cuneifolia ten. satureja hortensis l. 17 biologica nyssana ● 13 (1) september 2022: 11-25 saliaj et al. ● critical revision of lamiaceae family in albanian flora genera species scutellaria l. satureja montana l. satureja montana l. subsp. montana satureja montana l. subsp. variegata (host) p.e.ball. satureja subspicata bartl. ex vis. satureja subspicata bartl. ex vis. subsp. subspicata satureja parnassica heldr. & sart. ex boiss. satureja kitaibelii wierzb. ex heuff. scutellaria alpina l. scutellaria altissima l. scutellaria columnae all. scutellaria columnae all. subsp. columnae scutellaria galericulata l. scutellaria orientalis l. scutellaria orientalis subsp. pinnatifida j. r. edm. scutellaria rupestris boiss. & heldr. scutellaria rupestris subsp. adenotricha (boiss. & heldr.) greuter & burdet. scutellaria hastifolia l. scutellaria rubicunda hornem. scutellaria rubicunda hornem. subsp. rubicunda sideritis l. sideritis montana l. sideritis montana l. subsp. montana sideritis raeseri boiss. & heldr. sideritis raeseri boiss. & heldr. subsp. raeseri sideritis romana l. sideritis romana l. subsp. romana sideritis romana l. subsp. purpurea (talbot ex benth.) sideritis scardica griseb. stachys l. stachys albanica markgr. stachys alopecuros (l.) benth. stachys alopecuros (l.) benth. subsp. alopecuros stachys alpina l. stachys alpina l. subsp. alpina stachys alpina l. subsp. dinarica murb. stachys anisochila vis. & pancic. stachys annua (l.) l. stachys annua (l.) l. subsp. annua stachys arvensis (l.) l. stachys atherocalyx k. koch. stachys beckeana dörfl. & hayek stachys cretica l. genera species stachys l. stachys cretica subsp. cassia (boiss.) rech. f. stachys cretica l. subsp. cretica stachys cretica subsp. salviifolia (ten.) rech. f. stachys cretica subsp. bulgarica rech. f. stachys germanica l. stachys germanica l. subsp. germanica stachys germanica subsp. heldreichii (boiss.) hayek stachys germanica subsp. penicillata (heldr. & sart. ex boiss.) nyman stachys maritima gouan. stachys menthifolia vis. stachys mollissima willd. stachys obliqua waldst. & kit. stachys officinalis (l.) trevis. stachys officinalis (l.) trevis. subsp. officinalis stachys officinalis subsp. skipetarum jáv. stachys palustris l. stachys recta l. stachys recta subsp. baldaccii (k. malý) hayek. stachys recta subsp. doerfleri (hayek) hayek. stachys recta l. subsp. recta stachys recta subsp. subcrenata (vis.) briq. stachys recta subsp. labiosa (bertol.) briq. stachys scardica (griseb.) hayek. stachys serbica pancic. stachys sericophylla halácsy. stachys spinulosa sm. stachys sylvatica l. stachys tymphaea hausskn. stachys canescens bory & chaub. stachys parolinii vis. stachys plumosa griseb. teucrium l. teucrium arduinii l. teucrium capitatum l. teucrium capitatum l. subsp. capitatum teucrium chamaedrys l. teucrium chamaedrys l. subsp. chamaedrys teucrium flavum l. teucrium flavum l. subsp. flavum teucrium montanum l. teucrium montanum l. subsp. montanum 18 biologica nyssana ● 13 (1) september 2022: 11-25 saliaj et al. ● critical revision of lamiaceae family in albanian flora 19 biologica nyssana ● 13 (1) september 2022: 11-25 saliaj et al. ● critical revision of lamiaceae family in albanian flora genera species thymbra l. teucrium scordium l. teucrium scordium subsp. scordioides (schreb.) arcang. teucrium polium l. teucrium fruticans l. teucrium divaricatum sieber ex heldr. teucrium botrys l. thymbra capitata (l.) cav. thymus l. thymus boissieri halácsy. thymus doerfleri ronniger. thymus longicaulis c. presl. thymus longicaulis subsp. chaubardii (rchb. f.) jalas. thymus longicaulis c. presl subsp. longicaulis thymus parnassicus halácsy. thymus praecox opiz. thymus praecox subsp. jankae (čelak.) jalas. thymus praecox subsp. polytrichus (a. kern. ex borbás) jalas. thymus praecox subsp. zygiformis (heinr. braun ex wettst.) jalas. thymus pulegioides l. thymus pulegioides subsp. montanus (benth.) ronniger. thymus pulegioides l. subsp. pulegioides thymus sibthorpii benth. thymus striatus vahl. thymus teucrioides boiss. & spruner. thymus teucrioides boiss. & spruner subsp. teucrioides thymus thracicus velen. thymus × oblongifolius opiz. thymus ciliatopubescens (halácsy) halácsy. thymus dacicus borb. thymus degenii heinr. braun. thymus odoratissimus mill. thymus heterotrichus griseb. thymus roegneri k. koch thymus leucotrichus halácsy. thymus serpyllum l. thymus stojanovii degen. vitex l. vitex agnus-castus l. ziziphora l. ziziphora capitata l. ziziphora capitata l. subsp. capitata 4. clinopodium alpinum subsp. hungaricum (simonk.) govaerts. known as acinos alpinus subsp. majoranifolius (mill.) p.w.ball in “flora of albania”. biologica nyssana ● 13 (1) september 2022: 11-25 20 saliaj et al. ● critical revision of lamiaceae family in albanian flora 5. clinopodium alpinum subsp. meridionale (nyman) govaerts. in “flora of albania” 3rd edition it’s known as acinos alpinus subsp. meridionalis (nyman) p.w.ball. 6. clinopodium alpinum subsp. alpinum (not published in any of the volumes of “flora of albania”), according to barina et al., (2018), it’s present in albania under the genus acinos). 7. clinopodium grandiflorum (l.) kuntze. known as calamintha grandiflora (l.) moench. in “flora of albania” 3rd edition. 8. clinopodium grandiflorum (l.) kuntze subsp. grandiflorum. new subspecies according to the euro+med plantbase 2010, not published in any of the volumes of “flora of albania”. 9. clinopodium menthifolium (host) stace. known as calamintha sylvatica bromf. in “flora of albania”. 10. clinopodium menthifolium subsp. ascendens (jord.) govaerts. in “flora of albania” 3rd edition it’s known as calamintha sylvatica bromf. subsp. ascendens (jord.) p.w.ball. 11. clinopodium menthifolium (host) stace subsp. menthifolium. new subspecies according to the euro+med plantbase 2010, not published in any of the volumes of “flora of albania”. 12. clinopodium nepeta (l.) kuntze. known as calamintha nepeta (l.) savi (l.) moench in “flora of albania”. 13. clinopodium nepeta subsp. glandulosum (req.) govaerts. in “flora of albania” it’s known as calamintha nepeta (l.) savi subsp. glandulosa (req.) p.w.ball. 14. clinopodium nepeta (l.) savi subsp. nepeta. in “flora of albania” it’s known as calamintha nepeta (l.) savi subsp. nepeta. 15. clinopodium suaveolens (sm.) kuntze. new species according to the euro+med plantbase 2010, not published in any of the volumes of “flora of albania”. 16. clinopodium thymifolium (scop.) kuntze. in “flora of albania” it is known as micromeria thymifolia (scop.) fritsch. 17. clinopodium vardarense (icilic) govaerts. known as satureja vardarensis (šilić) greuter & burdet in “flora of albania” 3rd volume. 18. clinopodium vulgare subsp. orientale bothmer. known as clinopodium vulgare subsp. arundanum boiss. in “flora of albania”. 19. clinopodium serpyllifolium (m. bieb.) kuntze. it’s not published in any of the volumes of “flora of albania”, but according to barina et al. (2018), it’s known as micromeria fruticosa (l.) druce subsp. serpyllifolia (m.bieb.) p.h.davis. 20. clinopodium graveolens subsp. rotundifolium (pers.) govaerts. it’s not published in any of the volumes of “flora of albania”. according to barina et al. (2018), it is present in albania under the name acinos rotundifolius pers. 21. clinopodium suaveolens (sibth. & sm.) g. don. it’s not published in any of the volumes of “flora of albania”. according to barina et al. (2018), it’s present in albania under the genus acinos. genus hyssopus l. hyssopus genus consists in 1 species and 2 subspecies (tab. 1), of which in “flora of albania” only the species hyssopus officinalis l. is found, without its type and the other subspecies. 1. hyssopus officinalis subsp. aristatus (godr.) nyman (the euro+med plantbase, 2010). 2. hyssopus officinalis l. subsp. officinalis (the euro+med plantbase, 2010). genus lamium l. even the genus lamium has undergone many taxonomic changes over the last few years. it is now accepted that this genus includes 14 taxa (tab. 1), of which 8 taxa are not found published in any of the volumes of “flora of albania”. 1. lamium amplexicaule l. var. amplexicaule (the euro+med plantbase, 2010). 2. lamium galeobdolon (l.) crantz. in “flora of albania” 3rd edition it’s known as lamiastrum galeobdolon (l.), so the name of the genus has changed from lamiastrum to lamium. 3. lamium galeobdolon (l.) crantz subsp. galeobdolon. in “flora of albania” 3rd edition it is known as lamiastrum galeobdolon (l.) subsp. galeobdolon, so the name of the genus has changed from lamiastrum to lamium. 4. lamium galeobdolon subsp. montanum (pers.) hayek. in “flora of albania” 3rd edition it is known as lamiastrum galeobdolon (l.) subsp. montanum, so the name of the genus has changed from lamiastrum to lamium. 5. lamium garganicum subsp. striatum (sm.) saliaj et al. ● critical revision of lamiaceae family in albanian florabiologica nyssana ● 13 (1) september 2022: 11-25 21 hayek (the euro+med plantbase, 2010, barina et al., 2018). 6. lamium purpureum l. var. purpureum (the euro+med plantbase, 2010). 7. lamium flexuosum ten. (barina et al., 2018). 8. lamium orvala l. this species is most likely missing in albania and its presence needs further confirmation. according to barina et al. (2018), the presence of this species is reported only once (hayek, 1917). genus marrubium l. this genus in albania is already represented by 7 species (tab. 1), of which 3 are not found in “flora of albania” published editions. 1. marrubium anisodon k. koch. in the 3rd volume of “flora of albania”, it is known as marrubium alternidens rech. 2. marrubium thessalum boiss. & heldr. (barina et al., 2018). 3. marrubium × paniculatum desr. this hybrid species is most likely missing in albania and its presence needs further confirmation. according to barina et al. (2018), the presence of this species is reported only once (schütt, ~1939). genus mentha l. mentha genus has also undergone many taxonomic changes. it is now accepted that 11 taxa belong to this genus (tab. 1), of which 5 (2 types, 1 subspecies and 2 hybrid species) are not found in published editions of “flora of albania”. 1. mentha longifolia (l.) l. subsp. longifolia (the euro+med plantbase, 2010) 2. mentha spicata subsp. condensata (briq.) greuter & burdet. new name for albania according to the euro+med plantbase, in “flora of albania” 3rd edition, it’s known as mentha microphylla k. koch. 3. mentha spicata l. subsp. spicata (the euro+med plantbase, 2010) 4. mentha × dumetorum schult. the presence of this hybrid species in albania needs further confirmation, because according to barina et al. (2018), its presence is reported only once (schütt ,~1939). 5. mentha × rotundifolia (l.) huds. (barina et al., 2018) genus micromeria benth. the genus micromeria is already represented by 14 taxa (tab. 1), of which 8 taxa are not found in the “flora of albania”. 1. micromeria albanica (k. malý) šilic. (barina et al., 2018) 2. micromeria cremnophila boiss. & heldr. subsp. cremnophila (the euro+med plantbase, 2010) 3. micromeria cristata (hampe) griseb. subsp. cristata. according to barina et al. (2018), the presence of this subspecies in albania needs further confirmation, because it is reported only once (greuter et al., 1986). 4. micromeria cristata subsp. kosaninii (šilic) ined. (barina et al., 2018) 5. micromeria croatica (pers.) schott. (barina et al., 2018) 6. micromeria graeca subsp. fruticulosa (bertol.) guinea. new subspecies according to the euro+med plantbase 2010. 7. micromeria microphylla (d’urv.) benth. (barina et al., 2018) 8. micromeria nervosa (desf.) benth. (barina et al., 2018) genus nepeta l. nepeta genus is represented by 7 taxa (tab. 1), of which only 3 (1 type, 1 species and 1 subspecies) are not found in the volumes of “flora of albania”. 1. nepeta nuda l. subsp. nuda (the euro+med plantbase, 2010) 2. nepeta argolica baden (barina et al., 2018) 3. nepeta argolica baden subsp. malacotrichos (baden) a. strid & kit. tan. (barina et al., 2018) genus origanum l. in albania this genus is represented by 5 taxa (tab. 1), of which only 3 taxa (1 type and 2 subspecies) are not found in the “flora of albania” published editions. 1. origanum vulgare subsp. hirtum (link) ietsw. (barina et al., 2018) 2. origanum vulgare l. subsp. vulgare (barina et al., 2018) 3. origanum vulgare l. subsp. viridulum (martrindonos) nyman. (barina et al., 2018) genus phlomis l. in albania, nowadays, this genus is represented by 4 taxa (tab. 1), of which 2 (1 type and 1 species) are not published in “flora of albania”. 1. phlomis herba-venti l. subsp. herba-venti (the euro+med plantbase, 2010) 2. phlomis tuberosa l. (barina et al., 2018) biologica nyssana ● 13 (1) september 2022: 11-25 saliaj et al. ● critical revision of lamiaceae family in albanian flora genus prunella l. the genus prunella is already represented by 8 taxa (tab. 1), of which 5 (1 species, 2 types and 2 hybrid species) are not found in “flora of albania” published volumes. 1. prunella albanica pénzes (the euro+med plantbase, 2010) 2. prunella grandiflora (l.) scholler subsp. grandiflora (the euro+med plantbase, 2010) 3. prunella vulgaris l. subsp. vulgaris (the euro+med plantbase, 2010) 4. prunella × intermedia link. (barina et al., 2018) 5. prunella × surrecta dumort. according to barina et al. (2018), for this hybrid species there is lack of information (no reported location), so it may need further confirmation. genus rosmarinus l. in albania this genus already has as representative 1 species (rosmarinus officinalis l.) also found in “flora of albania”, but its type rosmarinus officinalis l. subsp. officinalis is missing (the euro+med plantbase, 2010). genus salvia l. salvia genus has 22 representative taxa found in albania (tab. 1), of which 8 (4 types and 4 species) are not published in the volumes of “flora of albania”. 1. salvia fruticosa mill. in “flora of albania” 3rd edition, it’s known as salvia triloba l.f. 2. salvia nemorosa l. subsp. nemorosa (the euro+med plantbase, 2010) 3. salvia officinalis l. subsp. officinalis (the euro+med plantbase, 2010) 4. salvia pratensis l. subsp. pratensis (the euro+med plantbase, 2010) 5. salvia tomentosa mill. new name for albania (the euro+med plantbase), in “flora of albania”, it’s known as salvia grandiflora etl. 6. salvia verticillata l. subsp. verticillata (the euro+med plantbase, 2010) 7. salvia aethiopis l. (barina et al., 2018) 8. salvia microphylla kunth. according to barina et al. (2018), this species is only found cultivated in albania. genus satureja l. the genus satureja in albania is represented by 9 taxa (tab. 1), of which 3 (2 types and 1 species) are not published in “flora of albania” and 2 species come with new accepted names by “the euro +med plantbase”. 1. satureja kitaibelii wierzb. ex heuff. new name for albania, in “flora of albania” 3rd volume, this species is known as satureja montana l. subsp. kitaibelii (wierzb.) p.w.ball. 2. satureja montana l. subsp. variegata (host) p.w.ball. (barina et al., 2018) 3. satureja subspicata bartl. ex vis. new name for albania (the euro+med plantbase), in “flora of albania” 3rd volume, this species is known as satureja montana l. subsp. illyrica. 4. satureja subspicata bartl. ex vis. subsp. subspicata (the euro+med plantbase, 2010) 5. satureja parnassica heldr. & sart. ex boiss. according to barina et al., 2018, the presence of this species in albania is mentioned only once (kárpáti & kárpáti, 1961), so it may need further confirmation. genus scutellaria l. this genus in albania is represented by 12 taxa (tab. 1), of which 3 (1 type and 2 subspecies) are not published in the “flora of albania” books and one has a new updated name according to “the euro+med plantbase”. 1. scutellaria columnae all. subsp. columnae (the euro+med plantbase, 2010) 2. scutellaria orientalis subsp. pinnatifida j. r. edm. (the euro+med plantbase, 2010, barina et al., 2018) 3. scutellaria rupestris boiss. & heldr. new name for albania, in “flora of albania” 3rd edition, this species is known as scutellaria rubicunda hornem. subsp. rupestris (boiss. & heldr.). 4. scutellaria rupestris subsp. adenotricha (boiss. & heldr.) greuter & burdet. (the euro+med plantbase, 2010) genus sideritis l. sideritis genus in albania is represented by 8 taxa (tab. 1), of which only 2 types are not published in “flora of albania”. 1. sideritis montana l. subsp. montana (the euro+med plantbase, 2010) 2. sideritis raeseri boiss. & heldr. subsp. raeseri (the euro+med plantbase, 2010) genus stachys l. the genus stachys has the largest number of species in the lamiaceae family. this genus has a total of 44 22 biologica nyssana ● 13 (1) september 2022: 11-25 saliaj et al. ● critical revision of lamiaceae family in albanian flora taxa (tab. 1), of which 16 (5 types, 8 subspecies and 3 species) are not found in “flora of albania” books and one has changed its name. 1. stachys alopecuros (l.) benth. subsp. alopecuros (the euro+med plantbase, 2010) 2. stachys alpina l. subsp. alpina (barina et al., 2018) 3. stachys alpina l. subsp. dinarica murb. (barina et al., 2018) 4. stachys annua (l.) l. subsp. annua (the euro+med plantbase, 2010) 5. stachys cretica subsp. cassia (boiss.) rech. f. (barina et al., 2018) 6. stachys cretica l. subsp. cretica. the presence of this subspecies in albania needs further confirmation due to lack of records (barina et al., 2018) 7. stachys cretica subsp. salviifolia (ten.) rech. f. (barina et al., 2018) 8. stachys cretica subsp. bulgarica rech.f. (barina et al., 2018) 9. stachys germanica subsp. penicillata (heldr. & sart. ex boiss.) nyman. (the euro+med plantbase, 2010) 10. stachys mollissima willd. new name for albania, in “flora of albania” 3rd book, this species is known as stachys decumbens pers. 11. stachys officinalis (l.) trevis. subsp. officinalis (the euro+med plantbase, 2010) 12. stachys officinalis subsp. skipetarum jáv. (barina et al., 2018) 13. stachys recta subsp. baldaccii (k. malý) hayek. (barina et al., 2018) 14. stachys recta subsp. doerfleri (hayek) hayek. (barina et al., 2018) 15. stachys canescens bory & chaub. (barina et al., 2018) 16. stachys parolinii vis. (barina et al., 2018) 17. stachys plumosa griseb. (barina et al., 2018) genus teucrium l. this genus is represented by 15 taxa (tab. 1), of which 6 (4 types and 2 species) are new to albanian flora and one has a new updated name according to “the euro+med plantbase”. 1. teucrium capitatum l. new name for albania, in “flora of albania” 3rd book, this species is known as subspecies: teucrium polium l. subsp. capitatum (l.) arcang. so it has passed from subspecies to species. 2. teucrium capitatum l. subsp. capitatum (the euro+med plantbase, 2010) 3. teucrium chamaedrys l. subsp. chamaedrys (the euro+med plantbase, 2010) 4. teucrium flavum l. subsp. flavum (the euro+med plantbase, 2010) 5. teucrium montanum l. subsp. montanum (the euro+med plantbase, 2010) 6. teucrium divaricatum sieber ex heldr. (the euro+med plantbase, 2010) 7. teucrium botrys l. according to barina et al. (2018), the presence of this species in albania is mentioned only once (demiri, 1983), so it may need further confirmation. genus thymbra l. this genus is mentioned only by the euro+med plantbase for the distribution of one species in albania. this genus is not found in any of the 4 published books of “flora of albania” thymbra capitata (l.) cav. in “flora of albania” the species is known as thymus capitata l. genus thymus l. this is also a genus that consists in a large number of taxa, a total of 28 (tab. 1), of which 19 taxa are new for albanian flora and 2 of the species come with new accepted names according to “the euro+med plantbase”. 1. thymus boissieri halácsy. in “flora of albania” 3rd volume, this species is known as thymus cherlerioides. 2. thymus doerfleri ronniger. (barina et al., 2018) 3. thymus longicaulis subsp. chaubardii (rchb. f.) jalas. (the euro+med plantbase, 2010) 4. thymus longicaulis c. presl subsp. longicaulis (the euro+med plantbase, 2010) 5. thymus parnassicus halácsy (the euro+med plantbase, 2010) 6. thymus praecox subsp. jankae (.elak.) jalas. in “flora of albania” 3rd volume, this species is known as thymus praecox subsp. scorpilii (vele.) jalas. 7. thymus pulegioides subsp. montanus (benth.) ronniger (the euro+med plantbase, 2010) 8. thymus pulegioides l. subsp. pulegioides (the euro+med plantbase, 2010) 9. thymus sibthorpii benth. (barina et al., 2018) 10. thymus teucrioides boiss. & spruner subsp. teucrioides (the euro+med plantbase, 23 biologica nyssana ● 13 (1) september 2022: 11-25 saliaj et al. ● critical revision of lamiaceae family in albanian flora 2010) 11. thymus thracicus velen. (barina et al., 2018) 12. thymus × oblongifolius opiz. according to barina et al. (2018), the presence of this hybrid species in albania is reported only once (jávorka, 1922), so it may need further confirmation. 13. thymus ciliatopubescens (halácsy) halácsy (barina et al., 2018) 14. thymus dacicus borb. according to barina et al. (2018), the presence of this species in albania is reported only once (hayek, 1924), so it may need further confirmation. 15. thymus degenii heinr. brown. according to barina et al. (2018), the presence of this species in albania is reported only once (jávorka, 1926), so it may need further confirmation. 16. thymus odoratissimus mill. according to barina et al. (2018), the presence of this species in albania is reported only once (greuter et al., 1986), so it may need further confirmation. 17. thymus heterotrichus griseb. (barina et al., 2018) 18. thymus roegneri k. koch. according to the euro+med plantbase 2010, this is the updated and accepted name for thymus hirsutus m. bieb. 19. thymus leucotrichus halácsy. according to barina et al. (2018), the presence of this species in albania is confirmed since 1933: (hayek (1933), rechinger (1939), mitrushi (1966), meyer (2011), rakaj et al. (2013), vangjeli (2015). 20. thymus serpyllum l. according to barina et al. (2018), the presence of this species in albania is reported only once (heldreich, 1879), so it may need further confirmation. 21. thymus stojanovii degen. the presence of this species in albania needs further confirmation. it is reported by barina et al. (2018) referred to other references: grimus (1871), bornmüller (1937), mitrushi (1966), tutin et al. (1972), qosja (1973), demiri (1983), greuter et al. (1986) and ball (2011) . some of the genera with a small number of species/subspecies have not undergone any taxonomic change over the years and are found the same in “flora of albania”, such as galeopsis l., glechoma l., leonurus l., lavandula l., lycopus l., melissa l., melittis l., ocimum l., and prasium l. vitex l. genus in the 3rd published edition “flora of albania” is not part of labiatae family, but is found under verbenaceae family (qosja et al., 1996). ziziphora l. genus has 2 representative taxa in albania, 1 species and 1 subspecies/the type, of which only the species (ziziphora capitata l.) is published in “flora of albania”, not its type (ziziphora capitata l. subsp. capitata). after finishing the revision of lamiaceae family for the flora of albania, referring all the time to the only documented books (4 volumes of “flora of albania”), a lot of changes should be highlighted. lamiaceae family is found in the 3rd volume of “flora of albania” with the name “labiatae” and has a total of 32 genera (qosja et al., 1996). lamiaceae family in albania (following the above methodology) is represented by a total of 32 genera and 276 taxa (species/subspecies/varieties) (barina et al., 2018). two genera: acinos miller and calamintha miller do not exist so far, this two genera are part now of clinopodium l genus. also lamiastrum heister ex fabr., genus does not exist anymore, it is now placed under the lamium l. genus. two new genera belong now to this family, thymbra and vitex, both with one representative species in albania. (http://ww2.bgbm.org/europlusmed/ query.asp). from 276 taxa of this family only 143 are also found unchanged like in the 3rd edition of “flora of albania”, whereas 133 taxa are new for the flora (with changed species/subspecies name or not even found in any of “flora of albania” published volumes). the total composition of lamiaceae family in albanian flora is found in tab. 1. references barina z., somogyi g., pifko d., rakaj m. 2018: checklist of vascular plants of albania. phytotaxa, 378(1): 1-339. barina, z., mullaj, a., pifkó, d., somogyi, g., meco, m., rakaj, m. 2017: distribution maps. – in: barina, z. (ed.): distribution atlas of vascular plants in albania. hungarian natural history museum. budapest. pp. 47–445. greuter, w., von raab-straube, e. (eds) 2008: med-checklist: a critical inventory of vascular plants of the circum-mediterranean countries. dicotyledones. optima secretariat. palermo. harley, r. m., atkins, s., budantsev, a. l., cantino, p. d., conn, b. j., grayer, r., harley, m. m., de kok, r., krestovskaja, t., morales, r., paton, a. j., ryding, o., upson, t. 2004: labiatae. in: kubitzki, k. (ed.): the families and genera of 24 biologica nyssana ● 13 (1) september 2022: 11-25 saliaj et al. ● critical revision of lamiaceae family in albanian flora vascular plants, vii. springer. germany. heywood, v. h., brummitt, r. k., culham, a., seberg, o. 2007: flowering plant families of the world. firefly books. canada. ontario. meço, m., mullaj, a. 2015: phenological aspects of albanian flora. proceedings of international conference on soil. agricultural university of tirana. paparisto, k., demiri, m., mitrushi, i., qosja, xh. 1988: flora e shqipërisë, 1. academy of sciences, center of biological research. albania. patwardhan, b., mashelkar, r. a. 2009: traditional medicine-inspired approaches to drug discovery: can ayurveda show the way forward? drug discovery today, 14(15-16): 804-811. qosja, xh., paparisto, k., demiri, i., vangjeli, j., balza, e. 1992: flora e shqipërisë, 2. academy of sciences, center of biological research. albania. qosja, xh., paparisto, k., vangjeli, j., ruci, b., mullaj, a. 1996: flora e shqipërisë, 3. academy of sciences, center of biological research. albania. the euro+med plantbase (https://ww2.bgbm.org/ europlusmed/ptaxondetail.asp?nameid=111505& ptreffk=8000000) vangjeli, j., ruci, b., mullaj, a., qosja, xh., paparisto, k. 2000: flora e shqipërisë, 4. academy of sciences, center of biological research. albania. 25 microsoft word 0302_karalija_et_al biologica nyssana 1 (1) december 2010: 111-121 stojanović-radić, z. et al. antimicrobial activity and cytotoxicity of… 57 original article ! somatic embryogenesis and in vitro plantlet regeneration of lilium martagon l. var. cattaniae vis. erna karalija, sanja trbojević, adisa parić* faculty of sciences sarajevo, university of sarajevo, zmaja od bosne 33 – 35, 71000 sarajevo, bosnia and herzegovina * e-mail: adisacausevic@hotmail.com abstract: karalija, e., trbojević, s., parić, a.: somatic embryogenesis and in vitro plantlet regeneration of lilium martagon l. var. cattaniae vis.. biologica nyssana, 1 (1-2), december 2010: 57-60. in this study organogenic capacity of two different explants type (leaves and whole bulbs) of lilium martagon l. var. cattaniae vis. was examined. for induction of in vitro somatic embryogenesis and adventitive regeneration different concentrations of 2,4-dichlorophenoxyacetic acid and 6-benzilaminopurine (from 0,25 mg/l to 8,00 mg/l) added to ms basal medium were used. our results indicate that concentration of 0,5 mg/l 2,4-dichlorophenoxyacetic acid and 4 mg/l 6benzilaminopurine promoted somatic embryogenesis from leaves of lilium martagon var. cattaniae, while all other concentrations promoted direct shoot regeneration from bulb explants. root formation was induced on ms basal medium with 0,2 mg/l indole butyric acid. these plantlets were acclimatized well in a greenhouse conditions. key words: lilium martagon var. cattaniae, somatic embryogenesis, regeneration introduction ! lilium martagon l. var. cattaniae vis. is a monocotyledonous plant from the liliaceae family. this taxon is a vulnerable endemic taxon of central dinarids distributed in warmer areas of mediterranean and submediterranean. in bosnia and herzegovina this taxon is also categorised as rare and endangered, and as such is listed in the checklist of plant species for the red book of bosnia and herzegovina (š i l i ć , 1996). in recent years, there is an increasing interest in somatic embryogenesis as a method for in vitro propagation of lilies and other plant species. this is probably because somatic embryogenesis produces a large number of individuals and can be used for production of artificial seeds and for the purposes of transformation as well. the method for induction of somatic embryogenesis has been established for many lilies including lilium martagon (k e d r a & b a c h , 2005), but such methods are unknown for lilium martagon var. cattaniae. therefore, the aim of this study was to investigate the regeneration potential and possibility of somatic embryogenesis induction in this taxon. material and methods ! plant material and in vitro conditions leaves and bulbs of in vitro grown lilium martagon var. cattaniae (hindija et al., 2007) were used for induction of somatic embryogenesis and plantlet regeneration. all media were adjusted to ph 5.7-5.8 using 1 n koh/hcl and 0.8% agar was used for solidification of media. media were autoclaved at 120°c for 20 min. all cultures were kept at 24°c (±2) and relative humidity 70%. during the first 15 days of the treatment the cultures were kept in the dark while for the remainder of the experiment cultures were kept in conditions of 16 hour photoperiod provided by cool, white, fluorescent tubes (3000 lux). 10th sfses • 17-20 june 2010, vlasina lake1 (1-2) • december 2010: 57-60 biologica nyssana 1 (1-2) december 2010: 57-60 karalija, e. et al. somatic embryogenesis and in vitro plantlet … 58 in vitro treatments and acclimatisation of plantlets for induction of somatic embryogenesis and plantlet regeneration, bulbs and leaves were cultivated on ms (m u r a s h i g e & s k o o g , 1962) medium containing different concentrations of 2,4-d and bap (tab. 1), including control, with no plant growth regulators (pgr) added. embryogenic nature of cultures was confirmed by visual identification. after 30 days of pgr treatment all cultures were then transferred to pgr free ms basal medium. regenerated shoots from bulb explants were multiplied on the ms medium containing 0,5 mg/l bap, 0,2 mg/l iba and 0,1 mg/l ga3, and they were subsequently cultivated on ms medium containing 0,2 mg/l iba for induction of rhizogenesis. acclimatisation was conducted in pots containing commercial soil for plant cultivation, under greenhouse conditions, with the temperature of 25°c and relative humidity of 70%. three replicates were taken for each treatment. data from all of the replicates was statistically analyzed using statistics program spss version 7. results and disscusion induction of somatic embryogenesis out of all of the tested concentrations of 2,4d and bap only 0,5 mg/l 2,4-d and 4 mg/l bap mg/l was successful in inducting somatic embryogenesis from leaf explants. the primary sign of induction of somatic embryogenesis was swelling of explants tissue (g e o r g e et al., 2008), which in our experiment was evident on the basal parts of leaf explants (fig. 1a), 15 days after inoculation. globular embryos appeared directly from the surface of the explants (fig. 1b). it seems that this combination provides suitable conditions for progression of totipotent somatic cells into somatic embryos. all other applied concentrations showed a reverse effect on embryogenesis. embryo development reached cotyledon state (fig. 1b) a month after inoculation, and 20 embryos per explant were formed. further development and germination of embryos was accomplished after 75 days of culture, with subsequent transfer of globular embryos on pgr free medium (fig. 1c). similar results were also described for somatic embryogenesis in other lilies as well as other species, where somatic embryos developed into plantlets when plated onto ms basal medium without pgrs (t a b i r a , 1994; t r i b u l a t o et al., 1997; h o et al., 2006; g e o r g e et al., 2008). plantlets were well acclimatised in greenhouse conditions. no induction of somatic embryogenesis was noticed from bulb explants. induction of plantlet regeneration from bulb explants regeneration was successful for all tested combinations of concentrations of 2,4-d and bap but showed different rate of regeneration (tab. 1). rate of regeneration was ranging from 50 to 100%, depending on pgrs applied. bulblets regeneration varied from 7, on medium containing 1 mg/l 2,4-d + 1 mg/l bap, up to 16 bulblets per explant, on medium containing 2 mg/l 2,4-d + 1 mg/l bap (tab. 1). however, the differences were not significant. regeneration of leaves also varied, from 2 to 20 leaves per explant (tab. 1). the highest number of regenerated leaves was on the ms medium containing 0,25 mg/l 2,4-d and 8 mg/l bap with 20 regenerated leaves on average (fig. 1d). statistically significant differences on p<0,05 level (lsd) were noticed for treatment containing 2 mg/l 2,4-d and 1 mg/l bap in comparison with results obtained on ms medium containing 0,25 mg/l 2,4-d and 8 mg/l bap and in comparison with control treatment. bulbs are the most commonly used explants in lily cultures as they show the highest organogenic potential (t a k a y a m a & m i s a w a , 1979). numerous authors studied the importance of interaction between auxins and cytokinins on formation of bulbs and shoots in vitro with higher concentrations of cytokinins promoting a higher regeneration of bulblets (t a k a y a m a & m i s a w a , 1979, 1982; n i i m i , 1995). results of our research also show that with the rise in the concentration of cytokinins there is an increase in the regeneration of leaves with regeneration rate peaking on the medium with the highest concentration of bap (8 mg/ml), but for best regeneration of bulblets auxin/cytokinine ratio was in favour of auxine (treatment with 2 mg/l 2,4-d + 1 mg/l bap). in our research 2,4 d induced callus formation in only one case, specifically on the medium containing 4 mg/l bap and 0,5 mg/l 2,4 d, which produced leaves and bulbs after being transferred onto a pgr free medium. o k a z a k i & k o i z u m i (1995) reported that for shoot production from callus it is necessary to cultivate callus on pgr free medium for a period of six months or by lowering the sucrose concentration from 3% to 1%. m o r i et al (2005) later showed that for a large number of lily species shoot induction biologica nyssana 1 (1-2) december 2010: 57-60 karalija, e. et al. somatic embryogenesis and in vitro plantlet … 59 table 1. effect of 2,4-d and bap on leaf and bulblet regeneration from bulb explants of l. martagon var. cattaniae pgr treatment: 2,4 d + bap (mg/l ) rate of regeneration (%) no. of regenerated leaves no. of regenerated bulblets 0,25 + 8,00 90 20a 14 0,50 + 4,00 70 12 13 1,00 + 2,00 80 11 12 2,00 + 1,00 100 2a,b 16 1,00 + 1,00 50 5 7 0,00 + 0,00 50 18b 15 values are mean of three replicates per each treatment. same letter within the column indicates a significant differences at p<0,05 by lsd test.values with no indicators (a or b) show no significant difference. a b c d figure 1. induction of somatic embryogenesis on ms basal medium containing 0,5 mg/l 2,4-d and 4 mg/l bap; a) globular embryos; b) cotyledon phase; c) germinated embryo d) regeneration from bulb explant on ms basal medium containing 0,25 mg/l 2,4-d and 8 mg/l bap. could be successful with only two months of cultivation on a pgr free medium. in our research differentiation of shoots was successful after only a month of callus cultivation on pgr free medium. regenerated shoots were cultivated on multiplication medium containing 0,5 mg/l bap, 0,2 mg/l iba and 0,1 mg/l ga3. multiplication was successful producing up to 30 shoots per explant. after induction of rhizogenesis on the ms medium containing 0,2 mg/l iba, plants were acclimatised in the greenhouse conditions. conclusion our research showed that induction of somatic embryogenesis could be successfully induced from leaf explants by cultivation on ms medium containing 0,5 mg/l 2,4-d + 4 mg/l bap. regeneration from bulb explants was successful biologica nyssana 1 (1-2) december 2010: 57-60 karalija, e. et al. somatic embryogenesis and in vitro plantlet … 60 with differences in bulblet and leaf regeneration. best treatment for leaf regeneration contained high cytokinins level (8 mg/l), while treatment with best results for bulblet regeneration contained more auxin then cytokinin in ratio 2:1. multiplication was very successful with production of large number of shoots with good acclimatisation rate. references george, e. f., hall, m.a., de geert-jan, k. (eds) 2008: plant propagation by tissue culture 3rd edition volume 1. the background. springerverlag, berlin, heidelberg. 508 p. hindija, j., muratović, e., parić, a. 2007: in vitro regeneration of lilium martagon l. var. cattaniae vis. v symposium of agriculture, veterinary, forestry and biotechnology, vlašić, travnik, bosna i hercegovina, abstracts: 216. ho, c.w., jian, w.t., lai, h.c. 2006: plant regeneration via somatic embryogenesis from suspension cell cultures of lilium x formolongi hort. using a bioreactor system. in vitro cellular & developmental biology -plant, 42 (3): 240– 246. kedra, m., bach, a. 2005: morphogenesis of lilium martagon l. explants in callus culture. acta biologica cracoviensia / botanica, 47 (1): 6573. mori, s., adachi, y., horimoto, s., suzuki, s., nakano, m. 2005: callus formation and plant regeneration in various lilium species and cultivars. in vitro cellular & developmental biology, 41 (6): 783-788. murashige, t., skoog, f. 1962: a revised medium for rapid growth and bioassays with tobacco tissue cultures. physiologia plantarum, 15 (3): 473-497. niimi, y. 1995: in vitro propagation and post-invitro establishment of bulblets of lilium japonicum thunb. journal of japanese society of horticultural sciences, 63 (4): 843-852. okazaki, k., koizumi, m. 1995: callus formation and regeneration of some species of lilium. acta horticulturae, 392: 97–106. šilić, č. 1996: spisak biljnih vrsta (pteridophyta i spermatophyta) za crvenu knjigu bosne i hercegovine. glasnik zemaljskog muzeja bosne i hercegovine, n.s., 31:323-367. tabira, h. 1994: development of micropropagation system of gentiana triflora. xxiv international horticultural congress, august 21–27, kyoto: 186. takayama, s., misawa, m. 1979: differentiation in lilium bulbscales grown in vitro. effect of various cultural conditions. physiologia plantarum, 46: 184-190. takayama, s., misawa, m. 1982: regulation of organ formation by cytokinin and auxin in lilium bulbscales grown in vitro. plant and cell physiology, 23 (1): 67-74. tribulato, a., remotti, p.c., loffler, h.j.m., van tuyl, j.m. 1997: somatic embryogenesis and plant regeneration in lilium longiflorum thunb. plant cell reports, 17 (2): 113–118. anticancer compounds from medicinal plants biologica nyssana 5 (1)  september 2014: 17-30 papović, o. et al.  analysis of the flora of rogozna… 17 original article received: 14 july 2014 revised: 21 august 2014 accepted: 10 september 2014 analysis of the flora of rogozna mountain in southwestern serbia olivera papović 1* , milica miljković 2 , novica ranđelović 2 , vladimir ranđelović 2 1 university of pristine, faculty of sciences and mathematics, department of biology, ive lole ribara 29, 38220 kosovska mitrovica, serbia 2 university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia * e-mail: olja.bio@open.telekom.rs abstract: papović, o., miljković, m., ranđelović, n., ranđelović, v.: analysis of the flora of rogozna mountain in southwestern serbia. biologica nyssana, 5 (1), september 2014: 17-30. as a result of the two-year investigation of the rogozna mt. flora, 795 plant taxa (species and subspecies) belonging to 337 genera and 77 families were recorded. floristic analysis of investigated area was performed in comparison to the floristic data of serbia, balkan peninsula and neighboring region of rogozna mt. (ibar river valley and high mountain region of kopaonik mt). key words: flora, floristic analysis, floristic spectrum. apstrakt: papović, o., miljković, m., ranđelović, n., ranđelović, v.: analiza flore planine rogozne u jugozapadnoj srbiji.. biologica nyssana, 5 (1), september 2014: 17-30. kao rezultat dvogodišnjih istraživanja flore planine rogozne utvrđeno je 795 biljnih taksona (vrsta i podvrsta) iz 337 rodova i 77 familija. floristička analiza istraživanog područja je sprovedena u poređenju sa florističkim podacima koji postoje za srbiju, balkansko poluostrvo i susedna područja planine rogozne (dolina reke ibar i visokoplaninski region kopaonika). ključne reči: flora, floristička analiza, floristički spektar. introduction rogozna mt. is situated in southwestern serbia in the triangle between the cities of kosovska mitrovica, raška and novi pazar. it is elongated in the northwestern-southeastern direction, bordered by the golija mt. massif on the northwest, slopes of the mokra mt. on the south, a great massif of kopaonik mt. on the east and northeast and stari kolašin on the west. on the south, east and northeast, rogozna mt. is mostly bordered by the ibar river, while on the northwest and west by raška and jošanica river. the main ridge of the mountain is slightly sinuous and starts from the highest mountain peak crni vrh (1479 m) 5 (1) • september 2014: 17-30 biologica nyssana 5 (1)  september 2014: 17-30 papović, o. et al.  analysis of the flora of rogozna… 18 figure 1. geographical position of investigated area followed by čador (1354 m), ravna glava (1369 m), smilov laza (1302), kašalj (1081 m), šanac (1292 m), bar (1207 m), zminjac (1111 m) and vinorog (1225 m). the geological structure of rogozna mt. is mostly represented by serpentine and eruptive stones. the pedological substrate consists of loamy soil. the climate of this area is between temperate and subalpine (b o ž a n i ć , 2006). up to date, rogozna mt. has not been well investigated and therefore, this paper gives a significant contribution to the knowledge of the flora of this mountain and serbia in general. biologica nyssana 5 (1)  september 2014: 17-30 papović, o. et al.  analysis of the flora of rogozna… 19 material and methods herbarium specimens collected during the two-year investigation are deposited at the herbarium of faculty of science and mathematics, university of niš (hmn). identification of the collected plants was performed according to flora europaea (t u t i n et al., 1964-1980) and the regional floras relevant for the investigated area (j o s i f o v i ć , ed., 1970-1977, v e l c h e v , ed., 1982-1989). the nomenclature follows med-checklist (g r e u t e r et al., 1984-1989), flora europaea (t u t i n et al., 1964-1980), and international organization for plant information (iopi) (http://plantnet.rbgsyd.nsw.gov.au/iopi/iopihome.htm). taxonomic structure of the rogozna mt. flora was compared to the flora of the balkan peninsula (t u r r i l l , 1929), serbia (s t e v a n o v i ć et al. 1994), kopaonik mt. (l a k u š i ć , 1993) and ibar river valley (p r o d a n o v i ć , 2007). generic coefficient was calculated using the formula ng/ns x 100, where ng is the number of genera and ns the number of species (j a c c a r d , 1912, a л ë х и н , 1944, j a n k o v i ć , 1985). results and discussion list of the recorded vascular flora of rogozna mt equisetopsida equisetaceae equisetum arvense l. equisetum telmateia ehrh. polypodiopsida aspidiaceae dryopteris filix-mas (l.) schott polystichum setiferum (forskål) woynar polypodiaceae polypodium vulgare l. adiantaceae notholaena maranthae (l.) desv. aspleniaceae asplenium adiantum-nigrum l. asplenium ceterach l. asplenium cuneifolium viv. asplenium ruta-muraria l. asplenium trichomanes l. asplenium septentrionale (l.) hoffm. woodsiaceae cystopteris fragilis (l.) bernh. pinopsida cupressaceae juniperus communis l. magnoliopsida aristolochiaceae asarum europaeum l. ranunculaceae anemone nemorosa l. clematis vitalba l. consolida regalis s. f. gray helleborus odorus waldst. & kit. helleborus serbicus adamović hepatica nobilis schreber ranunculus acris l. ranunculus bulbosus l. ranunculus illyricus l. ranunculus platanifolius l. ranunculus polyanthemos l. ranunculus repens l. ranunculus sardous crantz ranunculus strigulosus schur thalictrum aquilegiifolium l. berberidaceae epimedium alpinum l. papaveraceae fumaria officinalis l. papaver dubium l. papaver rhoeas l. ulmaceae ulmus glabra hudson fagaceae fagus sylvatica l. quercus cerris l. quercus petraea (mattuschka) liebl. subsp. medwediewii (a. camus) menitsky quercus petraea (mattuschka) liebl. subsp. petraea quercus pubescens willd. betulaceae alnus alnobetula (ehrh.) hartig corylaceae carpinus betulus l. carpinus orientalis miller http://plantnet.rbgsyd.nsw.gov.au/iopi/iopihome.htm biologica nyssana 5 (1)  september 2014: 17-30 papović, o. et al.  analysis of the flora of rogozna… 20 corylus avellana l. corylus colurna l. ostrya carpinifolia scop. caryophyllaceae agrostemma githago l. arenaria leptoclados (reichenb.) guss. arenaria serpyllifolia l. atocion armeria fourr. cerastium brachypetalum pers. cerastium decalvans schlosser & vuk. cerastium decalvans schlosser & vuk. cerastium fontanum baumg. subsp. vulgare (hartm.) greuter & burdet cerastium grandiflorum waldst. & kit. cerastium transsilvanicum schur cerastium sylvaticum waldst. & kit. dianthus armeria l. dianthus carthusianorum l. dianthus carthusianorum l. dianthus ciliatus guss. dianthus cruentus griseb. dianthus petraeus waldst. & kit. dianthus pinifolius sibth. & sm. subsp. pinifolius dianthus pinifolius sibth. & sm. subsp. serbicus wettst. dianthus serotinus waldst. & kit. dianthus sylvestris wulfen herniaria glabra l. herniaria hirsuta l. lychnis coronaria (l.) desr. lychnis flos-cuculi l. minuartia graminifolia (ard.) jáv. minuartia hirsuta (bieb.) hand.-mazz. subsp. falcata (griseb.) mattf. minuartia montana l. minuartia verna (l.) hiern. minuartia viscosa (schreber) schinz & thell. misopates orontium (l.) rafin. moehringia trinervia (l.) clairv. moenchia mantica (l.) bartl. paronychia cephalotes (bieb.) besser petrorhagia illyrica (ard.) p. w. ball & heywood petrorhagia saxifraga (l.) link sagina saginoides (l.) karsten saponaria glutinosa bieb. scleranthus annuus l. scleranthus perennis l. subsp. perennis scleranthus perennis l. subsp. dichotomus (schur) nyman scleranthus polycarpos l. silene bellidifoliajacq. silene bupleuroides l. subsp. bupleuroides silene bupleuroides l. subsp. staticifolia (sibth. & sm.) chowdhuri silene conica l. silene gallinyi heuffel ex reichenb. silene italica (l.) pers. silene noctiflora l. silene otites (l.) wibel silene paradoxa l. silene sendtneri boiss. silene viridiflora l. silene vulgaris (moench) garcke stellaria graminea l. stellaria holostea l. stellaria media (l.) vill. viscaria vulgaris bernh. amaranthaceae amaranthus crispus (lesp. & thév.) n. terracc. chenopodiaceae bassia prostrata (l.) a. j. scott chenopodium album l. chenopodium botrys l. chenopodium hybridum l. chenopodium polyspermum l. chenopodium vulvaria l. polygonaceae fallopia convolvulus (l.) å. löve fallopia dumetorum (l.) j. holub persicaria maculosa s. f. gray polygonum arenarium waldst. & kit. polygonum aviculare l. rumex acetosella l. plumbaginaceae goniolimon incanum (l.) hepper limonium gmelinii (willd.) o. kuntze hypericaceae hypericum annulatum moris hypericum barbatum jacq. hypericum elegans stephan ex willd. hypericum hirsutum l. hypericum humifusum l. hypericum montanum l. hypericum perforatum l. violaceae viola aetolica boiss. & heldr. subsp. kopaonikensis pančić ex tomović & niketić, ined. viola arvensis murray viola canina l. viola kitaibeliana schultes viola mirabilis l. viola sylvestris lam. viola tricolor l. biologica nyssana 5 (1)  september 2014: 17-30 papović, o. et al.  analysis of the flora of rogozna… 21 cistaceae fumana bonapartei maire & petitmengin helianthemum alpestre (jacq.) dc. helianthemum canum(l.) baumg. helianthemum nummularium (l.) miller brassicaceae aethionema saxatile (l.) r. br. subsp. graecum (boiss. & spruner) hayek alyssum alyssoides (l.) l. alyssum bertolonii desv. alyssum markgrafii o. e. schulz ex markgraf alyssum montanum l. subsp. serbicum novák alyssum murale waldst. & kit. subsp. murale alyssum repens baumg. arabis alpina l. subsp. caucasica (willd. ex schlecht.) briq. arabis turrita l. aurinia saxatilis (l.) desv. aurinia saxatilis (l.) desv. subsp. orientalis (ard.) t. r. dudley berteroa incana (l.) dc. berteroa mutabilis (vent.) dc. calepina irregularis (asso) thell. capsella bursa-pastoris (l.) medicus cardamine bulbifera (l.) crantz cardamine hirsuta l. cardamine resedifolia l. draba muralis l. erophila verna (l.) chevall. erucastrum gallicum (willd.) o. e. schulz erysimum carniolicum dolliner erysimum cuspidatum (bieb.) dc. erysimum diffusum ehrh. erysimum kuemmerlei jáv. fibigia clypeata (l.) medicus lepidium draba l. rorippa pyrenaica (all.) reichenb. rorippa sylvestris (l.) besser thlaspi alliaceum l. thlaspi arvense l. thlaspi perfoliatum l. resedaceae reseda lutea l. subsp. lutea salicaceae populus tremula l. salix fragilis l. salix purpurea l. salix viminalis l. ericaceae andromeda polifolia l. bruckenthalia spiculifolia (salisb.) reichenb. primulaceae anagallis arvensis l. anagallis foemina miller lysimachia nemorum l. lysimachia nummularia l. tiliaceae tilia platyphyllos scop. tilia tomentosa moench malvaceae malva moschata l. euphorbiaceae euphorbia barrelieri savi subsp. thessala (form.) bornm. euphorbia cyparissias l. euphorbia epithymoides l. euphorbia falcata l. euphorbia glabriflora vis. euphorbia helioscopia l. euphorbia salicifolia host euphorbia stricta l. euphorbia subhastata vis. & pančić euphorbia taurinensis all. euphorbia waldsteinii (soják) a. r. sm. mercurialis ovata sternb. & hoppe mercurialis perennis l. rosaceae agrimonia eupatoria ledeb. amelanchier ovalis medicus aremonia agrimonoides (l.) dc. cotoneaster tomentosus lindley crataegus monogyna jacq. cydonia oblonga miller filipendula ulmaria (l.) maxim. filipendula vulgaris moench fragaria vesca l. geum urbanum l. potentilla argentea l. potentilla erecta (l.) räuschel potentilla heptaphylla l. subsp. australis (krašan ex nyman) gams potentilla hirta l. potentilla incana p. gaertner, b. meyer & scherb. potentilla leucopolitana p. j. mueller potentilla recta l. potentilla reptans l. potentilla tommasiniana f. w. schultz potentilla visianii pančić prunus avium l. prunus spinosa l. pyrus communis l. rosa canina l. biologica nyssana 5 (1)  september 2014: 17-30 papović, o. et al.  analysis of the flora of rogozna… 22 rosa pendulina l. rosa spinosissima l. rubus hirtus waldst. & kit. rubus idaeus l. rubus ulmifolius schott sanguisorba minor scop. sanguisorba officinalis l. sorbus aria (l.) crantz sorbus graeca (spach) kotschy sorbus torminalis (l.) crantz spiraea cana waldst. & kit. spiraea media franz schmidt crassulaceae hylotelephium maximum (l.) holub sedum acre l. subsp. acre sedum hispanicum l. sedum ochroleucum chaix sedum rupestre l. sedum sartorianum boiss. sedum serpentini janchen sempervivum heuffelii schott sempervivum marmoreum griseb. saxifragaceae saxifraga rotundifolia l. parnassiaceae parnassia palustris l. fabaceae anthyllis vulneraria l. astragalus glycyphyllos l. astragalus onobrychis l. astragalus onobrychis l. colutea arborescens l. cytisus hirsutus l. subsp. ciliatus (wahlenb.) ascherson & graebner cytisus hirsutus subsp. hirsutus cytisus jankae velen. cytisus nigricans (schur) nyman cytisus pseudoprocumbens markgraf cytisus supinus l. dorycnium germanicum (gremli) rikli dorycnium herbaceum vill. genista depressa bieb. genista germanica l. genista januensis viv. genista ovata waldst. & kit. hippocrepis comosa l. hippocrepis emeroides (boiss. & spruner) czerep. lathyrus hallersteinii baumg. lathyrus latifolius l. lathyrus niger (l.) bernh. lathyrus nissolia l. lathyrus pratensis l. lathyrus sphaericus retz. lathyrus sylvestris l. lathyrus venetus (miller) wohlf. lathyrus vernus (l.) bernh. lens nigricans (bieb.) godron lotus corniculatus l. medicago arabica (l.) hudson medicago carstiensis jacq. medicago falcata l. medicago lupulina l. medicago minima (l.) bartal. medicago prostrata jacq. medicago sativa l. melilotus officinalis (l.) pallas onobrychis alba (waldst. & kit.) desv. onobrychis arenaria (kit.) dc. onobrychis viciifolia scop. ononis spinosa l. securigera elegans (pančić) lassen securigera varia (l.) lassen trifolium alpestre l. trifolium arvense l. trifolium aureum pollich trifolium badium schreber trifolium campestre schreber trifolium dalmaticum vis. trifolium diffusum ehrh. trifolium hirtum all. trifolium medium l. subsp. balcanicum velen. trifolium montanum l. trifolium ochroleucon hudson trifolium pannonicum jacq. trifolium pignantii fauché & chaub. trifolium pratense l. subsp. pratense trifolium pratense l. subsp. serotinum (witte) holub trifolium repens l. trifolium scabrum l. trifolium striatum l. trifolium sylvaticum gerard sec. c. visioso trifolium trichopterum pančić trifolium velenovskyi vandas vicia cassubica l. vicia cracca l. subsp. cracca vicia cracca l. subsp. incana (gouan) rouy vicia dumetorum l. vicia lathyroides l. vicia pannonica crantz vicia pisiformis l. vicia sativa l. subsp. sativa vicia sativa l. subsp. nigra (l.) ehrh. vicia sepium l. vicia tetrasperma (l.) schreber lythraceae lythrum salicaria l. biologica nyssana 5 (1)  september 2014: 17-30 papović, o. et al.  analysis of the flora of rogozna… 23 oenotheraceae circaea lutetiana l. epilobium angustifolium l. epilobium palustre l. epilobium dodonaei vill. epilobium hirsutum l. epilobium lanceolatum sebastiani & mauri epilobium montanum l. epilobium parviflorum schreber epilobium roseum schreber anacardiaceae cotinus coggygria scop. rutaceae dictamnus albus l. haplophyllum boissieranum vis. & pančić aceraceae acer campestre l. acer heldreichii orph. ex boiss. subsp. visianii k. malý acer hyrcanum fischer & c. a. meyer subsp. intermedium (pančić) bornm. acer platanoides l. acer pseudoplatanus l. acer tataricum l. linaceae linum austriacum l. linum catharticum l. linum flavum l. linum hirsutum l. linum hologynum reichenb. linum tauricum willd. subsp. serbicum (podp.) petrova linum tenuifolium l. balsaminaceae impatiens noli-tangere l. geraniaceae geranium columbinum l. geranium dissectum l. geranium lucidum l. geranium phaeum l. geranium purpureum vill. geranium pyrenaicum burm. fil. geranium robertianum l. geranium sanguineum l. geranium sylvaticum l. polygalaceae polygala alpestris reichenb. polygala comosa schkuhr polygala major jacq. polygala supina schreber cornaceae cornus mas l. cornus sanguinea l. apiaceae aegopodium podagraria l. angelica verticilaris l. astrantia major l. bifora radians bieb. bupleurum apiculatum friv. bupleurum falcatum l. subsp. cernuum (ten.) arcangeli bupleurum falcatum l. subsp. falcatum bupleurum praealtum l. bupleurum rotundifolium l. bupleurum veronense l. carum carvi l. cervaria rivinii gaertner chaerophyllum aureum l. chaerophyllum hirsutum l. daucus carota l. eryngium palmatum pančić & vis. eryngium serbicum pančić falcaria vulgaris bernh. ferulago sylvatica (besser) reichenb. geocaryum cynapioides (guss.) l. engstrand hacquetia epipactis (scop.) dc. holandrea carvifolia (vill.) reduron, charpin & pimenov laserpitium siler l. orlaya daucoides (l.) greuter orlaya grandiflora (l.) hoffm. pastinaca hirsuta pančić peucedanum austriacum (jacq.) koch peucedanum officinale l. peucedanum oreoselinum (l.) moench physospermum cornubiense (l.) dc. pimpinella saxifraga l. pimpinella serbica (vis.) bentham & hooker fil. ex drude sanicula europaea l. smyrnium perfoliatum l. tordylium apulum l. tordylium maximum l. torilis japonica (houtt.) dc. trinia glauca (l.) dumort. trinia ramosissima (fischer ex trev.) koch celastraceae evonymus europaeus l. evonymus verrucosus scop. rhamnaceae frangula dodonei ard. biologica nyssana 5 (1)  september 2014: 17-30 papović, o. et al.  analysis of the flora of rogozna… 24 rhamnus saxatilis jacq. salntalaceae thesium arvense horvátovszky thesium bavarum schrank thesium divaricatum jan. ex mert. & koch thesium linophyllon l. asclepiadaceae vincetoxicum hirundinaria medicus gentianaceae centaurium erythraea rafin. gentiana asclepiadea l. gentiana cruciata l. gentiana lutea l. gentiana pneumonanthe l. gentiana utriculosa l. oleaceae fraxinus ornus l. rubiaceae asperula cynanchica l. asperula hungarorum borbás asperula purpurea (l.) ehrend. subsp. apiculata (sibth. & sm.) ehrend. asperula purpurea (l.) ehrend. subsp. purpurea crucianella angustifolia l. cruciata laevipes opiz cruciata laevipes opiz cruciata pedemontana (bellardii) ehrend. galium album miller galium aparine l. galium mollugo l. galium odoratum (l.) scop. galium rivale (sibth. & sm.) griseb. galium rubrum l. galium sylvaticum l. galium verum l. rubia tinctorum l. caprifoliaceae lonicera caprifolium l. sambucus ebulus l. sambucus nigra l. valerianaceae valeriana officinalis l. dipsacaceae cephalaria laevigata (waldst. & kit.) schrader cephalaria leucantha (l.) roemer & schultes knautia dinarica (murb.) borbás knautia dipsacifolia kreutzer subsp. lancifolia (heuffel) ehrend. knautia drymeia heuffel scabiosa argentea l. scabiosa columbaria l. scabiosa fumarioides vis. & pančić scabiosa ochroleuca l. succisa pratensis moench convolvulaceae convolvulus arvensis l. convolvulus cantabricus l. cuscuta epithymum (l.) l. boraginaceae anchusa officinalis l. cerinthe minor l. echium vulgare l. halacsya sendtneri (boiss.) dörfler lithospermum purpureocaeruleum l. myosotis arvensis (l.) hill myosotis nemorosa besser myosotis scorpioides l. myosotis sparsiflora mikan ex pohl myosotis sylvatica hoffm. onosma echioides l. pulmonaria officinalis l. symphytum tuberosum l. solanaceae atropa bella-donna l. physalis alkekengi l. solanum nigrum l. scrophulariaceae chaenorhinum minus (l.) lange digitalis ferruginea l. digitalis grandiflora miller digitalis laevigata waldst. & kit. digitalis lanata ehrh. euphrasia picta subsp. kerneri (wettst). yeo euphrasia stricta d. wolff ex j. f. lehm. euphrasia tatarica fischer ex sprengel kickxia elatine(l.) dumort. linaria genistifolia (l.) miller linaria genistifolia (l.) miller subsp. sofiana (velen.) chater & d. a. webb linaria rubioides vis. & pančić linaria vulgaris miller melampyrum arvense l. melampyrum bihariense a. kerner melampyrum cristatum l. melampyrum fimbriatum vandas melampyrum heracleoticum boiss. & orph. melampyrum hoermannianum k. malý melampyrum nemorosum l. melampyrum pratense l. melampyrum scardicum wettst. biologica nyssana 5 (1)  september 2014: 17-30 papović, o. et al.  analysis of the flora of rogozna… 25 odontites luteus (l.) clairv. odontites vernus (bellardi) dumort. pedicularis comosa l. rhinanthus angustifolius c. c. gmelin scrophularia canina l. scrophularia nodosa l. scrophularia scopolii hoppe ex pers. scrophularia tristis (k. malý) šilić verbascum banaticum schrader verbascum lychnitis l. verbascum phlomoides l. verbascum thapsus l. veronica anagalloides guss. veronica austriaca l. veronica beccabunga l. veronica chamaedrys l. veronica cymbalaria bodard. veronica incana l. veronica jacquinii baumg. veronica officinalis l. veronica spicata l. orobanchaceae orobanche nowackiana markgraf orobanche reticulata wallr. acanthaceae acanthus hungaricus (borbás) baenitz plantaginaceae plantago altissima l. plantago argentea chaix subsp. liburnica v. ravnik plantago holosteum scop. subsp. holosteum plantago media l. verbenaceae verbena officinalis l. lamiaceae acinos alpinus (l.) moench subsp. albanicus (kümmerle & jáv.) niketić acinos arvensis (lam.) dandy acinos hungaricus (simonkai) šilić ajuga genevensis l. ajuga laxmannii (l.) bentham ballota nigra l. calamintha officinalis moench clinopodium menthifolium merino clinopodium thymifolium (scop.) kuntze clinopodium vulgare l. galeopsis speciosa miller glechoma hirsuta waldst. & kit. lamium galeobdolon (l.) l. lamium garganicum l. lamium maculatum l. lamium purpureum l. leonurus cardiaca l. lycopus europaeus l. melittis melissophyllum l. subsp. albida (guss.) p. w. ball mentha × piperita l. mentha aquatica l mentha longifolia (l.) hudson nepeta cataria l. origanum vulgare l. prunella laciniata (l.) l. prunella vulgaris l. salvia glutinosa l. salvia nemorosa l. salvia sclarea l. salvia verticillata l. scutellaria altissima l. scutellaria columnae all. scutellaria galericulata l. sideritis montana l. stachys alpina l. stachys alpina l. subsp. dinarica murb. stachys cretica l. subsp. cassia (boiss.) rech. fil. stachys officinalis (l.) trevisan stachys recta l. stachys recta l. subsp. baldaccii (k. malý) haye stachys recta l. stachys scardica (griseb.) hayek teucrium chamaedrys l. teucrium montanum l. thymus glabrescens willd. thymus lykae degen thymus praecox opiz subsp. jankae (čelak.) jalas thymus pulegioides l. ziziphora capitata l. campanulaceae asyneuma anthericoides (janka) bornm. asyneuma limonifolium (l.) janchen campanula bononiensis l. campanula cervicaria l. campanula glomerata l. campanula grossekii heuffel campanula lingulata waldst. & kit. campanula moesiaca velen. campanula patula l. campanula persicifolia l. campanula rapunculoides l. campanula rapunculus l. campanula sparsa friv. campanula sparsa friv. subsp. sphaerothrix (griseb.) hayek campanula trachelium l. legousia speculum-veneris (l.) chaix phyteuma orbiculare l. biologica nyssana 5 (1)  september 2014: 17-30 papović, o. et al.  analysis of the flora of rogozna… 26 asteraceae achillea crithmifolia waldst. & kit. achillea distans waldst. & kit. ex willd. achillea grandifolia friv. achillea millefolium l. anthemis arvensis l. anthemis ruthenica bieb. arctium lappa l. artemisia alba turra artemisia vulgarisl. aster alpinus l. aster amellus l. bidens cernua l. bidens tripartita l. bombycilaena erecta (l.) smolj. carduus candicans waldst. & kit. carduus personata (l.) jacq. carlina vulgaris l. centaurea jacea l. centaurea jacea l. subsp. angustifolia (dc.) gremli centaurea phrygia l. centaurea phrygia l. subsp. stenolepis a. kerner centaurea scabiosa l. centaurea stoebe l. subsp. australis (a. kerner) greuter centaurea stoebe l. subsp. stoebe cichorium intybus l. subsp. intybus cirsium arvense (l.) scop. cirsium eriophorum (l.) scop. cirsium grecescui rouy cirsium vulgare (savi) ten. cota austriaca (jacq.) schultz-bip. cota tinctoria (l.) j. gay crepis biennis l. crepis foetida l. subsp. rhoeadifolia crepis pulchra l. crepis sancta (l.) bornm. crepis setosa haller fil. crepis vesicaria l. subsp. taraxacifolia (thuill.) thell. crupina vulgaris cass. cyanus triumfettii (all.) á. löve & d. löve doronicum columnae ten. echinops exaltatus schrader echinops ritro l. subsp. ruthenicus (bieb.) nyman erigeron acris l. subsp. acris erigeron annuus (l.) desf. erigeron canadensis l. eupatorium cannabinum l. filago arvensis l. filago minima (sm.) pers. galatella albanica degen galatella linosyris (l.) bernh. gnaphalium sylvaticum l. gnaphalium uliginosum l. hieracium bifidum hornem. hieracium murorum l. hieracium racemosum willd. hieracium sabaudum l. hieracium tommasinianum k. malý hypochaeris illyrica k. malý hypochaeris maculata l. hypochaeris radicata l. inula britannica l. inula conyzae dc. inula ensifolia l. inula hirta l. inula oculus-christi l. inula salicina l. jurinea mollis (l.) reichenb. lactuca muralis (l.) gaertner lactuca saligna l. lactuca viminea (l.) j. presl & c. presl lapsana communis l. leontodon biscutellifolius dc. leontodon crispus vill. leontodon hispidus l. leucanthemum vulgare lam. picris hieracioides l. pilosella bauhinii (schultes) arv.-touv. pilosella sabina (sebastiani & mauri) f. w. schultz & schultz bip. fratt. pilosella cymosa (l.) f. w. schultz & schultz bip. fratt. pilosella hoppeana (schultes) f. w. schultz & schultz bip. fratt. pilosella officinarum f. w. schultz & schultz bip. fratt. podospermum canum c. a. meyer podospermum laciniatum (l.) dc. prenanthes purpurea l. pulicaria dysenterica (l.) bernh. reichardia dichotoma (dc.) freyn scorzonera austriaca willd. scorzonera hispanica l. senecio leucanthemifolius poiret subsp. vernalis (waldst. & kit.) greuter senecio squalidus l. subsp. squalidus senecio squalidus l. subsp. rupestris (waldst. & kit.) greuter ined. senecio vulgaris l. serratula tinctoria l. solidago virgaurea l. tanacetum corymbosum (l.) schultz bip. tanacetum macrophyllum (waldst. & kit.) schultz-bip. tanacetum parthenium (l.) schultz-bip. taraxacum officinale weber tephroseris crassifolia (schultes) griseb. & schenk biologica nyssana 5 (1)  september 2014: 17-30 papović, o. et al.  analysis of the flora of rogozna… 27 tephroseris papposa (reichenb.) schur tragopogon dubius scop. tragopogon pterodes pančić ex petrović tripleurospermum inodorum (l.) schultz-bip. tripleurospermum tenuifolium (kit.) freyn xeranthemum annuum l. liliopsida liliaceae anthericum liliago l. anthericum ramosum l. colchicum autumnale l. convallaria majalis l. lilium martagon l. ornithogalum gussonei ten. paris quadrifolia l. polygonatum multiflorum (l.) all. polygonatum odoratum (miller) druce allium carinatum l. subsp. pulchellum bonnier & layens allium flavum l. allium moschatum l. allium paniculatum l. allium scorodoprasum l. allium senescens l. subsp. montanum (f. w. schmidt) holub allium sphaerocephalon l. tulipa scardica bornm. tulipa serbica tatić & krivošej iridaceae iris graminea l. dioscoreaceae tamus communis l. orchidaceae cephalanthera damasonium (miller) druce cephalanthera longifolia (l.) fritsch epipactis helleborine (l.) crantz epipactis microphylla (ehrh.) swartz gymnadenia conopsea (l.) r. br. gymnadenia odoratissima (l.) l. c. m. richard neottia nidus-avis (l.) l. c. m. richard orchis ustulata l. platanthera bifolia (l.) l. c. m. richard juncaceae juncus articulatus l. juncus atratus krocker juncus bufonius l. juncus conglomeratus l. juncus effusus l. juncus inflexus l. juncus thomasii ten. luzula campestris (l.) dc. luzula luzuloides (lam.) dandy & wilmott luzula pilosa (l.) willd. luzula sylvatica (hudson) gaudin cyperaceae carex digitata l. carex distans l. carex divisa hudson carex divulsa stokes carex echinata murray carex hirta l. carex ovalis good. carex pairae f. w. schultz carex pallescens l. carex spicata hudson carex vulpina l. cyperus fuscus l. eleocharis palustris (l.) roemer & schultes scirpus sylvaticus l. poaceae achnatherum calamagrostis (l.) beauv. agropyron cristatum (l.) gaertner agrostis capillaris l. alopecurus pratensis l. apera spica-venti (l.) beauv. bothriochloa ischaemum (l.) keng brachypodium pinnatum (l.) beauv. brachypodium sylvaticum (hudson) beauv. bromus commutatus schrader bromus hordeaceus l. bromus pannonicus kummer & sendtner bromus ramosus hudson bromus riparius rehmann bromus squarrosus l. bromus sterilis l. calamagrostis epigejos (l.) roth chrysopogon gryllus (l.) trin. cynosurus cristatus l. cynosurus echinatus l. dactylis glomerata l. danthonia alpina vest. dasypyrum villosum (l.) p. candargy elymus repens (l.) gould festuca pratensis hudson festuca valesiaca schleicher ex gaudin festuca valesiaca schleicher ex gaudin subsp. parviflora (hack.) tracey glyceria notata chevall. holcus lanatus l. koeleria glauca (schrader) dc. lolium perenne l. melica ciliata l. melica uniflora retz. biologica nyssana 5 (1)  september 2014: 17-30 papović, o. et al.  analysis of the flora of rogozna… 28 molinia arundinacea schrank phleum bertolonii dc. phleum montanum c. koch phleum phleoides (l.) karsten phleum pratense l. piptatherum virescens (trin.) boiss. poa badensis haenke ex willd. poa bulbosa l. poa bulbosa l. subsp. pseudoconcinna (schur) domin poa compressa l. poa nemoralis l. poa pratensis l. setaria viridis (l.) beauv. stipa pennata l. stipa pulcherrima c. koch trisetum flavescens (l.) beauv. araceae arum maculatum l. taxonomic diversity flora of investigated area contains even 24.30% of the plant species recorded for the territory of serbia. significant quantitative indicator of floristic richness and taxonomic diversity is generic coefficient (42.51%). this value indicates a relatively low diversity of habitats on one hand, and relatively low level of autochthonous florogenesis tendency on the other hand. this phenomenon is a consequence of a pretty uniform geological structure and relatively small size of the investigated area. the most abundant family of the investigated area is, as expected, the most abundant family of the holarctic kingdom (in both species and genera), family asteraceae. at the investigated area, 106 species from 47 genera were recorded, which represents 13.33% of the total flora. beside the asteraceae family, the most numerous families considering the number of the species were fabaceae (75), caryophyllaceae (59), lamiaceae (49), poaceae (48) and scrophulariaceae (44), together with apiaceae (39), rosaceae (36) and brassicaceae (33). regarding the number of genera, the most numerous was, once again, family asteraceae, followed by poaceae (28), apiaceae (26), lamiaceae (22), caryophyllaceae (29), fabaceae (17) and rosaceae (17). floristic spectrum of the rogozna mt. deviates somewhat from the spectra of serbia and balkan peninsula (tab.1). the increased presence of the boreal and arctic families rosaceae and poaceae indicates a significant impact of these two horions on the rogozna mt. flora genesis. the presence of the families characteristic for mediteranneansubmediterannean region (fabaceae, caryophyllaceae) is increased. this can be explained by the geographical position of investigated area, floristic and florogenetic mediterranean impacts and the relatively low average elevation of the rogozna mt. the flora of rogozna mt. is differentiated from the flora of serbia by centraleuropean and temperate-boreal elements owing to reduced percentage of the families brassicace and poaceae (fig.2). table 1. comparative review of taxonomical structure of the most abundant families of rogozna mt., serbia (stevanović et al., 1995) and the balkan peninsula (turill, 1929) rogozna mt. serbia balkan peninsula family n % n % n % asteraceae 106 13.33 366 11.19 913 13.52 fabaceae 75 9.43 250 7.64 545 8.07 caryophyllaceae 59 7.42 205 6.27 418 6.19 lamiaceae 49 6.16 148 4.52 371 5.49 poaceae 48 6.04 250 7.64 358 5.30 scrophulariaceae 44 5.53 161 4.92 311 4.65 apiaceae 39 4.91 142 4.34 334 4.95 rosaceae 36 4.53 111 3.39 188 2.78 brassicaceae 33 4.15 194 5.93 344 5.09 rubiaceae 18 2.26 49 1.50 129 1.91 biologica nyssana 5 (1)  september 2014: 17-30 papović, o. et al.  analysis of the flora of rogozna… 29 figure 2. percentage deviation of the most abundant families in the flora of rogozna mt. from the families spectra of serbia and the balkan peninsula. taxonomical spectrum of the rogozna mt. flora was compared with spectra of neighboring kosovo’s part of ibar river valley (p r o d a n o v i ć , 2007) and the high mountain flora of kopaonik (l a k u š i ć , 1993). altitudinal range of the ibar river valley investigation area was 500-900 m and of the rogozna mt. was 800-1473, where similarity reflected in almost identical schedule of the 9 most abundant families is obvious (fig. 3). just like on rogozna mt., mediterranean impacts are more pronounced in ibar river valley in contrast to the high mountain area of kopaonik. on kopaonik, the second place in taxonomical spectrum belongs to the family of arctic and boreal regions, poaceae (9.34%) (l a k u š i ć , 1993), which is another proof of weaker mediteranian impact. investigation of the taxonomical spectrum showed that trifolium genera is the most abundant with 21 species (2.71%), followed by campanula with 13 species, dianthus and silene with 12 species, carex, euphorbia, potentilla, vicia with 11 species, melampyrum with 10 species, geranium, lathyrus and veronica with 9 species (tab. 2). the dominance of the genera trifolium is probably caused by the increased presence of xerothermic meadows and pastures. hieracium, the most abundant genera on the balkan peninsula, is even on the 33 rd place in the rogozna mt. taxonomical spectrum, which can only be explained by insufficient taxonomical research of this genera (jaccard, 1912). figure 3. overview of the most abundant families on the rogozna mt. and neighboring regions table 2. taxonomic structure of the most abundant genera in the flora of rogozna mt. genera species n° % trifolium 21 2.71 campanula 13 1.68 dianthus 12 1.55 silene 12 1.55 carex 11 1.42 euphorbia 11 1.42 potentilla 11 1.42 vicia 11 1.42 melampyrum 10 1.29 geranium 9 1.16 lathyrus 9 1.16 veronica 9 1.16 centaurea 8 1.03 epilobium 8 1.03 galium 8 1.03 ranunculus 8 1.03 stachys 8 1.03 references aлëхин, в.в., 1944: география растений.-изд. советская наука, москва. 455 p. božanić, s., 2006: ibarsko jezgro svetostefanskog vlastelinstva. filozofski fakultet novi sad-ia “srem”, novi sad-sremska mitrovica. 494 p. greuter, w., burdet, h.m., long, g. (ed.), 19841989: med-checklist, 1, 3, 4. gèneve. jaccard, p., 1912: the distribution of flora in the alpine zone. new phytologist, 11: 37-50. janković, m.m., 1985: fitogeografija. univerzitet u beogradu. 425 p. josifović, m. (ed.), 1970-1976: flora sr srbije, iix. sanu. beograd. biologica nyssana 5 (1)  september 2014: 17-30 papović, o. et al.  analysis of the flora of rogozna… 30 lakušić, d., 1993: visokoplaninska fl ora kopaonika ekološko fi togeografska studija. magistarski rad. biološki fakultet, beograd. prodanović, d., 2007: serpentinska flora kosovskog dela ibarske doline. doktorska disertacija. univerzitet u prištini, prirodno matematički fakultet, kosovska mitrovica. stevanović, v., vasić, v. (ed.), 1995: biodiverzitet jugoslavije sa pregledom vrsta od međunarodnog značaja. biološki fakultet i ekolibri, beograd. the international organisation for plant information http://plantnet.rbgsyd.nsw.gov.au/iopi/iopihome. htm)). turrill, w.b., 1929: the plant life of the balkan peninsula. a phytogeographical study. clarendon, oxford. tutin, t.g., heywood, v.h., burges, n.a., moore, d.m., valentine, d.h., walters, s.m., webb, d.a. (ed.), 1964-1980: flora europaea, i-v. cambridge, university press. london. velchev, v. (ed.), 1982–1989: flora reipubl. popularis bulgaricae, vols 8-9. in aedibus acad. sci. bulgaricae, serdicae (in bulgarian) http://plantnet.rbgsyd.nsw.gov.au/iopi/iopihome.htm) http://plantnet.rbgsyd.nsw.gov.au/iopi/iopihome.htm) anticancer compounds from medicinal plants biologica nyssana 5 (1)  september 2014: 1-10 stojković, m. et al.  antioxidant potential of tanacetum vulgare l. … 47 original article received: 3 september 2014 revised: 5 september 2014 accepted: 10 september 2014 antioxidant potential of tanacetum vulgare l. extracts milan b. stojković 1* , snežana s. mitić, jovana lj. pavlović, branka t. stojanović, dušan đ. paunović 1 department of chemistry, faculty of science and mathematics, university of niš, 33 višegradska street, 18 000 niš, serbia * e-mail: milan@svrljig.net abstract: stojković, m., mitić, s., pavlović, j., stojanović, b., paunović, d.: antioxidant potential of tanacetum vulgare l. extract. biologica nyssana, 5 (1), septmeber 2014: 47-51. antioxidant activity of tanacetum vulgare l. extracts was determined. areal plant parts (leaves and flowers) were dried, grinded and extracted with five different solvents: methanol, ethanol, acetone, water and isopropanol. total phenols and total flavonoids were determined and four in vitro antioxidant assays were applied. the best extraction medium (considering only phenols and flavonoids) was methanol and 62.7 mg galic acid equivalents per gram of dry weight were found (for leaves extract). comparing amounts of phenolic compounds found in the extracts and its antioxidant potential with other herbal teas and extracts, it may be concluded that t. vulgare is a plant species destitute with phenolic compounds. obtained results suggest that phenolic compounds, present in the plant tissues, are carriers of antioxidant properties. key words: antioxidant, flavonoid, extrac, phenol, tanacetum vulgare apstract: stojković, m., mitić, s., pavlović, j., stojanović, b., paunović, d.: antioksidantni potencijal ekstrakta biljne vrste tanacetum vulgare l. biologica nyssana, 5 (1), septmeber 2014: 47-51. određivana je antioksidantna aktivnost ekstrakta biljne vrste tanacetum vulgare l. nadzemni delovi ove biljne vrste (listovi i cvetovi) su sušeni, samleveni i ekstrakovani sa pet različitih rastvarača (metanol, etanol, aceton, voda i izopropanol). određen je sadržaj totalnih fenola i totalnih flavonoida kao i antioksidantna aktivnost pomoću četiri in vitro metoda. kao najbolje ekstrakciono sredstvo (za fenole i flavonoide) pokazao se metanol i nađeno je 62.7 mg ekvivalenata galne kiseline po gramu suve biljne mase (u ekstraktu lista). ako nađeni sadrđaj fenola i flavonoida uporedimo sa sličnim ekstraktima ili biljnim čajevima, možemo zaključiti da je biljna vrsta t. vulgare relativno siromašna fenolnim jedinjenjima. dobijeni rezultati sugeriču da su fenolna jedinjenja, prisutna u biljnim tkivima, nosioci antioksidantnih osobina. ključne reči: antioksidant, flavonoid, ekstrakt, fenol, tanacetum vulgare introduction tanacetum is a genus of about 160 species of flowering plants in the asteraceae family, native to many regions of the northern hemisphere. they are known commonly as tansies in english and povratič in serbian. tanacetum vulgare l. (common tansy) is perennial, herbaceous plant species native to temperate europe and asia but invasive in other parts of world. 5 (1) • september 2014: 47-51 biologica nyssana 5 (1)  september 2014: 47-51 stojković, m. et al.  antioxidant potential of tanacetum vulgare l. … 48 tansy is a well-known herbal plant widely used in traditional medicine in south-eastern serbia (k o j i ć et al., 1998). in recent times, preparing teas is contraindicated because of the toxic monoterpene, α-thujone, present in the plant tissue (b a r a n a u s k i e n e et al., 2014). tea prepared from t. vulgare is used as an antihelminthic, carminative, antispasmodic, stimulant to abdominal viscera, tonic, emmenagogue, antidiabetic, diuretic and antihypertensive (l a h l o u et al., 2008). various authors showed that the water and lipophilic extracts from t. vulgare possess antitumor, antiinflammatory, antioxidant and antimicrobial activity (l a h l o u et al., 2008). to explore potential health benefits, dried parts of t. vulgare (flowers and leaves) were extracted with five different solvents (methanol, ethanol, acetone, water and isopropanol) and total phenols and flavonoids were determined. series of in vitro antioxidant assays were performed (scavenging dpph radical, scavenging abts radical, iron(iii) to iron(ii) reduction assay and cupric ion reducing antioxidant capacity assay). material and methods plant material and extract preparation areal parts of tanacetum vulgare l. (asteraceae) were collected in mai 2012 from the mountain peak babin zub (latitude: 43.363 n; longitude: 22.585 e), south-east serbia. the plant material was air dried about two weeks, out of direct sunlight. flowers and leaves were separated from the stems and were grinded into fine powder. exact masses (0.25 g) of powdered flowers and leaves were extracted with five different solvents (methanol, ethanol, acetone, water and isopropanol) on ultrasonic bath. every batch was extracted three times for 30 minutes, with 20+20+10 ml of appropriate solvent. reagents 1,1-diphenyl-2-picrylhydrazyl radical (dpph), 2,2’azino-bis(3-ethylbenzothiazoline-6sulfonic acid) (abts), potassium-persulfate and catechin were obtained from sigma-aldrich (steinheim, germany), methanol, ethanol and acetone were purchased from j. t. baker (deventer, holland). gallic acid was obtained from carl roth (karlsruhe, germany). trolox (6-hydroxy-2,5,7,8tetramethylchroman-2-carboxylic acid) were obtained from acros organics (geel, belgium). folin-ciocalteu reagent, na2hpo4, nah2po4, na2co3, nano2, naoh, feso4 ∙ 7h2o, k3[fe(cn)6], trichloroacetic acid (tca) and alcl3 were purchased from merck (darmstadt, germany). all other chemicals were of analytical grade, and were used as received except that the solvents were distilled prior to use. determination of total phenolic (tp) content total phenolic content of the extracts were determined using folin-ciocalteau assay (m i t i ć e t a l ., 2011). briefly, 0.4 ml of extract were mixed with 2.0 ml of (20% w/v) na2co3 solution and 0.5 ml of fc reagent and made up to 10 ml with deionized water. the solution was mixed and, after aging for 120 min at 25˚c, absorbance was measured at 760 nm, using agilent 8453 uv-visible spectrophotometer (agilent technologies, usa). results were expressed as mg of gallic acid equivalents (gae) per g of the dry sample. determination of total flavonoid (tf) content the total flavonoid content of t. vulgare extracts was determined by a colorimetric method according to m i t i ć e t a l . (2012). a known volume of the samples was mixed with 2 ml of distilled water and subsequently with 0.3 ml of a nano2 solution (5%, w/w). after 5 min, 3 ml of alcl3 solution (1%, w/w) was added and the solution left for 5 min at room temperature. then, 2 ml of naoh solution (1 mol/l) was added to the mixture diluted with deionized water to the final volume of 10 ml. the mixture was thoroughly mixed and absorbance was immediately measured at 510 nm. results were expressed as mg catechin equivalent (ce) per g of dry weight (d.w.). dpph free radical-scavenging assay the antioxidant capacity of t. vulgare solvent extracts was studied through the evaluation of the extracts’ free radical-scavenging effect on 1,1diphenyl-2-picrylhydrazyl (dpph) radical (s t o j k o v i ć e t a l ., 2014). an aliquot (0.1 ml) of the different extracts was mixed with 2.5 ml of 100 µmol/l dpph methanol solution. the mixture was thoroughly vortex-mixed, kept out of light for 30 min and absorption was measured at 515 nm. the absorption of the blank containing the same amount of methanol and dpph solution was prepared and measured daily. the radical scavenging activity was calculated using the following formula: scavenging effect (%) = [1-(absorbance of the sample/absorbance of the blank)] ∙ 100 biologica nyssana 5 (1)  september 2014: 47-51 stojković, m. et al.  antioxidant potential of tanacetum vulgare l. … 49 the results were expressed as mg of trolox equivalents (te) per 1 g of sample. abts radical-scavenging capacity assay the abts radical cation (abts •+ ) solution was prepared by the reaction of solutions of 7 µmol/l abts and 2.45 µmol/l of potassium persufate, at 23°c in the dark for 16 h. the abts •+ solution was then diluted with 80% (v/v) aqueous ethanol to obtain a solution with the absorbance of 0.700 ± 0.020 at 734 nm (v e l j k o v i ć e t a l ., 2013). the abts •+ solution (3.9 ml) was added to 0.1 ml of the test sample and mixed thoroughly. the reaction mixture was left to stand at 23°c for 6 min and then the absorbance was measured at 734 nm. the total antioxidant activity of t. vulgare extracts was expressed as mg of te per g of dry weight. radical scavenging activity was calculated using the following formula: scavenging effect (%) = [1-(absorbance of sample/absorbance of blank)] ∙100 iron(iii) to iron(ii) reduction assay (ira) iron(iii) to iron(ii) reduction assay was performed according to s t o j k o v i ć et al. (2014). different dilutions of the extracts (0.5 ml) were added to the mixture of 1.25 ml of the phosphate buffer (0.2 mol/l, ph 6.6) and 1.25 ml of potassium ferricyanide (1%, w/w). the resultant solution was incubated at 50°c for 20 min. after that, trichloroacetic acid solution (1.25 ml, 10%, w/w) was added, diluted with 4.25 ml of water and 0.85 ml of ferric chloride solution (0.1%, w/w) was added. after 30 min, the absorbance was measured at 700 nm. ira of the extracts was expressed as mg gallic acid equivalents per g of dry weight of the sample. cupric ion reducing antioxidant capacity (cuprac) the cuprac method was applied as described by s t o j k o v i ć et al. (2014). a mixture comprised of 1 ml of 10 mmol/l copper(ii) chloride, 1 ml of 1mol/l ammonium acetate buffer at ph 7.0, and 1 ml of 7.5 mmol/l neocuproine solution was prepared, x ml sample solution and (1−x) ml distilled water were added, and well mixed (total volume: 4.0 ml). this final mixture in a stoppered test tube was left to stand at room temperature for 30 min. after that, the absorbance at 450 nm was measured against a blank. the total antioxidant activity of t. vulgare extracts was expressed as µmol of te per g of dry weight. statistical analysis all results were expressed as the mean ± standard deviation. statistically significant differences were determined by one-way analysis of variance (anova) followed by tukey´s post hoc test for multiple comparison (graph pad prism version 5.03, san diego, ca, usa). probability values (p) less than 0.05 were considered to be statistically significant. results and discussion phenolic compounds (phenolic acids and flavonoids) are the natural constituents of plants. they are secondary metabolites and produced by plants as a response to different environmental factors (light, chilling, pollution, etc.) and to defend injured plants. phenolic compounds are crucial for plant growth and reproduction. also, they have various roles in plants (protective, signal molecules, gives colours of flowers, fruits, and leaves, etc.) but they are not understood completely (g h a s e m z a d e h & g h a s e m z a d e h , 2011). polyphenols are well known for their antioxidant activity as radical scavengers and possible beneficial roles in human health, such as reducing the risk of cancer, cardiovascular and other diseases (m i t i ć et al., 2012). the main carriers of antioxidant properties in plant tissues are phenolic compounds. thus, total phenols and total flavonoids of t. vulgare’s flowers and leaves extracts were determined. the antioxidant capacity of the extracts were studied through four in vitro antioxidant assays: scavenging dpph radical, scavenging abts radical, iron(iii) to iron(ii) reduction assay (ira) and cupric ion reducing antioxidant capacity assay (cuprac). obtained results are shown in tab. 1. total phenols (tp) in various extracts are in the range from 62.7 to 12.2 mg of galic acid equivalents (gae) per g of dried weight (dw) for leaf extracts and from 42.1 to 7.2 mg gae/g dw for flower extracts. methanol was observed as the best extraction medium for total phenolic compounds among the others solvents used (ethanol, water, isopropanol and acetone). the lowest amounts of phenolic compounds were extracted with acetone. total flavonoids found in mentioned extracts are in the range from 18.2 to 4.7 mg of catechin equivalents (ce)/g dw for leaf extracts and from 10.5 to 4.0 mg ce/g dw for flower extracts. the best solvent for flavonoid extraction was water, dissimilar to total phenols where methanol was the best one, but the observed difference was small biologica nyssana 5 (1)  september 2014: 47-51 stojković, m. et al.  antioxidant potential of tanacetum vulgare l. … 50 table 1: total phenols, total flavonoids and in vitro antioxidant tests tp tf abts dpph ira cuprac mg gae/g dw mg ce/g dw mg te/g dw mg te/g dw mg gae/g dw mg te/g dw l e a v e s ethanol 31.6 ± 0.5 9.3 ± 0.3 25.5 ± 0.5 12.3 ± 0.5 18.7 ± 0.5 115 ± 3 methanol 62.7 ± 0.5 15.3 ± 0.5 41.2 ± 0.5 28.1 ± 0.5 34.2 ± 0.5 214 ± 5 acetone 12.2 ± 0.5 4.7 ± 0.1 7.6 ± 0.2 4.9 ± 0.1 10.2 ± 0.5 53 ± 1 water 47.8 ± 0.5 18.2 ± 0.5 22.2 ± 0.5 25.9 ± 0.5 35.2 ± 0.5 129 ± 3 isopropanol 12.3 ± 0.5 6.3 ± 0.1 5.5 ± 0.2 4.2 ± 0.1 9.9 ± 0.5 34 ± 1 f lo w e rs ethanol 19.0 ± 0.5 6.0 ± 0.1 12.2 ± 0.3 7.6 ± 0.3 13.9 ± 0.5 47 ± 1 methanol 42.1 ± 0.5 10.1 ± 0.3 27.6 ± 0.5 15.8 ± 0.5 27.0 ± 0.5 164 ± 3 acetone 7.2 ± 0.3 4.0 ± 0.1 4.5 ± 0.1 3.5 ± 0.1 9.2 ± 0.5 18.1 ± 0.5 water 42.3 ± 0.5 10.5 ± 0.3 19.9 ± 0.5 21.7 ± 0.5 25.8 ± 0.5 121 ± 3 isopropanol 10.3 ± 0.5 5.6 ± 0.1 4.8 ± 0.1 5.5 ± 0.2 10.0 ± 0.5 33 ± 1 table 2: correlation coefficient between total phenols, total flavonoids and different in vitro antioxidant assays tp tf abts dpph ira cuprac tp 1 0.9094 0.9474 0.9735 0.9737 0.9677 tf 1 0.7871 0.9470 0.9601 0.8158 abts 1 0.8613 0.8739 0.9790 dpph 1 0.9802 0.8943 ira 1 0.9118 cuprac 1 (leaf) or insignificant compared to the standard deviation (flower). phenol and flavonoid contents in various herbal teas (v e l j k o v i ć et al., 2013) are in the range from 26 to 241 mg gae/g dw and from 12 to 85 mg cae/g dw for phenols and flavonoids, respectively. according to this data, t. vulgare is a plant with small contents of phenolic compounds. 1,1-diphenyl-2-picrylhydrazyl radical (dpph) is a stable nitrogen-centered free radical whose color changes from violet to yellow upon reduction either by the process of hydrogen or electron-donation. antioxidant capacity were in the range from 28.1 mg trolox equivalents (te)/g dw (methanol extract of leaf) to 3.5 mg te/g dw (ethanol extract of flower). the abts assay method is based on the ability of antioxidant molecules to quench the longlived abts cation radicals (which ethanol solution is blue-green with characteristic absorption at 734 nm). the addition of antioxidants to the solution of prepared radical cation reduces it to abts, causing decolorizing. results were expressed as mg trolox equivalent per g of dw. antioxidant capacity, determined with this assay, was in the range from 41.2 mg trolox equivalents (te)/g dw (methanol extract of leaf) to 4.5 mg te/g dw (ethanol extract of flower). ira is based on reduction of ferricyanide ions to ferrocyanide in the presence of reductants (antioxidants) in the samples by donating an electron. the ferrocyanide ions then react with fe 3+ ions forming prussian blue which strongly absorbs on 700 nm. increasing absorbance at 700 nm indicates an increase in antioxidant ability of a sample. leaf extracts of both water and methanol showed the highest values for reduction capacity (35.2 and 34.2 mg gae/g dw, respectively). acetone and isopropanol extracts of leaf and flower showed the lowest antioxidant capacity and there is no significant difference between the values according to anova. another antioxidant assay used was cuprac. it is based on reduction of cu 2+ ion to cu + ion which reacts with neocuproine giving the complex with absorption maximum at 450 nm. the biologica nyssana 5 (1)  september 2014: 47-51 stojković, m. et al.  antioxidant potential of tanacetum vulgare l. … 51 best antioxidant capacity showed methanol extract of leaf (214 mg te/g dw) while isopropanol extracts of both leaf and flower had the lowest capacity (34 and 33 mg te/g dw, respectively). correlation coefficient between total phenols, total flavonoids and different in vitro antioxidant assays are calculated and showed in the tab. 2. the high values for the coefficients between tp and various in vitro tests suggest that phenolic compounds are the main carriers of antioxidant power in the plant tissues. also, the high values for the coefficients among various in vitro tests proposed that the experimental errors were minor. conclusion methanol extracts showed the best antioxidant potential among the other solvents used (ethanol, water, acetone and isopropanol). the best extraction medium considering only phenols and flavonoids was also methanol (62.7 mg galic acid equivalents per gram of dry weight were found for leaves extract). methanol is an extremely toxic solvent and it is not applicable for human usage, especially methanol extracts. on the other hand, water extracts are suitable for human intake and. obtained results suggest that antioxidant potential of water extracts were as high as the appropriate methanol ones. in some cases water extracts showed higher antioxidant potential. it can be concluded that the water extraction is a good way to obtain phenolic compounds from t. vulgare. comparing amounts of phenolic compounds found in the extracts and its antioxidant potential with other herbal teas and extracts, it may be concluded that t. vulgare is a plant species destitute with phenolic compounds. obtained results suggest that phenolic compounds, presents in the plant tissues, are carriers of antioxidant properties. acknowledgements. this work was supported by the ministry of education and science of serbia (project no 172061 and project no 31060). the authors are grateful for the financial support provided by this ministry. references baranauskiene, r., kazernaviciute, r., pukalskiene, m., mazdzieriene, r., venskutonis, p., 2014: agrorefinery of tanacetum vulgare l. into valuable products andevaluation of their antioxidant properties and phytochemicalcomposition. industrial crops and products, 60: 113–122. ghasemzadeh, a., ghasemzadeh, n., 2011: flavonoids and phenolic acids: role and biochemical activity in plants and human. journal of medicinal plants research, 5: 6697– 6708. kojić, m., stamenković, v., jovanović, d. 1998: lekovite biljke jugoistočne srbije. zavod za udžbenike i nastavna sredstva, beograd. lahlou, s., tangi, k., lyoussi, b., morel, n., 2008: vascular effects of tanacetum vulgare l. leaf extract: in vitro pharmacological study. journal of ethnopharmacology, 120: 98–102. mitic, s., stojkovic, m., pavlovic, j., mitic, m., stojanovic, b., 2012: antioxidant activity, phenolic and mineral content of stachys germanica l. (lamiaceae). oxidation communications, 35, (4): 1011–1020. stojkovic, n., stojkovic, m., marinkovic, m., chopra, g., kostic, d., zarubica, a., 2014: polyphenol content and antioxidant activity of anthemis cretica l. (asteraceae). oxidation communications, 37, (1): 237–246. veljković, j,. pavlović, a., mitić, s., tošić, s., stojanović, g., kaličanin, b., stanković, d., stojković, m., mitić, m., brcanović, j., 2013: evaluation of individual phenolic compounds and antioxidant properties of black, green, herbal and fruit tea infusions consumed in serbia: spectrophotometrical and electrochemical approaches. journal of food and nutrition research, 52: 12–24. biologica nyssana 5 (1)  september 2014: 47-51 stojković, m. et al.  antioxidant potential of tanacetum vulgare l. … 52 mahmutović-dizdarević et al. 2023, biologica nyssana 14(1) 14 (1) june 2023: 47-56 doi: 10.5281/zenodo.8027148 essential oils of selected citrus fruits and spice plants as potential antibacterial and antibiofilm agents original article irma mahmutović-dizdarević department of biology, faculty of science, university of sarajevo, zmaja od bosne 33-35, 71000 sarajevo, bosnia and herzegovina irma.m@pmf.unsa.ba (corresponding author) nejra bektaš department of genetics and bioengineering, international burch university, francuske revolucije bb, 71210 ilidža, bosnia and herzegovina šabanija gazić department of genetics and bioengineering, international burch university, francuske revolucije bb, 71210 ilidža, bosnia and herzegovina anesa jerković-mujkić department of biology, faculty of science, university of sarajevo, zmaja od bosne 33-35, 71000 sarajevo, bosnia and herzegovina mirsada hukić academy of sciences and arts of bosnia and herzegovina, center for disease control and geohealth studies, bistrik 7, 71000 sarajevo, bosnia and herzegovina institute for biomedical diagnostics and research nalaz, čekaluša 69, 71000 sarajevo, bosnia and herzegovina monia avdić department of genetics and bioengineering, international burch university, francuske revolucije bb, 71210 ilidža, bosnia and herzegovina academy of sciences and arts of bosnia and herzegovina, center for disease control and geohealth studies, bistrik 7, 71000 sarajevo, bosnia and herzegovina received: december 22, 2022 revised: april 07, 2023 accepted: april 17, 2023 abstract: this study evaluates the antibacterial and antibiofilm properties of essential oils (eos) from citrus lemon (l.) osbeck, lemon; citrus reticulata blanco, mandarine; nigella sativa l., black cumin, and foeniculum vulgare mill., fennel, using the disk-diffusion, broth microdilution, and tissue culture plate methods on 11 bacterial strains, including the multidrug-resistant (mdr). results showed that tested eos exhibit antibacterial effects, that are stronger in gram-positive bacteria. the widest inhibition zones were achieved with the black cumin eo against mdr staphylococcus aureus. values of the minimum inhibitory concentrations ranged from 250 to 750 µg/ml, while minimum bactericidal concentrations were 500-1000 µg/ml. black cumin eo performed strong antibiofilm features, with the total elimination of the biofilm in the case of different s. aureus strains (including methicillin-resistant, mrsa), and pseudomonas aeruginosa. all investigated eos exhibited strain-specific and dose-dependent antibacterial and antibiofilm activity. key words: bacterial biofilms, antibiofilm agents, essential oils, citrus lemon (l.) osbeck, citrus reticulata blanco, nigella sativa l., foeniculum vulgare mill. apstrakt: eterična ulja ploda odabranih citrusa i začinskih biljaka kao potencijalni antibakterijski i antibiofilm agensi ova studija evaluira antibakterijska i antibiofilm svojstva eteričnih ulja iz citrus lemon (l.) osbeck, limuna; citrus reticulata blanco, mandarine; nigella sativa l., crnog kima i foeniculum vulgare mill., komorača, korišćenjem disk-difuzione, mikrodilucijske i “tissue culture plate” metode na 11 sojeva bakterija, uključujući multirezistentne (mdr) sojeve. rezultati su pokazali da testirana eterična ulja ispoljavaju antibakterijski efekat, koji je jači kod gram-pozitivnih bakterija. najšire zone inhibicije su postignute delovanjem eteričnog ulja crnog kima na mdr staphylococcus aureus. vrednosti minimalne inhibitorne koncentracije su se kretale od 250 do 750 µg/ml, dok su minimalne baktericidne koncentracije bile 500-1000 µg/ml. eterično ulje crnog kima je ispoljilo jako antibiofilm dejstvo, sa totalnom eliminacijom biofilma u slučaju različitih sojeva s. aureus (uključujući meticilin-rezistentni, mrsa) i pseudomonas aeruginosa. sva ispitivana eterična ulja su ispoljila antibakterijsku i antibiofilm aktivnost koja je bila specifična za soj, te dozno-zavisna. ključne reči: bakterijski biofilmovi, antibiofilm agensi, eterična ulja, citrus lemon (l.) osbeck, citrus reticulata blanco, nigella sativa l., foeniculum vulgare mill. introduction antimicrobial resistance is considered one of the major global health challenges of the 21st century (hernando-amado et al., 2019). according to urbanchmiel et al. (2022), antibiotic resistance is acquired via several mechanisms such as active removal of the antibiotic from the cell, enzymatic modifications of the drug, modifications of cell components which are the target of the antibiotic, overexpression of an enzyme inactivated by the antibiotic, a change in the permeability of bacteria cell membranes, production of an alternative metabolic pathway, an increase in the concentration of a metabolite which is an antagonist of the antibiotic, a reduction in the amount or activity of an enzyme activating the precursor of the antibiotic, modifications in regulatory systems not associated with the direct mechanism © 2023 mahmutović-dizdarević et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 47 of action of the antibiotic, or a reduction in the demand for the product of the inhibited metabolic pathway. bacterial biofilm represents a structured consortium of microorganisms embedded in a self-produced matrix made from polysaccharides, proteins, and dna, characterized by increased resistance to antimicrobial agents (høiby et al., 2010). studies suggest that microbial cells within the biofilm have 10–1000 times more antibiotic resistance in comparison to planktonic cells (mah, 2012). furthermore, bacterial biofilms are involved in approximately 80% of chronic and recurrent microbial infections in humans (sharma et al., 2019). numerous investigations have shown that different plant products as well as plant active ingredients can inhibit the formation and development of bacterial biofilms, and eradicate mature biofilms (cheng et al., 2022). essential oils (eos) are the volatile secondary metabolites of plants, produced by more than 17.500 plant species from many angiosperm families, but only about 300 of them are commercialized (wińska et al., 2019). numerous biological activities such as antiseptic, antibacterial, antiviral, antioxidant, antiparasitic, antifungal, and insecticidal effects are previously reported for eos. also, eos are considered a powerful tool in the reduction of bacterial resistance (chouhan et al., 2017). essential oils as well as other bioactive products of medicinal plants have received excessive attention for their low toxicity, pharmacological activities, and economic viability (auddy et al., 2003). therefore, eos and their components are naturally occurring antimicrobial compounds with the potential to prevent the limitations of conventional antimicrobial agents (orhan-yanıkan et al., 2019). lemon, citrus limon (l.) osbeck (rutaceae), is a well-known and frequently used plant for different purposes, especially in cooking, but studies suggest its various biological activities such as anticancer, antioxidant, antimicrobial, etc. lemon is also proven for many beneficial effects on the nervous, cardiovascular, respiratory, and skeletal systems (klimek-szczykutowicz et al., 2020). bioactive properties such as antimicrobial, anticancer, antioxidant, antigenotoxic, hepatoprotective, etc. are also related to citrus reticulata blanco (rutaceae), the mandarin orange (musara et al., 2020). nigella sativa l. (ranunculaceae), known as black cumin, is a worldwide distributed plant with many curative effects on human health. the pharmacological significance of this species is mainly illustrated by its capacity to act as an antimicrobial and antioxidative agent (tabassum et al., 2018). fennel or foeniculum vulgare mill. (apiaceae), is a seasonal medicinal plant, grown by humans in nearly every region. due to its great aromaticity and spiciness, this is a popular cooking ingredient but nevertheless, pharmacological studies revealed that fennel possesses bioactive potential and could be used in the treatment of different diseases (tripathi et al., 2012). the main goal of this study was to evaluate the antibacterial and antibiofilm potential of essential oils made from well-known edible and spice plants: lemon, c. lemon (lemon), c. reticulata (mandarin orange), n. sativa (black cumin), and f. vulgare (fennel). materials and methods essential oils in this investigation pure eos derived from citrus lemon (l.) osbeck, citrus reticulata blanco, nigella sativa l., and foeniculum vulgare mill. (dea flores d.o.o., rijeka, croatia) were used. stock solutions of the test substances were prepared in 0.1% dimethyl sulfoxide ≥99% (dmso) (sigma-aldrich) and kept at room temperature in the dark. bacterial species investigation of antibacterial and antibiofilm properties of selected eos comprised a total of 11 bacteria, including the multidrug-resistant (mdr), strains: staphylococcus aureus atcc 6538 (sa1); s. aureus atcc 25923 (sa2); methicillin-resistant s. aureus (mrsa): s. aureus atcc 33591 (sa3), and s. aureus nctc 12493 (sa4); enterococcus faecalis atcc 29212 (ef); bacillus subtilis atcc 6633 (bs); escherichia coli 14169 (ec1); e. coli atcc 25922 (ec2); extended-spectrum beta-lactamase-producing (esbl) e. coli atcc 35218 (ec3); pseudomonas aeruginosa atcc 27853 (pa); salmonella enterica nctc 6017 (se). investigated bacterial strains were purchased from the american type culture collection (atcc) and national collection of type cultures (nctc) (microbiologics, st. cloud, minnesota, usa). inoculums were set in accordance with eucast (2017). overnight cultures were diluted in sterile saline solution to the final turbidity of 0.5 mcfarland standard, which corresponds to the bacterial concentration of 1.5 × 108 cfu/ml. determination of the inhibition zones the antibacterial activity of investigated essential oils was evaluated by using pure eo (100%), as well as different concentrations of eo (250, 500, and 750 µg/ml) aseptically dissolved in 0.1% dmso. for this part of the study, the disk diffusion method was applied (bauer et al., 1966). bacterial strains were cultured in mueller hinton (mh) medium (fluka biochemica; buchs, 48 biologica nyssana ● 14 (1) june 2023: 47-56 mahmutović-dizdarević et al. ● essential oils of selected citrus fruits and spice plants as potential antibacterial and antibiofilm agents biologica nyssana ● 14 (1) june 2023: 47-56 mahmutović-dizdarević et al. ● essential oils of selected citrus fruits and spice plants as potential antibacterial and antibiofilm agents with bacterial inoculum. inoculums were prepared as described above, i. e turbidity of 0.5 mcfarland standard and bacterial concentration of 1.5 × 108 cfu/ml. the adherence of bacteria in the presence of tsb was used for the determination of the biofilm formation. after the overnight incubation, the plates were emptied, washed in phosphate buffered saline, pbs (sigma-aldrich, usa), and stained with 0.1% crystal violet solution for 10 minutes. after washing, 96% ethanol was added to each well. results were analysed on the microplate reader (biochrom ez read 400) at 595 nm. this experiment was done in four replications, and results were given as the means ± stdev. the biofilm-forming category was determined according to stepanović et al. (2007) and by the biofilm classifier software ver 1.1. the optical density cut-off value (odc) was calculated as three standard deviations above the mean od of the negative control, while the biofilm categories were determined as follows: od ≤ odc: non-adherent (na), odc < od ≤ 2 x odc: weakly adherent (w), 2 x odc < od ≤ 4 x odc: moderately adherent (m), and 4 x odc < od: strongly adherent (s). statistical analysis for the calculation of the descriptive statistical parameters (mean values and standard deviation) and the percentage of biofilm inhibition microsoft office 2019 excel (microsoft corporation, usa) was used. data were further analyzed by one-way anova and post-hoc fisher’s lsd test (statistica 10; statsoft. inc.) at the significance level of p<0.05. results and discussion the antibacterial activity of essential oils is mainly associated with their hydrophobic properties, which ensure their distribution in the lipids of the cell membranes and mitochondria, which further affects the structural integrity and function of those structures and after all results in leakage of cell contents (tang et al., 2020). such events interfere with the atp balance and among others, have an impact on ph, protein synthesis, coagulation of the cytoplasmic material, dna disruption, and quorum sensing inhibition (el-tarabily et al., 2021). obtained results regarding the antibacterial activity of investigated eos through the measuring of inhibition zones are presented in table 1. overall observation showed that all investigated eos exhibited antibacterial potential against all tested bacteria except e. coli atcc 14169, e. coli atcc 35218 (esbl strain), and s. enterica nctc 6017. the most sensitive bacterial species was b. subtilis, with achieved inhibition zones with all tested eos, in all concentrations except the lowest dilution switzerland), and after adjusting the final density of inoculum (1.5 × 108 cfu/ml), spread over the growth medium plates. an amount of 10 µl of each concentration of eos was impregnated into the paper disk which was enough to achieve saturation. a total of six disks (four concentrations of eo, positive and negative control included) were placed on one 150 mm plate. after the application of eo, plates were incubated at 35±2 °c for 16-18 hours. as the positive control, antibiotics ampicillin (10µg), streptomycin (10µg), and colistin (10µg), all made by oxoid™ (great britain) were used, while 0.1% dmso served as the solvent control. the antibacterial effect was evaluated based on the diameter (mm) of inhibition zones. all tests were performed in triplicate, and the mean value ± standard deviation (stdev) was taken for further analysis. minimum inhibitory and minimum bactericidal concentration the minimum inhibitory concentration (mic) of eos was determined through the broth microdilution method (clsi, 2018). pure eos (100%) were dissolved in 0.1% dmso to achieve the stock concentration of 1000 µg/ml. the final volume of 100 µl of two-fold dilutions of eos ranging from 500 to 1.95 µg/ml, was applied in a 96-well microtiter plate containing the mueller hinton broth (sigma-aldrich). an amount of 10 µl of bacterial inoculum (concentration of 1.5 × 108 cfu/ml) was then added to each well. pure bacterial culture was taken as the growth control, and uninoculated media with the dmso was used as the negative control. after the overnight incubation, results were read on a microplate reader (biochrom ez read 400) at 595 nm. experiments were performed in quadruplets, and mic concentrations were determined based on the generated absorbance values. minimum bactericidal concentration (mbc) was evaluated by replating the inoculums from the wells without signs of growth on a sterile mueller hinton medium and observing the presence of growth after overnight incubation. evaluation of the antibiofilm activity in order to determine the biofilm formation in the presence of tested eos, the tissue culture plate method (tcp) in 96 well plates (merritt et al., 2005) was used, with the tryptic soy broth, tsb (sigmaaldrich) as the growing medium. the stock solution of eo in dmso was two-fold diluted in tsb up to the end concentration of 1.95 µg/ml. an amount of 100 µl of such dilution was added to each well, followed by the inoculation with 10 µl of the bacterial suspension. uninoculated media with dmso was used as the solvent control, while wells described as the untreated biofilms controls contained media 49 sa m pl e b ac te ri al s tr ai ns sa 1 sa 2 sa 3* sa 4* e f b s e c 1 e c 2 e c 3* p a se g ro w th in hi bi ti on (m m ) pure eo l em on n ia n ib n ic, d 19 .0 0± 0. 0e 11 .0 0± 0. 0f 20 .0 0± 0. 0 a, b, c, e, f,g ,h ,i, j n ig n ih ,k ,l n ic, k n ii n id ,j, l m an da ri ne 13 .3 0± 0. 6a n ib n ic, d n ie n if 12 .3 0± 1. 2 a, b, c, e, f,g ,h ,i, j n ig n ih ,k ,l n ic, k n ii n id ,j, l b la ck c um in 19 .7 0± 1. 2a 21 .0 0± 0. 0b 38 .3 0± 2. 9c ,d 32 .0 0± 1. 7e n if 30 .5 0± 0. 7 a, b, c, e, f,g ,h ,i, j n ig 30 .0 0± 0. 0h ,k ,l n ic, k 12 .0 0± 1. 0i n id ,j, l fe nn el n ia n ib 8. 50 ±1 .4 c, d n ie n if 11 .3 0± 1. 5 a, b, c, e, f,g ,h ,i, j n ig n ih ,k ,l n ic, k n ii n id ,j, l 250 µg/ml l em on n ia n ib n ic, d n ie n if n i a ,b ,c ,e ,f, g, h, i,j n ig n ih ,k ,l n ic, k n ii n id ,j, l m an da ri ne n ia n ib n ic, d n ie n if n i a ,b ,c ,e ,f, g, h, i,j n ig n ih ,k ,l n ic, k n ii n id ,j, l b la ck c um in n ia n ib 17 .3 0± 2. 5c ,d n ie n if n i a ,b ,c ,e ,f, g, h, i,j n ig n ih ,k ,l n ic, k n ii n id ,j, l fe nn el n ia n ib n ic, d n ie n if n i a ,b ,c ,e ,f, g, h, i,j n ig n ih ,k ,l n ic, k n ii n id ,j, l 500 µg/ml l em on n ia n ib n ic, d n ie 10 .0 0± 0. 0f 13 .0 0± 0. 0 a, b, c, e, f,g ,h ,i, j n ig n ih ,k ,l n ic, k n ii n id ,j, l m an da ri ne 10 .7 0± 0. 6a n ib n ic, d n ie n if 8. 30 ±0 .6 a ,b ,c ,e ,f, g, h, i,j n ig n ih ,k ,l n ic, k n ii n id ,j, l b la ck c um in 7. 30 ±0 .6 a 9. 00 ±2 .6 b 30 .3 0± 2. 8c ,d 20 .3 0± 2. 3e n if 27 .0 0± 1. 4 a, b, c, e, f,g ,h ,i, j n ig 21 .7 0± 2. 9h ,k ,l n ic, k 9. 30 ±0 .6 i n id ,j, l fe nn el n ia n ib n ic, d n ie n if 7. 00 ±1 .7 a ,b ,c ,e ,f, g, h, i,j n ig n ih ,k ,l n ic, k n ii n id ,j, l 750 µg/ml l em on n ia n ib n ic, d 16 .5 0± 0. 7e 9. 00 ±0 .0 f 14 .0 0± 0. 0 a, b, c, e, f,g ,h ,i, j n ig n ih ,k ,l n ic, k n ii n id ,j, l m an da ri ne 8. 00 ±1 .7 a n ib n ic, d n ie n if 8. 70 ±0 .6 a ,b ,c ,e ,f, g, h, i,j n ig n ih ,k ,l n ic, k n ii n id ,j, l b la ck c um in 16 .7 0± 1. 2a 17 .3 0± 2. b 35 .2 0± 1. 7c ,d 26 .3 0± 2. 5e n if 29 .0 0± 2. 6 a, b, c, e, f,g ,h ,i, j n ig 28 .3 0± 1. 5h ,k ,l n ic, k 10 .0 0± 0. 0i n id ,j, l fe nn el n ia n ib 6. 60 ±1 .1 c, d n ie n if 8. 30 ±1 .1 a ,b ,c ,e ,f, g, h, i,j n ig n ih ,k ,l n ic, k n ii n id ,j, l a m pi ci lli n 11 .0 0± 0. 0a 12 .0 0± 0. 0b n ic, d n ie 9. 00 ±0 .0 f 22 .0 0± 0. 0 a, b, c, e, f,g ,h ,i, j 14 .0 0± 0. 0g 16 .0 0± 0. 0h ,k ,l n ic, k n ii n id ,j, l st re pt om yc in 9. 00 ±0 .0 a 10 .0 0± 0. 0b n ic, d n ie 15 .0 0± 0. 0f 20 .0 0± 0. 0 a, b, c, e, f,g ,h ,i, j 15 .0 0± 0. 0g 17 .0 0± 0. 0h ,k ,l 12 .0 0± 0. 0c ,k 14 .0 0± 0. 0i 15 .0 0± 0. 0d ,j, l c ol is ti n n ia n ib n ic, d n ie n if 10 .0 0± 0. 0 a, b, c, e, f,g ,h ,i, j 16 .0 0± 0. 0g 15 .0 0± 0. 0h ,k ,l 10 .0 0± 0. 0c ,k 17 .0 0± 0. 0i 11 .0 0± 0. 0d ,j, l ta bl e 1. d ia m et er o f i nh ib iti on z on es o f i nv es tig at ed e ss en tia l o ils a nd s ta nd ar d an tib io tic s s a 1: s ta ph yl oc oc cu s au re us a tc c 6 53 8; s a 2: s . a ur eu s a tc c 2 59 23 ; s a 3: s . a ur eu s a tc c 3 35 91 (m r s a ); s a 4: s . a ur eu s n c tc 1 24 93 (m r s a ); e f: e nt er oc oc cu s fa ec al is a tc c 2 92 12 ; b s : b ac ill us s ub til is a tc c 6 63 3; e c 1: e sc he ric hi a co li a tc c 1 41 69 ; e c 2: e . c ol i a tc c 2 59 22 ; e c 3: e . c ol i a tc c 35 21 8 (e s b l) ; p a : p se ud om on as a er ug in os a a tc c 2 78 53 ; s e : s al m on el la e nt er ic a n c tc 6 01 7. * m d r s tra in s. le tte rs in s up er sc rip t i nd ic at e st at is tic al ly s ig ni fic an t d iff er en ce s af te r p er fo rm in g po st -h oc f is he r’s l s d te st . v al ue s w ith th e sa m e le tte r d iff er s ig ni fic an tly at p <0 .0 5. r es ul ts a re m ea n± s d e v . n i= n o in hi bi tio n. d m s o =n i. mahmutović-dizdarević et al. ● essential oils of selected citrus fruits and spice plants as potential antibacterial and antibiofilm agents biologica nyssana ● 14 (1) june 2023: 47-56 50 mahmutović-dizdarević et al. ● essential oils of selected citrus fruits and spice plants as potential antibacterial and antibiofilm agents (250 µg/ml). in comparison to the antibacterial activity of standard antibiotics, the black cumin eo performed the greater growth inhibition of b. subtilis (tab.1). black cumin is recognized for its antimicrobial, immunomodulatory, antiinflammatory, and antioxidant features (ugur et al., 2016). dalli et al. (2021) investigated the chemical profile of the black cumin eo from different geographical areas. as the major components α-phellandrene, β-pinene, β-cymene, and 4-caranol were identified, and the antibacterial activity of this plant could be associated with them. according to previous findings (oussalah et al., 2006; saad et al., 2013), volatile compounds possess the capacity to inhibit the synthesis of structural macromolecules and growth regulators. in general, gram-positive bacteria included in this study were more susceptible to the activity of tested eos. growth inhibition zones observed in gram-positive strains ranged from 6.60±1.10 (s. aureus atcc 33591, mrsa strain; achieved by the activity of fennel eo at 750 µg/ml) to 38.30±2.90 (same strain; inhibited by the pure eo of black cumin). a possible explanation of such results could be related to the more complex and rigid outer membrane of gram-negative bacteria, where the lipopolysaccharide layer limits the diffusion of hydrophobic constituents, unlike the gram-positive bacteria, where peptidoglycan cell wall provides less resistance (patterson et al., 2019). suggested mechanisms of the antibacterial activity of eos are increased cell permeability and toxic effects on membrane structure (swamy et al., 2016; chouhan fig. 1. inhibited bacterial strains after the application of essential oils and antibiotics et al., 2017). furthermore, there should be mentioned that eos are composed of 20 to 60 different compounds (nazzaro et al., 2013), and specific ones could be responsible for the particular antibacterial activity. adequate diffusion of the active molecules through the agar medium requires optimization of medium concentration to achieve optimum activity of some chemical compounds. the results of uzair et al. (2017) suggest that essential oils exhibit a strong synergism with certain antibiotics, even in mdr bacterial strains. therefore, some essential oils that weren’t so effective alone, could be discussed as synergistic candidates. both mrsa strains included in this study were susceptible to eos, while the same strains were resistant to all standard antibiotics (tab. 1). the number of bacterial strains inhibited by the activity of investigated eos and antibiotics is presented in fig. 1. broth microdilution protocol was performed after all the results from the disk-diffusion method were gained. only those bacterial species where the inhibition with the investigated eo was achieved, were further analyzed in the sense of determining the minimum inhibitory concentration. minimum inhibitory concentrations of investigated eos against tested bacterial species ranged from 250 µg/ml, recorded in the case of the black cumin eo against s. aureus atcc 33591 (mrsa strain), while the highest mic values (750 µg/ml) are related to the fennel and lemon eos against s. aureus atcc 33591 and s. aureus nctc 12493, both mrsa strains. mic values of the fennel and lemon eos against mrsa strains (s. aureus atcc 33591 and s. aureus nctc 12493, respectively) were extrapolated from the disk-diffusion method since there was bacterial growth at 500 µg/ml, and no growth at 1000 µg/ml (pure oil in dmso), but a particular concentration of 750 µg/ml was not included in the decimal dilutions used in the broth microdilution method. obtained values are presented in tab. 2. in order to establish the mbc value, the results of the previously performed methods were taken into account. the mbc was determined for the eos with known mic. with the exception of the black cumin eo against the s. aureus atcc 33591 (mrsa), where mbc was determined at 500 µg/ml, other samples biologica nyssana ● 14 (1) june 2023: 47-56 51 52 biologica nyssana ● 14 (1) june 2023: 47-56 performed bactericidal activity as the pure eo, at 1000 µg/ml of dmso. for the evaluation of the antibiofilm potential of tested eos, the first step included the determination of the biofilm-forming category of investigated bacterial strains based on the results generated in positive controls. obtained results showed that strong biofilm-formers were: s. aureus atcc 6538 (sa1), s. aureus nctc 12493 (sa4; mrsa), e. coli atcc 35218 (ec3; esbl), and p. aeruginosa atcc 27853 (pa). moderately adherent biofilm was detected in s. aureus atcc 25923 (sa2), s. aureus atcc 33591 (sa3; mrsa), b. subtilis atcc 6633 (bs), and e. coli 14169 (ec1). weakly adherent biofilm was formed by e. faecalis atcc 29212 (ef), e. coli atcc 25922 (ec2), and s. enterica nctc 6017 (se). mean absorbance values with standard deviations are presented in fig. 2. mahmutović-dizdarević et al. ● essential oils of selected citrus fruits and spice plants as potential antibacterial and antibiofilm agents table 2. minimum inhibitory concentration (µg/ml) of tested essential oils and standard antibiotics essential oils bacterial strains sa1 sa2 sa3 sa4 ef bs ec1 ec2 ec3 pa se lemon 750* 500 500 mandarine 500 black cumin 500 500 250 500 500 500 500 fennel 750* 500 sa1: staphylococcus aureus atcc 6538; sa2: s. aureus atcc 25923; sa3: s. aureus atcc 33591 (mrsa); sa4: s. aureus nctc 12493 (mrsa); ef: enterococcus faecalis atcc 29212; bs: bacillus subtilis atcc 6633; ec1: escherichia coli atcc 14169; ec2: e. coli atcc 25922; ec3: e. coli atcc 35218 (esbl); pa: pseudomonas aeruginosa atcc 27853; se: salmonella enterica nctc 6017. *mic is extrapolated according to the results of the disk-diffusion method. fig. 2. biofilm-forming categories of investigated bacteria (red columns, strong biofilm-formers: sa1, staphylococcus aureus atcc; sa4, methicillin-resistant (mrsa) s. aureus nctc 12493; ec3, extendedspectrum beta-lactamase-producing (esbl) e. coli atcc 35218; pa, pseudomonas aeruginosa atcc 27853. yellow columns, moderate biofilm-formers: sa2, s. aureus atcc 25923; sa3, methicillin-resistant s. aureus (mrsa) atcc 33591; bs, bacillus subtilis atcc 6633; ec1, escherichia coli 14169. green columns, weak biofilm-formers: ef, enterococcus faecalis atcc 29212; ec2, e. coli atcc 25922; se, salmonella enterica nctc 6017) subinhibitory concentrations of tested eos were examined for antibiofilm properties. changes in the biofilm-forming capacity of investigated bacteria due to the activity of eos are presented in tab. 3. lemon eo decreased the biofilm-forming capacity of s. aureus nctc 12493 (mrsa), with full inhibition of the biofilm formation at a concentration of 62.5 µg/ml. the same sample removed the weakly adherent biofilm of e. faecalis atcc 29212 and the moderately adherent biofilm of b. subtilis atcc 6633 at 125 µg/ml. lemon eo is known for its antibacterial potential, but recently there are also studies regarding its antibiofilm effects (sun et al., 2018; luciardi et al., 2021; jamil et al., 2022). our investigation confirmed that lemon eo has the ability to inhibit bacterial growth, including the mdr strains. furthermore, antibiofilm activity is recorded against gram-positive bacteria. as with other eos, biological activity is typically associated with the most abundant compound. lawrence & palombo (2009) noted that particular constituents of eo don’t show the same level of antibacterial activity as the complete oils, which suggests the involvement of the minor components, that can act synergistically with major compounds. in the lemon eo the most abundant monoterpene hydrocarbons are d-limonene, γ-terpinene, and β-pinene, while among the oxygenated monoterpenes, the most abundant are α-terpineol, nerol, and geraniol (yazgan et al., 2019). mandarin orange eo leads to a reduction of biofilm formation of s. aureus atcc 6538 which is otherwise a good biofilm producer, and in b. subtilis atcc 6633 which has a moderate biofilm-forming capacity, to weakly adherent at 125 µg/ml. mandarin orange eo consists mainly of limonene and γ-terpinene, while other compounds include α-and β-pinene, β-myrcene, o-cymene, and b-thujene, with the limonene being considered the main constituent involved in bioactive and antimicrobial properties (song et al., 2020). song et al. (2021) noted that mandarin orange eo can inhibit biofilm formation and destroy mature biofilms, but these authors also emphasize the lower activity of eo against gram-negative bacteria. in accordance with other results of this study, black cumin eo showed great antibiofilm potential, and eradication of biofilm was recorded in the case of s. aureus atcc 6538, s. aureus atcc 33591 (mrsa), s. aureus nctc 12493 (mrsa), and p. aeruginosa atcc 27853. at the concentration of 125 µg/ ml of black cumin eo, there was no bacterial cell adherence in the abovementioned strains. bourgou et al. (2010) also noted differences in the chemical composition of the black cumin eo from various geographical areas, while chaieb et al. (2011) defined thymoquinone as the active principle of the black cumin with anti-biofilm potential. a review by forouzanfar et al. (2014) stands out the antimicrobial properties of black cumin, with an accent of wide medicinal use of this plant without reported side effects. fennel eo leads to the eradication of preformed biofilms of s. aureus atcc 33591 (mrsa) and b. subtilis atcc 6633 at the concentration of 125 µg/ml. antibacterial properties of fennel investigated earlier (diao et al., 2014; mutlu-ingok, 2021; alam et al., 2022) are recently supported by antibiofilm studies of this plant and its derivates (kwiatkowski et al., 2020). the major ingredient of the fennel eo is p-metoxypropenylobenzene, which is a by-product of terpene synthesis (kwiatkowski table 3. impact of the tested eos on the biofilm-forming capacity of investigated bacteria essential oil bacterial strains sa1 sa2 sa3 sa4 ef bs ec1 ec2 ec3 pa se lemon +++* +* ++* mandarine ++ + black cumin +++* + ++* +++* + + +++* fennel ++* ++* sa1: staphylococcus aureus atcc 6538; sa2: s. aureus atcc 25923; sa3: s. aureus atcc 33591 (mrsa); sa4: s. aureus nctc 12493 (mrsa); ef: enterococcus faecalis atcc 29212; bs: bacillus subtilis atcc 6633; ec1: escherichia coli atcc 14169; ec2: e. coli atcc 25922; ec3: e. coli atcc 35218 (esbl); pa: pseudomonas aeruginosa atcc 27853; se: salmonella enterica nctc 6017. changes in the biofilm-forming category: +++ decreasing up to three biofilm-forming categories ++ decreasing up to two biofilm-forming categories + decreasing in one biofilm-forming category * elimination of the biofilm in presence of a particular concentration of tested eo no antibiofilm activity mahmutović-dizdarević et al. ● essential oils of selected citrus fruits and spice plants as potential antibacterial and antibiofilm agents biologica nyssana ● 14 (1) june 2023: 47-56 53 biologica nyssana ● 14 (1) june 2023: 47-56 mahmutović-dizdarević et al. ● essential oils of selected citrus fruits and spice plants as potential antibacterial and antibiofilm agents et al., 2019). conclusion essential oils represent bioactive compounds of natural origin, where the proportion of active constituents vary due to geographical distribution and many environmental conditions. these slight differences in the chemical profile of the essential oil could be crucial in terms of the occurrence of bacterial resistance. the antibacterial potential of eos derived from four edible and spice plants: lemon, mandarin orange, black cumin, and fennel is emphasized through the results of this research. performed investigation showed that essential oils derived from c. lemon, c. reticulata, n. sativa, and f. vulgare exhibited antibacterial properties which are proved through different microbiological assays. gram-positive bacteria were more susceptible to the activity of eos. for instance, mdr s. aureus treated with black cumin eo had the greatest growth inhibition, which was not achieved by the activity of tested antibiotics. additionally, b. subtilis was successfully inhibited by all four eos. besides inhibition, investigated eo performed bactericidal properties. the antibiofilm activity was noted for all examined eos, with the most prominent activity of the black cumin eo, where complete inhibition of the biofilm formation was noted for strong biofilm-formers, including mdr strains. it could be concluded that all investigated essential oils displayed strain-specific and dose-dependent antibacterial activity. references alam, a., foudah, a. i., salkini, m. a., raish, m. & sawale, j. (2022). herbal fennel essential oil nanogel: formulation, characterization and antibacterial activity against staphylococcus aureus. gels, 8(11), 736. auddy, b., ferreira, m., blasina, f., lafon, l., arredondo, f., dajas, f., tripathi, p. c., seal, t. & mukherjee, b. (2003). screening of antioxidant activity of three indian medicinal plants, traditionally used for the management of neurodegenerative diseases. journal of ethnopharmacology, 84(2-3), 131-138. bauer, a. w., kirby, w. m., sherris, j. c. & turck, m. (1966). antibiotic susceptibility testing by a standardized single disk method. american journal of clinical pathology, 45(4), 493-496. bourgou, s., pichette, a., marzouk, b. & legault, j. (2010). bioactivities of black cumin essential oil and its main terpenes from tunisia. south african journal of botany, 76(2), 210-216. chaieb, k., kouidhi, b., jrah, h., mahdouani, k. & bakhrouf, a. (2011). antibacterial activity of thymoquinone, an active principle of nigella sativa and its potency to prevent bacterial biofilm formation. bmc complementary and alternative medicine, 11(1), 1-6. cheng, p., xiong, j., li, h., wang, s., zhang, y., mei, c. & chen, h. (2022). using plant extracts and their active ingredients to inhibit bacterial biofilms. sheng wu gong cheng xue bao= chinese journal of biotechnology, 38(5), 1753-1767. chouhan, s., sharma, k. & guleria, s. (2017). antimicrobial activity of some essential oils present status and future perspectives. medicines, 4(3), 58. clsi (clinical and laboratory standards institute) (2018). methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 11th ed. clsi standard m07. clinical and laboratory standards institute, wayne, pennsylvania, usa. retrieved from https://clsi.org/ media/1928/m07ed11_sample.pdf dalli, m., azizi, s., benouda, h., azghar, a., tahri, m., bouammali, b., maleb, a. & gseyra, n. (2021). molecular composition and antibacterial effect of five essential oils extracted from nigella sativa l. seeds against multidrug-resistant bacteria: a comparative study. evidence-based complementary and alternative medicine, 2021, 1–9. diao, w.-r., hu, q.-p., zhang, h. & xu, j.-g. (2014). chemical composition, antibacterial activity and mechanism of action of essential oil from seeds of fennel (foeniculum vulgare mill.). food control, 35(1), 109–116. el-tarabily, k. a., el-saadony, m. t., alagawany, m., arif, m., batiha, g. e., khafaga, a. f., elwan, h. a. m., elnesr, s. s., e. & abd elhack, m. (2021). using essential oils to overcome bacterial biofilm formation and their antimicrobial resistance. saudi journal of biological sciences, 28(9), 5145–5156. forouzanfar, f., bazzaz, b. s. & hosseinzadeh, h. (2014). black cumin (nigella sativa) and its constituent (thymoquinone): a review on antimicrobial effects. iranian journal of basic medical sciences, 17(12), 929–938. hernando-amado, s., coque, t. m., baquero, f. & martínez, j. l. (2019). defining and combating antibiotic resistance from one health and global health perspectives. nature microbiology, 4(9), 1432-1442. 54 55 biologica nyssana ● 14 (1) june 2023: 47-56 mahmutović-dizdarević et al. ● essential oils of selected citrus fruits and spice plants as potential antibacterial and antibiofilm agents & karbancioglu‐guler, f. (2021) antioxidant and antimicrobial activities of fennel, ginger, oregano and thyme essential oils. food frontiers, 2(4), 508– 518. nazzaro, f., fratianni, f., de martino, l., coppola, r. & de feo, v. (2013). effect of essential oils on pathogenic bacteria. pharmaceuticals, 6(12), 1451–1474. orhan-yanıkan, e., da silva-janeiro, s., ruizrico, m., jiménez-belenguer, a. i., ayhan, k. & barat, j. m. (2019). essential oils compounds as antimicrobial and antibiofilm agents against strains present in the meat industry. food control, 101i, 29–38. oussalah, m., caillet, s. & lacroix, m. (2006). mechanism of action of spanish oregano, chinese cinnamon, and savory essential oils against cell membranes and walls of escherichia coli o157: h7 and listeria monocytogenes. journal of food protection, 69(5), 1046-1055. patterson, j. e., mcelmeel, l. & wiederhold, n. p. (2019). in vitro activity of essential oils against gram-positive and gram-negative clinical isolates, including carbapenem-resistant enterobacteriaceae. open forum infectious diseases, 6(12), 1-4. saad, n. y., muller, c. d. & lobstein, a. (2013). major bioactivities and mechanism of action of essential oils and their components. flavour and fragrance journal, 28(5), 269-279. sharma, d., misba, l. & khan, a. u. (2019). antibiotics versus biofilm: an emerging battleground in microbial communities. antimicrobial resistance & infection control, 8(1), 1-10. song, x., liu, t., wang, l., liu, l., li, x. & wu, x. (2020). antibacterial effects and mechanism of mandarin (citrus reticulata l.) essential oil against staphylococcus aureus. molecules, 25(21), 4956. song, x., wang, l., liu, t., liu, y., wu, x. & liu, l. (2021). mandarin (citrus reticulata l.) essential oil incorporated into chitosan nanoparticles: characterization, anti-biofilm properties and application in pork preservation. international journal of biological macromolecules, 185, 620– 628. stepanović, s., vuković, d., hola, v., di bonaventura, g., djukić, s., cirković, i. & ružička, f. (2007). quantification of biofilm in microtiter plates: overview of testing conditions and practical recommendations for assessment of biofilm production by staphylococci. apmis: acta pathologica, microbiologica, et immunologica scandinavica, 115(8), 891–899. høiby, n., bjarnsholt, t., givskov, m., molin, s. & ciofu, o. (2010). antibiotic resistance of bacterial biofilms. international journal of antimicrobial agents, 35(4), 322-32. jamil, a. h., abdulhussien, a., alsharifi, m. (2022). the anti-biofilm activity of lemon oil against methicillin resistance of staphylococcus aureus and staphylococcus haemolyticus. aip conference proceedings, 2386 (1): 020001. klimek-szczykutowicz, m., szopa, a., ekiert, h. (2020). citrus limon (lemon) phenomenon-a review of the chemistry, pharmacological properties, applications in the modern pharmaceutical, food, and cosmetics industries, and biotechnological studies. plants 9(1), 119. kwiatkowski, p., pruss, a., masiuk, h., mnichowska-polanowska, m., kaczmarek, m., giedrys-kalemba, s., dołęgowska, b., zielińskabliźniewska, h., olszewski, j. & sienkiewicz, m. (2019). the effect of fennel essential oil and trans-anethole on antibacterial activity of mupirocin against staphylococcus aureus isolated from asymptomatic carriers. advances in dermatology and allergology, 36(3), 308–314. kwiatkowski, p., grygorcewicz, b., pruss, a., wojciuk, b., giedrys-kalemba, s., dołęgowska, b., zielińska-bliźniewska, h., olszewski, j., sienkiewicz, m. & kochan, e. (2020). synergistic effect of fennel essential oil and hydrogenperoxide on bacterial biofilm. advances in dermatology and allergology, 37(5), 690–698. lawrence, h. a. & palombo, e. a. (2009). activity of essential oils against bacillus subtilis spores. journal of microbiology and biotechnology, 19(12), 1590–1595. luciardi, m. c., blázquez, m. a., alberto, m. r., cartagena, e. & arena, m. e. (2021). lemon oils attenuate the pathogenicity of pseudomonas aeruginosa by quorum sensing inhibition. molecules, 26(10), 2863. mah, t.-f. (2012). biofilm-specific antibiotic resistance. future microbiology, 7(9), 1061–1072. merritt, j. h., kadouri, d. e. & o’toole, g. a. (2011). growing and analyzing static biofilms. current protocols in microbiology, 22(1), 1b-1. musara, c., aladejana, e. b. & mudyiwa, s. m. (2020): review of the nutritional composition, medicinal, phytochemical and pharmacological properties of citrus reticulata blanco (rutaceae). f1000 research, 9, 1387. mutlu‐ingok, a., catalkaya, g., capanoglu, e. biologica nyssana ● 14 (1) june 2023: 47-56 sun, y., chen, s., zhang, c., liu, y., ma, l. & zhang, x. (2018). effects of sub-minimum inhibitory concentrations of lemon essential oil on the acid tolerance and biofilm formation of streptococcus mutans. archives of oral biology, 87, 235–241. swamy, m. k., akhtar, m. s. & sinniah, u. r. (2016). antimicrobial properties of plant essential oils against human pathogens and their mode of action: an updated review. evidence-based complementary and alternative medicine, 2016, 1–21. tabassum, h., ahmad, a. & ahmad, i. z. (2018). nigella sativa l. and its bioactive constituents as hepatoprotectant: a review. current pharmaceutial biotechnology, 19(1), 43-67. tang, c, chen, j., zhang, l., zhang, r., zhang, s., ye, s., zhao, z. & yang, d. (2020). exploring the antibacterial mechanism of essential oils by membrane permeability, apoptosis and biofilm formation combination with proteomics analysis against methicillin-resistant staphylococcus aureus. international journal of medical microbiology, 310(5). tripathi, p., tripathi, r., patel, r. k. & pancholi, s. s. (2012). investigation of antimutagenic potential of foeniculum vulgare essential oil on cyclophosphamide induced genotoxicity and oxidative stress in mice. drug and chemical toxicology, 36(1), 35–41. ugur, a. r., dagi, h. t., ozturk, b., tekin, g. & findik, d. (2016). assessment of in vitro antibacterial activity and cytotoxicity effect of nigella sativa oil. pharmacognosy magazine, 12(47), 471. urban-chmiel, r., marek, a., stępień-pyśniak, d., wieczorek, k., dec, m., nowaczek, a.& osek, j. (2022). antibiotic resistance in bacteria-a review. antibiotics, 11(8), 1079. uzair, b., niaz, n., bano, a., ali khan, b., zafar, n., iqbal, m. & fasim, f.(2017). essential oils showing in vitro anti mrsa and synergistic activity with penicillin group of antibiotics. pakistan journal of pharmaceutical sciences,30(5), 1997-2002. wińska, k., mączka, w., łyczko, j., grabarczyk, m., czubaszek, a. & szumny, a. (2019). essential oils as antimicrobial agents—myth or real alternative? molecules, 24(11), 2130. yazgan, h., ozogul, y. & kuley, e. (2019). antimicrobial influence of nanoemulsified lemon essential oil and pure lemon essential oil on food-borne pathogens and fish spoilage bacteria. international journal of food microbiology, 306. mahmutović-dizdarević et al. ● essential oils of selected citrus fruits and spice plants as potential antibacterial and antibiofilm agents 56 stanojević et al 2020, biologica nyssana 11(2) 11 (2) december 2020: 109-114 doi: 10.5281/zenodo.4393961 copper(ii) compounds as effective agents against colletotrichum acutatum causing anthracnose of strawberry original article ivana stanojević faculty of agriculture, university of niš, kosančićeva 4, 37000 kruševac, serbia stanojevic.ivana85@gmail.com biljana glišić faculty of science, department of chemistry, university of kragujevac, r. domanovića 12, 34000 kragujevac, serbia bglisic@kg.ac.rs sanja živković faculty of agriculture, university of niš, kosančićeva 4, 37000 kruševac, serbia gajicsanja43@gmail.com sonja filipović faculty of agriculture, university of niš, kosančićeva 4, 37000 kruševac, serbia sonjafilipovic86@yahoo.com snežana andjelković institue for forage crops, kruševac, 37251, globoder, serbia snezana.andjelkovic@ikbks.com tanja vasić faculty of agriculture, university of niš, kosančićeva 4, 37000 kruševac, serbia tanjavasic82@gmail.com (corresponding author) received: may 21, 2020 revised: september 18, 2020 accepted: november 30, 2020 abstract: the aim of this study was to evaluate the in vitro antifungal effects of copper(ii) compounds on the growth of the fungus colletotrichum acutatum isolated from strawberry. the growth inhibition activity of na2[cu(1,3pddadp)].6h2o complex (1,3-pddadp is 1,3-propanediamine-n,n’-diacetaten,n’-di-3-propionate anion) against this fungus was compared to those of cuso4.5h2o and ba2(1,3-pddadp).8h2o. additionally, the effects of these compounds on sporulation level and the number of conidia per 1 mm2 of colony were investigated. the obtained results showed that the highest percentage of inhibition for mycelium growth was achieved at a concentration of 100 and 200 µg/ml for all applied compounds. the biological activities of the investigated compounds were compared with those for the commercial formulation of fungicide metod 480 sc (captan). key words: antifugal activity, colletotrichum acutatum, copper(ii) compounds, strawberry apstract: jedinjenja bakra(ii) kao efikasni agensi prema gljivi colletotrichum acutatum, uzročniku antraknoze jagoda cilj ovog rada je ispitivanje in vitro antifungalne aktivnosti jedinjenja bakra(ii) prema gljivi colletotrichum acutatum koja je izolovana iz jagode. ispitivana je aktivnost na2[cu(1,3-pddadp)].6h2o kompleksa (1,3-pddadp je 1,3-propandiamin-n,n’-diacetat-n,n’-di-3-propionat), pri čemu su dobijeni rezultati poređeni sa aktivnošću cuso4.5h2o i ba2(1,3-pddadp).8h2o. dodatno je analiziran je uticaj različitih koncentracija ovih jedinjenja na intenzitet sporulacije i broj konidija po 1 mm2 kulture c. acutatum. dobijeni rezultati su pokazali da sva ispitivana jedinjenja imaju najveću aktivnost pri koncentraciji od 100 i 200 µg/ml. biološka aktivnost ispitivanih jedinjenja je poređena sa odgovarajućom aktivnošću komercijalnog fungicida metod 480 sc kaptan. ključne reči: antifungalna aktivnost, colletotrichum acutatum, jedinjenja bakra(ii), jagoda introduction strawberry (fragaria ananassa) is commercially the most important fruit, which is grown worldwide and is predominant species cultivated for production globally (fao, 2000). one of the species that has been reported to cause wide spread disease in strawberry, namely anthracnose, is the fungus colletotrichum acutatum (sutton, 1992; maas and palm, 1997; freeman et al., 1998; wedge et al., 1999). this fungus can attack the plant at all stages of development, including flowers, leaves, stems and roots. however, it causes the greatest damage to fruits, causing significant losses in strawberry production (mertely et al., 2018). there are only a few available fungicides which are effective against c. acutatum, nevertheless a long and frequent use of these fungicides led to the © 2020 stanojević et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 109 resistance development (maas, 2004). considering the heavy burden for the strawberry production sector worldwide that anthracnose is causing, development of advanced and efficient therapeutic options became of great importance. with the increasing emergence of drug-resistant c. acutatum species and due to a lack of potent agent against anthracnose, a special attention has been devoted to the evaluation of antimicrobial potential of metal compounds (schatzschneider, 2019). among them, copper(ii) compounds have received a particular interest, due to their wellknown application in agriculture as fungicides, nutritionals and algicides (richardson, 1997). the importance of copper(ii)-based compounds as fungicides in viticulture has been recognized after discovery of the bordeaux mixture, which consists of copper(ii) sulfate, lime and water. nowadays, the pharmaceutical companies have been produced different copper(ii)-based fungicides in soluble forms of sulfates, oxychlorides, acetates, carbonates, oleates, silicates and hydroxides. copper(ii) oxychloride has been widely used due to its good crop tolerance and effectiveness against different phytopathogenic fungi such as rhizoctonia solani, r. bataticola, botrytis cinerea, fusarium semitectum, f. culmorum, f. moniliforme, f. solani, f. oxysporum, stemphylium radicinum, sclerotinia sclerotiorum and colletotrichum gloeosporioides (gharieb et al., 2004). copper(ii)-based fungicides have a broad spectrum of activity against different fungal and bacterial microorganisms as a consequence of their ability to be attached on the plant surface. at the same time, they are economically acceptable and do not lead to resistance development (müller et al., 2000). with the aim to selectively deliver cu(ii) ion to the diseased tissues, different copper(ii) complexes with various ligands have been synthesized and reported to possess significant antibacterial and antifungal activities (singh et al., 2008; singh et al., 2009; patel et al., 2010; ng et al., 2016). beside copper(ii) compounds, diaminopolycarboxylate ligands which belong to edta family (ethylenediamine-n,n,n’,n’-tetraacetate anion) and their metal complexes have shown a remarkable antimicrobial activity (anderegg, 1987; bulman, 1987; douglas and radanović, 1993). for instance, edta has shown activity against different gramnegative and gram-positive bacteria, yeasts, amoeba and fungi (brown and richards, 1965; kite and hatton, 2014). considering the importance of copper(ii) compounds and diaminopolycarboxylate ligands in design of antifungal agents, in the present study, we have synthesized and biologically evaluated na2[cu(1,3-pddadp)].6h2o complex (1,3-pddadp is 1,3-propanediamine-n,n’-diacetate-n,n’-di-3propionate anion) against the fungus c. acutatum. the obtained biological results for this complex was compared to those for cuso4.5h2o and ba2(1,3pddadp).8h2o. material and methods materials barium salt of diaminopolycarboxylate ligand from edta-family, ba2(1,3-pddadp).8h2o, and copper(ii) complex na2[cu(1,3-pddadp)].6h2o were prepared according to a procedure published in the literature (radanović et al., 1992; radanović et al., 1997). other reagents were obtained commercially and used without further purification. test organism in this experiment, the isolate of c. acutatum was used. it was isolated from strawberries grown in serbia. the isolate was determined on the basis of morphological, pathogenic and molecular characteristics and maintained on a potato-dextrose agar (pda) at 25 °c. antifungal activity antifungal activity of the three compounds cuso4.5h2o, ba2(1,3-pddadp).8h2o and na2[cu(1,3-pddadp)].6h2o was tested on pda in petri dishes of 90 mm diameter. substrates were inoculated by mycelial fragments of c. acutatum isolates taken from the edge of 7-days old culture. tested compounds were dissolved in distilled water at three different concentrations: 100, 200 and 300 µg/ml. the solutions were incorporated into the autoclaved pda, which was cooled to 55 °c. cultures prepared in this way were grown for 7 days in a thermostat at 25 °c. the commercial formulation of fungicide metod 480 sc (captan, galenika fitofarmacija) was used as a positive control and applied at a concentration recommended for practical application. the negative control variant, fungal isolates grown on pda, were used under identical conditions without described treatments (zang et al., 2012). after seven days, the linear increase in the studied cultures was measured. the inhibition of the fungal growth expressed in percentage terms was determined from the growth in the test plate relative to the respective control plate as given below: inhibition (%) = (c-t) 100 / c where, c = diameter of fungal growth in the control plate and t = diameter of fungal growth in the test plate. sporulation level ten days after treatment, the effects of cuso4.5h2o, ba2(1,3-pddadp).8h2o and na2[cu(1,3-pddadp)].6h2o 110 biologica nyssana ● 11 (2) december 2020: 109-114 stanojevic et al. ● copper(ii) compounds as effective agents against colletotrichum acutatum causing anthracnose of strawberry 111 on c. acutatum sporulation levels were determined. determination of sporulation levels was performed using a tahoma haemocytometer. for this purpose, a spore suspension was prepared by adding 5 ml of distilled water to the petri dish with a culture of isolate c. acutatum. the sporulation level was expressed according to the scale by quesada and lopez (1980), where: + = poor sporulation (<5.000 spores/ml), ++ = medium sporulation (5.000-10.000 spores/ml) and +++ = abundant sporulation (>10.000 spores/ml). the experiment was set in three repetitions. the number of conidia per 1 mm2 of colony was determined based on the number of conidia/ ml and the area of the colony, which was calculated from the proportion of paper weight on which the contours of colonies were drawn and paper weight of 1 dm2 (stojanović, 1997). the results obtained during the research were processed by anova analysis with the statistical program statistica 8.0 (statsoft inc., usa). duncan’s test was conducted to analyze the difference between various pre-treatments. a value of p = 0.05 was considered statistically significant. results and discussion biological activity all tested compounds showed a remarkable growth inhibition of c. acutatum mycelia. the inhibition of mycelial growth was observed at concentrations of 100 μg/ml and higher, for all tested compounds, na2[cu(1,3-pddadp)].6h2o, cuso4.5h2o and ba2(1,3-pddadp).8h2o, and was dependent on the applied concentration of the compounds. moreover, a significant percentage inhibition of c. acutatum mycelium compared to the commercial captan formulation (metod 480 sc, galenika fitofarmacija) was achieved at all applied concentrations (100, 200 and 300 µg/ml) of the tested compounds (tab. 1 and figs. 1 and 2). the linear growth of colonies of the tested c. acutatum isolate was statistically significantly influenced by the concentrations of the applied compounds. during the study, it was found that the fig. 1. influence of two copper(ii) compounds, na2[cu(1,3pddadp)].6h2o and cuso4.5h2o on the growth inhibition of c. acutatum mycelium biologica nyssana ● 11 (2) december 2020: 109-114 stanojevic et al. ● copper(ii) compounds as effective agents against colletotrichum acutatum causing anthracnose of strawberry table 1. the percentage of growth inhibition of c. acutatum by na2[cu(1,3-pddadp)].6h2o, cuso4.5h2o and ba2(1,3-pddadp).8h2o compounds compound 100 µg/ml 200 µg/ml 300 µg/ml na2[cu(1,3-pddadp)].6h2o 36.3b,c 38.7 b 31.6 c cuso4.5h2o 35.3b,c 34.7 c 39.3 b ba2(1,3-pddadp).8h2o 39.7 b 34.3 b 40.0 b control c. acutatum 53.3a 53.3 a 53.3 a metod 480 sc 100d 100d 100d * the data in rows marked by the same letter are not statistically significantly different based on duncan test (p = 0.05) 112 highest linear growth of the colony of c. acutatum was achieved after treatment with cuso4.5h2o and ba2(1,3-pddadp).8h2o at a concentration of 300 µg/ ml relative to the negative control (pure culture of c. acutatum) (tab. 1, fig. 2b and 2c). contrary to this, the smallest increase in mycelium of the fungus c. acutatum was after treatment with na2[cu(1,3pddadp)].6h2o and cuso4.5h2o at concentrations of 100 and 200 µg/ml (fig. 2a and 2b). considering this, it can be concluded that the minimum inhibitory concentrations (mics) against the c. acutatum are at 100 and 200 µg/ml for the tested compounds (tab. 1 and fig. 2). oziengbe and osazee (2012) evaluated the inhibitory effect of copper(ii) sulfate on the linear growth of c. gloeosporioides mycelium. their results showed that this copper(ii) salt reduced the linear growth of this fungus in all applied concentrations (0.2, 0.4, 0.6 and 0.8 mg/l). on the other hand, everett and timudotorrevilla (2007) tested four commercial copperbased preparations (champ (copper-hydroxide, 37.5% copper), kocide (copperhydroxide, 35% copper), kocide (copper-hydroxide, 46.1% copper) and cuprofix disperss (copper-hydroxosulphate, 20% copper)) against the most important pathogens on avocado, colletotrichum acutatum, c. gloeosporioides, botryosphaeria parva, b. dothidea and phomopsis spp. in new zealand. the results showed that four copper formulations inhibited the germination of spores of all tested fungi, but at high concentrations (i.e. 40.9 µg/ml for copper hydroxide (46.1% copper) against c. gloeosporioides). the copper formulation that was effective at the lowest concentrations against the germination of botryosphaeria parva spores was copper hydroxide (37.5% copper), the most effective against both species of colletotrichum was copper hydroxosulfate (20% copper), while the most table 2. influence of the investigated compounds on the number of conidia per mm2 of c. acutatum compound 100 µg/ml 200 µg/ml 300 µg/ml na2[cu(1,3-pddadp)].6h2o + ++ + cuso4.5h2o +++ ++ ++ ba2(1,3-pddadp).8h2o ++ + +++ control c. acutatum +++ +++ +++ metod 480 sc * sporulation: + = poor sporulation, ++ = medium sporulation, +++ = abundant sporulation fig. 2. the effect of na2[cu(1,3-pddadp)].6h2o complex (a), cuso4.5h2o (b) and ba2(1,3-pddadp).8h2o (c) on the growth of c. acutatum isolate in vitro after seven days. biologica nyssana ● 11 (2) december 2020: 109-114 stanojevic et al. ● copper(ii) compounds as effective agents against colletotrichum acutatum causing anthracnose of strawberry effective against b. dothidea and phomopsis sp. was copper-hydroxide (46.1% copper) (everett and timudo-torrevilla, 2007). sporulation level the number of conidia formed per mm2 of colony was statistically significantly conditioned by the applied concentrations of the tested compounds in c. acutatum culture. the lowest number of conidia per mm2 was found for na2[cu(1,3-pddadp)].6h2o at 100 and 300 µg/ml, for cuso4.5h2o at 200 and 300 µg/ml, and for ba2(1,3-pddadp).8h2o at 100 and 200 µg/ml. the highest number of spores per mm2 was observed for cuso4.5h2o and ba2(1,3-pddadp).8h2o at concentrations of 100 and 300 µg/ml, respectively (tab. 2). as can be seen from tab. 3, the ability of the fungus c. acutatum to form conidia is affected by different concentrations of the applied compounds, na2[cu(1,3-pddadp)].6h2o, cuso4.5h2o and ba2(1,3-pddadp).8h2o. in the treatment with captan which was used as a positive control, no sporulation of c. acutatum occurred. in the case of ba2(1,3pddadp).8h2o at 200 µg/ml and for na2[cu(1,3pddadp)].6h2o complex at 100 and 300 µg/ml, a lower and medium level of sporulation was noticed. contrary to this, the abundant sporulation was observed for the cuso4.5h2o at 100 µg/ml, as well as for ba2(1,3-pddadp).8h2o at 300 µg/ml (tab. 3). spores are very important for the spread of the disease on strawberry plantations. thus, the number of spores and the intensity of sporulation play a significant role in the spread of pathogens and the progress of infection. spores of the pathogen are passively dispersed by spraying rain or irrigation water which causes infection and pathogen develop on immature fruits and young tissues, and these spores germinate and penetrate the cuticle and epidermis to multiply through the tissues (babović, 2003). symptoms include small black spots and/ or large black lesions on the tissue surface, which coalesce and penetrate deep into the fruit, resulting in fruit rot. borkow and gabbay (2009) found that different copper formulations have inhibited a spore germination of colletotrichum gloeosporioides at different concentrations (0.2, 0.4, 0.6 and 0.8 mg/l). similar conclusions were drawn by oziengbe and osazee (2012), who tested different concentrations of copper compounds against the isolates of colletotrichum gloeosporioides originated from mango. conclusion in conclusion, the safety of copper for humans and its powerful biocidal properties allow the use of copper and its compounds for different applications. this study showed that copper(ii) compounds in the form of complex or simple inorganic salt could inhibit the growth of colletotrichum acutatum mycelium, by affecting the intensity of sporulation and the number of spores per mm2 in vitro, while strongly causing defensive reactions on the strawberry fruit. references anderegg, g., 1987: complexones. in: wilkinson g, editor. comprehensive coordination chemistry vol. 2, pergamon press, oxford. babović m. 2003: osnovi patоlogije biljaka. poljoprivredni fakultet, univerzitet u beogradu, beograd. 651 p. borkow g. and gabbay j. 2009: copper, an ancient remedy returning to fight microbial. fungal and viral infections current chemical biology, 3: 272-278. brown, m.r., richards, r.m.e., 1965. effect of ethylenediamine tetraacetate on the resistance of pseudomonas aeruginosa to antibacterial agents. nature, 207, 1391-1393. bulman, r. a. 1987: the chemistry of chelating agents in medical sciences. in coordination compounds: synthesis and medical application (pp. 91-141). springer, berlin, heidelberg. douglas, b. e., radanović, d. j. 1993: coordination chemistry of hexadentate edta-type ligands with m (iii) ions. coordination chemistry reviews, 128 (1-2): 139-165. everett, k. r. and timudo-torrevilla o. e. 2007: in vitro fungicide testing for control of avacado fruit rots. new zealand plant protection, 60: 99-103. fao (2000) food and agriculture organization 113 compound 100 µg/ml 200 µg/ml 300 µg/ml na2[cu(1,3-pddadp)].6h2o + ++ + cuso4.5h2o +++ ++ ++ ba2(1,3-pddadp).8h2o ++ + +++ control c. acutatum +++ +++ +++ metod 480 sc * sporulation: + = poor sporulation, ++ = medium sporulation, +++ = abundant sporulation table 2. influence of the investigated compounds on the sporulation level of c. acutatum biologica nyssana ● 11 (2) december 2020: 109-114 stanojevic et al. ● copper(ii) compounds as effective agents against colletotrichum acutatum causing anthracnose of strawberry of the united nations. statistical databases. available online: http://www.fao.org freeman, s., katan, t., shabi, e. 1998: characterization of colletotrichum species responsible for anthracnose diseases of various fruits. plant disease, 82(6): 596-605. gharieb m. m., ali i. m. and el-shoura a. a. (2004): transformation of copper oxychloride fungicide into copper oxalate by tolerant fungi and the effect of nitrogen source on tolerance. biodegradation 15: 49–57. kite p., hatton d., 2014: u.s. patent no. 8,703,053. washington, d c: u.s. patent and trademark office maas, j. l., palm, m. e. 1997: occurrence of anthracnose irregular leafspot, caused by colletotrichum acutatum, on strawberry in maryland. advances in strawberry research, 16: 68-70. maas, j. l. (2004). strawberry disease management. in diseases of fruits and vegetables: volume ii (pp. 441-483). mertely, j. c., forcelini, b. b. and peres, n. a. 2018: anthracnose fruit rot of strawberry. ifas ekstension, university of florida, report number: pp-207: 1-4. müller, f., ackermann, p., margot, p. 2000: fungicides, agricultural. ullmann’s encyclopedia of industrial chemistry. ng, n. s., wu, m. j., jones, c. e., aldrichwright, j. r. 2016: the antimicrobial efficacy and dna binding activity of some copper (ii) complexes of 3, 4, 7, 8-tetramethyl-1, 10-phenanthroline, 4, 7-diphenyl-1, 10-phenanthroline and 1, 2-diaminocyclohexane. journal of inorganic biochemistry, 162, 62-72. oziengbe e.o. and osazee j.o 2012: antifungal activity of copper sulphate against colletotrichum gleosporioides. journal of asian scientific research 2(12):835-839. patel, m. n., parmar, p. a., gandhi, d. s. 2010: square pyramidal copper (ii) complexes with forth generation fluoroquinolone and neutral bidentate ligand: structure, antibacterial, sod mimic and dna-interaction studies. bioorganic and medicinal chemistry, 18(3): 1227-1235. quesada, l. g. and lopez, e. h. 1980: forma sexual medios de cultivopara collerotrichum gleospotioides, patogenodel mango en cuba. ciencias de la agricultura, 7: 11-17. radanović, d.j., prelesnik, b.v., radanović, d.d., matović, z.d., douglas, b.e. 1997: the favored trans (o6) geometry in hexadentate copper (ii) complexes of 1,3-propanediamine-n, n′-diaceticn,n′-di-3-propionic acid. crystal structure of trans (o6)-na2[cu (1,3-pddadp)]· nano3·2h2o. strain analysis and spectral assignments of complexes. inorganica chimica acta, 262(2): 203-211. radanović, d.j., trifunović, s.r., cvijović, m.s., maricondi, c., douglas, b.e. 1992: synthesis and characterization of hexadentate cobalt (iii) complexes with novel edta-type ligands part 2. circular dichroism of the trans (o5o6) and trans (o6) isomers of a cobalt (iii) complex of 1,3-propanediamine-n, n′-diacetic-n, n′-di-3-propionic acid. inorganica chimica acta, 196(2): 161-169. richardson, h.w. 1997: handbook of copper compounds and applications. crc press. schatzschneider, u. 2019: antimicrobial activity of organometal compounds: past, present, and future prospects. in: hirao, t., moriuchi, t. (eds.): advances in bioorganometallic chemistry, 173-192. elsevier. singh, b. k., bhojak, n., mishra, p., garg, b. s. 2008: copper (ii) complexes with bioactive carboxyamide: synthesis, characterization and biological activity. spectrochimica acta part a: molecular and biomolecular spectroscopy, 70(4): 758-765. singh, a. p., kaushik, n. k., verma, a. k., hundal, g., gupta, r. 2009: synthesis, structure and biological activity of copper (ii) complexes of 4-(2-pyridylmethyl)-1, 7-dimethyl-1, 4, 7-triazonane-2, 6-dione and 4-(2-pyridylethyl)-1, 7-dimethyl-1, 4, 7-triazonane-2, 6-dione. european journal of medicinal chemistry, 44(4), 1607-1614. stojanović, s. (1997): epidemiological and ecological studies of colletotrichum gleosporioides, a superparasitic stroma of polystigma rubrum. doctoral dissertation, faculty of agriculture, university of novi sad. sutton, b. c. 1992: the genus glomerella and its anamorph colletotrichum. in: bailey, j.a., jeger, m.j. (eds.): colletotrichum: biology, pathology and control. cab international, wallingford, oxon, uk, 1-26 wedge, d. e., riley, m. b., tainter, f. h. 1999: phytotoxicity of discula destructiva culture filtrates to cornus spp. and the relationship to disease symptomology. plant disease, 83(4): 377-380. zhang, c.q., liu, y.h., wu, h.m., xu, b.c. sun, p.l., and xu, z.h. 2012: baseline sensitivity of pestalotiopsis microspora, which causes black spot disease on chinese hickory (carya cathayensis), to pyraclostrobin. crop protection 42: 256-259. 114 biologica nyssana ● 11 (2) december 2020: 109-114 stanojevic et al. ● copper(ii) compounds as effective agents against colletotrichum acutatum causing anthracnose of strawberry janaćković et al., 2019, biologica nyssana 10(2) 10 (2) december 2019: 77-85 doi: 10.5281/zenodo.3600177 micromorphology and anatomy in systematics of asteraceae. an old-fashioned approach? review article peđa janaćković university of belgrade, faculty of biology, department of morphology and systematics of plants, studentski trg 16, belgrade, serbia pjanackovic@bio.bg.ac.rs (corresponding author) alfonso susanna botanic institute of barcelona (ibb, csic-icub), pg. del migdia s. n., 08038 barcelona, spain asusanna@ibb.csic.es petar d. marin university of belgrade, faculty of biology, department of morphology and systematics of plants, studentski trg 16, belgrade, serbia pdmarin@bio.bg.ac.rs received: october 27, 2019 revised: november 28, 2019 accepted: december 17, 2019 abstract: the comparative study of plant morphology, intertwined with anatomy, has always been the basis for plant systematics, which strives to explain diversity, evolution and phylogeny of plants. in the molecular era, some authors diminish importance of morphology and especially anatomy in systematic and phylogenetic studies of plants. however, are molecular data exclusively a primary and self-sufficient approach in taxonomic research of plants? this review paper addresses this issue through specific examples. studies of some asteraceae taxa showed that morphological, micromorphological and anatomical data are extremely important in systematics. new opportunities for systematic morphology, micromorphology and anatomy in case of asteraceae taxonomy, but certainly also in other plant groups, that were not present in the premolecular era, are opening regarding synergistic multidisciplinary taxonomic, evolutionary and phylogenetic studies that combine molecular with morphological, anatomical and other analyses (e.g. chemophenetics describes a given taxon phenetically using specialized metabolites as phytochemical characters), keeping in the throne these “old fashioned” approaches. key words: morphology, taxonomy, characters, synergy apstract: mikromorfologija i anatomija u sistematici familije asteraceae. staromodan pristup? uporedna studija morfologije isprepletene sa anatomijom, uvek je bila osnova za sistematiku biljaka koja teži da objasni raznolikost, evoluciju i filogeniju biljaka. u molekularnom dobu, u sistematskim i filogenetskim studijama biljaka, neki autori umanjuju značaj morfologije, a posebno anatomije biljaka. međutim, da li su molekularne metode i podaci isključivo primaran i samodovoljan pristup u taksonomskom istraživanju biljaka? ovaj pregledni rad, kroz određene primere, daje odgovor na ovo pitanje. istraživanja određenih taksona iz familije asteraceae pokazala su da su morfološki, mikromorfološki i anatomski podaci izuzetno važni u sistematici. novi pristupi u sistematskoj morfologiji, mikromorfologiji i anatomiji u slučaju taksonomije asteraceae, ali sigurno i u slučaju taksonomije drugih grupa biljaka, koji nisu bili mogući u premolekularnom dobu, otvaraju se sada kao sinergija multidisciplinarnih taksonomskih, evolucionih i filogenetskih studija koje kombinuju molekularne sa morfološkim, anatomskim i drugim analizama i pristupima (npr. hemofenetika koja opisuje dati takson fenetički, upotrebom specijalizovanih metabolita kao fitohemijskih karaktera), držeći na prestolu ove „staromodne“ pristupe. ključne reči: morfologija, taksonomija, karakteri, sinergija introduction the comparative study of plant structure has always been the backbone of plant systematics, which strives to elucidate plant diversity, phylogeny and evolution (endress et al., 2000). plant taxonomic studies traditionally use morphological and karyological (stebbins, 1953), as well as micromorphological characters (hayat et al., 2009; bak & ozcan, 2018). micromorphological characters are of decisive importance in unring taxonomic and phylogenetic relationships of various plant groups and have been successfully used in plant systematic studies for decades (endress et al., 2000). for more than a © 2019 janaćković et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 77 13th symposium on the flora of southeastern serbia and neighboring regions century, comparative anatomy is used as a tool in the plant systematics. anatomical characters are very important in perceiving systematic and phylogenetic relationships of particular plant groups. indeed, anatomical features can provide useful characters which could help in identification of problematic plant taxa, as well as establishing their taxonomic relationships (metcalfe & chalk, 1957; scatena et al., 2005; makbul et al., 2011; sosa et al., 2014; karanović et al., 2015, gavrilović et al., 2019a, b; janaćković et al., 2019). on the other hand, the data provided by the phytochemists are extremely difficult to interpret in a cladistic context. even so, integration of phytochemical and macro-molecular characters can be of prominent benefit, and can help, for example, in the delineation of clades so far only supported by dna sequence data (enke et al., 2012). in order to overcome the confusion in the interpretation of phytochemical characters (specialized metabolites that actually describe the given taxon phenetically) as phylogenetic characters and “under the umbrella” of the term chemotaxonomy (which is mistakenly identified with chemosystematics), the new term chemophenetics has been proposed by zidorn, (2019). nevertheless, chemophenetic studies contribute to the phenetic description of taxa, similarly to anatomical, morphological and karyologycal approaches, which have already been recognized as of major importance for establishing “natural” systems and which continue to be of the highest importance for the description of organisms classified with the help of modern molecular methods (zidorn, 2019). asteraceae (compositae), habitually known as the daisy or sunflower family, represent one of three mega-diverse families which jointly count more than 25% of all extant angiosperm species (mandel et al., 2019). asteraceae, counting 25000–35000 species, comprise 10% of all flowering plant species. members of the sunflower family occur on every continent including antarctica (smith & richardson, 2011) and inhabit nearly every type of habitat on earth with the largest concentration of species in deserts, prairies, steppes, montane regions, and areas with mediterranean-like climates (mandel et al., 2019). according to newer fossil data and recent molecular clock dating, asteraceae likely originated during the late cretaceous: ∼83 mya (mandel et al., 2019). according to mandel et al. (2019) the family consists of 13 subfamilies and 47 tribes. asteraceae also includes species of wide economic interest, e.g. vegetables, sources of oil, medicinal plants, insecticides and many horticultural and garden ornamentals. however, some species of asteraceae constitute a big problem for agriculture as noxious weeds. as a one of the largest, natural (with a combination of several specialized morphological characteristics e.g., capitula, highly reduced and modified flowers, syngenesious anthers, inferior ovaries and very unique in the plant kingdom fruit cypsela) and economically most important families of flowering plants asteraceae has been researched for centuries. characters related to form, whether gross morphology, micromorphology, anatomy, embryology, palinology and so forth, regarded as morphologycal data, have larger impact on the cladistics and classification of the asteraceae than other characters e.g. chemical and molecular data (schönberger, 2002; stuessy, 2009). molecular data are significant but also insufficient, that is why, in some cases, better phylogenetic reconstructions of the asteraceae are obtained taking into account also a morphological data (pornpongrungrueng et al., 2007; gruenstaeudl et al., 2009; wang et al., 2013). more recent, modern-day studies of asteraceae taxa have shown that morphological, micromorphological and anatomical data are still extremely important in systematics of asteraceae family (makbul et al., 2011; wang et al., 2013; sosa et al., 2014; karanović et al., 2015; bombo et al., 2016; ginko et al., 2016; batista and de souza, 2017; gavrilović et al., 2017; gavrilović et al., 2018a, b; gavrilović et al., 2019a, b; janaćković et al., 2019). brief history and modern-day micromorphology and anatomy in systematics of asteraceae micromorphology a different sets of characters has been used to demarcate the asteraceae. certainly, one of the pioneer work regarding micromorpholgical approaches to asteraceae classification was done by cassini (1821). these summarize many of the microscopic traits on which cassini based his tribes. of these, pronate versus recurved mature style branches, stigmatic surfaces, truncate to enlarged style appendages, bases of the anther thecae with or without tails, shape of the anther collar and form of the corolla are most important. the stigmatic surfaces of many tribes (mutisieae, lactuceae, vernonieae, arctotideae, eremothamneae, cardueae) are consistently continuous over the inner surface of the style branch. in other tribes (eupatorieae, anthemideae, astereae, most inuleae, most heliantheae, most senecioneae) the stigmatic surface is divided into two lines. the nature of endothecial tissue of the stamens, which could be polarized or radial, showed to be good character in taxonomy within senecioneae (dormer, 1962). in subtribes senecioninae and othonninae, the endothecium is radial in all genera except dauresia, graphistylis, and perhaps synotis. in 78 biologica nyssana ● 10 (2) december 2019: 77-85 janaćković et al. ● micromorphology and anatomy in systematics of asteraceae. an old-fashioned approach? tussilagininae s.str. a polarized endothecium is the rule, but the radial type has been recorded in several genera (tephroseris, nemosenecio, psacaliopsis, psacalium, arnoglossum); in sinosenecio both types and an intermediate pattern seem to occur (jeffrey & chen, 1984). moreover, the anthers of cichorieae members vary considerably in length, but this variation probably occurs repeatedly within many genera and is therefore only of taxonomic relevance at the species level (kilian et al., 2009). in arctotideae morphological and micromorphological characters confirmed close relationships between the gorteria clade and berkheya clade (karis et al., 2009). microcharacters of involucral bracts are considered very helpful for delimitation in certain taxonomic groups of asteraceae (e.g., for subtribes of cardueae, with spiny pectinate-fimbriate appendages in cardopatiinae; usually spiny, innermost exappendiculate or with rudimentary appendages in carduinae; inner often conspicuous and coloured in carlininae; scarious, fimbriate, pectinate, spiny or unarmed appendage in centaureinae; and in many rows in echinopsinae (robinson, 2009; susanna & garcia-jacas, 2009). certain floral microcharacters (anther size, shape of the anther apical appendage, configuration of stigmatic areas on the inner surface of the style branch, and configuration of the endothecial thickenings and of the filament collar) of 36 taxa of sinosenecio showed that these floral characters are highly consistent with evidence from molecular systematics and cytology and provide the most important diagnostic characters in the tribe senecioneae, as in the family at large and strongly suggest a polyphyletic nature of this genus, as well as the need of a taxonomic change at generic level (liu & yang, 2011). still nowadays, micromorphological investigations of asteraceae could provide some novel characters (erbar & leins, 2015; gavrilović et al. 2017, 2019b). investigating style morphology of 395 species of 258 genera (covering all, in that time, 44 tribes of the asteraceae), erbar & leins (2015) found a new microstructural feature, namely, often conspicuous cuticular patterns on the stylar hairs (involved in secondary pollen presentation) and stylar appendages. they determined five different patterns of cuticular striation and when they put these patterns onto a generalized phylogenetic tree (based on molecular data), they concluded that there is considerable homoplasy in these features. nevertheless, cuticular patterns are still useful in characterizing some clades within the family. gavrilović et al. (2017) investigating involucral bract micromorphology found, for the first time, a large number of densely packed crystals on the involucral bract surface. also, the presence of nonglandular, curly trichomes and biologica nyssana ● 10 (2) december 2019: 77-85 janaćković et al. ● micromorphology and anatomy in systematics of asteraceae. an old-fashioned approach? 79 biseriate glandular trichomes on the bract surface, as well as the sylvite crystals on the petal surface of x. cylindraceum, clearly differentiates this species from x. annuum (gavrilović et al., 2017). comparative micromorphological analyses were conducted on five members of the xerantheminae, both perennial (amphoricarpos exsul and shangwua masarica) and annual (chardinia orientalis, siebera pungens and xeranthemum inapertum), showing that micromorphological traits link together perennial species, some link annual ones, some are species-specific, and some are common to all taxa (gavrilović et al., 2019b). we could conclude that morphology and micromorphology of florets (e.g., style base, anther appendages, trichomes on corollas), and inflorescence (involucral bracts characters, e.g., crystals and glandular and nonglandular trichomes on their wall) were used as major distinguishing features for subtribal and generic delimitation, even though these characters can sometimes be significant at the species level. anatomy at the beginning of the twentieth century col (18991901), in light of anatomy, reviewed in considerable detail distribution of laticiferous versus resiniferous tissue throughout the asteraceae. taxa with latex in canals or sacs occur in several tribes of the subfamily cichorioideae and consistently in the lactuceae, but very rarely in the asteroideae, where resin sacs and resin canals are common. carlquist (1966) investigated the basic plan of the wood anatomy of asteraceae (focusing on four tribes, anthemideae, ambrosieae, calenduleae, and arctotideae), which provided useful tribal characters and minor intertribal variation. even though carlquist (1966) stated that asteraceae members share a basically specialized wood plan and that wood anatomy is not likely to reward one with tribal or subtribal characters, certain characters are of systematic value within anthemideae, ambrosieae, calenduleae, and arctotideae (e.g., carbonized resins in intercellular spaces, secretory canals in rays, patterns of crystal occurrence are characters which may be of specific or generic value). metcalfe & chalk (1957) noted some particular anatomical traits, which showed to have taxonomic importance within the family, e.g., presence of secretory and laticiferous canals, types of nonglandular and glandular trichomes, occurrence of medullar and cortical vascular bundles and presence of anomalous secondary thickening. also, anatomical characteristics observable in asteraceae are: (a) presence of various types of glandular and non-glandular tri80 chomes; (b) papillae on the abaxial leaf epidermis; (c) anomocytic, anisocytic and rarely heliocytic stomata types; (d) presence of hydathodes; (e) presence of hypodermis; (f) homogeneous or heterogeneous mesophyll and (g) vascular bundles with a parenchymatic sheath composed of large cells (metcalfe & chalk, 1979). anatomy of several members of the tribe senecioneae showed that they possess resin ducts in stems, leaves and roots, sometimes also in floral parts, and even in cotyledons. the resin production is noted as stickiness and exudates on vegetative parts. this occurs in the tussilaginoid as well as in senecioid group (nordenstam et al., 2009). ginko et al. (2016) investigated suitability of anatomical characters of root and rhizome of 59 species belonging to 34 genera and 12 subtribes from tribes cardueae and cichorieae for taxonomic classification and phylogenetic reconstruction. in this case, anatomy is demonstrated as valuable to discriminate tribes and many species but not so for subtribes and genera. however, most anatomical traits seems to be homoplastic, which limits their application as phylogenetically informative characters. bombo et al. (2016) stated that anatomical features can help in resolving taxonomical problems within the genus aldama la llave, especially among brazilian members, which are difficult to identify taxonomically. on the basis of their findings, the authors concluded that anatomy is able to provide data which assist with the taxonomic problems within the four analysed species. the systematic value of leaf epidermal characters in asteraceae has been proven by numerous studies, as leaf surface is under strong genetic control (adedeji & jewoola, 2008; karanović et al., 2015). some leaf blade characters (e.g., epidermal anticlinal cell walls, epicuticular wax and trichome type) have shown to be diagnostic to separate aster l., galatella cass. and tripolium nees (karanović et al., 2015). a comparative study of the leaf epidermis in 12 species of asteraceae showed that the type and shape of trichomes, nature of cuticular striations and stomatal type are taxonomically important for the delimitation of species (adedeji & jewoola, 2008). qualitative anatomical characters (e.g., shape of the young stem and peduncle cross-sections, type of glandular trichomes and occurrence of cortical vascular bundles) were shown to be useful in delimitating x. annuum from x. cylindraceum (gavrilović et al., 2019a). moreover, some of the anatomical characters found in xeranthemum (secondary growth in roots and dorsiventral leaves) suggested that adaptation from mesophytic to xeric habitats (gavrilović et al., 2019a) are important for phylogenetic relationships within xerantheminae. anatomical data can also contribute in resolving complex taxonomy of certain genera, e.g. artemisia. in anatomical investigation of five artemisia species, janaćković et al. (2019) showed that some characters link together a. absinthium and a. arborescens from the same section; some other connect species belonging to different sections (a. campestris and a. arborescens; a. absinthium and a. judaica; a. judaica and a. herba-alba), while some could be considered as species-specific. we could summarize that certain anatomical characters, such us distribution of laticiferous versus resiniferous tissue is useful on subfamily an tribal level, while patterns of crystal occurrence might be significant at species or genus level. occurrence of cortical vascular bundles seems to be important on species level. root and rhizome anatomical traits have proven to be useful on tribal and species, but not on the genus level. leaf anatomical epidermal character are diagnostic and can be used for delimiting species. also, qualitative anatomical characters may have role in understanding and solving phylogenetic relationships, which are reflected in the systematics of given taxa. micromorphology and anatomy of cypsela the cypsela is a special form of dry indehiscent fruit in which the seed coat (testa) and fruit wall (pericarp) are tightly attached to one another and is exclusive characteristic of the family asteraceae (roth, 1977). as an exclusive fruit of the family cypsela and its features have been attracted by tournefort (1694), vaillant (1719), cassini (1819), lessing (1832), bentham (1873), hoffman (1894), cronquist (1955), robinson (1977), bremer (1994), nordenstam (1994), rao & datt (1996), robinson (1999), nordenstam et al. (2006), lack (2007) and mukherje & nordenstam (2004, 2010). cypsela morphology (macroand micromorphology) and anatomy have been widely used in illuminating taxonomic relationships in asteraceae and still represents a source of valid taxonomic characters (lavialle, 1912; stebbins, 1953; wagenitz, 1976; dittrich 1977; barthlott, 1984; singh & pandey, 1984; dittrich, 1985; bruhl & quinn, 1990; glynis, 1993; geng et al., 1994; blanca & díaz de la guardia, 1997; petit, 1997; häffner, 2000; zhu et al., 2006; garg & sharma, 2007; pandey & kumari, 2007; zarembo and boyko, 2008; abid & qaiser, 2009; abid & ali, 2010; inceer et al., 2012; ozcan & akinci, 2019). this is why micromorphology and anatomy of cypsela are separated herein. bremer (1987) stated the importance of cypselae characters at lower taxonomic levels but not at the tribal level. in asteraceae, the anatomy and micromorphology of biologica nyssana ● 10 (2) december 2019: 77-85 janaćković et al. ● micromorphology and anatomy in systematics of asteraceae. an old-fashioned approach? 81 biologica nyssana ● 10 (2) december 2019: 77-85 janaćković et al. ● micromorphology and anatomy in systematics of asteraceae. an old-fashioned approach? cypselas are taxonomically significant at both genus and species levels (abid & qaiser, 2009, kulkarni, 2013, hussein & eldemerdash, 2016; karanović et al., 2016; gavrilović et al., 2019b). cypselae size and shape, number of ribs, presence of prickles, ornamentation of the intercostal gaps, tapering (sharp or gradual) of the body/beak junction, shape of the beak, degree of swelling at its apex and pubescence of the annulus are diagnostic in tragopogon l. (blanca & díaz de la guardia, 1997). micromorphological characters of the cypselae (shape, surface, colour, size), pappus (stucture, shape, number, colour, size) and carpopodium (shape, position, diameter) in the tribes senecioneae and anthemidae are useful for assessing the relationship and delimitation at both generic and specific levels (abid & qaiser, 2009; abid & ali, 2010). as a result of morphological and anatomical investigations of the cypselae in east asian species of rhaponticum vaill., klasea cass., serratula l. and synurus iljin in the tribe cardueae s.l., zarembo & boyko (2008) clarified the following diagnostic traits at the species level: topography of epidermal cells of the pericarp, presence of phlobaphenes, occurrence, topography and localization of calcium oxalate crystals, and occurrence and location of secretory ducts in the mesocarp. karanović et al. (2016) showed that, besides receptacle characters, organisation of sclerenchymatous tissue in a fruit is a feature that tend to be diagnostic for genera inula, pulicaria, dittrichia and limbarda. also, some fruit features have been demonstrated to be especially useful in distinguishing certain similar species (e.g., inula britannica from i. oculus-christi). moreover, authors stated that i. helenium should be separated from the inula genus, as its authenticity is evident base on cypsela characters. silva et al. (2017) showed that features of the cypselae of the subtribe disynaphiinae such as the carpopodium, floral disc, pappus, outer mesocarp, sclerenchyma, phytomelanin layer, ribs and trichomes are valuable at both generic and specific levels. moreover, authors revealed the presence of a multiplicative pericarp only in a few symphyopappus spp., rare trait in asteraceae, which probably evolved independently in the family. cypsela structure also supports the exclusion of disynaphia praeficta from the subtribe, since this species posesses several different characters comparing with other representatives of disynaphiinae. characters of cypselae are shown to be very informative at generic level within xerantheminae (gavrilović et al., 2019). for instance, shangwua is distinguished from other genera in having glabrous cypsela, while chardinia only possesses papillose cypsela surface. moreover, cypsela features of xerantheminae taxa are significant for phylogeny of the subtribe (some characters share all members, while some characters separates annual from perennial genera). ozcan & akinci (2019) evaluated feasibility of cypsela characters as taxonomic markers investigating 21 taxa representing 12 genera of the tribe cardueae. they observed considerable variability in surface sculptures of pappus and cypselae, as well as in pericarp and testa structures. authors concluded that micromorphological and anatomical cypsela characters are distinct between the genera and are also useful for delimiting species. conclusion and future prospects the breakthrough of molecular tools in plant systematics and its contribution to phylogenetic frameworks was and it is still a tremendous stimulus for comparative morphology and anatomy. one should have in mind that the structure and biology of a majority of asteraceae members are far from sufficiently investigated, thus combining morpho-anatomical, phytochemical, and molecular studies are necessary to explore them. although this overview represents only a glimpse of a role of micromorphological and anatomical approaches to asteraceae systematics, it gives an valuable insight and perspective of this topic. thus, the accumulated knowledge and permanent investigation of asteraceae taxa using micromorphological and anatomical methods will put light on branching topologies of phylogenetic trees which molecular data established. new opportunities for systematic morphology, micromorphology and anatomy in case of asteraceae taxonomy, but certainly also in other plant groups, which were not present in the premolecular era, are now opening regarding synergistic multidisciplinary taxonomic, evolutionary and phylogenetic studies which combine molecular with morphological, anatomical and other approaches (e.g. chemophenetics), keeping in the throne these “old fashioned” approaches. acknowledgements. we acknowledge the financial support provided by the serbian ministry of education, science and technological development, project no. 173029. references abid, r., ali, n. 2010: cypsela morphology and its taxonomic significance for the tribe senecioneae (asteraceae) from pakistan. pakistan journal of botany, 42: 117–133. 82 abid, r. d., qaiser, m. 2009: taxonomic significance of the cypsela morphology in the tribe anthemideae (asteraceae) from pakistan and kashmir. pakistan journal of botany, 41: 555–579. adedeji, o., jewoola, o. a. 2008: importance of leaf epidermal characters in the asteraceae family. notulae botanicae horti agrobotanici cluj-napoca, 36:7–16 bak, f. e., ozcan, m. 2018: pollen morphology of endemic ne anatolian cirsium taxa (asteraceae). pakistan journal of botany, 50 (3): 1181–1185. barthlott, w. 1984: microstructural features of seed surfaces. in: heywood, v. h., moore, d. m. (eds.), current concepts in plant taxonomy. academic press, cambridge, pp. 95–105. batista, m. f., de souza, l. a. 2017:. flower structure in ten asteraceae species: considerations about the importance of morpho-anatomical features at species and tribal level. brazilian journal of botany, 40(1): 265–279. bentham, g. 1873: notes on the classification, history, and geographical distribution of compositae. botanical journal of the linnean society, 13(70-72): 335–577. blanca, c. c., guardia, c. d. 1997: fruit morphology in tragopogon l. (compositae: lactuceae) from the iberian peninsula. botanical journal of the linnean society, 125: 319–329. bombo, a. b., filartiga, a. l., appezzato-daglória, b. 2016: solving taxonomic problems within the aldama genus based on anatomical characters. australian journal of botany, 64(6): 501–512. bremer, k. 1987: tribal interrelationships of the asteraceae. cladistics 3: 210–253. bremer, k. 1994: asteraceae. cladistics and classification. timber press, portland. bruhl, j. j., quinn, c. j. 1990:. cypsela anatomy in the ‘cotuleae’ (asteraceae–anthemideae). botanical journal of the linnean society, 102(1): 37–59. carlquist, s. 1966: wood anatomy of anthemideae, ambrosieae, calenduleae and articotideae (compositae). aliso, 6:1–23 cassini, a. h. g. 1819: suite du sixième mémoire sur la famille des synanthérées, contenant les caractères des tribus. journal de physique, de chimie, d‘histoire naturelle et des arts, 88: 196– 198. cassini, h. 1821. hélianthées [with discussion of tribes]. in: cuvier, f. (ed.), dictionnaire des sciences naturelles, ed. 2, vol. 20. le normant, paris. pp. 354–385 col, a. 1889-1901:. quelques recherches sur l’appareil sécréteur des composées. journal de botanique (morot), 17: 252–318, 18: 110–133, 153– 175. columbia university press, new york. cronquist, a. 1955:. phylogeny and taxonomy of the compositae. american midland naturalist, 478– 511. dittrich, m. 1977. cynareae – systematic review. in: heywood, v.h., harborne, j.b., turner, b.l. (eds.), the biology and chemistry of the compositae 2. academic press, london, new york, san francisco, pp. 999–1015. dittrich, m. 1985. morphologische und anatomische untersuchungen an blüten und früchten der gattung carlina (compositae). botanische jahrbücher fur systematik, pflanzengeschichte und pflanzengeographie, 107: 591–609. dormer, k. j. 1962: the fibrous layer in the anthers of compositae. the new phytologist, 61(2): 150– 153. endress, p. k., baas, p., gregory, m. 2000: systematic plant morphology and anatomy ‐ 50 years of progress. taxon, 49(3): 401–434. enke, n., gemeinholzer, b., zidorn, c. 2012: molecular and phytochemical systematics of the subtribe hypochaeridinae (asteraceae, cichorieae). organisms diversity & evolution, 12(1): 1–16. erbar, c., leins, p. 2015: cuticular patterns on stylar hairs in asteraceae: a new micromorphological feature. international journal of plant sciences, 176(3): 269–284. garg, s. k., sharma, k. c. 2007: taxonomical significance of the micromorphological and scanning electron microscopic surface patterns of cypselas in some members of the tribe heliantheae (asteraceae). feddes repertorium, 118: 165–191. gavrilović, m., erić, s., marin, p. d., garciajacas, n., susanna, a., janaćković, p. 2017: scanning electron microscopy coupled with energy dispersive spectrometric analysis reveals for the first time weddellite and sylvite crystals on the surface of involucral bracts and petals of two xeranthemum l. (compositae) species. microscopy and microanalysis, 23(3): 679–686. gavrilović, m., tešević, v., đorđević, i. s., rajčević, n., bakhia, a., jacas, n. g., susanna, a., janaćković, p. t. 2018a: leaf micromorphology, antioxidative activity and a new record of 3-deoxyamphoricarpolide of relict and biologica nyssana ● 10 (2) december 2019: 77-85 janaćković et al. ● micromorphology and anatomy in systematics of asteraceae. an old-fashioned approach? 83 biologica nyssana ● 10 (2) december 2019: 77-85 janaćković et al. ● micromorphology and anatomy in systematics of asteraceae. an old-fashioned approach? limestone endemic amphoricarpos elegans albov (compositae) from georgia. archives of biological sciences, 70(4): 613–620. gavrilović, m., de oliveira, a. f. m., barbosa, m. o., garcia-jacas, n., susanna, a., marin, p. d., janaćković, p. 2018b: micromorphology and fatty acid composition of the cypselae of xeranthemum cylindraceum sm.(asteraceae, cardueae). botanica serbica, 42 (2): 241–250. gavrilović, m., rančić, d., škundrić, t., dajićstevanović, z., marin, p., garcia-jacas, n., susanna, a., janaćković, p. 2019a: anatomical characteristics of xeranthemum l. (compositae) species: taxonomical insights and evolution of life form. pakistan journal of botany, 51: 1007–1019. gavrilović, m., jacas, n. g., susanna, a., marin, p. d., janaćković, p. 2019b: how does micromorphology reflect taxonomy within the xeranthemum group (cardueae-asteraceae)?. flora, 252, 51–61. geng, s. l., an, z. x., tian, y. w. 1994: the studies of fruit anatomy and systematic classification of tribe senecioneae in xinjiang. journal of agriculture & life sciences, 17: 9–17. ginko, e., dobeš, c., saukel, j. 2016: suitability of root and rhizome anatomy for taxonomic classification and reconstruction of phylogenetic relationships in the tribes cardueae and cichorieae (asteraceae). scientia pharmaceutica, 84: 585–602. glynis, v. c. 1993. the anatomy of the cypselae of species of cineraria l (asteraceae–senecioneae) and its taxonomic signif-icance. botanical journal of the linnean society, 112: 319–334. gruenstaeudl, m., urtubey, e., jansen, r. k., samuel, r., barfuss, m. h., stuessy, t. f. 2009: phylogeny of barnadesioideae (asteraceae) inferred from dna sequence data and morphology. molecular phylogenetics and evolution, 51(3): 572–587. häffner, e. 2000: on the phylogeny of the subtribe carduinae (tribe cardueae, compositae). englera, 21: 1–208. hayat, m. q., ashraf, m., khan, m. a., yasmin, g., shaheen, n., jabeen, s. 2009: diversity of foliar trichomes and their systematic implications in the genus artemisia (asteraceae). international journal of agriculture and biology, 11: 542–546. hoffmann o. 1890-1894: compositae. in: engler, a., prantle, k. (eds.), dienatürlichen pflanzenfamilien, engelmann, leipzig 4(5): 87–387. hussein, h. a., eldemerdash, m. m. 2016: comparative morphology and surface microsculpture of cypsela in some taxa of the asteraceae and their taxonomic significance. egyptian journal of botany, 56: 409–422. inceer, h., bal, m., ceter, t., pinar, n.m. 2012: fruit structure of 12 turkish endemic tripleurospermum sch. bip. (asteraceae) taxa and its taxonomic implications. plant systematics and evolution, 298: 845–855. janaćković, p., gavrilović, m., rančić, d., dajićstevanović, z., giweli, a. a., marin, p. d. 2019: comparative anatomical investigation of five artemisia l. (anthemideae, asteraceae) species in view of taxonomy. brazilian journal of botany, 42(1): 135–147. jeffrey, c., chen, y. l. 1984: taxonomic studies on the tribe senecioneae (compositae) of eastern asia. kew bulletin, 39: 205–446. karanović, d., luković, j., zorić, l., anačkov, g., boža, p. 2015: taxonomic status of aster, galatella and tripolium (asteraceae) in view of anatomical and micro-morphological evidence. nordic journal of botany, 33: 484–497. karanović, d., zorić, l., zlatković, b., boža, p., luković, j. 2016: carpological and receptacular morpho-anatomical characters of inula, dittrichia, limbarda and pulicaria species (compositae, inuleae): taxonomic implications. flora, 219, 48– 61. karis, p.o., funk, v., mckenzie, r.j., barker, n.p., chan, r. 2009: arctotideae. in: funk, v. a., susanna, a., stuessy, t.f., bayer, r.j. (eds.), systematics, evolution and biogeography of compositae. iapt, vienna, pp. 385–411. kilian, n., gemeinholzer, b., walter lack, h. 2009: cichorieae in: funk, v. a., susanna, a., stuessy, t.f., bayer, r.j. (eds.), systematics, evolution and biogeography of compositae. iapt, vienna, pp. 343–383. kulkarni, s. v. 2013: sem studies of achenes in some taxa of asteraceae. international journal of environmental rehabilitation and conservation, 4: 87–97. lack, h.w. 2007: cichorieae. in: kadereit, j.w., jeffrey, c. (eds.), the families and genera of vascular plants, vol. 8, flowering plants, eudicots. asterales. springer, berlin. pp. 180–199. lavialle, p. 1912: recherches sur le développement de l’ovaire en fruit chez les composées. masson. lessing, c. f. 1832: synopsis generum compositarum. duncker & humblot, berlin. 84 liu, y., yang, q. e. 2011: floral micromorphology and its systematic implications in the genus sinosenecio (senecioneae-asteraceae). plant systematics and evolution, 291(3-4): 243–256. makbul, s., güler, n. s., durmuş, n., güven, s. 2011: changes in anatomical and physiological parameters of soybean under drought stress. turkish journal of botany, 35(4): 369–377. mandel, j. r., dikow, r. b., siniscalchi, c. m., thapa, r., watson, l. e., funk, v. a. 2019: a fully resolved backbone phylogeny reveals numerous dispersals and explosive diversifications throughout the history of asteraceae. proceedings of the national academy of sciences, 116 (28) 14083– 14088. metcalfe, c. r., chalk, l. 1957: anatomy of the dicotyledons. clarendon press, oxford. metcalfe, c. r., chalk, l. 1979: anatomy of dicotyledons: systematic anatomy of the leaf and stem, with a brief history of the subject, vol 1. claredon press, oxford. mukherjee, s. k., nordenstam, b. 2010: distribution of calcium oxalate crystals in the cypselar walls in some members of the compositae and their taxonomic significance. compositae newsletter, 48: 63–88. mukherjee, s. k., nordenstam, b. 2004: diversity of carpopodial structure in the asteraceae and its taxonomic significance. compositae newsletter, 41: 29–50. nordenstam, b. 1994: tribe calenduleae. asteraceae. cladistics and classification. timber press, portland, 365-376. nordenstam, b. 2007: tribe calenduleae cass. (1819). in: kadereit, j. w., jeffrey, c. (eds), the families and genera of vascular plants, vol. 8. springer, berlin, heidelberg, pp. 241–245. nordenstam, b., pelser, p.b., kadereit, j.w., watson, l.e. 2009: senecioneae. in: funk, v. a., susanna, a., stuessy, t.f., bayer, r.j. (eds.), systematics, evolution and biogeography of compositae. iapt, vienna, pp. 503–525. ozcan, m., akinci, n. 2019: micromorphoanatomical fruit characteristics and pappus features of representative cardueae (asteraceae) taxa: their systematic significance. flora, 256: 16–35. pandey, a. k., kumari, a. 2007: anatomical patterns of pericarp in asteraceae. in: chauhan, s. v. s., rana, a., chauhan, s. (eds), plant reproductive biology and biotechnology. aavishkar publisher, jaipur, pp. 64–77. petit, d. p. 1997: generic interrelationships of the cardueae (compositae): a cladistic analysis of morphological data. plant systematics and evolution, 207: 173–203. pornpongrungrueng, p., borchsenius, f., englund, m., anderberg, a. a., gustafsson, m. h. 2007: phylogenetic relationships in blumea (asteraceae: inuleae) as evidenced by molecular and morphological data. plant systematics and evolution, 269(3–4): 223–243. rao, r. r., dutt, b. 1996: diversity and phytogeography of indian compositae. in: hind, d. j. n., beentje, h. j. (eds.), compositae: systematics. proceedings of the international compositae conference 1, royal botanic garden, kew. pp. 445– 461. robinson, h. 1977: an analysis of the characters and relationships of the tribes eupatorieae and vernonieae (asteraceae). systematic botany, 2(3): 199–208. robinson, h. 2009: an introduction to microcharacters of compositae. in: funk, v. a., susanna, a., stuessy, t.f., bayer, r.j. (eds.), systematics, evolution and biogeography of compositae. iapt, vienna, pp. 89–100. robinson, h. 1999: two new subtribes stokesiinae and pacourininae, of the vernonieae (asteraceae). proceedings of the biological society of washington, 112: 216–219. roth, i. 1977: fruits of angiosperms. gebrüder borntraeger, berlin. scatena, v. l., giulietti, a. m., borba, e. l., van den berg, c. 2005: anatomy of brazilian eriocaulaceae: correlation with taxonomy and habitat using multivariate analyses. plant systematics and evolution, 253: 1–22. schönberger, i. 2002: biosystematics and taxonomy of the ozothamnus leptophyllus (compositae) complex in new zealand. doctorate. university of canterbury. silva, t. d., marzinek, j., hattori, e. k., nakajima, j. n., de-paula, o. c. 2017: comparative cypsela morphology in disynaphiinae and implications for their systematics and evolution (eupatorieae: asteraceae). botanical journal of the linnean society, 186(1): 89–107. singh, r. p., pandey, a. k. 1984: development and structure of seeds and fruits in compositae cynareae. phytomorphology, 34: 1–10. smith, r. i. l., richardson, m. 2011: fuegian plants in antarctica: natural or anthropogenically biologica nyssana ● 10 (2) december 2019: 77-85 janaćković et al. ● micromorphology and anatomy in systematics of asteraceae. an old-fashioned approach? assisted immigrants? biological invasions, 13(1): 1–5. sosa, m. m., via do pico, g. m., dematteis, m. 2014: comparative anatomy of leaves and stems in some species of the south american genus chrysolaena (vernonieae, asteraceae) and taxonomic implications. nordic journal of botany, 32: 611–619. stebbins, g. l. 1953: a new classification of the tribe cichorieae, family compositae. madroño, 12(3): 65–81. stuessy, t. f. 2009: plant taxonomy: the systematic evaluation of comparative data.–2nd ed. columbia university press, new york, chichester, west sussex. susanna, a., garcia-jacas, n. 2009: cardueae (carduoideae), in: funk, v. a., susanna, a., stuessy, t.f., bayer, r.j. (eds.), systematics, evolution and biogeography of compositae. iapt, vienna, pp. 293–313. tournefort, j.p. 1694: élemens de botanique. 3 vols. imprimeric royale, paris. vaillant, s. 1719: establishment de nouveau caracteres de triosfamillesou classes de plantes a fleur composes: scavoir, des cynarocephales, des corymbiferes, et des cichoracees. histoire de l’ academie royale des sciences avec les memoires de mathematique&des physique (paris 4) 1718: 143–191. wang, y. j., von raab-straube, e., susanna, a., liu, j. q. 2013: shangwua (compositae), a new genus from the qinghai-tibetan plateau and himalayas. taxon, 62(5): 984–996. wang, z. h., peng, h., kilian, n. 2013: molecular phylogeny of the lactuca alliance (cichorieae subtribe lactucinae, asteraceae) with focus on their chinese centre of diversity detects potential events of reticulation and chloroplast capture. plos one, 8(12): p.e82692. zarembo, e.v., boyko, e.v. 2008: carpology of some east asian cardueae (asteraceae). anales del jardín botánico de madrid, 65: 129–134. zhu, s.x., qin, h.n., shih, c. 2006: achene wall anatomy and surface sculpturing of lactuca l. and related genera (compositae: lactuceae) with notes on their systematic significance. journal of integrative plant biology, 48: 390–399. zidorn, c. 2019: plant chemophenetics − a new term for plant chemosystematics/plant chemotaxonomy in the macro-molecular era. phytochemistry, 163: 147–148. biologica nyssana ● 10 (2) december 2019: 77-85 janaćković et al. ● micromorphology and anatomy in systematics of asteraceae. an old-fashioned approach? 85 džamić & alimpić aradski 2022, biologica nyssana 13(2) 13 (2) december 2022: 83-87 doi: 10.5281/zenodo.7437204 in memoriam professor sonja duletić-laušević (1962-2021) original article ana m. džamić university of belgrade, faculty of biology, institute of botany and botanical garden “jevremovac”, studenski trg 16, 11000 belgrade, serbia simicana@bio.bg.ac.rs (corresponding author) ana z. alimpić aradski university of belgrade, faculty of biology, institute of botany and botanical garden “jevremovac”, studenski trg 16, 11000 belgrade, serbia professor sonja duletić-laušević suddenly passed away on december 18, 2021, leaving a large void behind. her passing left us in disbelief and sadness. as a full professor at the university of belgrade, faculty of biology, she made great contributions both as a teacher and scientist. sonja n. duletić-laušević was born on february 14, 1962, in belgrade where she finished primary school and gymnasium. she enrolled in the department of biology of the faculty of science and mathematics, university of belgrade, and graduated in 1986 in the group of general biology. she received a master’s degree at the university of belgrade faculty of biology in 1990 defending the thesis entitled: “investigation of the production of t-2 toxin and its effect on rec mutants of escherichia coli”. in 1995, she defended her doctoral thesis with the title “comparative anatomical analysis of the vascular system of the genus helianthus l. genotypes” at the university of belgrade faculty of biology. her first job was at the department of plant physiology (mycology laboratory) of the institute for biological research „siniša stanković” in belgrade (1987-1988) where she was a junior assistant. in 1989 she was employed as a teaching assistant at the chair of plant morphology and systematics, institute of botany, university of belgrade faculty of biology. she was promoted to assistant professor in 1997, associate professor in 2005, and full professor in 2019 at the chair of plant morphology and systematics, at the faculty of biology. she participated in the preparation and realization of practical and theoretical courses organized by the chair of plant morphology and systematics at all study levels. she taught the morphology of plants and general botany course to numerous generations. in recent years, she has been teaching anatomy and morphology of plants and botany at the undergraduate level. she is a co-author of numerous textbooks and practicums. she is the coauthor of the digital content visual dictionary of plant anatomy and morphology. she was the mentor of three doctoral dissertations and a member of nine defense comissions, as well as numerous master’s, and diploma theses. her scientific opus includes over 230 bibliographic units, © 2022 džamić & alimpić aradski. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 83 14th symposium on the flora of southeastern serbia and neighboring regions 84 biologica nyssana ● 13 (2) december 2022: 83-87 džamić & alimpić aradski ● in memoriam professor sonja duletić-laušević (1962-2021) over 730 citations, and an h-index of 16. she worked in a specific scientific field of morphology, phytochemistry, and systematics of plants and extensively carried out anatomical, histochemical, and micromorphological analyses of plants. she placed special emphasis on the analysis of plant organs of representatives of the lamiaceae family. special focus of her research was glandular trichomes as the main secretory structures synthesizing diverse bioactive compounds. she was also involved in phytochemical and biological activity research on plants. at the same time, she worked in the field of mycology and collaborates with a group coordinated by professor jelena vukujević. professor solomon wasser. apart from the scientific research work that takes place at the faculty of biology, she continued work with colleagues from mycology and collaborated with many colleagues from our or other institutions. she was a member of several scientific associartions: serbian biological society and serbian society for microscopy; serbian chemical society; international sunflower association; optima; amapseec. also, she was a member of the editorial board of the international scientific journal botanica serbica. she served as a reviewer of many scientific journals and student books and was a commitee member for many teaching and scientific promotion. professor sonja with the members of mycology research group (december 2009, institute of botany and botanical garden „jevremovac” since the beginning of her career, she has been engaged in numerous national and international projects. as part of the international project “higher education reform in biological sciences”, tempus jep 40094 (2006-2009), dedicated to the reform of study programs at the faculty of biology, she stayed at the universities of turin and nice in december 2006. in 2004, she stayed at the university of haifa (israel) in cooperation with in addition, she was working on the popularization of botany and was co-author of botany for kids which is translated into spanish, chinese, and turkish languages, and after that as a longtime editor of textbook editions of the publishing house creative center. we will remember her as an enthusiastic scientist and teacher, a careful person who enjoyed in work with young scientists and students. we worked 85 biologica nyssana ● 13 (2) december 2022: 83-87 džamić & alimpić aradski ● in memoriam professor sonja duletić-laušević (1962-2021) with her, discussed, made and achieved goals. she suggested to the future generation that „the challenge is to be a botanist and know that you belong to the few who feel at home among plants”. this sentence represents sonja’s thinking and we will keep her in our memory for a long time. bibliography books and practicums vukojević, j., duletić-laušević, s. 2004: patogene gljive povrća i voća u srbiji. nnk international, beograd. isbn 86-83635-30-9. petković, b., merkulov, lj., duletić-laušević, s. 2005: anatomija biljaka sa praktikumom. nnk international, beograd. isbn 86-907471-1-7. petković, b., merkulov, lj., duletić-laušević, s. 2005: morfologija biljaka sa praktikumom. nnk international, beograd. isbn 86-907471-2-5. duletić-laušević, s., janošević, d. 2008: botanika, edicija saznaj i probaj, kreativni centar, belgrade. petković, b., duletić-laušević, s. (2001): praktikum iz anatomije i morfologije biljaka. nnk international, beograd. isbn 86-83635-066. (14) duletić-laušević, s., janaćković, p., džamić, a., veljić, m., grujić, s., rajčević, n. 2019: praktikum iz botanike. biološki fakultet univerziteta u beogradu, beograd. isbn: 978-86-7078-153-5. petković, b., merkulov, lj., duletić-laušević, s. 2012: anatomija i morfologija biljaka sa praktikumom. biološki fakultet univerziteta u beogradu. isbn 978-86-7078-084-2. duletić-laušević, s., janaćković, p., grujićjovanović, s., marin, m., džamić, a., rajčević, n., gradojević, j. (2009): vizuelni rečnik anatomije i morfologije biljaka. biološki fakultet univerziteta u beogradu. digitalni sadržaj. chapters in monographs vukojević, j., muntañola-cvetković, m., duletićlaušević, s. 2002: diaporthe/phomopsis helianthi, the sunflower pathogen: twenty years of research. in: environment and crop production (dris, r., jain, s. m., khan, i. a. (eds.), crc press, taylor & francis group. pp. 99-110. isbn: 9781578082575. professor sonja with the closest collaborators (september 2014, university of belgrade) 86 biologica nyssana ● 13 (2) december 2022: 83-87 džamić & alimpić aradski ● in memoriam professor sonja duletić-laušević (1962-2021) duletić-laušević, s, vukojević, j., soković, m. 2005: fungi as causative agents of diseases of vegetables and fruits. in: crops, growth, quality & biotechnology. dris, r. (ed.) wfl publisher science and technology. pp. 405-424. isbn: 95291-8601-0. vukojević, j., duletić-laušević, s., glamočlija, j. 2005: pathogenic fungi of vegetables and rosaceae fruits. in: crops, growth, quality & biotechnology. dris, r. (ed.) wfl publisher science and technology. pp. 425-448. isbn: 95291-8601-0. alimpić aradski, a., oalđe, m., duletić-laušević, s. 2021: salvia officinalis: traditional medicinal plant with novel therapeutical perspectives. in: salvia officinalis: production, cultivation and uses, cameron ashton (ed.). nova science publishers, new york, usa. pp. 1-72. isbn: 978-1-53619-531-6. selected papers marin, p., sajdl, v., kapor, s., tatić, b., petković, b., duletić, s. 1992: fatty acids of the stachyoideae. biochemical systematics and ecology, 20(4): 389392. marin, p.d., petković, b., duletić, s. 1994: nutlet sculpturing of selected teucrium species (lamiaceae): a character of taxonomic significance. plant systematics and evolution, 192: 199-214. marin, m., koko, v., duletić-laušević, s., marin, p.d., rančić, d., dajić stevanović z. 2006: glandular trichomes on the leaves of rosmarinus officinalis: morphology, stereology and histochemistry. south african journal of botany, 72: 378-382. stajić, m., persky, l., hadar, y., friesem, d., duletić-laušević, s., wasser, s., nevo, e. 2006: effect of copper and manganese ions on activities of laccase and peroxidases in three pleurotus species grown on agricultural wastes. applied biochemistry and biotechnology 128(1): 87-96. marin m., koko, v., duletić-laušević, s., marin, p.d. 2008: micromorphology of trichomes of thymus malyi (lamiaceae). journal of microscopy, 232(3): 406-409. stajić, m., vukojević, j., duletić-laušević, s. 2009: biology of pleurotus eryngii and role in biotechnological processes: a review. critical reviews in biotechnology, 29(1): 55-66. stajić, m., kukavica, b., vukojević, j. simonić, j., veljović-jovanović, s., duletić-laušević, s. 2010: wheat straw conversion by enzymatic system of ganoderma lucidum. bioresources 5(4): 23622373. ćilerdžić, j., stajić, m., vukojević, j., duletićlaušević, s., knežević a. 2011: potential of trametes hirsuta to produce ligninolytic enzymes during degradation of agricultural residues. bioresources, 6(3): 2885-2895. stajić, m., vukojević, j., knežević, a., duletićlaušević, s., milovanović, i. 2013: antioxidant protective effects of mushroom metabolites. current topics in medicinal chemistry, 13(21): 2660-2676. al sheef, n.b., duletić-laušević, s., janošević, d., budimir, s., marin, m., alimpić, a., giweli, a.a.m., marin, p.d. 2013: micromorphology аnd ultrastructure of trichomes of libyan salvia fruticosa mill. archives of biological sciences, 65(1): 239-248. alimpić, a., pljevljakušić, d., šavikin, k., knežević, a., ćurčić, m., veličković, d., stević, t., petrović, g., matevski, v., vukojević, j., marković, s., marin, p. d., duletić-laušević, s. 2015: composition and biological effects of salvia ringens (lamiaceae) essential oil and extracts. industrial crops and products. 76: 702-709. alimpić, a., knežević, a., milutinović, m., stević, t., šavikin, k., stajić, m., marković, s., marin, p.d., matevski, v., duletić-laušević, s. 2017: biological activities and chemical composition of salvia amplexicaulis lam. extracts. industrial crops and products. 105: 1-9. alimpić, a., knežević, a., šavikin, k., ćurčić, m., veličković, d., stević, t., matevski, v., stajić, m., marković, s., marin, p.d., duletić-laušević, s. 2017: composition and biological activities of different extracts of salvia jurisicii, a rare and endemic macedonian species. plant biosystems, 151(6): 1002-1011. živković, j. č., barreira, j. c. m., šavikin, k., alimpić, a., stojković, d., dias, m. i., santosbuelga, c., duletić-laušević, s., ferreira, i. c. f. r. 2017: chemical profiling and assessment of antineurodegenerative and antioxidant properties of veronica teucrium l. and veronica jacquinii baumg. chemistry and biodiversity, 14(8): 1-11. šavikin, k., živković j., alimpić, a., zdunić, g., janković, t., duletić-laušević, s., menković, n. 2018: activity guided fractionation of pomegranate extract and its antioxidant, antidiabetic and antineurodegenerative properties. industrial crops and products. 113: 142-149. ćilerdžić, j. l., sofrenić, i.v., tešević, v. v., biologica nyssana ● 13 (2) december 2022: 83-87 džamić & alimpić aradski ● in memoriam professor sonja duletić-laušević (1962-2021) brčeski, i. d., duletić-laušević, s. n., vukojević. j. b., stajić, m. m. 2018: neuroprotective potential and chemical profile of alternatively cultivated ganoderma lucidum basidiocarps. chemistry and biodiversity, 15(5): e1800036. duletić-laušević, s. alimpić aradski, a., šavikin, k., knežević, a. milutinović, m., stević, t., vukojević, j., marković, s., marin, p. d. 2018: composition and biological activities of libyan salvia fruticosa mill. and s. lanigera poir. extracts. south african journal of botany, 117: 101-109. savić, a., jarić, s., dajić stevanović, z., duletićlaušević, s. 2019: ethnobotanical survey and traditional use of local pear varieties (pyrus communis l.) in polimlje, southwest serbia. genetic resources and crop evolution, 66(3): 589-609. alimpić aradski, a., janošević, d., pećinar, i., budimir, s., dajić stevanović, z., matevski, v., marin, p.d., duletić-laušević, s. 2021: micromorphological and anatomical characteristics of salvia amplexicaulis lam., s. jurisicii košanin and s. ringens sibth. & sm. (lamiaceae). plant biosystems, 155(1): 92-108. oalđe, m., kolarević, s., živković, j., alimpić aradski, a., marić, j. j., kolarević, m. k., đorđević, j., marin, p. d., šavikin, k., vuković-gačić, b., duletić-laušević, s. 2021: a comprehensive assessment of the chemical composition, antioxidant, genoprotective and antigenotoxic activities of lamiaceae species using different experimental models in vitro. food and function, 12: 3233-3245. oalđe pavlović, m., kolarević, s., đorđević, j., jovanović marić, j., lunić, t., mandić, m., kračun kolarević, m., živković, j., alimpić aradski, a., marin, p. d., šavikin, k., vuković-gačić, b., božić nedeljković, b., duletić-laušević, s. 2021: a study of phytochemistry, genoprotective activity, and antitumor effects of extracts of the selected lamiaceae species. plants, 10(11): 2306. 87 tusevski et al., 2019, biologica nyssana 10(2) 10 (2) december 2019: 159-168 doi: 10.5281/zenodo.3600195 antioxidant activity and phenolic compounds in hypericum perforatum l. wildgrowing plants collected in the republic of macedonia original article oliver tusevski department of plant physiology, institute of biology, faculty of natural sciences and mathematics, university „ss. cyril and methodius“, arhimedova str. 3, 1000 skopje, macedonia oliver.tusevski@pmf.ukim.mk (corresponding author) marija todorovska department of plant physiology, institute of biology, faculty of natural sciences and mathematics, university „ss. cyril and methodius“, arhimedova str. 3, 1000 skopje, macedonia todorovskabt96@gmail.com mirko spasenoski department of plant physiology, institute of biology, faculty of natural sciences and mathematics, university „ss. cyril and methodius“, arhimedova str. 3, 1000 skopje, macedonia mirkoms@pmf.ukim.mk sonja gadžovska simić department of plant physiology, institute of biology, faculty of natural sciences and mathematics, university „ss. cyril and methodius“, arhimedova str. 3, 1000 skopje, macedonia sonjag@pmf.ukim.mk received: september 29, 2019 revised: december 11, 2019 accepted: december 17, 2019 abstract: the aim of this study was to evaluate phenolic compounds composition and antioxidant activity in roots (ro), non-flowering shoots (nfs) and flowering shoots (fs) of hypericum perforatum l. wild-growing plants collected in the republic of macedonia. the analyses of total phenolic compounds included quantification of phenolics, flavonoids, catechin derivatives, flavan-3-ols, condensed tannins and hypericin. antioxidant activity was determined by the cupric ions reducing antioxidant capacity, phosphomolybdenum test, reducing power and dpph scavenging. the contents of phenolics in fs and nfs were significantly higher than those found in ro extracts. hypericin content in fs was 2-fold increased in comparison to nfs, while ro showed low capability for the accumulation of naphthodianthrones. also, fs and nfs exhibited markedly higher antioxidant activities compared to ro. in summary, aerial parts of h. perforatum accumulated significant levels of antioxidant phenolic compounds that were characterized with hydrogen atom donation, radical scavenging and participation in redox reactions. key words: antioxidant activity, flowering shoots, hypericum perforatum l., non-flowering shoots, phenolic compounds, roots apstract: antioksidativna aktivnost i fenolna jedinjenja u divljerastućim biljkama hypericum perforatum l. sakupljenim u republici makedoniji cilj ove studije bio je da utvrdi sastav fenolnih jedinjenja i antioksidativnu aktivnost korena (ro), necvetajućih ((nfs) i cvetajućih izdanaka (fs) divljerastućih biljaka hypericum perforatum l., sakupljenih u republici makedoniji. analize totalnih fenolnih jedinjenja uključivale su kvantifikaciju fenola, flavonoida, derivata katehina, flavan-3-ola, kondenzovanih tanina i hipericina. antioksidativna aktivnost određivana je kapacitetom redukcije bakarnih jona, fosfomolibdenskim testom, redukcionim potencijalom i dpph testom. sadržaj fenola u fs i nfs bili su značajno viši nego onaj nađen u ekstraktima ro. sadržaj hipericina u fs bio je dvostruko veći u poređenju sa nfs, dok je ro pokazao nisku sposobnost akumulacije naftodiantrona. takođe, fs i fns su ispoljili značajno veću antioksidativnu aktivnost u poređenju sa ro. u zaključku, nadzemni delovi h. perforatum akumulirali su značajni nivo antioksidantnih fenolnih jedinjenja okarakterisanih dinacijom vodonikovih atoma, hvatanjem slobodnih radikala i učešćem u redoks reakcijama. ključne reči: antioksidativno delovanje, cvetajući izdanci, hipericum perforatum l., necvetajući izdanci, fenolna jedinjenja, korenovi introduction hypericum perforatum (st john’s wort) is an important medicinal plant with long and worldwide usage in traditional medicine. the crude drug (hyperici herba) consists of dried flowering tops or aerial parts of the wild-growing plants. the phytochemistry and pharmacology of this species have been extensively studied (greeson et al., 2001; nahrstedt & butterweck, 2010). phenolic compounds (naphthodianthrones, acyl-phloroglucinols and flavonoids) that contributed to the biological activities of h. perforatum are usually accumulated in the leaves and flowers. naphthodianthrones are chemotaxonomic marker compounds of the genus hypericum that is © 2019 tusevski et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 159 13th symposium on the flora of southeastern serbia and neighboring regions mainly localized in the dark glands dispersed over the margins of leaves and flower petals (zobayed et al., 2006). the naphthodianthrones presented by hypericin and pseudohypericin, as well as their protoforms (protohypericin and protopseudohypericin) are included in the analytical term “total hypericins” (williams et al., 2006). the cytotoxic and photodynamic properties of hypericin are responsible for antiviral, antitumor and antibacterial effects of h. perforatum extracts (delaey et al., 2001). two closely related acyl-phloroglucinol derivatives, hyperforin and its homolog adhyperforin have been detected in the translucent glands of the leaves, flowers and fruits of h. perforatum (soelberg et al., 2007). it has been considered that hyperofrin is the major constituent responsible for the antidepressant activity of h. perforatum extracts through the inhibition of synaptic re-uptake of neurotransmitters (chatterjee et al., 1998). the h. perforatum is widely known for its richness in flavonoids especially by the flavonols quercetin and kaempferol, as well by the catechin derivatives represented with monomeric flavan-3ols and oligomeric proanthocyanidins (nahrstedt & butterweck, 2010). flavonoids are mainly presented in the leaves as water-soluble glycosides in the vacuoles of epidermal cells due to their uv-protective function (germ et al., 2010). the flavonoid-containing fractions of h. perforatum have been shown to exhibit powerful antioxidant activity and radical scavenging capacity (barnes et al., 2001). even that flowers from h. perforatum wildgrowing plants have been the subject of many research studies concerning the production of total phenolics, flavonoids and hypericins (germ et al., 2010; altun et al., 2013; becker et al., 2016), the biosynthesis of various groups of phenolic compounds in different plant parts are not yet fully understood. recent phytochemical investigations revealed that roots from h. perforatum wild-growing plants represented a promising source of phenolics with potential biological activities (crockett et al., 2011; tusevski et al., 2018). however, the roots of h. perforatum wild-growing plants have never been evaluated for their capacity for the production of total phenolic compounds and antioxidant activities. therefore, it would be of great interest to evaluate the distribution of certain groups of phenolic compounds and antioxidant properties in various plant organs of h. perforatum. the main objective of this study was to determine the contents of six groups of phenolic compounds and antioxidant capacity in h. perforatum roots (ro), non-flowering shoots (nfs) and flowering shoots (fs). for the realization of this objective, the following topics were included: (1) quantification of total phenolics, flavonoids, catechin derivatives, flavan-3-ols, condensed tannins and hypericin, (2) evaluation of antioxidant activity by cupric ion reducing antioxidant capacity, reducing power, phosphomolybdenum assay and dpph radical scavenging and (3) determination of relationship between phenolic compounds contents and antioxidant activities. material and methods phenolic compound contents wild-growing plants of hypericum perforatum (voucher number 060231) collected in the national park pelister (1394 m a.s.l.) were separated into three plant sections: roots (ro), non-flowering shoots (nfs) and flowering shoots (fs). phenolic compounds were extracted from powdered plant material (0.2 g) with 80% (v/v) ch3oh in ultrasonic bath for 30 min at 4 ºc. thereafter, methanolic extracts were centrifuged at 12.000 rpm for 15 min, and the supernatants were used for the determination of phenolic compound contents. the phenolic compound contents in plant extracts included determination of total phenolics (tp), flavonoids (tf), catechin derivatives (tcd), flavan3-ols (tfa), condensed tannins (tct) and hypericin (th). spectrophotometric analyses were performed on spectramax 190 microplate reader (molecular devices corp., sunnyvale, ca) supported with softmax pro (v. 5.4.1) software. total phenolics (tp). the tp contents were determined when plant extracts were mixed with folin–ciocalteau reagent and 0.7 m na2co3 (singleton & rossi, 1965). the samples were incubated at 50 ºc for 15 min and then cooled at room temperature. then, absorbance was measured spectrophotometrically at 765 nm. the results for tp concentration were expressed as milligrams of gallic acid equivalents (ga) per gram of dry weight (mg ga·g-1 dw). total flavonoids (tf). the tf contents in plant extracts were determined using the assay described by zhishen et al. (1999). an aliquot of the extract was mixed with 5% nano2 and allowed to react for 5 min. following this, 10% alcl3 was added and the mixture stood for further 5 min. then, 1 m naoh and distilled water were added to the sample and the absorbance was measured spectrophotometrically at 510 nm. the concentration of tf was expressed as milligrams of catechin equivalents (c) per gram of dry weight (mg c·g-1 dw). total catechin derivatives (tcd). the tcd contents in plant extracts were determined by the vanillin method (sun et al., 1998). a diluted plant extract was mixed with 1% vanillin-hcl solution. the samples were incubated at room temperature 160 biologica nyssana ● 10 (2) december 2019: 159-168 tusevski et al. ● antioxidant activity and phenolic compounds in hypericum perforatum l. wild-growing plants collected in the republic of macedonia) room temperature and the absorbance was recorded at 450 nm. the molar extinction coefficient of trolox (ε535=1.67x10 4 l·mol-1·cm-1) was used for the determination of cuprac. the cuprac values were expressed as micromoles of trolox equivalents (t) per gram of dry weight (μmol t·g-1 dw). reducing power (rp). the rp of plant extracts was determined according to the method of oyaizu (1986), with the following modifications. the reaction mixture contained plant extract, phosphate buffer (0.2 m kh2po4/k2hpo4, ph 6.6) and 1% k3[fe(cn)6]. after incubation at 50 ºc for 20 min, an aliquot of 10% tca was added to the mixture followed by centrifugation at 1000 rpm for 10 min. finally, the collected supernatant was mixed with ch3oh and 0.1% fecl3 and incubated at room temperature for 10 min. the absorbance of the samples was measured at 700 nm. the rp was expressed as micromoles of ascorbic acid equivalents (aa) per gram of dry weight (μm aa·g-1 dw). phosphomolybdenum (pm) assay. the pm assay was evaluated using the procedure of prieto et al. (1999). the plant extract was mixed with pm reagent [0.6 m h2so4, 28 mm na3po4 and 4 mm (nh4)2moo4] and the samples were incubated at 95°c for 90 min. after cooling at room temperature, the absorbance of the green pm complex was measured at 695 nm. the pm capacity was expressed as milligrams of ascorbic acid equivalents (aa) per gram of dry weight (mg aa·g-1 dw). dpph radical scavenging activity. the ability of plant extracts to scavenge stable free radical 2,2-diphenyl-1-picrylhydrazyl (dpph•) was determined with the method of brand-williams et al. (1995). the reaction mixture consisted of plant extract and 0.25 mm dpph in ch3oh. in the control sample, the extract was replaced with ch3oh. the reaction for dpph• scavenging was carried out at room temperature in the dark for 10 min, and then decrease in absorbance was recorded at 518 nm. the dpph radical scavenging activity was expressed as micromoles of trolox equivalents (t) per gram of dry weight (μm t·g-1 dw). results phenolic compound contents and antioxidant activities in h. perforatum wild-growing plants the contents of total phenolic compounds in h. perforatum extracts (ro, nfs and fs) are summarized in tab. 1. the tp contents in fs and nfs were about 2.9-fold higher than that of ro. the amounts of tf were significantly higher in nfs and fs (2.7and 2.4-fold, respectively), compared to ro extracts. also, fs and nfs showed higher tcd contents (2.7and 2.3-fold, respectively) in comparison to ro sample. similar amounts of for 30 min and the absorbance was measured spectrophotometrically at 500 nm. the concentration of tcd was expressed as milligrams of catechin equivalents (c) per gram of dry weight (mg c·g-1 dw). total flavan-3-ols (tfa). the tfa in plant extracts were estimated by dmaca (4-dimethylaminocinnamaldehyde) assay (arnous et al., 2002). the 0.1% dmaca reagent in ch3oh/ hcl (91.4/8.6, v/v) was added to the extracts. samples were incubated for 10 min at room temperature and the absorbance was measured at 640 nm. the tfa concentration was expressed as milligrams of catechin equivalents (c) per gram of dry weight (mg c·g-1 dw). total condensed tannins (tct). the tct contents in plant extracts were determined by the method of porter et al. (1985) with the following modifications. the samples were prepared by mixing the diluted plant extracts with n-butanol/hcl (95:5, v/v) and 2% nh4fe(so4)2 x 12h2o. the mixture was incubated at 95°c for 50 min and the absorbance was measured at 535 nm. the molar extinction coefficient of cyanidin-3-glucoside (ε535=34700 l·mol -1·cm-1) was used for determination of tct in the extracts. the results were expressed as milligrams of cyanidin-3glucoside equivalents (cg) per gram of dry weight (mg cg·g-1 dw). total hypericin (th). the th contents in plant samples were determined according to european pharmacopoeia (2008). the powdered plant sample was extracted with 80% tetrahydrofuran at 65 ºc for 30 min. samples were centrifuged at 3000 rpm for 10 min and the collected supernatant was lyophilized under vacuum (0.22 mbar). the dry residue was dissolved with ch3oh in an ultrasonic bath and then centrifuged at 12.000 rpm for 10 min. the absorbance of the supernatant was measured at 590 nm. the concentration of th was expressed as milligrams of hypericin equivalents (h) per gram of dry weight (mg h·g-1 dw). antioxidant capacity assays the antioxidant capacity of plant extracts was determined by the following methods: cupric ion reducing antioxidant capacity (cuprac), reducing power (rp), phosphomolybdenum assay (pm) and dpph radical scavenging activity. those methods were performed in the same extracts used for the determination of phenolic compound contents. cupric ion reducing antioxidant capacity (cuprac). the cuprac assay of plant extracts was determined by the method of apak et al. (2004). the reaction mixture consisted of plant extract, 10 mm cucl2, 7.5 mm neocuproine and 1 m ch3coonh4 buffer (ph 7.0). samples were incubated 30 min at 161 biologica nyssana ● 10 (2) december 2019: 159-168 tusevski et al. ● antioxidant activity and phenolic compounds in hypericum perforatum l. wild-growing plants collected in the republic of macedonia) tfa and tct were observed between nfs and fs, (about 2.5-fold higher) compared to ro extracts. the th concentration in fs was about 2-fold higher than that found in nfs extracts, while ro showed significantly lower capability for the accumulation of hypericin derivatives. the antioxidant activities in h. perforatum ro, nfs and fs extracts are presented in tab. 2. the results for cuprac assay showed that fs and nfs extracts had significantly higher antioxidant activities (3-fold) compared to ro. similar results were observed for rp, where nfs and fs had markedly higher values for antioxidant activity (4.4and 3.8-fold, respectively) compared to ro extracts. the antioxidant activity measured by pm assay was significantly higher in nfs and fs (2.2 and 1.9-fold, respectively) than ro extracts. also, fs and nfs showed markedly higher dpph radical scavenging activity (3.1and 2.6-fold, respectively) in comparison to ro extracts. principal component analysis of phenolic compounds production and antioxidant activities in h. perforatum wild-growing plants principal component analysis (pca) was performed to define the correlation between total 162 phenolic contents and antioxidant activities among different parts of h. perforatum wild-growing plants. in order to find out if any ordination of the analyzed ro, nfs and fs extracts exists, a pca (fig. 1) was applied using the following variables: total contents of phenolics (tp, tf, tcd, tfa, tct, th) and antioxidant activities (cuprac, rp, pm, dpph). two main principal components (pc) were used to characterize phenolic compound composition and antioxidant activities in ro, nfs and fs extracts. the pca data showed that the pc1 and pc2 explained 100% of the total variation. the pca plot (fig. 1) indicated that pc1 explained the variance of 96.19% that was positively related to all variables concerning the phenolic compound contents and antioxidant activities. the pc2 showed a variance of 3.81% and it was not connected to the tested parameters. the pca plot showed that tested h. perforatum samples are well separated on pc1 (fig. 1). in this context, fs and nfs extracts exhibited positive scores on pc1 and they were characterized with high contents of phenolic compounds and antioxidant activities. on the other hand, ro extracts had a negative score on pc1 and possessed a low capacity for phenolic compound production and antioxidant table 1. the content of total phenolic compounds in h. perforatum wild-growing plants* phenolic compounds ro nfs fs tp (mg ga·g-1 dw) 36.51±1.02 a 103.88±4.97 b 107.38±0.90 b tf (mg c·g-1 dw) 28.14±1.56 a 76.70±7.66 b 68.59±0.26 b tcd (mg c·g-1 dw) 15.97±0.46 a 36.42±4.58 b 42.52±1.10 b tfa (mg c·g-1 dw) 9.98±0.78 a 22.78±2.09 b 24.82±0.45 b tct (mg cg·g-1 dw) 6.52±0.51 a 15.58±3.03 b 17.60±0.57 b th (μg h·g-1 dw) 7.00±0.25 a 581.86±14.45 b 1150.28±7.86 c * ro: roots; nfs: non-flowering shoots; fs: flowering shoots; tp: phenolics; tf: flavonoids; tcd: catechin derivatives; tfa: flavan-3-ols; tct: condensed tannins; th: hypericin; ga: gallic acid; c: catechin; cg: cyanidin-3-glucoside; h: hypericin; dw: dry weight. a the values in one row marked with lower-cases denoted significant differences between samples at p<0.05 (duncan's multiple range test). table 2. the antioxidant activities in hypericum perforatum wild-growing plants* antioxidant activities ro nfs fs cuprac (μм т·g-1 dw) 230.11±19.24 a 685.60±17.33 b 708.30±12.58 b rp (μм aa·g-1 dw) 425.05±13.91 a 1865.21±78.81 b 1584.39±163.80 b pm (mg aa·g-1 dw) 57.89±3.55 a 108.96±4.99 b 128.46±0.51 c dpph (μм т·g-1 dw) 158.81±0.10 a 407.86±2.36 b 496.67±11.67 c * ro: roots; nfs: non-flowering shoots; fs: flowering shoots; cuprac: cupric ions reducing antioxidant capacity; rp: reducing power; pm: phosphomolybdenum test; dpph: dpph radical scavenging activity; t: trolox; aa: ascorbic acid; dw: dry weight. a the values in one row marked with lower-cases denoted significant differences between samples at p<0.05 (duncan's multiple range test). biologica nyssana ● 10 (2) december 2019: 159-168 tusevski et al. ● antioxidant activity and phenolic compounds in hypericum perforatum l. wild-growing plants collected in the republic of macedonia) activities. in addition, fs and nfs extracts were located on the opposite side on pc2 and the major parameters responsible for the separation of these two aerial plant samples were th content with positive score on pc2, as well tf content and antioxidant activity measured by rp assay with negative scores on pc2. according to these results, fs extracts showed significantly higher th contents, but lower tf amount and rp value in comparison to nfs extracts. pearson’s correlation matrix (tab. 3) demonstrated significant positive correlations between phenolic compound contents and antioxidant activities in tested samples. in this view, the tp contents were in significant positive correlation with cuprac assay. a significant positive correlation was also found between tf and rp methods. similarly, tcd amounts were positively related to the antioxidant activity measured by pm and dpph assays. these results clearly indicated that phenolic compounds greatly contributed to the observed antioxidant activities of plant extracts. discussion production of phenolic compounds in h. perforatum wild-growing plants present data showed that aerial plant parts of h. perforatum were an excellent source of phenolics and flavonoids, while root tissues showed lower capability for accumulation of these phenolic compounds. outgoing results for total phenolics and flavonoids are in accordance with those previously reported for aerial parts of h. peforatum and other hypericum species (germ et al., 2010; hernandez et al., 2010). for instance, gioti et al. (2009) have noticed that shoots, non-flower and flower branches of h. peforatum possess significant amounts of phenolic compounds. taking into account literature data, kalogeropoulos et al. (2010) reported lower amounts of total phenolics and flavonoids for h. peforatum plants collected in greece in comparison to the results presented here. on the other hand, altun et al. (2013) observed higher values of these compounds for hyperici herba from turkey. the variation of phenolic and flavonoid contents in h. fig. 7. canonical discriminant analysis of the six g. pratensis populations from serbia and montenegro table 3. correlation analysis between phenolic compounds production and antioxidant activities in hypericum perforatum wild-growing plants (n = 3 samples)* r tp tf th tcd tfa tct cuprac rp pm tf 0.980 th 0.889 0.779 tcd 0.984 0.929 0.956 tfa 0.996 0.960 0.924 0.996 tct 0.992 0.946 0.940 0.999 0.999 0.999 0.980 0.888 0.984 0.996 0.992 rp 0.974 0.999 0.761 0.919 0.952 0.937 0.974 pm 0.974 0.910 0.969 0.998 0.990 0.995 0.974 0.898 dpph 0.978 0.916 0.965 0.999 0.992 0.996 0.977 0.904 0.999 * r: pearson’s coefficient; tp: phenolics; tf: flavonoids; tcd: catechin derivatives; tfa: flavan-3-ols; tct: condensed tannins; th: hypericin; cuprac: cupric ions reducing antioxidant capacity; rp: reducing power; pm: phosphomolybdenum test; dpph: dpph radical scavenging activity. the values in bold indicate significance at p < 0.05. 163 biologica nyssana ● 10 (2) december 2019: 159-168 tusevski et al. ● antioxidant activity and phenolic compounds in hypericum perforatum l. wild-growing plants collected in the republic of macedonia) peforatum could be attributed to the genotypic and environmental factors, various plant organs, as well harvesting time (gioti et al., 2009; germ et al., 2010). however, the direct comparison of phenolic and flavonoid contents is a complex task due to the differences in solvents and procedures for plant extraction, the usage of reference compounds for quantification and the methods for determination of phenolic compounds. even that some non-phenolics (ascorbic acid, amino acids, proteins, organic acids and sugars) could react with folin-ciocalteu reagent (prior et al. 2005), the procedure for h. peforatum plant extraction used in this study eliminated the possibility for interference of these compounds. taking into account the richness of h. perforatum extracts with monomeric flavan-3-ols, as well as oligomeric and polymeric tannins (nahrstedt & butterweck, 2010), the different assays were applied for determination of catechin derivatives (tcd, tfa and tct) in plant samples. present results clearly demonstrated that aerial parts of h. perforatum have greater capability for accumulation of those catechin derivatives compared to root samples. the literature data for total contents of flavan-3-ols or tannins in h. perforatum are rather scarce. in this view, germ et al. (2010) have shown higher values for catechin derivatives in h. perforatum leaves and flowers than those reported here. the heterogeneity in the results for total catechin derivatives may be due to the high sensitivity of vanillin-hcl assay, as well the solvents, reaction time, temperature and vanillin concentrations used for analysis (sun et al., 1998). the ratio tcd/tct could be used as a rough estimation of the polymerization degree of monomeric flavan-3-ols into oligomeric and polymeric tannins (ribéreau-gayón & stonestreet, 1966). in this study, the ratio tcd/tct in analyzed samples was from 2 to 3 indicating that polymeric tannins in h. perforatum are probably represented by proanthocyanidin dimers and trimers. moreover, the summation of total flavan-3-ol and condensed tannin contents accurately represented the amounts of total catechin derivatives in the analyzed samples. therefore, vanillin-hcl assay for total catechin derivatives could be proposed as a routine procedure for determination of total catechin derivatives including flavan-3-ol monomers and polymeric condensed tannins in h. perforatum. the measurement of total hypericin is commonly used for quality control of h. perforatum standardized extracts and phytopharmaceuticals (williams et al., 2006). the results for total hypericin contents showed that flowering shoots accumulated markedly higher contents of hypericin compared to non-flowering shoots, while minor amounts were noticed for roots. in this view, naphthodianthrone accumulation has been connected with the growth and development of h. perforatum reproductive parts (southwell & bourke, 2001). in addition, the correlation between dark glands formation and hypericin levels in leaves, stems, flower petals, stamens, carpels and sepals of h. perforatum has been reported (zobayed et al., 2006). therefore, the dark glands could be considered as the limiting factors for hypericin accumulation due to necessity of these glandular structures for deposition of hypericin (pasqua et al., 2003). even that aerial parts of h. perforatum in full bloom have been proposed as excellent sources of hypericin (southwell & bourke, 2001), the roots have never been explored as a hypericin-producing tissues. however, recent histochemical analysis showed the presence of darkred globules with hypericin in h. perforatum leaves, stems and roots (qian et al., 2012). these findings suggested that dark glands are not the only site of naphthodianthrone accumulation and hypericin is probably synthesized either in mesophyll, root or stem cells and subsequently is transported to the dark glands. all these observations suggested that aerial parts of h. perforatum accumulated higher contents of phenolic compounds (flavonoids, catechin derivatives and hypericin) compared to roots. antioxidant activity of h. perforatum wild-growing plants the cuprac assay has been widely used to determine the antioxidant capacity of plant extracts as it requires relatively standard equipment and delivers fast and reproducible results (apak et al., 2004). the cuprac assay is useful for determination of antioxidant capacity in a wide variety of antioxidant compounds, such as phenolic acids, flavonoids, carotenoids, anthocyanins, as well for thiols (glutathione), synthetic antioxidants, vitamins c and e. results from this study demonstrated very strong cuprac activity for the aerial parts, while root extracts possessed the lowest cuprac value. the relevance of cuprac method for evaluation of antioxidant potential of aerial parts from h. perforatum and other hypericum species has also been reported (maltas et al., 2013; boga et al., 2016). the results obtained from cuprac in vitro measurements could be efficiently extended to the in vivo antioxidant activity because the cuprac reaction is carried out at physiological ph (apak et al., 2004). the non-flowering and flowering shoots of h. perforatum demonstrated high antioxidant activity measured by rp assay, thereby could be proposed as efficient reductones. the reductones terminated free radical chain reactions and thus, rp could be related to the antioxidant activity of plant extracts 164 biologica nyssana ● 10 (2) december 2019: 159-168 tusevski et al. ● antioxidant activity and phenolic compounds in hypericum perforatum l. wild-growing plants collected in the republic of macedonia) (gordon, 1990). phenolic compounds as effective reductones have previously been confirmed for h. perforatum (zou et al., 2004; gioti et al., 2009), as well as other hypericum species (şerbetçi et al., 2012; rainha et al., 2013). in this study, aerial parts showed high rp values, which indicated that h. perforatum is characterized by abundance of antioxidant compounds that reduce fe3+/ferricyanide complex into ferrous form. present results showed that flowering shoots exhibited the highest value for pm assay, followed by non-flowering shoots, while the lowest antioxidant capacity was found for root extracts. these results for aerial parts were consistent with those previously reported for h. perforatum flower, leaf and stem extracts (radulović et al., 2007). in contrast, raghu chandrashekhar et al. (2009) showed that roots have greater antioxidant capacity determined by pm method than those found for aerial parts of h. hookerianum wild-growing plants. the discrepancy in the results for pm assay among different plant organs could be related to the interspecies variability concerning the distribution of antioxidant compounds. despite polyphenols, many other non-phenolic hydrophilic and lipophilic compounds could significantly contribute to the antioxidant activity of plant extracts measured by pm assay (prieto et al., 1999). in this study, dpph method was selected as the most effective assay for evaluation of radical scavenging capacity of plant extracts by chainbreaking mechanism (tusevski et al., 2014). present results demonstrated that flowering and non-flowering shoots were better dpph scavengers compared to root extracts. accordingly, there are many studies that confirm the strong dpph radical scavenging activity of h. perforatum extracts (silva et al., 2008; gioti et al., 2009; kalogeropoulos et al., 2010; becker et al., 2016). taken together, the variation in the results for antioxidant activities of h. perforatum extracts could be due to the structural features, antioxidant mechanism, as well as synergistic effects of different classes of phenolic compounds. it is widely accepted that the antioxidant activity of h. perforatum extracts is correlated to the content of phenolic compounds. although present results were focused on this topic, there is still a lack of evidence to determine the contribution of various classes of phenolic compounds to the antioxidant activity of h. perforatum. since the methods used in this study enabled the quantification of various groups of phenolic compounds, it was possible to establish the correlation between those metabolites and antioxidant activity in h. perforatum. correlation between phenolic compound contents and antioxidant activity in h. perforatum wild-growing plants the establishment of a relationship between antioxidant activity and phenolic contents is a very difficult approach because plant extracts represented complex mixtures of various metabolites with antioxidant and prooxidant properties that exhibit synergistic actions. in this study, the principal component analysis (pca) as a statistical tool showed that flowering shoot and non-flowering shoot extracts represented the richest source of phenolics with strong antioxidant activity. on the other side, roots were characterized by low capacity for phenolic compound production and antioxidant activity. the correlation analyses performed in this study demonstrated significant positive correlation between tp and cuprac. these results suggested that phenolics in h. perforatum wild-growing plants could be proposed as preeminent constituents that exhibit cupric ion reducing capacities. in this view, antioxidant activity of h. perforatum and other hypericum species measured with cuprac assay has been well correlated with total phenolic and flavonoid contents (maltas et al., 2013; moein et al., 2015). moreover, results presented here showed a significant correlation between tf and rp in h. perforatum extracts indicating that high reducing power could be attributed to the total flavonoid contents. such a correlation between total flavonoids and reducing power of h. perforatum extracts has also been reported by zou et al. (2004). the strong positive correlations of tcd with dpph and pm assays found in this study were expected since monomeric flavan-3-ols and polymeric proanthocyanidins have already been shown as the most active dpph scavengers (froehlicher et al., 2009). in addition, raghu chandrashekhar et al. (2009) confirmed that total flavan-3-ol contents in h. hookerianum correlated with total antioxidant capacity measured by pm assay. outgoing results showed a significant positive correlation between pm and dpph indicating that these assays could be proposed as suitable and reliable methods for evaluation of antioxidant activity of h. perforatum extracts. the th contents did not show any significant correlation with antioxidant methods used in this study. these data could be ascribed to the small percentage distribution of hypericin (up to 1%) in phenolic profile that resulted in their minor contribution to the antioxidant activity of h. perforatum extracts. 165 biologica nyssana ● 10 (2) december 2019: 159-168 tusevski et al. ● antioxidant activity and phenolic compounds in hypericum perforatum l. wild-growing plants collected in the republic of macedonia) conclusion in this study, the capability for the production of phenolic compounds and antioxidant activities of roots, non-flowering shoots and flowering shoots of h. perforatum wild-growing plants was presented as detailed for the first time. the aerial plant samples represented the richest source of phenolics, flavonoids, catechins (flavan-3-ols and condensed tannins) and hypericin with strong antioxidant activities. noteworthy, root extracts from h. perforatum demonstrated satisfactory amounts of various groups of phenolics with moderate antioxidant activities that make them potential candidates as a natural source of antioxidant and radical scavenging compounds. the correlation analysis used in this study indicated that antioxidant activity of h. perforatum wild-growing plants is related to the phenolic compounds that are characterized by hydrogen atom donation, radical scavenging and participation in redox reactions. the phytochemical profiling and initial screening of antioxidant activity of h. perforatum extracts could be a step forward in the preparation of new phytoproducts in food and pharmaceutical industry. references altun, m.l., yılmaz, b.s., orhan, i.e., citoglu, g.s. 2013: assessment of cholinesterase and tyrosinase inhibitory and antioxidant effects of hypericum perforatum l. (st. john’s wort). industrial crops and products, 43: 87-92. apak, r., güçlü, k., özyürek, m., karademir, s.e. 2004: novel total antioxidant capacity index for dietary polyphenols and vitamins c and e, using their cupric ion reducing capability in the presence of neocuproine: cuprac method. journal of agricultural and food chemistry, 52(26): 79707981. arnous, a., makris, d.p., kefalas, p. 2002: correlation of pigment and flavanol content with antioxidant properties in selected aged regional wines from greece. journal of food composition and analysis, 15(6): 655-665. barnes, j., anderson, l.a., phillipson, j.d. 2001: st john’s wort (hypericum perforatum l.): a review of its chemistry, pharmacology and clinical properties. journal of pharmacy and pharmacology, 53(5): 583-600. becker, l., zaiter, a., petit, j., zimmer, d., karam, m.c., baudelaire, e., scher, j., dicko, a. 2016: improvement of antioxidant activity and polyphenol content of hypericum perforatum and achillea millefolium powders using successive grinding and sieving. industrial crops and products, 87: 116-123. boga, m., ertas, a., eroglu-ozkan, e., kizil, m., ceken, b., topcu, g. 2016: phytochemical analysis, antioxidant, antimicrobial, anticholinesterase and dna protective effects of hypericum capitatum var. capitatum extracts. south african journal of botany, 104: 249-257. brand-williams, w., cuvelier, m.e., berset, c.l.w.t. 1995: use of a free radical method to evaluate antioxidant activity. lwt-food science and technology, 28(1): 25-30. chatterjee, s.s., bhattacharya, s.k., wonnemann, m., singer, a., müller, w.e. 1998: hyperforin as a possible antidepressant component of hypericum extracts. life sciences, 63(6): 499-510. crockett, s.l., poller, b., tabanca, n., pferschy‐wenzig, e.m., kunert, o., wedge, d.e., bucar, f. 2011: bioactive xanthones from the roots of hypericum perforatum (common st john’s wort). journal of the science of food and agriculture, 91(3): 428-434. delaey, e.m., obermueller, r., zupko, i., de vos, d., falk, h., de witte, p.a.m. 2001: in vitro study of the photocytotoxicity of some hypericin analogs on different cell lines. photochemistry and photobiology, 74(2): 164-171. european pharmacopoeia 2008: st. john’s wort, in european pharmacopoeia (6th ed.). strasbourg, france: council of europe, 2958–2959. froehlicher, t., hennebelle, t., martin-nizard, f., cleenewerck, p., hilbert, j.l., trotin, f., grec, s. 2009: phenolic profiles and antioxidative effects of hawthorn cell suspensions, fresh fruits, and medicinal dried parts. food chemistry, 115(3): 897-903. germ, m., stibilj, v., kreft, s., gaberščik, a., kreft, i. 2010: flavonoid, tannin and hypericin concentrations in the leaves of st. john’s wort (hypericum perforatum l.) are affected by uv-b radiation levels. food chemistry, 122(3): 471-474. gioti, e.m., fiamegos, y.c., skalkos, d.c., stalikas, c.d. 2009: antioxidant activity and bioactive components of the aerial parts of hypericum perforatum l. from epirus, greece. food chemistry, 117(3): 398-404. gordon, m.h. 1990: the mechanism of antioxidant action in vitro. in: food antioxidants (pp. 1-18). springer netherlands. greeson, j.m., sanford, b., monti, d.a. 2001: st. john’s wort (hypericum perforatum): a review of the current pharmacological, toxicological, and clinical 166 biologica nyssana ● 10 (2) december 2019: 159-168 tusevski et al. ● antioxidant activity and phenolic compounds in hypericum perforatum l. wild-growing plants collected in the republic of macedonia) literature. psychopharmacology, 153(4): 402-414. hernandez, m.f., falé, p.l., araújo, m.e.m., serralheiro, m.l.m. 2010: acetylcholinesterase inhibition and antioxidant activity of the water extracts of several hypericum species. food chemistry, 120(4): 1076-1082. kalogeropoulos, n., yannakopoulou, k., gioxari, a., chiou, a., makris, d.p. 2010: polyphenol characterization and encapsulation in β-cyclodextrin of a flavonoid-rich hypericum perforatum (st john’s wort) extract. lwt-food science and technology, 43(6): 882-889. maltas, e., uysal, a., yildiztugay, e., aladag, m.o., yildiz, s., kucukoduk, m. 2013: investigation of antioxidant and antibacterial activities of some hypericum species. fresenius environmental bulletin, 22: 862-869. moein, m., farmani, f. 2015: free radical scavenging activity and lipid peroxidation inhibition of hypericum helianthemoides (spach) boiss. trends in pharmaceutical sciences, 1(2): 111-114. nahrstedt, a., butterweck, v. 2010: lessons learned from herbal medicinal products: the example of st. john’s wort. journal of natural products, 73(5): 1015-1021. oyaizu, m. 1986: studies on products of browning reaction--antioxidative activities of products of browning reaction prepared from glucosamine. the japanese journal of nutrition and dietetics, 44(6): 307-315. pasqua, g., avato, p., monacelli, b., santamaria, a.r., argentieri, m.p. 2003: metabolites in cell suspension cultures, calli, and in vitro regenerated organs of hypericum perforatum cv. topas. plant science, 165(5): 977-982. porter, l.j., hrstich, l.n., chan, b.g. 1985: the conversion of procyanidins and prodelphinidins to cyanidin and delphinidin. phytochemistry, 25(1): 223-230. prieto, p., pineda, m., aguilar, m. 1999: spectrophotometric quantitation of antioxidant capacity through the formation of a phosphomolybdenum complex: specific application to the determination of vitamin e. analytical biochemistry, 269(2): 337-341. prior, r.l., wu, x., schaich, k. 2005: standardized methods for the determination of antioxidant capacity and phenolics in foods and dietary supplements. journal of agricultural and food chemistry, 53(10): 4290-4302. qian, j., wu, j., yao, b., lu, y. 2012: preparation of a polyclonal antibody against hypericin synthase and localization of the enzyme in red-pigmented hypericum perforatum l. plantlets. acta biochimica polonica, 59: 639-64. radulović, n., stankov-jovanović, v., stojanović, g., šmelcerović, a., spiteller, m., asakawa, y. 2007: screening of in vitro antimicrobial and antioxidant activity of nine hypericum species from the balkans. food chemistry, 103(1): 15-21. raghu chandrashekhar, h., venkatesh, p., ponnusankar, s., vijayan, p. 2009: antioxidant activity of hypericum hookerianum wight and arn. natural product research, 23(13): 1240-1251. rainha, n., koci, k., coelho, a.v., lima, e., baptista, j., fernandes-ferreira, m. 2013: hplc-uv-esi-ms analysis of phenolic compounds and antioxidant properties of hypericum undulatum shoot cultures and wild-growing plants. phytochemistry, 86: 83-91. ribéreau-gayón, p., stonestreet, e. 1966: dógase des tannins du vin rouges et détermination du leur structure. chemia analityczna, 48: 188-196. şerbetçi, t., özsoy, n., demirci, b., can, a., kültür, ş., başer, k. c. 2012: chemical composition of the essential oil and antioxidant activity of methanolic extracts from fruits and flowers of hypericum lydium boiss. industrial crops and products, 36(1): 599-606. silva, b.a., malva, j.o., dias, a.c. 2008: st. john’s wort (hypericum perforatum) extracts and isolated phenolic compounds are effective antioxidants in several in vitro models of oxidative stress. food chemistry, 110(3): 611-619. singleton, v.l., rossi, j.a. 1965: colorimetry of total phenolics with phosphomolybdicphosphotungstic acid reagents. american journal of enology and viticulture, 16(3): 144-158. soelberg, j., jørgensen, l.b., jäger, a.k. 2007: hyperforin accumulates in the translucent glands of hypericum perforatum. annals of botany, 99(6): 1097-1100. southwell, i.a., bourke, c.a. 2001: seasonal variation in hypericin content of hypericum perforatum l.(st. john’s wort). phytochemistry, 56(5): 437-441. sun, b., ricardo-da-silva, j.m., spranger, i. 1998: critical factors of vanillin assay for catechins and proanthocyanidins. journal of agricultural and food chemistry, 46(10): 4267-4274. tusevski, o., kostovska, a., iloska, a., trajkovska, l., gadzovska simic, s. 2014: phenolic production and antioxidant properties of some macedonian 167 biologica nyssana ● 10 (2) december 2019: 159-168 tusevski et al. ● antioxidant activity and phenolic compounds in hypericum perforatum l. wild-growing plants collected in the republic of macedonia) medicinal plants. open life sciences, 9(9): 888-900. tusevski, o., krstikj, m., stanoeva, j. p., stefova, m., gadzovska simic, s. 2018: phenolic profile and biological activity of hypericum perforatum l.: can roots be considered as a new source of natural compounds?. south african journal of botany, 117: 301-310. williams, f.b., sander, l.c., wise, s.a., girard, j. 2006: development and evaluation of methods for determination of naphthodianthrones and flavonoids in st. john’s wort. journal of chromatography a, 1115(1): 93-102. 168 biologica nyssana ● 10 (2) december 2019: 159-168 tusevski et al. ● antioxidant activity and phenolic compounds in hypericum perforatum l. wild-growing plants collected in the republic of macedonia) zhishen, j., mengcheng, t., jianming, w. 1999: the determination of flavonoid contents in mulberry and their scavenging effects on superoxide radicals. food chemistry, 64(4): 555-559. zobayed, s.m.a., afreen, f., goto, e., kozai, t. 2006: plant-environment interactions: accumulation of hypericin in dark glands of hypericum perforatum. annals of botany, 98(4): 793-804. zou, y., lu, y., wei, d. 2004: antioxidant activity of a flavonoid-rich extract of hypericum perforatum l. in vitro. journal of agricultural and food chemistry, 52(16): 5032-5039. vasić et al 2020, biologica nyssana 11(2) 11 (2) december 2020: 115-119 doi: 10.5281/zenodo.4393965 resistance to anthracnose (colletotrichum linicola) on different cultivars of red clover (trifolium pratense) in serbia original article tanja vasić faculty of agriculture, university of niš, kosančićeva 4, 37000 kruševac, serbia tanjavasic82@gmail.com (corresponding author) sanja živković faculty of agriculture, university of niš, kosančićeva 4, 37000 kruševac, serbia gajicsanja43@gmail.com mitra debasis division of soil science and agricultural chemistry, icar indian institute of horticultural research, karnataka 560 089, india debasismitra3@gmail.com ivana stanojević faculty of agriculture, university of niš, kosančićeva 4, 37000 kruševac, serbia stanojevic.ivana85@gmail.com sonja filipović faculty of agriculture, university of niš, kosančićeva 4, 37000 kruševac, serbia sonjafilipovic86@yahoo.com biljana anđelić faculty of agriculture, university of niš, kosančićeva 4, 37000 kruševac, serbia andjelic.biljana@ni.ac.rs jordan marković institute for forage crops, kruševac, 37251, globoder, serbia jordan.markovic@ikbks.com received: february 05, 2020 revised: november 23, 2020 accepted: november 30, 2020 abstract: in addition to alfalfa, red clover (trifolium pratense l.) is the most important perennial forage legume grown in serbia. it is rich in proteins that are important for the nutrition of livestock. red clover, like many legumes, is parasitized by fungi of the genus colletotrichum, and the consequence is significant economic damage in the form of reduced yields and reduced hay quality. in this study, sixteen cultivars of red clover of different geographical origin were tested for the resistance to the isolate coll-44 (c. linicola). the susceptibility of different cultivars of red clover to the tested c. linicola isolate was determined using the scale of 1-5. six cultivars (wilo, repio, viglana, kolubara, k-39 and k-17) showed high levels of resistance to the tested isolate of c. linicola. based on the severity index, the most resistant to the isolate of c. linicola was the american variety wilo with a severity index of 1.6 (50%), while the lowest severity index of the serbian variety k-39 was 2.6 (20%). the purpose of this research was to evaluate the susceptibility and tolerance level of 16 red clover cultivars to anthracnose causative agents, in this case, c. linicola. key words: colletotrichum linicola, cultivar, resistance, trifolium pratense apstract: otpornost na antraknozu (colletotrichum linicola) kod različitih sorti crvene deteline (trifolium pratense) u srbiji pored lucerke, crvena detelina (trifolium pratense l.) je najvažnija višegodišnja krmna leguminoza koja se gaji u srbiji. bogata je proteinima koji su važni za ishranu domaćih životinja. crvenu detelinu kao i mnoge leguminoze parazitiraju gljive iz roda colletotrichum, nanoseći značajne ekonomske štete u vidi smanjenja prinosa i kvaliteta sena. u ovom radu testirano je šesnaest sorti crvene deteline različitog geografskog porekla na otpornost prema izolatu coll-44 (c. linicola). osetljivost različitih sorti crvene deteline prema ispitivanom izolatu c. linicola određena je prema skali od 1-5. šest sorti wilo, repio, viglana, kolubara, k-39 and k-17 pokazalo je visok nivo otpornosti na testirani izolat c. linicola. na osnovu indeksa otpornosti najotpornijom prema izolatu c. linicola se pokazala američka sorta wilo sa indeksom otpornosti 1.6, dok je najniži indeks otpornosti kod srpske sorte k-39 iznosio 2.6. cilj ovog istraživanja bio je da se oceni stepen osetljivosti i tolerantnosti kod 16 sorti crvene deteline prema prouzrokovačima antraknoze, u ovom slučaju c. linicola. ključne reči: colletotrichum linicola, sorta, otpornost, trifolium pratense introduction red clover (trifolium pratense l.) is one of the most important forage crops in serbia. as such, it represents an essential element in the crop rotations. in recent years, it has been observed that the life span and quality of red clover hay decreased significantly, and, as a reason for this occurrence, fungal species of the genus colletotrichum (in particular, c. trifolii bain et essary, c. destructivum o’gara, c. coccodes (wallr.) hughes, c. truncatum (schw.) andrus and moore, c. dematium (pers.) grove and c. linicola pethybr. and laff) were cited (schubiger et al., 2004; jacob et al., 2010; vasić et al., 2014). anthracnose © 2020 vasić et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 115 decreases the vigour of the individual plants and thins out the plant population. during the summer and autumn, diseased plants start to appear in the red clover and alfalfa fields. these diseased plants have typical appearance. plants, in the advanced stages of the disease, develop deformations of the upper part of the stem. stem infection results in wilting and death of the upper portion of the stem and thus making the characteristic “shepherd’s crook” symptom (fig. 1). this is considered as the typical symptom of the disease. as the disease progresses, the infection spreads and passes from the stem to the root neck and the root area. crown and root infection is characterized by dry rot with a change in the color of the affected tissue to blue-black (vasić et al., 2016). damage caused by presence and development of pathogen agent cause the reduction of quantity and quality of fresh mass of 10% up to 30% depending on cultivar of red clover and alfalfa, species of pathogen, climatic and edaphic factors (stuteville and erwin, 1990; vasić, 2010b). the diseased plants can, thus, decay during one growing season or be severely weakened, often succumbing to frost in winter conditions (schubiger et al., 2003). since the growing importance of red clover crops in serbia, the aim of this experiment was to determine the susceptibility and tolerance level of different red clovers cultivars to anthracnose agents, in this case, c. linicola, and to find the right sources of resistance. material and methods in june-july, 2018, sixteen commercial cultivars of red clover were tested for resistance to the anthracnose causative agent c. linicola. the experiment was done in the greenhouse of the institute for forage crops in kruševac. commercial red clover cultivars of different origin were used for the experiment. cultivars were as following: k-9, k-17, k-27, k-32, k-38, k-39 (institute for forage crops, kruševac, serbia), una and kolubara (institute of field and vegetable crops, novi sad, serbia), diplomat (deutsche saatveredelung ag – dsv, lippstadt, germany), nigula (estonian crop research institute, estonia), longevo (istituto sperimentale per le colture foraggere, lodi, italy), viglana, manuela (research institute of plant production in piestany, slovakia) and wilo, repio and diana (us dairy forage research dairy farm, wisconsin, usa). one isolate from serbia, coll44, was used in this study and was identified as c. linicola originated from alfalfa from serbia, based on morphological, pathological and molecular traits. colonies of isolate c. linicola were light to dark olive green. in cultures on pda medium, acervuli were formed. conidia from acervuli were released in mucous masses that were orange to cream-pink in color (vasić et al., 2014). the sowing of the red clover seeds was done using plastic containers at a depth of 2 cm in sterile substrate in a greenhouse. the conidial suspension was prepared from cultures of the tested isolate. it was grown for ten days on pda medium at 25 °c in the dark. the concentration of spores was determined by hemocytometer by thoma and it was 4-6 x 104 spores/ml. inoculated red clover plants were 7 weeks old and they were scarred prior to inoculation by cutting and sprayed with a spore suspension. the experiment was conducted in five repetitions, 5 plants per genotype and red clover plants inoculated with sterile water used as a negative control. plants were categorized in 5 score classes, based on the anthracnose reaction using the 1 to 5 scale (1 = no stem lesions or only a few small water-soaked or black spots; 2 = stems with elongated black lesions but without acervuli; 3 = stems with long, wide, but non-gridling lesions, with acervuli present; 4 = large, coalescing and sporulating lesions which kill 116 fig. 1. “shepherd’s crook” symptom on red clover biologica nyssana ● 11 (2) december 2020: 115-119 vasić et al. ● resistance to anthracnose (colletotrichum linicola) on different cultivars of red clover (trifolium pratense) in serbia 117 upper part of seedling; 5 = seedling dead) (ivanović and ivanović, 2005). based on the results, the severity index was calculated according to the following formula: i severity index ntotal number of seedling n number of seedlings in class k number of categories k number of individual categories s sum of products according to ostazeski et al. (1969), where 1 and 2 are assigned to resistant plants, plants were scored individually, four weeks after inoculation. disease intensity or severity of infected plants was calculated using the severity index. when the severity index is used as a criterion, all cultivars are classified with severity index from 2.72 to 2.25 can be classified as resistant cultivars (ostazeski et al., 1979). results the studied cultivars showed a clear difference in sensitivity to the tested isolate of c. linicola. the number of surviving plants varied over a wide range. four weeks after the inoculation of the tested plants, primarily necrotic lesions occurred on the stem and there was slight twist of the top of the upper third of the stem. necrotic lesions did spread further throughout the plant and it led to the complete drying and decay of some plants. the intensity of disease development was evaluated using the scale of 1-5. the response of the tested red clover cultivars after inoculation with the tested c. linicola isolate as well as the severity index score are shown in tab. 1. in the conditions of this experiment, the most resistant was the american cultivar of red clover wilo, which had the severity index of 1.6 and 50% of resistant plants. furthermore, vinglana cultivar, with the severity index 2.4, and repio, with the severity index 2.6, showed high levels of resistance. also, serbian varieties k-39, k-17 and kolubara showed significant resistance, and their severity indices ranged from 2.6 to 2.8, while the percentage of resistant plants for all tested cultivars was 20. the most sensitive was cultivar diana, with the severity index 3.6 and 10% of the healthy plants, while cultivars k-27 and diplomat had severity indices 3.4 without healthy plants. discussion according to results of ostazeski et al. (1969), sensitive cultivars include cultivars with 10% of healthy plants, while resistant cultivars include cultivars with over 65% of healthy plants. vasić et al. (2015) tested thirteen (k-27, k-39, biologica nyssana ● 11 (2) december 2020: 115-119 vasić et al. ● resistance to anthracnose (colletotrichum linicola) on different cultivars of red clover (trifolium pratense) in serbia table 1. evaluation of sensitivity in commercial cultivars of red clovers (trifolium pratense l.) to isolate c. linicola (coll-44) cultivars plants categorised by ostazeski scale (%) severity index resistant plants (1+2%)1 2 3 4 5 k-9 0 20 10 20 0 3.0 20 k-17 0 20 20 10 0 2.8 20 k-27 0 0 30 20 0 3.4 0 k-32 0 10 30 10 0 3.0 10 k-38 0 0 30 20 0 3.4 0 k-39 0 20 30 0 0 2.6 20 diplomat 0 0 30 20 0 3.4 0 nigula 0 10 30 20 0 3.0 10 viglana 10 10 30 0 0 2.4 20 manuela 0 10 20 10 20 3.4 10 longevo 0 10 20 10 0 3.4 10 kolubara 0 20 20 10 0 2.8 20 una 0 10 30 10 0 3.0 10 wilo 20 30 0 0 0 1.6 50 repio 0 20 30 0 0 2.6 20 diana 0 10 10 20 0 3.6 10 control 118 k-32, k-38, una, kolubara, čiroku, lea, virgiana, valentine, wilo, repio and diana) commercial cultivars of red clover for resistance to the anthracnose causative agent c. destructivum. the reactions of the tested cultivars were different. the most resistant to c. destructivum was the cultivar wilo with severity index 1.9, while cultivar valentina showed the highest sensitivity to the tested isolate. despite the large number of studies, which indicated that resistance to anthracnose is controlled by major dominant or recessive genes and other resistance mechanisms, resistance to c. trifolii is not clear enough (schubiger et al., 2004). schubiger et al. (2003) found that the degree of resistance to colletotrichum trifolii was different in different cultivars of red clover. the anthracnose resistance of the red clover plants ranged from 3% to 52% (schubiger et al., 2004). according to boller et al. (2004), merula and pavo were red clover cultivars of the mattenklee type resistant to the causative agents of anthracnose, c. trifolii. according to jacob et al. (2010), the german cultivars of red clover showed a wide range of resistance to colletotrichum trifolii with plant survival from 29% to 87%. schubiger et al. (2004) examined twelve swiss cultivars of red clover (leisi, renova, ruettinova, milvus, formica, corvus, merula, pavo, temara, vanessa, larus, astur) considering the resistance to c. destructivum. in their research, they stated that none of the red clover cultivars were severely affected by c. destructivum. also, jacob et al. (2015) tested thirtyeight red clover cultivars listed in germany, for the resistance to c. trifolii and found that resistance of red clover to anthracnose was highly heritable. vasić et al. (2010b) reported that different cultivars of red clover (manuela, margot, k-39, k-32 and breed population l-50) show different levels of resistance depending on different c. trifolii isolates. the degree of resistance of the red clover cultivar depended on the virulence of the c. trifolii isolates. vasić et al. (2010a) showed that alfalfa clones had different resistance to c. trifolii. by comparing american and serbian cultivars, it can be noted that the american alfalfa cultivars had the highest resistance level (64.5% of plant survival). the most susceptible tested cultivar had the plant survival rate of 56.4%. conclusion it can be concluded, from the presented assessment, that red clover cultivars showed variations in their resistance response to c. linicola. six cultivars of wilo, repio, viglana, kolubara, k-39 and k-17 showed resistance to the tested isolate, and could be listed for cultivation under integrated production systems and used in the development of new resistant red clover cultivars. references boller, b., tanner, p., schubiger, f.x. 2004: merula und pavo: neue, ausdauernde mattenkleesorten. agrarforschung, 11(5): 162-167. ivanović, m., ivanović, d. 2005: bolesti voćaka i vinove loze i njihovo suzbijanje. poljoprivredni fakultet. beograd. jacob i., hartmann s., schubiger f.x., struck c. 2010: genetic diversity of red clover varieties listed in germany concerning the resistance to southern anthracnose. grassland science in europe, 15: 344346. jacob, i., hartmann, s., schubiger, f. x., struck, c. 2015: resistance screening of red clover cultivars to colletotrichum trifolii and improving the resistance level through recurrent selection. euphytica, 204: 303-310. ostazeski, s.a., barnes, d.k., hanson, c.h. 1969: laboratory selection of alfalfa for resistance to anthracnose, colletotrichum trifolii. crop science, 9: 351-354. ostazeski, s.a., elgin, j.h., jr., mcmurtray, j.e. 1979: occurrence of anthracnoses on formerly anthracnose-resistant “arc” alfalfa. plant disease report, 63: 734-736. stuteville d.l., erwin d.c. 1990: compendium of alfalfa diseases, second edition. the american phytopathological society st. paul, minnesota. schubiger f.x., streckeisen p., boller b. 2003: resistance to southern anthracnose (colletotrichum trifolii) in cultivars of red clover (trifolium pratense). czech journal of genetics and plant breeding, 39 (special issue): 309-312. schubiger, f.x., alconz, e., streckeisen, p., boller, b. 2004: resistance to southen anthracnoses (colletotrichum trifolii) in red clover (trifolium pratense). agrarforschung, 11(5): 168-173. vasić, t., radović, j., lugić, z., marković, j., jevtić, g., gajić, s. 2010a: occurrence of colletotrichum trifolii (bain et essary), the inducer of alfalfa anthracnose in serbia. in: c. huyghe (ed.): sustainable use of genetic diversity in forage and turf breeding. springer, 53: 369-374. vasić, t., lugić, z., anđelković, s., štrbanović, r., marković, j., gajić, s, anđelković, b. 2010b: the impact of colletotrichum trifolii isolates on resistance in different red clover cultivars. proceedings of the xii international symposium on forage crops of republic of serbia, 26-28. may, kruševac, serbia, biotechnology in animal husbandry, 26 (spec.issue), book 2: 51-56. biologica nyssana ● 11 (2) december 2020: 115-119 vasić et al. ● resistance to anthracnose (colletotrichum linicola) on different cultivars of red clover (trifolium pratense) in serbia vasić, t., bulajić, a., krnjaja, v., jevremović, d., živković, s., andjelković, b. 2014: first report of anthracnose on alfalfa caused by colletotrichum linicola in serbia. plant disease, 98, (9): 1276. vasić, t., lugić, z., terzić, d., milenković, j., marković, j., živković, s. 2015: the impact of colletotrichum destructivum on resistance in different red clover cultivars. book of proceedings/ sixth international scientific agricultural symposium “agrosym 2015”, 15th-18th october; mountain jahorina, bosnia and herzegovina, 972976. vasić, t., jevremović, d., andjelković, s., marković, j., zornić, v., babić, s., leposavić, a. 2016: morphological and molecular identification of a new alfalfa parasite-colletotrichum linicola in serbia. book of proceedings vii international scientific agriculture symposium “agrosym 2016” jahorina, october 06 09, pp. 1480-1485. 119 biologica nyssana ● 11 (2) december 2020: 115-119 vasić et al. ● resistance to anthracnose (colletotrichum linicola) on different cultivars of red clover (trifolium pratense) in serbia mašić et al. 2020, biologica nyssana 11(2) 11 (2) december 2020: 93-101 doi: 10.5281/zenodo.4393955 new data on distribution of hydrurus foetidus (villars) trevisan in freshwater habitats on vranica mountain (bosnia and herzegovina) original article ermin mašić university of sarajevo, faculty of science, department of biology, zmaja od bosne 33-35, 71000 sarajevo, bosnia and herzegovina erminmasic@hotmail.com (corresponding author) senka barudanović university of sarajevo, faculty of science, department of biology, zmaja od bosne 33-35, 71000 sarajevo, bosnia and herzegovina sebarudanovic@gmail.com sabina žero university of sarajevo, faculty of pharmacy, department of natural sciences in pharmacy, zmaja od bosne 8, 71000 sarajevo, bosnia and herzegovina sabina_zero@hotmail.com emina ramić university of sarajevo, faculty of science, department of biology, zmaja od bosne 33-35, 71000 sarajevo, bosnia and herzegovina emina.ramich@gmail.com armin macanović university of sarajevo, faculty of science, department of biology, zmaja od bosne 33-35, 71000 sarajevo, bosnia and herzegovina arminecology@gmail.com senaid fejzić secondary technical school „kemal kapetanovic“ kakanj, šehida 32, 72240 kakanj, bosnia and herzegovina senaidfejzic@yahoo.com received: april 08, 2020 revised: august 13, 2020 accepted: october 11, 2020 abstract: freshwater habitats such as springs, streams and lakes at mountain area represent extremely fragile ecosystems. these types of habitats occur in the wide area of vranica mountain. during our research (august and september 2018) of freshwater habitats in this area, rare golden macroalgae hydrurus foetidus (villars) trevisan was found. it has been already detected in bosnia and herzegovina, but in this study new localities are presented. water temperature, ph, dissolved oxygen, specific electrical conductivity, turbidity and total dissolved solids were measured on sampling sites. in addition to the analysis of ecological characteristics of habitats, diatom assemblages were analysed. in this study a total of 48 diatom taxa belonging to 25 genera were identified. the results of diatom indices confirmed the good water quality of the studied sites on which h. foetidus was found. this study represents a contribution to algological research in bosnia and herzegovina. key words: bioindicators, conservation ecology, macroalgae, springs, streams, water quality apstract: novi podaci o rasprostranjenosti hydrurus foetidus (villars) trevisan u slatkovodnim staništima na planini vranici (bosna i hercegovina) slatkovodna staništa kao što su izvori, potoci, reke i jezera na planinskim područjima predstavljaju izuzetno osetljive ekosisteme. ovi tipovi staništa su zastupljeni na širem području planine vranice. tokom istraživanja (avgust i septembar, 2018) slatkovodnih tipova staništa na ovom području, utvrđena je retka zlatna makroalga hydrurus foetidus (villars) trevisan. vrsta je već konstatovana u bosni i hercegovini, ali u ovom istraživanju predstavljeni su novi lokaliteti. temperatura vode, ph, rastvoreni kiseonik, elektroprovodljivost, mutnoća i ukupna količina rastvorenih organskih materija su izmereni na mestu uzorkovanja. pored analize ekoloških karakteristika staništa, analiziran je i sastav diatomeja. tokom ove studije ukupno je identifikovano 48 dijatomeja iz 25 rodova. rezultati dijatomskih indeksa potvrdili su dobar kvalitet vode na lokalitetima gdje je utvrđen h. foetidus. ova studija predstavlja značajan doprinos algološkim istraživanjima bosne i hercegovine. ključne reči: bioindikatori, konzervacijska ekologija, makroalge, izvori, potoci, kvalitet vode introduction the species hydrurus foetidus (villars) trevisan is a macroscopic golden alga with mucilaginous, branched and bushy to feathery, dark brown thallus up to 30 cm long. the thallus and other characteristics are well described in various publications (john et al., 2003; wehr et al., 2015). this species inhabit mountain streams during early spring and lowland rivers at latitudes where seasonal conditions are appropriate for this coldwater species (klaveness et al., 2011; klaveness, 2017). according to guiry & guiry (2019), this alga is widely distributed in europe and it’s presence was reported in austria (pfister, 1992), britain (kristiansen, 2002; whitton et al., 2003; john et al., 2011), croatia (stanković & leitner, 2016; koletić et al., 2017), georgia (barinova & kukhaleishvili, 2017), germany (mauch & schmedtje, 2003), © 2020 mašić et al.. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 93 norway (klaveness et al., 2011; klaveness & lindstrøm, 2011; klavness, 2012), romania (caraus, 2017), russia (europe) (voloshko, 2007; patova & demina, 2007; patova et al., 2014), serbia (krizmanić et al., 2008), slovakia (hindák & hindáková, 2016) and turkey (europe) (aysel, 2005). on the territory of bosnia and herzegovina h. foetidus was reported only from a few localities (fig. 1), and these data are found in various publications (protić, 1904; blagojević, 1966; kosorić, 1977; aganović, 1981; ratković, 1985; redžić, 1988; hafner, 1991; redžić, 1991). until now, several algological studies were performed on vranica mountain concerning different algal groups and habitats (protić, 1926; kapetanović & hafner, 2007; barudanović et al., 2017) but did not demonstrate the occurrence of h. foetidus in any studied sites. the main aim of this study was to present new localities where h. foetidus occurs considering the ecological characteristics of habitat in which the species was found. material and methods study area vranica mountain has very heterogeneous geology and petrography. the area is rich in numerous springs, brooks and small rivers, which are active throughout the year. unfortunately, in recent years due to growing tourism activity anthropogenic impact has increased. these effects are manifested mostly through the construction and expansion of road communication, drainage of wet habitats, springs capturing and the construction of new accommodation facilities (redžić, 2007). sampling sites are listed in tab. 1. sampling and algae analysis physical and chemical parameters of water were measured directly on sampling sites. water temperature (°c), ph, dissolved oxygen (mgl-1) and specific electric conductivity (μscm-1) were measured with portable multimeter orion star a329, while turbidity (ntu) was measured with portable turbidimeter aq3010 and total dissolved solids (ppm) with pce-cm 41. live algological material was collected from six microlocalities in 2018, during summer and autumn seasons, photographed and transported to the laboratory of algae and fungi at faculty of science, university of sarajevo. phytobenthos was collected 94 table 1. the main physical characteristics of the sampling sites (sp – spring; st – stream) biologica nyssana ● 11 (2) december 2020: 93-101 mašić et al. ● new data on distribution of hydrurus foetidus (villars) trevisan in freshwater habitats on vranica mountain (bosnia and herzegovina) site habitat types date coordinate [n] coordinate [e] altitude [m] anthropogenic disturbance 1. sp1 11/08/2018 43.95353 17.75757 1682 moderate 2. st1 11/08/2018 43.95353 17.75757 1682 moderate 3. st2 11/08/2018 43.95440 17.75760 1677 moderate 4. st3 25/08/2018 43.95731 17.74518 1727 moderate 5. st4 20/10/2018 43.95563 17.75786 1465 moderate 6. st5 20/10/2018 43.95544 17.75767 1652 moderate fig. 1. utm map of distribution of hydrurus foetidus in bosnia and herzegovina 95 from small stones by scraping or by squeezing of mosses growing on rocks and stones. the collected material was fixed with a 4% formalin. small portion of h. foetidus was collected and observed under the microscope from temporary slides under 40 x magnification. the identification of h. foetidus was supported by the following references: starmach (1985), john et al. (2003) and wehr et al. (2015). laboratory processing of diatoms was carried out applying methods used by hustedt (1930). light microscope observation was conducted using best scope 2020 microscope. species composition and the quantitative relationship of diatoms are estimated from the permanent slides under 1000 x magnification. the identification of diatoms was done according to lange-bertalot & metzeltin (1996) and cantonati et al. (2017). the nomenclature of identified algae was adjusted according to the internet base of guiry & guiry, 2019. at least 300 valves in each slide were counted for all samples. species abundance of diatoms are estimated on a five-degree scale as follows: 1-rare (single valve or frustule), 2-sparse (up to 10% of the sample), 3-frequent (11-15% of the sample), 4-very frequent (51-75% of the sample), 5-common (in more than 75% of the sample) (tab. 3). omnidia software (lecointe et al., 1993) version 6.0.8, was used to calculate diatom indices, including ecological and taxonomic data. water quality of the localities where h. foetidus was assessed based on the following diatom indices: ips (coste, 1982), slad (sládeček, 1986), tid (rott et al., 1999) and sid rott (rott et al., 1997). range of diatom indices varied from 1 to 20 and corresponding to the water quality as follows: bad (1-4), poor (5-8), moderate (9-12), good (13-16) and very good (1720) (solak et al., 2020). results description of sampling sites the species of h. foetidus (chrysophyceae) was found near a reocren spring of mountain stream and on five localities downstream. it lives as a benthic species attached to the mesolithic and megalithic stones on the bottom of the water body an is often associated with mosses. although it tolerates occasional water fluctuations, in most cases it has been found in running water, but also in places where the water is a little deeper and slower. the altitude of the sites where the h. foetidus was recorded, ranged from 1465 to 1727 meters. the spring and mountain stream are covered with dense macrophytes and helophytes vegetation. two types of habitats where h. foetidus is recorded are showed on fig. 2, while in fig. 3 and fig. 4 the macroscopic and microscopic appearance of the thallus are shown. physical and chemical parameters of water a comparison of the physical and chemical parameters of water in the mountain spring and stream revealed the following: the water temperature on sampling sites during the investigated period varied from 6.2 to 19.9 °c. the lowest ph of the water was 7.92 while the highest ph value was 8.50. the highest concentration of dissolved oxygen in water was measured at the site 2 (10.82 mgl-1), while the smallest value was measured at the site 4 (7.56 mgl-1). value of specific conductivity varied from 162 to 330 μscm-1. the lowest value of turbidity was measured at the site 4 (0.45 ntu), while at the site 6 the highest value of turbidity was measured (2.81 ntu). fig. 2. two types of habitats where hydrurus foetidus was recorded: mountain spring (left) and mountain stream (right) biologica nyssana ● 11 (2) december 2020: 93-101 mašić et al. ● new data on distribution of hydrurus foetidus (villars) trevisan in freshwater habitats on vranica mountain (bosnia and herzegovina) 96 values of physical and chemical parameters are presented in tab. 2. characteristics of habitats of hydrurus foetidus and diversity of diatoms hydrurus foetidus has been identified at six sites located in the area of vranica mountain. it inhabits mainly spring and fast and clean mountain streams. together with mosses and other groups of algae, it grows on mesolithic and megalithic bases, and represents a significant product component of these freshwater habitats. the presence of this species indicates good water quality and good ecological status of freshwater ecosystems. in addition to h. foetidus, diatoms are second the most prominent group of algae which have been found in this mountain freshwater habitats. a total of 48 diatom taxa belonging to 25 genera were determined in the collected material (tab. 3). the number of recorded species of diatoms in the study sites ranged from 11 to 22. genera with the highest number of species were pinnularia (5), gomphonema (5), diatoma (4) and cocconeis (4). altogether 47 diatom taxa mostly belonging to pennate diatoms were found. the centric species, aulacoseira crenulata (ehrenberg) thwaites, appeared in only one sample. the most common species were odontidium mesodon (ehrenberg) kützing, cocconeis lineata ehrenberg, meridion circulare (gréville) c. agardh, cocconeis placentula ehrenberg, gomphonema minusculum krasske and achnanthidium minutissimum (kützing) czarnecki. water qualities of investigated sites according to diatom indices values of ips diatom index varied from 15.0 to 18.3. according to the ips diatom index, all studied sites have very good water quality. saprobic index at four sites indicates good, while on the other studied sites it indicates very good water quality. trophic index at all investigated sites indicates moderate water quality. saprobic index according to rott et al. (1997) ranged from 16.4 to 17.1. high values of diatom indices correspond to a very good water quality of investigated freshwater habitats on vranica mountain (tab. 3). discussion hydrurus foetidus was found in the mountain spring (1682 m a.s.l.) and in the mountain streams with table 2. value of the water temperature, ph, dissolved oxygen, specific electric conductivity, turbidity and tds [t1 – water temperature, do – dissolved oxygen, sc – specific electric conductivity, t2 – turbidity, tds – total dissolved substance]. site group t1 [°c] ph do [mgl -1] sc [μscm-1] t2 [ntu] tds [ppm] 1. sp1 6.8 7.92 10.35 330 0.80 n/a 2. st1 6.2 7.97 10.85 162 1.12 n/a 3. st2 7.4 8.23 10.25 208 0.73 n/a 4. st3 19.9 8.31 7.56 171 0.45 121 5. st4 8.1 8.47 10.51 n/a 0.91 267 6. st5 7.4 8.50 10.29 219 2.81 161 *n/a-not available fig. 3. thallus of h. foetidus (villars) trevisan fig. 4. microscopic appearance of the thallus of h. foetidus (villars) trevisan (40 x) biologica nyssana ● 11 (2) december 2020: 93-101 mašić et al. ● new data on distribution of hydrurus foetidus (villars) trevisan in freshwater habitats on vranica mountain (bosnia and herzegovina) 97 table 3. records of hydrurus foetidus and diatom taxa at sampling sites no. taxa / site 1 2 3 4 5 6 h hydrurus foetidus (villars) trevisan + + + + + + 1. odontidium mesodon (ehrenberg) kützing 4 6 4 1 1 3 2. cocconeis lineata ehrenberg 1 2 5 2 3 4 3. meridion circulare (gréville) c. agardh 1 2 1 2 1 1 4. cocconeis placentula ehrenberg 1 3 2 1 . 3 5. gomphonema minusculum krasske 1 1 1 1 1 . 6. achnanthidium minutissimum (kützing) czarnecki 6 1 . 1 1 1 7. encyonema minutum (hilse) d.g. mann 4 . 1 1 1 . 8. tetracyclus rupestris (braun) grunow in van heurck . 1 1 1 1 . 9. diatoma ehrenbergii kützing . . 1 4 4 4 10. ulnaria ulna (nitzsch) compère . . 1 6 1 1 11. encyonema silesiacum (bleisch) d.g. mann . 1 1 1 . . 12. hannea arcus (ehrenberg) r.m.patrick in patrick & reimer . . 5 . 4 3 13. encyonema ventricosum (c.agardh) grunow in a. schmidt et al. 1 1 . . . . 14. pinnularia borealis ehrenberg 1 1 . . . . 15. planothidium lanceolatum (brébisson ex kützing) lange-bertalot 1 1 . . . . 16. gomphonema parvulum (kützing) kützing . 1 2 . . . 17. denticula tenuis kützing . 1 1 . . . 18. caloneis alpestris (grunow) cleve . 1 . 1 . . 19. diploneis krammeri lange-bertalot et reichardt . 1 . 1 . . 20. epithemia turgida (ehrenberg) kützing . 1 . 1 . . 21. gomphonema acuminatum ehrenberg . . 1 . 1 . 22. cocconeis pediculus ehrenberg 1 . . . . . 23. cymbopleura naviculiformis (auerswald) krammer 1 . . . . . 24. gomphonema productum (grunow) lange-bertalot et reichardt in l-b 1 . . . . . 25. navicula lanceolata (c.agardh) ehrenberg 1 . . . . . 26. nitzschia fonticola grunow in cleve & möller 1 . . . . . 27. pinnularia viridis (nitzsch) ehrenberg 1 . . . . . 28. diatoma vulgaris bory . 1 . . . . 29. diploneis ovalis (hilse) cleve . 1 . . . . 30. eunotia sp. 1 . 1 . . . . 31. diatoma elongatum (lyngbye) agardh . 1 . . . . 32. pinnularia rupestris hantzsch in rabenhorst . 1 . . . . 33. amphora copulata (kützing) schoemann et archibald . 1 . . . . 34. aulacoseira crenulata (ehrenberg) thwaites . . 1 . . . 35. cymbella aspera (ehrenberg) h. peragallo . . 1 . . . 36. navicula tripunctata (o.f. müller) bory . . 1 . . . 37. cymbella neolanceolata w.silva . . . 1 . . 38. cymbopleura amphicephala (nägeli) krammer . . . 1 . . 39. diatoma moniliformis (kützing) williams . . . 1 . . 40. gomphonema subclavatum (grunow) grunow . . . 1 . . 41. pinnularia neomajor krammer . . . 1 . . 42. staurosirella leptostauron (ehrenberg) d.m. williams et round . . . 1 . . 43. staurosirella pinnata (ehrenberg) d.m. willams et round . . . 1 . . 44. surirella spiralis kützing . . . 1 . . 45. nitzschia acidoclinata lange-bertalot . . . . 1 . 46. pinnularia subrupestris krammer . . . . 1 . 47. cocconeis pseudolineata (geitler) lange-bertalot . . . . . 1 48. cymbopleura austriaca (grunow) krammer . . . . . 1 a total number of taxa (n) 16 22 17 22 13 10 b ips/20 18.3 18.3 17.7 15.0 17.0 17.0 c sla/20 16.4 17.1 17.7 14.7 17.2 16.5 d rott ti/20 (tid) 11.6 8.5 11.5 6.5 11.6 12.0 e rott ti/20 (sid) 16.5 16.9 16.5 16.7 16.4 17.1 biologica nyssana ● 11 (2) december 2020: 93-101 mašić et al. ● new data on distribution of hydrurus foetidus (villars) trevisan in freshwater habitats on vranica mountain (bosnia and herzegovina) altitude ranged between 1465 and 1727 meters a.s.l. in all studied sites, the species was well developed. according to literature data (klaveness, 2017, 2019) h. foetidus appears under different ecological conditions. this species is a rheophile, preferring swiftly flowing water. it is also a psychrophile and dependent on low temperature. the species hydrurus foetidus is visibly a large freshwater alga found in cold rivers. generally, the species may be found during late winter months, sometimes forming dense cover on pebbles and rocks at the river bottom and less prominent in the spray zone. the speci es is usually well developed during the colder months of the year but disappears in summer with the increasing flow (klaveness, 2019). according to klaveness (2017) this species is an indicator of clean water and good ecological status, but there is a various observation that some population tolerate mesosaprobic or oligomesosaprobic condition. in some occasions, h. foetidus thrives well under seasonal climatic conditions, where there is snow in the winter, cold meltwater and moderate summer temperatures (klaveness, 2019). by comparing our results, with the results of other literature data, a certain similarity of ecological characteristics of habitas can be obtained. the results of our study are correlated with other research conducted in the balkan peninsula dealing with ecology of h. foetidus (blagojević & hafner, 1979; redžić, 1988; krizmanić et a., 2008; stanković & leitner, 2016) especially in terms of habitats characteristics and physical and chemical parameters of water. on the site in the river neretva (redžić, 1988) water temperature ranged from to 11.5 – 14.9 °c, ph value varied between 7.5 – 8.3 and dissolved oxygen ranged between 10.6 – 12.3 mgl-1. the distribution and ecological characteristics of h. foetidus in serbia are given by the authors krizmanić et al. (2008). at two investigated sites, water temperature ranged from 4.6 to 9.6 °c, while the oxygen concentration ranged from 11.0 to 15.1 mgl-1. by summarizing a large number of data, but also on the basis of the study carried out, the authors concluded that the measured physical and chemical parameters of water confirm the rheophilic nature of this species. stanković & leitner (2016) recorded h. foetidus in croatia for the first-time during the environmental study of the bijela rijeka located within the plitvice lakes national park. the water temperature on the studied site was 9.0 °c, ph value 8.3 while the specific electrical conductivity was 441 μscm-1. the concentration of oxygen was 11.02 mgl-1, cod 1.32 mgo2l -1, and the alkalinity had the values of 259.5 mgcaco3l -1. in terms of measured nutrients, ammonium was not found in the studied sites, the nitrates had low values (0.003 mgnl-1), while the nitrates had somewhat higher values (0.759 mgnl1). on the studied site orthophosphates were also measured, and their value was 0.025 mgpl-1. the average water velocity was 0.60 ms-1 and the water depth ranged from 20 to 15 cm. during investigation of algal assemblages in springs of the konjuh mountain golden algae phaeodermatium rivulare hansgirg (chrysophyceae) was identified (kamberović et al., 2019). unfortunately, species which are very common in alpine springs, such as h. foetidus was not recorded in this study. the main reason for absence of this species was due to the lower water velocity and different geological substratum (kamberović et al., 2019). in addition to h. foetidus which indicates clean water and good ecological status of freshwater habitats according to literature data (klaveness, 2019), ascertained diatoms indicate the same or similar environmental conditions. the results of diatom indices confirmed the good water quality of the studied sites on which h. foetidus was found. along with h. foetidus, the authors stanković & leitner (2016) found certain diatoms and bluegreen algae in the bijela rijeka which is located in the plitvice lakes national park. in addition to h. foetidus same authors identified two macroalgae, namely zygnema sp. and vaucheria sp. during complex ecological studies on algal assemblages in the krivaja river (bosnia and herzegovina) authors blagojević & hafner (1979) noted the presence of h. foetidus. in all investigated samples 40 taxa are common and the most prominent are chamaesiphon incrustans, nostoc sphaericum, phormidium favosum, phormidium autumnale, vaucheria sp., diatom ehrenbergii, cocconeis pediculus, achnanthidium minutissimum, navicula tripunctata and encyonema ventricosa. despite previously conducted research on vranica mountain, the presence of rare and endangered species h. foetidus has not been noted. the main reason lies in the fact that the weather conditions that prevail in the area of the vranica mountain are very severe, and that earlier research was carried out during favourable climatic conditions and at a time when the species was no longer present. the species h. foetidus inhabits freshwater habitats in high altitudes that are under weak or moderate anthropogenic impacts. as it indicates the good ecological status of mountain springs and streams it can be used in bioindication of oligotrophy and good ecological status of freshwater habitat types 98 biologica nyssana ● 11 (2) december 2020: 93-101 mašić et al. ● new data on distribution of hydrurus foetidus (villars) trevisan in freshwater habitats on vranica mountain (bosnia and herzegovina) in the mountain area. in this respect, the habitats that provide conditions for the development of this alga have great conservation importance. according to the red list of slovakia, the species h. foetidus is classified into endangered categories (en), while the category of vulnerability is not included in the iucn red list (feráková et al., 2001). hydrurus foetidus develops thick mat on solid river bottom, therefore, it is a valuable food source for emerging waterborne insect larvae (milner et al., 2001, 2009; zah et al., 2001 in klaveness, 2017). hydrurus foetidus appears to be of specific importance for the early emerging aquatic larvae of chironomids (klaveness, 2019). in a study conducted by the authors moog & janecek (1991) in the rivers of the central alps, it was determined that the distribution of biomass macrozoobenthos varies significantly if different substrates are compared, and that a positive correlation is determined by the biomass of the benthic macroalgae h. foetidus, and negative correlation is determined by the flow of water. conclusions the presented study suggests that the species h. foetidus prefers freshwater habitats located at higher altitudes. it usually occurs during the cold months, but if the conditions permit, it can occur even during the warm months when the thallus starts to disintegrate slightly. the physical and chemical characteristics measured on the investigated sites corresponded to the literature data. habitats providing conditions for the development of h. foetidus on vranica mountain have a good water quality. in our study distributional data are presented, and also environmental characteristics of habitats in which h. foetidus usually occures. in order to expand knowledge about the ecological characteristics of macroalgae in the future, it is necessary to conduct more detailed research not only in the area of vranica mountain but also on the other mountains of bosnia and herzegovina. in addition to measuring the basic physical and chemical parameters of water, it is necessary to take into account other parameters, such as nutrients and heavy metals. this study contributes to algological research in bosnia and herzegovina and also give good basis for future investigation of macroalgae in this area. acknowledgements. this study is a part of a project entitled as: “conservation of freshwater habitat types on vranica mountain and establishment of long-term monitoring of biodiversity” which is funded by rufford foundation (application id 24578-1). we would like to thank the two anonymous reviewers for their suggestions and comments. references aganović, m. 1981: istraživanje ekosistema na području srednjeg toka vrbasa područje uticaja “he bočac”. blagojević, s., hafner, d. sastav algi. elaborat. biološki institut univerziteta u sarajevu. aysel, v. 2005: check-list of the freshwater algae of turkey. journal of black sea/mediterranean environment, 11: 1-124. barinova, s., kukhaleishvili, l. 2017: diversity and ecology of algae and cyanobacteria in the aragvi river, georgia. elixir bio sciences, 104: 45934-45947. barudanović, s., mašić, e., macanović, a. 2017: tresetišta na bosanskim planinama. prirodnomatematički fakultet. sarajevo. 1-184. blagojević, s. 1966: prilog poznavanju algi kraških izvorišta u bosni i hercegovini. i. chrysophyceae, xanthophyceae, bacillariophyceae. godišnjak biološkog instituta, sarajevo, 29: 5-22. blagojević, s., hafner, d. 1979: ekološka istraživanja na cijanofitama i algama rijeke krivaje. godišnjak biološkog instituta, sarajevo, 32: 13-31. cantonati, m., kelly, g. m., lange-bertalot, h. 2017: freshwater benthic diatoms of central europe: over 800 common species used in ecological assesment. english edition with updated taxonomy and added species. koeltz botanical book. caraus, i. 2017: algae of romania. a distributional checklist of actual algae. version 2.4. studii si cercetari biologie, 7: 1-1002, 1 map. coste, m. 1982: étude des méthodes biologiques d’appréciation quantitative de la qualité des eaux. rapport cemagref qe lyon-af bassin rhône méditerranée corse. feráková, v., maglocký, š., marhold, k. 2001: červený zoznam paprodostov a semenných raslín slovenska (december 2001) – in: baláž, d., marhold, k. & urban, p. eds., červený zoznam rastlín slovenska, ochrana prírody, 20 (suppl.): 48-81. guiry, m. d., guiry, g. m. 2019: algaebase. worldwide electronic publication, national university of ireland, galway. http://www.algaebase.org; searched on 30 march 2020. 30 april 2019. hafner, d. 1991: floristička istraživanja mikrofita rijeke une. floristic research microphytes on 99 biologica nyssana ● 11 (2) december 2020: 93-101 mašić et al. ● new data on distribution of hydrurus foetidus (villars) trevisan in freshwater habitats on vranica mountain (bosnia and herzegovina) the river una. bilten društva ekologa bosne i hercegovine, ser. b., 6: 177-185. hindák, f., hindáková, a. 2016: algae. in: zoznam nižších a vyšších rastlín slovenska [list of lower and upper plants of slovakia]. version 1.1. slovakia: on-line list. hustedt, f. 1930: bacillariophyta (diatomeae). in: die süswasser flora mitteleuropas (a. pascher, ed.), 10, gustav fischer, jena. 468 pp. john, d. m, whitton, b. a., brook a. j. 2003: the freshwater algae flora of the british isle. an identification guide to freshwater and terrestrial algae. the natural history museum and the british phycology society, 2002, cambridge. john, d. m., whitton, b. a., brook, a. j. 2011: the freshwater algal flora of the british isles. an identification guide to freshwater and terrestrial algae. second edition. pp. i-xvii, 1-878. cambridge: cambridge university press. kamberović, j., plenković-moraj, a., kralj borojević, k., gligora udovič, m., žutinić, p., hafner, d., cantonati, m. 2019: algal assemblages in springs of different lithologies (ophiolites vs. limestone) of the konjuh mountain (bosnia and herzegovina). acta botanica croatica, 78(1), 6681. kapetanović, t., hafner, d. 2007: diatoms of wet habitats in the subalpine belt of mt. vranica (bosnia and herzegovina). in proceedings of the 1st central european diatom meeting, berlin-dahlem, 73-78. klaveness, d., lindstrøm, e. a. 2011: hydrurus foetidus (chromista, chrysophyceae): a large freshwater chromophyte alga in laboratory culture. phycological research, 59: 105-112. klaveness, d. 2012: en kuldekjær biofilm-regissør hydrurus foetidus. biolog, oslo, 30(3): 20-26. klaveness, d. 2017: hydrurus foetidus (chrysophyceae) an inland macroalga with potential. journal of applied phycology, 29: 14851491. klaveness, d. 2019: hydrurus foetidus (chrysophyceae): an update and request for observations. algae, 34(1), 1-5. klaveness, d., brate, j., patil, v., shalchiantabrizi, k., kluge, r, gislerod, h. r., jakobsen, k. s. 2011: the 18s and 28s rdna identity and phylogeny of the common lotic chrysophyte hydrurus foetidus. european journal of phycology, 46(3): 282-291. koletić, n., alegro, a., šegota, v., vuković, n., rimac, a., vilović, t. 2017: new sites of rare coldwater golden algae hydrurus foetidus (villaris) trevisan (ochrophyta: chrysophyceae) in croatia. natura croatica, 26(2): 305-311. kosorić, đ. 1977: sastav i karakteristike životnih zajednica neretve (od mostara do granica sa sr hrvatskom) za period do ljeta 1976. godine. blagojević i hafner. sastav algi. elaborat. kristiansen, j. 2002: phylum chrysophyta (golden algae). in: the freshwater algal flora of the british isles. an identification guide to freshwater and terrestrial algae. (john, d.m., whitton, b. a., brook, a. j. (eds), pp. 214-244. cambridge: cambridge university press. krizmanić, j., subakov-simić, g., karadžić, v. 2008: supplementary notes on the distribution of hydrurus foetidus (vill.) trevisan (chrysophyta) in serbia. archives of biological sciences, 13-14. lange-bertalot, h., metzeltin, d. 1996: oligotrophie-indicatoren. 800 taxa repräsentative fur drei diverse see-typen. iconographia diatomologica, 2: 1-390. lange-bertalot, h., steindorf, a. 1996: rote liste der limnischen kieselalgen (bacillariophyceae) deutschlands. schriftenreihe für vegetationskunde, 28: 633-677. lecointe, c., coste, m. prygiel, j. 1993: “omnidia”: sofware for taxonomy, calculation of diatom indices and inventories management. h. van dam (ed.), twelfth international diatom symposium. hydrobiologia, 269/270: 509-513. mauch, e., schmedtje, s. 2003: taxaliste der gewässerorganismen deutschlands zur kodierung biologischer befunde. informationsberichte des bayerischen landsamtes für wasserwirtschaft heft 01/03. pp. 1-388. münchen: bayerisches landesamt für wasserwirtschaf. milner, a. m., brittain, j. e., castellas, e. petts, g. e. 2001: trends of macroinvertebrate community structure in glacier-fed rivers in relation to environmental conditions: a synthesis. freshwater biology, 46: 1833–1847. milner, a. m, brown, l. e., hannah, d. m. 2009: hydroecological response of river systems to shrinking glaciers. hydrological processes, 23: 62–77. moog, o., janecek, b. f. u. 1991: river flow, substrate type and hydrurus density as major determinants of benthic macroinvertebrate abundance, composition, and distribution. verhandlungen internationale vereinigung für theoretische und angewandte 100 biologica nyssana ● 11 (2) december 2020: 93-101 mašić et al. ● new data on distribution of hydrurus foetidus (villars) trevisan in freshwater habitats on vranica mountain (bosnia and herzegovina) limnologie, 24: 1888-1896. patova, e., sterlyagova, i., shabalina, y. 2014: rare macroscopic algae species in the pechora and vychegda river basins (north-eastern part of european russia). botanica lithuanica, 20(2): 7786. patova, e. n., demina, i. v. 2007: algae of other divisions. in: biodiversity of the polar ural ecosystems. (getsen, m.v. eds), pp. 69-89. syktyvkar: komi science center ural div. ras. pfister, p. 1992: artenspektrum des algenaufwuchses in 2 tiroler bergbachen -teil 1: cyanophyceae, chrysophyceae, chlorophyceae, rhodophyceae phytobenthos communities from 2 tyrolean mountain streams. part 1: cyanophyceae, chrysophyceae, chlorophyceae, rhodophyceae. algological studies, 65: 43-61. predojević, d., kljajić, ž., kovačević, e., milosavljević, j., papić, p., lazić, m., simić, g. s. 2014: diversity of chrysophyceae (heterokontophyta) in the zasavica river (serbia). archives of biological sciences, 66(3), 1195-1204. protić, đ. 1904: prilog k poznavanju flore kriptogama (tajnocvjetaka) okoline sarajeva. glasnik zemaljskog muzeja, 1: 61-72. protić, đ. 1926: hidrobiološke i plankton-studije na jezerima bosne i hercegovine. iii dio glečerska jezera. glasnik zemaljskog muzeja, 1: 47-78. ratković, v. 1985: dinamika biocenoza ekosistema rijeke vrbas (od banja luke do ušća). blagojević, s., hafner, d. naselje cijanobakterija i algi u donjem toku vrbasa. elaborat. srbih, biološki institut univerziteta u sarajevu. redžić, a. 1988: fitobentos rijeke neretve kao pokazatelj kvaliteta voda. godišnjak biološkog instituta univerziteta u sarajevu, 41: 49-62. redžić, a. 1991: uticaj onečišćenja na distribuciju fitobentosa u rijeci uni. effect of the pollution on the phytobenthos distribution in the una river. bilten društva ekologa bosne i hercegovine, ser. b, 6: 187-199. redžić, s. 2007: syntaxonomic diversity as an indicator of ecological diversity—case study vranica mts in the central bosnia. biologia, 62(2), 173-184. rott, e., hofmann, g., pall, k., pfister, p., pipp, e. 1997: indikationslisten für aufwuchsalgen in österreichischen fliessgewässern, 1: saprobielle indikation. bundesministerium für landund forstwirtschaft, wasserwirtschaftskataster, wien. rott, e., pipp, e., pfister, p., van dam, h., ortler, k., binder, n., pall k. 1999: indikationslisten für aufwuchsalgen in österreichischen fliessgewässern, 2: trophie-indikation sowie geochemische präferenz; taxo-nomische und toxikologische anmerkungen. bundesministerium für landund forstwirtschaft, wasserwirtschaftskataster. wien. sládeček, v. 1986: diatoms as indicators of organic pollution. acta hydrochimica et hydrobiologica, 14(5), 555-566. solak, c. n., peszek, ł., yilmaz, e., ergül, h. a., kayal, m., ekmekçi, f., várbíró g, yüce a.m, canli, o., binici, m.s., ács, é. (2020): use of diatoms in monitoring the sakarya river basin, turkey. water, 12(3), 703. stanković, i., leitner, p. 2016: the first record of hydrurus foetidus (villars) trevisan (ochrophyta: chrysophyceae) in croatia with ecological notes. natura croatica, 25(2): 223-231. starmach, k. 1985: süßwasserflora von mitteleuropa, bd. 1. chrysophyceae und haptophyceae. gustav fischer verlag, stuttgart. 322 p. voloshko, l.n. 2007: golden algae. in: biodiversity of the polar ural ecosystems. (anon. eds), 57-68. wehr, j. d., sheath, r. g., kociolek, j. p. 2015: freshwater algae of north america: ecology and classification. elsevier. whitton, b. a., john, d. m., kelly, m. g., haworth, e. y. 2003: a coded list of freshwater algae of the british isles. second edition. worldwide web electronic publication. zah, r., burgherr, p., bernasconi, s. m., uehlinger, u. 2001: stable isotope analysis of macroinvertebrates and their food sources in a glacier stream. freshwater biology, 46: 871–882. 101 biologica nyssana ● 11 (2) december 2020: 93-101 mašić et al. ● new data on distribution of hydrurus foetidus (villars) trevisan in freshwater habitats on vranica mountain (bosnia and herzegovina) terzić et al. 2022, biologica nyssana 13(2) 13 (2) december 2022: 129-139 doi: 10.5281/zenodo.7437258 genetic potential of novi sad winter wheat varieties on smonica-type soil original article dragan terzić faculty of agriculture, university of niš, kosančićeva 4, 37000 kruševac, republic of serbia vera rajičić faculty of agriculture, university of niš, kosančićeva 4, 37000 kruševac, republic of serbia verarajicic@yahoo.com (corresponding author) milan biberdžić faculty of agriculture, university of priština-kosovska mitrovica, kopaonička bb, 38228 lešak, serbia vesna perišić faculty of agriculture, university of niš, kosančićeva 4, 37000 kruševac, republic of serbia marijana dugalić faculty of agriculture, university of niš, kosančićeva 4, 37000 kruševac, republic of serbia dragoslav đokić faculty of agriculture, university of niš, kosančićeva 4, 37000 kruševac, republic of serbia snežana branković university of kragujevac, faculty of science, department of biology and ecology, radoja domanovića 12, kragujevac, republic of serbia received: september 01, 2022 revised: october 27, 2022 accepted: november 04, 2022 abstract: the field experiment with the wheat varieties simonida, ns 40 s, zvezdana, ns ilina, ns futura, ns mila and ns obala was set up on the smonica-type soil during the 2016/17 and 2017/18 growing seasons. the aim of the research was to analyse the yield, weight of 1000 grains and hectolitre weight of seven novi sad wheat varieties grown on slightly acidic soil. the highest values of the observed properties were established in the 2017/18 vegetation year with moderate temperatures and a higher amount of precipitation. varieties ns futura, ns obala and ns 40 s had the highest grain yield during the two-year study. the simonida variety was characterized by the highest hectolitre weight. the highest values of hectolitre weight were found in the zvezdana variety in both study years. the analysis of variance established a highly significant influence of the interaction of agro climatic conditions and genotype on the weight of 1000 grains and the hectolitre weight of the tested varieties of winter wheat. key words: grain yield, hectolitre weight, variety, wheat apstrakt: genetski potencijal novosadskih sorti ozime pšenice na zemljištu tipa smonice poljski ogled sa sortama pšenice simonida, ns 40 s, zvezdana, ilina, futura, mila i obala postavljen je na zemljištu tipa smonica tokom vegetacionih sezona 2016/17 i 2017/18 godine. cilj istraživanja je bio da se kod sedam novosadskih sorti pšenice gajene na slabo kiselom zemljištu analizira prinos, masa 1000 zrna i hektolitarska masa. najveće vrednosti ispitivanih osobina ustanovljene su u godini sa umerenim temperaturama i većom količinom padavina u vegetacionoj 2017/18 godini. sorte ns futura, ns obala i ns 40 s imale su najveći prinos zrna tokom dvogodišnjeg istraživanja. sorta simonida odlikovala se najvećom hektolitarskom masom. najveće vrednosti hektolitarske mase ustanovljene su kod sorte zvezdana u obe ispitivane godine. analizom varijanse ustanovljen je visoko značajan uticaj interakcije agroklimatskih uslova i genotipa na masu 1000 zrna i hektolitarsku masu kod ispitivanih sorti ozime pšenice. ključne reči: hektolitarska masa, prinos zrna, pšenica, sorta introduction winter wheat (triticum aestivum l.) is one of the most important agricultural crops in serbia. according to fao data, in year 2017 in the republic of serbia, wheat was harvested from 556,115 hectares with an average yield of 4.092 t/ha, while in 2018 it was harvested from 643,083 hectares with an average yield of 4.574 t/ha. statistical data show that in the past two-year period, wheat in the world has been grown on over 216,120,193 hectares with an average yield of 3,480 t/ha, while in the republic of serbia in the same period, the area under wheat has amounted to 599,599 ha with an average yield of 4,333 t/ha (fao, 2021). the average yields of wheat for the last 10 years in the main production areas of serbia have ranged from 4.5-8.0 t/ha. wheat production in serbia largely depends on external environmental factors. the average grain yields of winter wheat in our country and the fertility potential of cultivated varieties differ significantly, especially in the mountainous regions of serbia (jelić et al., 2015, 2016; jevtić & đekić, 2018). the yield and yield components of © 2022 terzić et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 129 winter wheat vary significantly depending on the cultivation system, applied nitrogen doses, variety and conditions present during growing season, as well as their complex interactions (đekić et al., 2012; tmušić et al., 2021; rajičić et al., 2021). in addition to the genotype, the grain yield of winter wheat is greatly influenced by the fertilization system, as one of the key factors that affects the formed yield and its quality, but it should be harmonized with climatic and soil conditions, as well as the requirements of the variety (đekić et al., 2014; terzić et al., 2018; rajičić et al., 2019; biberdžić et al., 2020b; đurić et al., 2020; luković et al., 2020). in the production of wheat, proper zoning of varieties is very important, and it can contribute to less variation in realized yields and achieving better average results (đurić et al., 2018; terzić et al., 2018; rajičić et al., 2020; grčak et al., 2020). with all that in mind, it is necessary that the climatic conditions are in accordance with the biological requirements of the plants. in the last few years, extreme temperatures and disturbances in the amount and distribution of precipitation have significantly influenced the reduction of the total production of organic matter and the reduction of yields (đurić et al., 2016; luković et al., 2017; grčak et al., 2018; đekić et al., 2019; popović et al., 2020; biberdžić et al., 2021b). the aim of the study was to analyse grain yield and some yield traits and examine the influence of varieties and ecological and environmental factors on the differences in stability and adaptability of the tested winter wheat genotypes. during two growing seasons, in field trials, seven winter wheat genotypes were examined in order to determine the selection of better varieties for production conditions in western serbia. materials and methods materials and field trials the trial was carried out on the experimental field of the secondary agricultural school with the student home “ljubo mićić” in požega (43°50´41´´n latitude, 20°02´11´´e longitude, 310 m above sea level), town and municipality in the zlatibor district of western serbia. during two growing seasons (2016/17 and 2017/18), in conditions of dry crop production, tests were carried out with the aim to analyse the yield and quality of winter wheat grains and determine and choose better varieties for production conditions in western serbia. the experiment was set up according to the random block system in four repetitions, with the size of the elementary plot 100 m2 (10 x 10 m). the soil on which the experiment was set up was the smonica-type soil. the main crop (preceding crop) in both years was corn. sowing was done with a small mechanical seeder in both years in the middle of the third decade of october, at an inter-row distance of 12.5 cm and 3 cm in a row. the amount of seeds per square meter was 400-450 germinated seeds, depending on the characteristics of the variety. the total amount of fertilizer 300 kg/ ha npk 15:15:15, where one third of nitrogen was distributed by hand on the ploughing surface before the pre-sowing soil preparation. at the beginning of intensive plant growth, in the 2-3 leaf phase, at the beginning of march, 200 kg/ha of kan (cancalcium-ammonium-nitrate, 27% n) was applied. wheat varieties simonida, ns 40 s, zvezdana, ns ilina, ns futura, ns mila and ns obala of the institute of field and vegetable crops, novi sad, were selected as material for the trial. the following properties were analysed: grain yield (t/ha), weight of 1000 grains (g) and hectolitre weight (kg/hl). at the stage of full maturity, a sample of 30 plants from five plots was taken to determine the weight of 1000 grains. after harvesting, the grain yield from each plot was measured and converted to yield in t/ha based on 14% grain moisture, after which a sample was taken for analysis of the hectolitre weight of wheat. soil conditions the trial was carried out on the experimental field of the secondary agricultural school with the student home “ljubo mićić” in požega, on a plot of smonicatype soil. the soil on which the research was carried out has unfavourable physical properties, poor water-air regime with frequent water deficiency. the soil is slightly acidic (ph<6.5), compact, with a high content of silt and clay particles and slow percolation of water. the soil is well supplied with humus (3.1%) and has good microbiological activity, due to the introduction of manure. it is characterized by a low content of available phosphorus (10.7 mg/100 g of soil) and an optimal content of potassium (20 mg/100 g of soil). meteorological conditions the požega area is characterized by a moderately continental climate, with an uneven monthly distribution of precipitation. the latitude of the meteorological station in požega is 43°50´41´´n, the longitude is 20°02´11´´e, and the altitude is 310 m above sea level. on the basis of the data provided by the meteorological station, the years in which the surveys were conducted differed from the multiannual average for the given area. the average air temperature in 2016/17 was lower by 0.33 ºc, while in 2017/18 it was higher by 0.91 ºc compared to the 130 biologica nyssana ● 13 (2) december 2022: 129-139 terzić et al. ● genetic potential of novi sad winter wheat varieties on smonica-type soil 131 multi-annual average (tab. 1). the data presented in tab. 1 for the studied vegetation period (2016-2018) clearly indicate that the years in which the tests were performed differed in terms of meteorological conditions from the multiannual average characteristic of this area. the total amount of precipitation during the trial was below the multi-annual average in the 2016/17 vegetation season, with a rather uneven distribution by month (501.1 mm), while in the same period during the second vegetation season years 2017/18, the amount of precipitation was higher and amounted to 605.9 mm, with an even monthly distribution (tab. 1). weather conditions in the growing season in 2016 and 2017 were marked by a high amount of precipitation in october, while in may 2016 and 2017 there was less precipitation compared to the multi-annual average. may 2017 and 2018 and june 2017 were characterized by changeable and moderately warm weather with less precipitation than average. the vegetation period 2016/17 was rainy, especially from the beginning of heading until harvest (may–june). in the same year, precipitation was lower than the multi-annual average from december to march. during the tillering period, which took place during march, the applied amounts of nitrogen fertilizers in the soil were not sufficiently used due to insufficient moisture, as the nitrogen fertilizer did not change into easily accessible forms for the plant and was not used by the plants. nitrogen, due to insufficient moisture, did not play the role of yield builder, which resulted in a decrease in yield in year 2017. in april, precipitation was above the multi-annual average. the total amount of precipitation was 39.4 mm less than the multiannual average (tab. 1). conditions for germination and initial growth of plants in the first year (2017/18) were significantly more favourable. the period from november to february was favourable for germination, sprouting and tillering of wheat. the continuation of tillering and the beginning of stem elongation during the month of march was with a slightly higher amount of precipitation compared to the multi-annual average. in the month of march, the precipitation was above the multi-annual average, which had a favourable effect on the period of fertilization and filling of grains (tab. 1). changeable and moderately warm weather with more precipitation than average marked june 2018. in the growing season 2017/18, the sum of precipitation was higher than the multiannual average by 65.4 mm. based on the fact that the amount of precipitation and temperature in the period march–june are very important for the development of wheat, the second year can be characterized as more favourable in terms of the distribution and amount of precipitation. temperature variations on average were greater in the first compared to the second growing season. vegetation period of wheat during testing in 2017/18 mostly took place in conditions of higher temperatures compared to the multi-annual average (tab. 1). relatively warm weather during the entire vegetation period with a relatively sufficient amount of precipitation enabled intensive germination, sprouting/shooting and normal development of all phases of winter wheat plant development. in the final stages of growth and development, during the month of june, high temperatures contributed to the early drying of leaves and accelerated ripening. statistical analysis experimental data were analysed by descriptive and analytical statistics using the statistics module analyst program genstat (2013) for pc/windows 7. the usual variational statistical indicator was calculated, average value (x), as well as standard deviation (s) and standard error (sx). evaluation of significance was made on the basis of the anova test at 5% and 1% significance levels. relative biologica nyssana ● 13 (2) december 2022: 129-139 terzić et al. ● genetic potential of novi sad winter wheat varieties on smonica-type soil table 1. mean monthly air temperatures and precipitations in požega, serbia months interval x xi xii i ii iii iv v vi average mean monthly air temperature (oc) 2016/17 9.7 4.6 -1.5 -6.6 2.9 8.3 9.4 15.3 20.4 6.94 2017/18 10.1 4.5 2.1 0.4 0.8 4.4 14.5 17.5 19.3 8.18 average 10.4 4.9 -0.1 -1.3 1.0 5.7 10.6 15.2 19.0 7.27 the amount of precipitation (mm) 2016/17 81.0 85.9 9.1 20.6 30.9 35.9 76.7 76.8 84.2 501.1 2017/18 108.1 29.8 68.9 48.5 76.7 109.6 20.4 48.5 95.4 605.9 average 60.6 54.3 54.3 41.1 46.1 52.7 60.1 82.2 89.1 540.5 132 dependence was defined through correlation analysis (pearson’s coefficient), and the values of coefficient that were obtained were tested at the 5% and 1% levels of significance. results grain yield and yield components the average values of the yield, weight of 1000 grains and hectolitre weight of the tested wheat varieties of the institute of field and vegetable crops from novi sad are shown in tab. 2. grain yield: in regard to grain yield, differences were observed between the tested varieties. the average yield of wheat for all tested genotypes in the experiment was 5.188 t/ha. the higher average grain yield of 5.585 t/ha of the tested wheat varieties was achieved in the 2017/18 growing season and was higher by 793 kg/ha compared to the yield in vegetation season 2016/17 (4.792 t/ha), which can mainly be related to the adequate distribution of precipitation during the second vegetation period (tab. 2). the highest grain yield in the 2016/17 vegetation season was realized with the ns futura variety (5.175 t/ha), but a good yield was also achieved with the ns obala variety (5.025 t/ha). in the second study year, the ns futura variety had the highest average grain yield (6.087 t/ha), but a good yield was also achieved with the ns obala (5.802 t/ha) and ns 40 s (5.697 t/ha) varieties. the variety ns fubiologica nyssana ● 13 (2) december 2022: 129-139 terzić et al. ● genetic potential of novi sad winter wheat varieties on smonica-type soil table 2. average values of investigated winter wheat varieties traits (x-average value, s-standard deviation and sx-standard error) varieties 2016/17 2017/18 average x s sx x s sx x s sx yield, t/ha simonida 4.253 0.251 0.126 5.447 0.329 0.165 4.850 0.693 0.245 ns 40 s 4.876 0.517 0.259 5.697 0.517 0.259 5.286 0.650 0.230 zvezdana 4.523 0.468 0.234 5.556 0.444 0.222 5.040 0.695 0.246 ns ilina 4.892 0.450 0.225 5.326 0.540 0.270 5.109 0.515 0.182 ns futura 5.175 0.192 0.096 6.087 0.580 0.290 5.631 0.631 0.223 ns mila 4.799 0.502 0.251 5.178 0.319 0.160 4.989 0.439 0.155 ns obala 5.025 0.126 0.063 5.802 0.580 0.290 5.414 0.569 0.201 average 4.792 0.451 0.085 5.585 0.516 0.097 5.188 0.625 0.083 1000 grain weight, g simonida 43.45 1.072 0.536 50.73 0.348 0.174 47.09 3.965 1.402 ns 40 s 42.72 0.644 0.322 44.96 1.173 0.586 43.84 1.485 0.525 zvezdana 41.03 1.757 0.879 43.52 1.683 0.841 42.28 2.076 0.734 ns ilina 36.72 0.769 0.384 43.22 1.408 0.704 39.97 3.632 1.284 ns futura 42.54 0.853 0.426 44.36 0.844 0.422 43.45 1.251 0.442 ns mila 42.03 0.755 0.378 41.44 0.779 0.390 41.73 0.778 0.275 ns obala 41.64 1.134 0.567 44.77 0.505 0.253 43.20 1.860 0.658 average 41.44 2.296 0.434 44.71 2.899 0.548 43.08 3.072 0.410 hectolitre weight, kg/hl simonida 75.05 0.816 0.408 77.00 0.252 0.126 76.03 1.183 0.418 ns 40 s 74.50 0.443 0.222 78.16 0.278 0.139 76.33 1.987 0.703 zvezdana 77.35 0.476 0.238 79.00 0.503 0.252 78.18 0.992 0.351 ns ilina 71.90 2.253 1.127 78.18 0.708 0.354 75.04 3.693 1.306 ns futura 74.60 0.755 0.378 77.55 0.258 0.129 76.08 1.661 0.587 ns mila 70.90 1.969 0.984 76.70 1.112 0.556 73.80 3.435 1.215 ns obala 73.65 0.231 0.115 73.80 0.252 0.126 73.73 0.237 0.084 average 73.99 2.280 0.431 77.20 1.668 0.315 75.60 2.556 0.341 133 biologica nyssana ● 13 (2) december 2022: 129-139 terzić et al. ● genetic potential of novi sad winter wheat varieties on smonica-type soil tura had the highest average grain yield during the two-year study (5.631 t/ha), and varieties simonida (4.850 t/ha) and ns mila (4.989 t/ha) the lowest. 1000 grain weight: the grain of the tested wheat varieties had good physical properties, especially the 1000 grain weight. the weight of 1000 grains in 2017/18 was significantly higher than in 2016/17 (tab. 2). the vegetation period in year 2017, at the time of grain filling, was marked by drought and high temperatures, which affected the reduction of 1000 grain weight. the highest 1000 grain weight during the two-year research period was recorded for the simonida variety (47.09 g), and the lowest for the ns ilina variety (39.97 g). the simonida variety had the highest 1000 grain weight in the 2017 vegetation year (43.45 g), and the ns ilina variety had the lowest (36.72 g). in the second year of research, the simonida variety had the highest 1000 grain weight (50.73 g), and the lowest was recorded for the ns mila variety (41.44 g). test weight: hectolitre weight is an indicator of grain quality, especially its monetary value. the obtained data on the hectolitre weight, regardless of the year, showed that there was a significant difference between the genotypes, with the zvezdana variety having the highest hectolitre weight (78.18 kg/hl) on average for the trial years. the average value of hectolitre weight during the two-year research was 75.60 kg/hl. the higher average value of hectolitre weight was achieved in the 2017/18 growing season (77.20 kg/hl) compared to the in-vegetation season 2016/17 (73.99 kg/hl), (tab. 2). in the first research year, the zvezdana variety had the highest hectolitre weight (77.35 kg/hl), while in the second year, the highest hectolitre weight was observed for the zvezdana variety (79.00 kg/hl) and with a slightly lower value for the varieties ns ilina (78.18 kg/hl) and ns 40 s (78.16 kg/hl). analysis of variance between observed traits of wheat the analysis of yield variance, weight of 1000 grains and hectolitre weight of the tested winter wheat varieties from novi sad during two vegetation seasons are shown in tab. 3. the influence of year and variety and their interactions on the tested properties of winter wheat are shown in tab 3. based on the analysis of variance, it can be concluded that the interaction of variety and year in tested wheat varieties did not significantly affect the grain yield, while it had a very significant effect on 1000 grain weight (fexp=13.456**) and hectolitre weight (fexp=10.742**) in tested varieties of winter wheat. the influence of year on the yield, 1000 grain weight and hectolitre weight of tested winter wheat varieties was highly significant. no significant influence of the variety on grain yield was found, among the tested wheat genotypes, while highly significant differences were found in the weight of 1000 grains and hectolitre weight. correlation dependence between tested wheat traits the average values of pearson’s correlation coeftable 3. analysis of variance of the tested parameters (anova) *statistically significant (p<0.05); **statistically high significant (p<0.01) effect of year on the traits analyzed traits mean sqr effect mean sqr error f (1.54) p-level grain yield (t/ha) 8.801 0.234 37.524 0.000** 1000 grain weight (g) 149.635 6.839 21.879 0.000** hectolitre weight (kg/hl) 143.840 3.990 36.052 0.000** effect of genotype on the traits analyzed traits mean sqr effect mean sqr error f (6.49) p-level grain yield (t/ha) 0.585 0.366 1.597 0.168 ns 1000 grain weight (g) 38.595 5.865 6.581 0.000** hectolitre weight (kg/hl) 19.525 4.942 3.951 0.003** effect of the year x genotype interaction traits mean sqr effect mean sqr error f (6.42) p-level grain yield (t/ha) 0.178 0.192 0.924 0.488 ns 1000 grain weight (g) 15.101 1.122 13.456 0.000** hectolitre weight (kg/hl) 9.920 0.923 10.742 0.000** ficient (r) of the tested traits of winter wheat are shown in tab. 4. correlations between grain yield and hectolitre weight of the tested genotypes of winter wheat at the požega location show a negative value in both investigated vegetation seasons. correlations between grain yield and 1000 grain weight show a negative value in the first year of research (2016/17), while correlations between 1000 grain weight and hectolitre weight show a negative value in the vegetation year 2017/18. correlations between grain yield and 1000 grain weight and between the 1000 grain weight and hectolitre weight of winter wheat genotypes tested at the požega location during the two-year study show a positive and highly significant value. correlations between grain yield and hectolitreweight, during the two-year study, show a positive and significant value. discussion adverse weather conditions such as frost and temperature stress, and short periods with high temperatures, have a strong negative impact on plant production and represent a great risk (rajičić et al., 2020; biberdžić et al., 2021a; ljubičić et al., 2021). according to lalić et al. (2014), the negative impact of high temperatures on winter wheat is reflected in a reduced number of grains, an increase in the intensity of grain filling (up to a certain limit), as well as a shortening of the duration of the filling period, which results in a decrease in yield when temperatures exceed the critical level above 35 oc. in numerous studies, soil drying or shortening of phenophases during the growing season are reported as the cause of winter wheat yield reduction with increasing temperatures (mohamed et al., 2013). in their research, ottman et al. (2012), report that wheat yield decreased by 6.9% with each degree increase in temperature during the season when it exceeded 16.3 °c. a 2.5 °c increase in night-time temperatures in northern china resulted in a 27% drop in wheat yields (fang et al., 2010). the significant deviation of precipitation and temperature from the multi-annual average is becoming more pronounced (dimitrijević et al., 2011; jocković et al., 2014). it has been established that the newly created high-yielding varieties of wheat react less to temperature deviations (except for extremes), compared to precipitation (milovanović et al., 2012; đekić et al., 2015, 2017). namely, the total amount of precipitation is maintained at the multi-annual average, but the distribution, especially in critical stages of development, is significantly disturbed (đekić et al., 2013; zafaranaderi et al., 2013). it has been established that winter precipitation significantly affects the realization of the production potential of wheat (laghari et al., 2011; luković et al., 2016). in addition to the necessary reserve for the spring part of the vegetation, winter precipitation greatly affects the distribution of easily accessible nitrogen in the soil (biberdžić et al., 2014; terzić et al., 2018; rajičić et al., 2019). the variety, as an autonomous genetic, biological, and agronomic entity, is one of the decisive factors on both the quantitative and qualitative level of production (đurić et al., 2020). the increase in 134 biologica nyssana ● 13 (2) december 2022: 129-139 terzić et al. ● genetic potential of novi sad winter wheat varieties on smonica-type soil table 4. correlation coefficients by studied environments in winter wheat *statistically significant (p<0.05); **statistically high significant (p<0.01) grain yield, gy 1000 grain weight, gw hectolitre weight, hw correlations between the traits analysed in the 2016/17 grain yield, gy 1.00 -0.037 -0.232 1000 grain weight, gw 0.295 hectolitre weight, hw 1.00 correlations between the traits analysed in the 2017/18 grain yield, gy 1.00 0.045 -0.129 1000 grain weight, gw -0.066 hectolitre weight, hw 1.00 correlations between the traits analysed in the 2016-2018 grain yield, gy 1.00 0.351** 0.297* 1000 grain weight, gw 0.416** hectolitre weight, hw 1.00 135 biologica nyssana ● 13 (2) december 2022: 129-139 terzić et al. ● genetic potential of novi sad winter wheat varieties on smonica-type soil wheat yield primarily depends on the cultivated variety, climatic conditions and applied cultivation technology (jelić et al., 2015; đekić et al., 2018). the introduction into production of varieties with increased genetic potential for yield and improved agronomic and technological properties represents the contribution of breeding to achieving higher production per unit area (perišić et al., 2016; branković et al., 2018; đurić et al., 2018). the genetic potential for yield can be increased in different ways: better use of genetic variability, better use of solar energy, increasing the number and weight of grains, increasing the total biomass of the plant, using heterosis, i.e., wheat hybrid (đurić et al., 2016). grain yield: grain yield is a complex property because it depends on several factors, primarily: genotype, external environment, and their interaction. a high and stable yield, first of all, must be economically justified, which can be achieved, by respecting varietal agrotechnics and with favourable agro climatic conditions. the results of our two-year study show that the grain yield of the tested wheat varieties was 5.188 t/ha (tab. 2). the 2018 production year had a higher average grain yield compared to the 2017 production year, which was mostly contributed by favorable weather conditions. the yield of winter wheat in 2018 was 5.585 t/ha and varied in the range from 5.178 t/ha to 6.087 t/ha. the lower quality and quantity of the yield in the 2017 production year was influenced by unfavourable temperature conditions and uneven distribution of precipitation in periods of the year that coincided with the sensitive stages of wheat development, which affected the yield, which was 4.792 t/ha (tab. 2). temperature, precipitation, and sufficient amount of water in the soil are the three most important reasons for yield instability in our area. in the ecological conditions of serbia, high temperatures and water deficit during the may-june period led to a reduction in yield and a deterioration in the technological properties of grain, which is why by extending the total vegetation period the period of grain filling for the purpose of yield increase cannot be extended. živanović et al. (2017), show slightly lower wheat yields on vertisol-type land in šumadija (from 4.31 t/ha to 4.44 t/ha), obtained during year 2017. đurić et al. (2013), state that the yields in the trial were very high and ranged from 8.00 t/ha of grain in case of the variety esperia to 9.60 t/ha in pobeda and talas varieties. however, in the first three varieties talas, victory and ns 40 s, difference in yield was about 400 kg and that is negligible considering the total yield. hristov et al. (2013) state that in a threeyear period, all varieties, except pobeda, achieved a higher yield at a higher density. the lower yield of the pobeda variety was primarily the result of longstanding water on the plot. also, with the exception of the problematic year 2010, varieties pobeda (10.76 t/ha) and renesansa (11.05 t/ha) achieved higher yields in 2011, indicating that in order to make correct conclusions, it is necessary to analyse a larger number of localities over a longer period of time. đekić et al. (2013), examined five winter wheat varieties (takovčanka, kg 100, kg 56s, ana morava and lazarica). the average grain yield of the tested wheat varieties ranged from 3.011 t/ha to 3.774 t/ha. in regard to the physical characteristics of the grain, the ana morava variety achieved the highest average yield in both growing seasons (4.499 t/ha and 3.049 t/ha). terzić et al. (2018) point out that the average yield of wheat in the trial was 5.091 t/ha, ranging from 4.630 t/ha in the second year to 5.553 t/ha in the first year of the study. the highest grain yield in the 2010/11 vegetation year was established with the vizija variety (6.127 t/ha), but a good yield was also achieved with the kg 56s variety (5.863 t/ha). in the second year of research, the variety kg 56s had the highest average grain yield (5.159 t/ha). the highest average grain yield in the two-year period was observed for the varieties kg 56s (5.511 t/ha) and vizija (5.485 t/ha), and the lowest for the variety aleksandra (4.628 t/ha). đurić et al. (2016) point out that the pkb vizantija variety achieved an average grain yield of 8.356 t/ ha. rajičić et al. (2021) have found that the highest winter wheat yield of 4.673 t/ha was obtained with the renesansa variety. analysis of variance showed a very significant influence of vegetation on wheat yield. 1000 grain weight: the 1000 grain weight is a direct component of yield and at the same time an important indicator of grain quality. it represents a highly variable property which depends to a high degree on the conditions of the external environment. numerous authors (milovanović et al., 2011; đekić et al., 2012; đurić et al., 2013) have pointed out that the 1000 grain weight is a varietal characteristic and that there is a significantly greater variation between different genotypes than between applied treatments or external environmental factors. during the two-year trial, the grain of the tested wheat varieties was characterized by good physical properties, especially the 1000 grain weight. the 1000 grain weight during the two-year research period was 43.08 g, ranging from 39.97 g to 47.09 g. the obtained values are close to the values obtained by jevtić and đekić (2018) and terzić et al. (2018), and slightly higher than the results obtained by biberdžić et al. (2020a). đurić et al. (2013) state that the weight of 1000 grains ranged from 38.80 biologica nyssana ● 13 (2) december 2022: 129-139 terzić et al. ● genetic potential of novi sad winter wheat varieties on smonica-type soil g in the variety ns 40 s to 46.50 g in the variety pobeda. đekić et al. (2013) point out that the average value of the 1000 grain weight in the tested wheat varieties ranged from 36.23 to 42.70 g. examining different variants of fertilization in the wheat variety takovčanka, đekić et al. (2014) have found that the weight of 1000 grains was the highest with balanced fertilization with all three nutrients in the variant n80p60k60 (44.46 g) and variant n80p100k60 (43.38 g). đekić et al. (2015a), point out that in the first year of research, the vizija variety had the highest weight of 1000 grains (42.65 g), while the kruna variety had a slightly lower value (41.63 g). in the second year of research, the weight of 1000 grains of the vizija variety was by 1.036 g higher than the kruna variety. đekić et al. (2015b), have examined six winter wheat varieties from kragujevac, with the aim of determining and choosing the best varieties for serbia’s production conditions. based on a twoyear study, they concluded that the highest two-year average value of the weight of 1000 grains was observed in the variety planeta (44.22 g), while the lowest was reported for the variety vizija (37.53 g). the results of the research by jelic et al. (2015), show that the highest 1000 grain weight of 47.06 g was achieved with the complete application of mineral npk, lime and manure with a higher dose of caco3 of 5 t/ha and phosphorus of 100 kg/ha p2o5. terzić et al. (2018) point out that the average weight of 1000 grains was 43.13 g, ranging from 42.05 g to 44.21 g. in the study of five varieties of wheat (kruna, renesansa, pobeda, ns 40 s and takovčanka) during two growing seasons in the agro ecological conditions of western serbia, rajičić et al. (2021) have found that the highest weight of 1000 grains was obtained in case of the variety renesansa (43.72 g). by analysis of variance, the same authors have found a highly significant influence of genotype on the weight of 1000 grains. hectolitre weight: hectolitre weight is a complex quantitative trait that is determined by the action of a large number of genes under the strong influence of the external environment (đurić et al., 2013). the average value of hectolitre weight during the twoyear research was 75.60 kg/hl, ranging from 73.73 kg/hl to 78.18 kg/hl. the highest value of hectolitre weight in both examined years was achieved by the variety zvezdana (77.35 kg/hl and 79.00 kg/hl). the obtained hectolitre weight values were slightly higher than the values obtained by jelic et al. (2015), jevtić and đekić (2018) and biberdžić et al. (2020a), and lower than the results obtained by đurić et al. (2013). it is generally considered that grain with a higher hectolitre weight is of better quality compared to that with lower values (đurić et al., 2013; đekić et al., 2015b; rajičić et al., 2021). đurić et al. (2013) state that the obtained hectolitre weight values are relatively high, which indicates a good marketable quality, ranging from 78.9 kg/hl for the ns 40 s variety to 84.4 kg/hl for the merkur variety. đekić et al. (2014) have found the highest value of hectolitre weight in the variant with individual fertilization with 60 kg/ha of phosphorus (71.57 kg/hl) and in the variant that was fertilized with 80 kg/ha of nitrogen, 60 kg/ha of phosphorus and 60 kg/ha of potassium (71.49 kg/hl). by evaluating the significance, they have found statistically very significant differences for the hectolitre weight between the tested fertilization variants. đekić et al. (2015b) by using the analysis of variance, have established highly significant differences for the hectolitre weight values between the tested wheat varieties. according to the results of jelić et al. (2015), observed by fertilization variants, the highest hectolitre weight of grain was recorded for variant f5 (70.91 kg/hl), which was fertilized with 120 kg/ha n, 100 kg/ha p2o5, 60 kg/ha k2o, 5.0 t/ha of caco3 and with 20 t/ha of manure. jevtić and đekić (2018) point out that the hectolitre grain weight of the tested wheat varieties, during the two-year study, ranged from 69.5 to 71.03 kg/hl. regarding the optimal wheat sowing date, biberdžić et al. (2020a) have found that the hectolitre weight of grain was 71.42 kg/hl, while in the case of later sowing it was 69.96 kg/hl, which does not represent a statistically significant difference. rajičić et al. (2021) have found that the highest two-year average value of hectolitre weight was observed for varieties kruna and pobeda (77.52 kg/hl and 77.31 kg/hl, respectively). they established a very significant influence of vegetation and genotype on the hectolitre weight of wheat. conclusions based on the obtained results, it can be concluded that the winter wheat variety ns futura achieved the best results in terms of grain yield, while the simonida variety had the highest of 1000 grain weight, and the variety zvezdana had the highest hectolitre weight in the observed two-year period. the highest values for yield of wheat grains, 1000 grain weight and hectolitre weight, were observed in the 2017/18 vegetation season with moderate temperatures at the time of grain filling and a large amount of precipitation evenly distributed in the second part of the vegetation season. the grain yield of the tested wheat varieties in the two-year period ranged from 4.850 t/ha (simonida) to 5.631 t/ha (ns futura). the average hectolitre weight during the two-year research was 75.60 kg/hl, with 73.99 kg/hl in the 2016/17 growing season and 77.20 kg/hl in the 136 2017/18 vegetation season. grain yield showed a tendency to increase in years with higher sum of precipitation and better distribution of rainfall during critical stages of plant development. the analysis of variance established a very significant influence of the interaction of year x variety on the weight of 1000 grains and the hectolitre weight of the examined wheat varieties, while the influence of the vegetation season on all the examined traits was statistically justified. many traits play a decisive role in the formation of grain yield. the contribution of each individual trait can be different in different genotypes and in different environmental conditions. based on the obtained results, it can be concluded that the sowing structure should be based on more than one variety, in order to reduce the risk of the unpredictability of each individual vegetation, regardless of the reliability of the criteria for selection of variety for sowing in a particular research year. acknowledgements. ph.d. dragan terzić and authors sincerely thanks to the secondary agricultural school with the student dormitory “ljubo mićić” in požega in serbia, which has enabled the use and analysis of traits of the wheat. investigations necessary for this paper are part of the projects agreement number 451-03-09/202114/200383 financed by the ministry of education, science and technology development of republic of serbia. references biberdžić, m., jelić, m., knežević, b., barać, s., lalević, d. 2014: yield of wheat in pseudogley in dependence of fertilisation with mineral fertilisers, lime fertiliser and manure. agriculture & forestry, 60(4): 7-13. biberdžić, m., barać, s., deletić, n., stojković, s., madić, m., lalević, d., đekić, v., stojiljković, j. 2020a: the effect of sowing time on the yield of some wheat varieties. proceedings of the 2st international symposium “modern trends in agricultural production and environmental protection”, 01-04 july, tivat, montenegro, 66-75. biberdžić, m., barać, s., lalević, d., đikić, a., prodanović, d., rajičić, v. 2020b: influence of soil tillage system on soil compactionand winter wheat yield. chilean journal of agricultural research, 80(1): 80-89. biberdžić, m., lalević, d., barać, s., stojiljković, j., madić, m., prodanović, s., rajičić, v. 2021a: yield of some wheat varieties depending on fertilization with a combination of mineral fertilizers and zeolites. proceedings of the 3st international symposium “modern trends in agricultural production, rural devalopment and environmental protection”, 01-03 july, vrnjacka banja, serbia, 188-197. biberdžić, m., lalević, d., barać, s., đikić, a., deletić, n., stojković, s., rajičić, v. 2021b: influence of acid soil fertilization on the yield of some wheat varieties. proceedings of the 3st international symposium “modern trends in agricultural production, rural devalopment and environmental protection”, 01-03 july, vrnjacka banja, serbia, 198-205. branković, g., dodig, d., pajić, v., kandić, v., knežević, d., đurić, n., živanović, t. 2018: genetic parameters of triticum aestivum and triticum durum for technological quality properties in serbia. zemdirbyste-agriculture, 105(1): 39-48. dimitrijević, m., knežević, d., petrović, s., zečević, v., bošković, j., belić, m., pejić, b., banjac, b. 2011: stability of yield components in wheat (triticum aestivum l.). genetika, 43(1): 2939. đekić, v., milovanović, m., staletić, m., stevanović, v., milivojević, j. 2012: influence of growing season on some agronomic characteristics of six winter wheat cultivars grown in acidic soil. proceedings of 47th croatian and 7th international symposium on agriculture, 13.-17. februar, opatija, croatia, 478-482. đekić, v., staletić, m., jelić, m., popović, v., branković, s. 2013: the stability properties of wheat productionon acid soil. proceedings of 4th international symposium “agrosym 2013”, 03-06. oktober, jahorina, 84-89. đekić, v., glamočlija, đ., jelić, m., simić, d., perišić, v., perišić, v., mitrović, m. 2014: uticaj đubrenja na prinos pšenice. zbornik naučnih radova instituta pkb agroekonomik, 20(1-4): 41-48. đekić, v., milovanović, m., milivojević, j., staletić, m. popović, v., simić, d., mitrović, m. 2015a: uticaj godine na prinos i kvalitet zrna ozime pšenice. zbornik naučnih radova instituta pkb agroekonomik, beograd, 21(1-2): 79-86. đekić, v., milovanović, m., milivojević j., jelić, m., popović, v., branković, s., perišić, v. 2015b: uticaj godine na prinos i kvalitet zrna ozimih sorti pšenice. zbornik radova xx savetovanja o biotehnologiji sa međunarodnim učešćem, 13-14. mart, čačak, 20 (22): 39-44. đekić, v., milivojević, j., jelić, m., popović, v., branković, s., biberdžić, m., terzić, d. 2017: effects of fertilization on production traits of takovčanka cultivar of winter wheat. proceedings of the 2nd international and 14th national congress terzić et al. ● genetic potential of novi sad winter wheat varieties on smonica-type soil biologica nyssana ● 13 (2) december 2022: 129-139 137 of soil science society of serbia “solutions and projections for sustainable soil management”. nsoil, 25-28 september 2017, novi sad, serbia, 4954. đekić, v., milivojević, j., popović, v., jovović, z., branković, s., terzić, d., ugrenović, v. 2018: effects of fertilization on production traits of winter wheat. proceedings of the green room sessions international gea conference “geo eco-eco agro 2018”, 1-3 november, podgorica, montenegro, 2531. đekić, v., milivojević, j., madić, m., popović, v., branković, s., perišić, s., terzić, d. 2019: grain yield and its components in winter barley grown. journal of central european agriculture, 20(1): 238-250. đurić, n., trkulja, v., simić, d., prodanović, s., đekić, v., dolijanović, ž. 2013: analiza prinosa zrna i kvaliteta brašna nekih sorata ozime pšenice u proizvodnoj 2011-2012. godini. zbornik naučnih radova instituta pkb agroekonomik, 19(1-2): 1521. đurić, n., cvijanović, g., dozet, g., matković, m., branković, g., đekić, v. 2016: correlation analysis of more significant production traits of certain winter wheat pkb varieties. agronomy journal, 78(2-3): 85-96. đurić, n., prodanović, s., branković, g., đekić, v., cvijanović, g., zilić, s., dragičević, v., zečević, v., dozet, g., 2018: correlation-regression analysis of morphological-production traits of wheat varieties. romanian biotechnological letters, 23(2): 13457-13465. đurić, n., grčić, v., cvijanović, g., rajičić, v., branković, g., poštić, d. 2019: the influence of year and locality on yield of grain and some characteristics of winter wheat brand. agronomy journal, 81(5): 291-304. đurić, n., cvijanović g., rajičić, v., branković g., poštić d., cvijanović, v. 2020: analysis of grain yield and flour quality of some winter wheat varieties in the 2020 production. agronomski glasnik, croatia, 82(5-6): 253-262. fang, s., tan, k., ren, s. 2010: winter wheat yields decline with spring higher night temperature by controlled experiments. science agriculture sinica, 43: 3251-3258. faostat 2021: available online: http://faostat. fao.org (accessed on 8 jul 2021). genstat release 16.2 (pc/windows 7) 2013: genstat procedure library. release pl24.2. vsn international ltd. rothamsted, uk. biologica nyssana ● 13 (2) december 2022: 129-139 terzić et al. ● genetic potential of novi sad winter wheat varieties on smonica-type soil m., đekić v., aksić m. 2018: comparison of maize and wheat production in serbia during the 20072016 periods. agro-knowledge journal, 19(3), 199210. grčak, m., grčak, d., penjišević, a., simjanović, d., orbović, b., đukić, n., rajičić, v. 2020: the trends in maize and wheat production in the republic of serbia. acta agriculturae serbica, 25(50), 121127. hristov, n., mladenov, n., kondić-špika, a., jocković, b. 2013: uticaj padavina i temperature na prinos ozime pšenice pri različitim gustinama setve. zbornik naučnih radova instituta pkb agroekonomik, 19(1-2): 31-38. jelić, m., milivojević, j., nikolić, o., đekić, v., stamenković, s. 2015: effect of long-term fertilization and soil amendments on yield, grain quality and nutrition optimization in winter wheat on an acidic pseudogley. romanian agricultural research, 32: 165-174. jelić, m., milivojević, j., paunović, a., madić, m., đekić, v. 2016: adaptation of winter wheat genotypes to low ph and high mobile al content in the soil. proceedings of vii international scientific sympozium “agrosym jahorina 2016”, 06-09 october, jahorina, 762-767. jevtić, a., đekić v. 2018: influence of growing season on some agronomic characteristics of winter wheat cultivars. biologica nyssana, 9(2): 133-139. jocković, b., mladenov, n., hristov, n., aćin, v., đalović, i. 2014: interrelationship of grain filling rate and other traits that affect the yield of wheat (triticum aestivum l.). romanian agricultural research, 31: 1-7. lalić, b., eitzinger, j., thaler, s., vučetić, v., nejedlik, p., eckersten, h., jaćimović, g., nikolićđorić, e. 2014: can agrometeorological indices of adverse weather conditions help to improve yield prediction by crop models? atmosphere, 5(4): 1020-1041. laghari, g.m., oad, f.c., tunio, s., chachar, q., gandahi, a.w., siddiqui, m.h. 2011: growth and yield attributes of wheat at different seed rates. sarhad journal of agriculture, 27: 177-183. luković, k., milovanović, m., prodanović, s., perović, d., perišić, v., staletić, m., đekić v. 2016: kg variety olimpija contribution to biodiversity of spring wheat in serbia. proceedings of xx international eco-conference® 2016, 9th ecoconference® on safe food, 28-30. september 2016, novi sad, 73-82. 138 luković, k., prodanović, s., perišić, v., milovanović, m., perišić, v., rajičić, v., zečević, v. 2020: multivariate analysis of morphological traits and the most important productive traits of wheat in extreme rainfall conditions. applied ecology and environmental research, 18 (4), 58575871. ljubičić, n., popović, v., ćirić, v., kostić, m., ivošević, b., popović, d., pandžić, m., el musafah, seddiq, janković, s. 2021: multivariate interaction analysis of winter wheat grown in environment of limited soil conditions. plants-basel, 10(3): 604. milovanović, m., staletić, m., đekić, v., nikolić, o., luković, k. 2011: seed production and contribution of kg varieties to biodiversity of small grains in the period 2006-2010. 01.-02. december 2011, beograd, economics of agriculture, ii(58): 103-111. milovanović, m., staletić, m., rajičić, v., nikolić, o., perišić, v. 2012: actualities of hard winter wheat breeding in center for small grains in kragujevac. xvi international eco-conference, novi sad, proceedings safe food, 115-123. mohamed, n., said, a., amein, k. 2013: additive main effects and multiplicative interaction (ammi) and gge-biplot analysis of genotype × environment interactions for grain yield in bread wheat (triticum aestivum l.). african journal of agricultural research, 8(42): 5197-5203. ottman, m.j., kimball, b.a., white, j.w., wall, g.w. 2012: wheat growth response to increased temperature from varied planting dates and supplemental infrared heating. agronomy journal, 104: 7-16. perišić, v., perišić, v., živanović, t., milovanović, m., đekić, v., staletić, m., luković, k. 2016: comparative stability analysis of yield components of wheat genotypes. proceedings of xx international eco-conference® 2016, 9th eco-conference® on safe food, 28-30. september 2016, novi sad, 63-72. popović, v., ljubičić, n., kostić, m., radulović, m., blagojević, d., ugrenović, v., popović, d., ivošević, b. 2020: genotype x environment interaction for wheat yield traits suitable for selection in different seed priming conditions. plants, 9(12): 1804. rajičić, v., milivojević, j., popović, v., branković, s., đurić, n., perišić, v., terzić, d. 2019: winter wheat yield and quality depending on the level of nitrogen, phosphorus and potassium fertilization. agriculture and forestry, 65 (2): 79-88. rajičić, v., terzić, d., perišić, v., dugalić, m., madić, m., dugalić, g., ljubičić, n. 2020: impact of long-term fertilization on yield in wheat grown on soil type vertisol. agriculture & forestry, 66 (3): 127-138. rajičić, v., terzić, d., babić, v., perišić, v., dugalić, m., đokić, d., branković, s. 2021: yield components and genetic potential of winter wheat on pseudogley soil of western serbia. biologica nyssana, 12(2): 141-150. terzić, d., đekić, v., jevtić, s., popović, v., jevtić, a., mijajlović, j., jevtić, a. 2018: effect of long term fertilization on grain yield and yield components in winter triticale. the journal of animal and plant sciences, 28(3): 830-836. terzić, d., đekić, v., milivojević, j., branković, s., perišić, v., perišić, v., đokić, d. 2018: yield components and yield of winter wheat in different years of research. biologica nyssana, 9(2): 119-131. tmušić, n., ćirić, s., nikolić, k., knežević, j., rajičić, v. 2021: effects of different fertilization treatments on the yield performance, yield parameters and grain quality of winter wheat grown on vertisol soil type. applied ecology and environmental research, 19(6): 4611-4627. zafaranaderi, n., aharizad, s., moha-mmadi, s.a. 2013: relationship between grain yield and related agronomic traits in bread wheat recombinant inbred lines under water deficit condition. annals of biological research, 4(4): 7-11. živanović, lj., popović, v., ikanović, j., kolarić, lj. 2017: uticaj količine i oblika azota na produktivnost ozime pšenice. “xxii savetovanje o biotehnologiji” zbornik radova, knjiga 1. čačak. terzić et al. ● genetic potential of novi sad winter wheat varieties on smonica-type soil biologica nyssana ● 13 (2) december 2022: 129-139 139 mohammadi et al. 2020, biologica nyssana 11(1) 11 (1) september 2020: 45-54 doi: 10.5281/zenodo.4060294 a real-time pcr assay for evaluation of drought tolerance in a new tea clone original article narjes mohammadi department of biology, faculty of science, university of guilan, rasht, iran mohammadi_narges32@yahoo.com akbar norastehnia department of biology, faculty of science, university of guilan, rasht, iran norasteh@guilan.ac.ir (corresponding author) mohannad m. sohani department of biology, faculty of science, university of guilan, rasht, iran msohani@guilan.ac.ir korosh falakroo tea research institute, lahijan, iran k.falakro@areo.ir received: september 14, 2019 revised: february 24, 2020 accepted: march 23, 2020 abstract: drought stress induces oxidative stress with subsequent increases in reactive oxygen species ros in plants. to evaluate oxidative stress intensity with re-irrigation, changes in superoxide dismutase (sod) expression were investigated by real-time pcr in two tea clones, dn and 100. results showed that sod expression and other measured factors, except for carotenoids, increased under drought stress in clone dn, but the changes were not significant in clone 100, compared to control. all of the meaningful increases in evaluated enzymatic and non-enzymatic antioxidant factors were eliminated with re-irrigation. the observed changes indicate that clone dn activated its antioxidant defense system in response to drought. in contrast, the lack of response in clone 100 indicates higher levels of tolerance of this new clone against drought. key words: drought, oxidative stress, proline, superoxide dismutase, tea apstract: ukupan sadržaj fenola i antioksidativni potencijal različitih varijeteta brassica oleracea stres izazvan sušom indukuje oksidativni stres sa naknadnim povećanjem ros-a u biljkama. da bi se procenio intenzitet oksidativnog stresa ponovnim navodnjavanjem, ispitivane su promene ekspresije superoksid dismutaze (sod) pomoću pcr-a u realnom vremenu u 2 čajna klona dn i 100. rezultati su pokazali da ekspresija sod-a i drugih merenih faktora, osim karotenoida, povećava stres izazvan sušom u klonu dn, dok promene nisu bile značajne u klonu 100, u poređenju sa kontrolom. sva značajna povećanja procenjenih enzimskih i neenzimskih antioksidativnih faktora eliminisana su ponovnim navodnjavanjem. uočene promene pokazuju da je klon dn aktivirao svoj antioksidativni odbrambeni sistem kao odgovor na sušu. suprotno tome, nedostatak odgovora u klonu 100 ukazuje na viši nivo tolerancije ovog novog klona na sušu. ključne reči: suša, oksidativni stres, prolin, superoksid dizmutaza, čaj introduction plants are often exposed to many stresses, such as drought, high temperatures, and high salt levels, heavy metals and finally oxidative stress. drought is one of the environmental stresses which has harmful effects on all stages of growth and triggers the production of ros in plants (farooq et al., 2009). the amount of damage caused by water shortage depends on the plant species, genotype, duration and intensity of water deficit, soil characteristics, age and developmental stage of the plant (sánchez et al., 2001). in addition to the physiological changes that occur in plants due to water shortages, stress also inhibits photochemical activity and reduces calvin cycle activity (monakhova & chernyad’ev, 2004) accumulation of ros is one of the many biochemical changes which occurs in plants under stress, as an inevitable and normal product of cell metabolism and oxidative damage caused by ros is the major limiting factor for plant growth (allen & ort, 2001). while there are several sources of ros production, and whereas some of the most common sources of ros are the electron transport chains in mitochondria and chloroplasts, chloroplasts are the main source of ros production in plants (noctor et al., © 2020 mahammadi et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 45 2014). plants respond to increased ros amounts with activation of their enzymatic and non-enzymatic defense systems (zhu, 2001; mukhopadhyay ,2014). among the most important crops subjected to stress in iran is tea. the total area under tea cultivation in the north of iran is over 40 thousand hectares. 4-4,5 percent of the total tea in the world is produced in iran. it is essential to elevate the amount of product produced per unit to match the increasing consumption of tea. one important strategy for achieving this goal is reducing the environmental damage caused by stresses such as dehydration. although most tea gardens in iran are located in areas with high rainfall, reduced crop yields are nevertheless encountered due to the unequal distribution of rainfall in different seasons, especially in the growing season. research has shown that in areas where the annual rainfall is less than 1150 mm, tea plant growth is reduced due to stress. to reduce losses due to water deficits, varieties should be carefully selected to resist this stress, because damage to the plant is dependent greatly on the plants’ stress tolerance. the first intracellular line of defense is sod. phospholipid membranes are non-permeable to o2 (asada, 2006). it is therefore essential that sod be present to remove them in places where these compounds are formed. based on required metal co-factors, sod enzymes are classified into 4 groups: fesod, mn-sod, cu/zn-sod, and ni-sod. these isoforms are located in different cellular compartments: fe-sod is in chloroplasts as is cu/zn-sod, the latter of which is also present in cytosol and extracellular spaces. mn-sod is found in mitochondria and peroxisomes (jamal et al., 2006), and the fourth isoform of sod, ni-sod, which contains nickel in its active site has been found in several types of soil bacteria (bannister et al., 1987; bowler et al., 1992). many attempts have been made to produce transgenic plants, variously using all 3 isoforms of sod enzymes. most of these reports have noted that sod over-expression led to increased tolerance towards oxidative stress (faize et al., 2011). however, only in transgenic plants containing mn-sod protection was induced against, for example, reduced biomass and leaf damage (van breusegem et al., 1999; samis et al., 2002). many studies have been consistent with this data and mn-sod has been purified from pea (sevilla et al., 1980; palma et al., 1998), corn (baum & scandalios, 1981), pine (streller et al., 1994) and tea under cold stress (vyas & kumar, 2005). however, research has not been carried out on the role of this isoform in the improvement of drought tolerance in tea. in light of the results in other plants, mn-sod is expected to play a crucial role in the improvement of drought tolerance in tea. hence, in this study, the role of this isoform in resistance to drought was examined. complex responses of plants to abiotic stresses are manifested in the expression of multiple genes and various biochemical and molecular pathways. understanding these differences will lead to the identification of genes and molecular processes that play roles in a variety of conditions within biological systems (moody, 2001). in this study, the expression levels of sod transcripts were measured by real-time pcr. to evaluate the adaptation capacity of the two clones investigated 100 and dn against oxidative stress caused by drought, some indicators of oxidative stress, such as malondialdehyde (mda), carotenoid and proline content, chlorophyll a and total chlorophyll were also investigated. material and methods sampling two selected genotypes belonging to the tea research center, namely dn and 100, were used in this study. farm sampling was done from 8th july till 25th july for 17 days at the fashalam research station, iran. the experiment was arranged with a randomized complete block design with 3 replicates. 10-year-old vegetatively propagated tea plants (70-80 cm, height) were used under farm conditions (at ~24-33°c day/night and relative humidity of 75-80%) at longitude and latitude 49°55ʹ10ʺ and 37°8ʹ20ʺ, respectively. soil was sandy-loam with ph 5.8 and electric conductivity = 270. based on data obtained previously, a 10-day drought stress was imposed on the plants. this was followed by a one week irrigation period (twice a week, according to the usual practice of the research station) to exit from the stress condition. soil moisture content (smc) was measured on days 0, 5, 10 and 15th after withholding water began (black, 1965). sampling from the third terminal leaf was carried out in 2 steps, at day 10 and day 17. these samples provided data showing the effects of drought stress and exit from drought conditions, respectively. leaves were moved in liquid nitrogen and kept at -70 °c until needed. total rna extraction fresh leaf tissue, 0.1 g, was used for rna extraction according to kit instructions (cinnagen co.), with slight modification. because high levels of secondary metabolites in tea prevent high-quality rna extraction, isoamyl chloroform (1:24) was used to minimize the deposition of these compounds in rna isolates, and sodium acetate (2 m) was applied to optimize rna precipitation. after extraction, the resulting pellet was dissolved in 50 μl depc-treated water and maintained at -70 °c. to determine the 46 biologica nyssana ● 11 (1) september 2020: 45-54 mohammadi et al. ● a real-time pcr assay for evaluation of drought tolerance in a new tea clone 47 biologica nyssana ● 11 (1) september 2020: 45-54 mohammadi et al. ● a real-time pcr assay for evaluation of drought tolerance in a new tea clone quality of the extracted rna, 1% agarose gel electrophoresis was used. to determine the quantity and the degree of contamination of the rna, absorbance was read at a260/280 and a260/230 nm. rnas concentration in µg/µl was calculated as: cdna synthesis by the protocol included with the accupower rt premix kit (bioneer), one microgram of rna was used for cdna synthesis. first, 1 µg of rna was mixed with 0.5 µg of oligo dt primer and the mixture was incubated at 70 °c for 5 min to complete annealing. the product was then transferred to accupower rt premix microtube and the volume was brought to 20 µl with depc-treated water. this was vortexed for a few seconds and placed for 60 min at 42 °c. finally, the mixture was placed at 94 °c for 5 min and the synthesized cdna was maintained at -20 °c. primer design primers used for mn-sod gene and the 18s rrna reference gene were designed using oligo7 software (molecular biology insights, inc) (tab. 1). amplifications were performed in a total reaction volume of 25 µl containing 0.5 mm of dntp mix, 1 µl of each primer, 2.5 µl of 10x pcr buffer, 0.75 mm mgcl2 25mm, 0.3 unit taq dna polymerase and 4µl of template cdna and 14.95 µl sterile distilled water with an initial denaturing step of 95 °c for 5 min, followed by 40 amplification cycles of 95 °c for 45 s, 60.5±5 °c for 45 s, and 72 °c for 90 s and a final extension step of 72 °c for 5 min. finally 60.5 °c was selected for annealing tempreture. rtpcr was used to determine the appropriate annealing temperature of the primers. pcr products were electrophoresed on a 2% agarose gel. real-time pcr various approaches can be used for understanding gene expression in different circumstances. using of real-time pcr allows precise quantization of gene expression with a minimum amount of nucleic acid sample. measurement of mn-sod gene expression was performed on treated and control, untreated samples. the reaction mixture contained 12.5 μl real-time master mix, 2 μl each of forward and reverse primers, 6 μl template cdna and 2.5 μl sterile distilled water, with a final volume of 25 μl by 35 amplification cycles. 3 replicates were prepared from each sample and, for standardization, the housekeeping gene (18s rrna) was used. finally, after standardizing the data using the expression level of genes in the samples, the actual amount of target gene expression (mn-sod) was determined using model 2ctδδ (livak & schmittgen, 2001) at the level of mrna synthesis. statistical analysis using one way anova and duncan test were performed in spss 18 and charts exhibiting the changes in gene expression were plotted in excel. measurement of leaf relative water content relative water content (rwc) was measured on days 0 (control), 5, 10 and 17 based on barrs and weatherley (1962). after calculating the wet (or fresh) weight (fw), dry weight (dw) and saturated weight (sw), the percentage of leaf rwc in the two clones was obtained in triplicate and finally, rwc percentage was calculated by following formula: rwc = (fw dw/sw dw) × 100 measurement of proline proline measurement was carried out with ninhydrin reagent according to bates et al. (1973). for this purpose, 0.5 g of fresh leaf tissue, glacial acetic acid and toluene were used. after preparation of the extracts, absorbance of the upper layer (containing toluene and proline) was read at 520 nm. by using the standard curve in terms of micrograms per gram fresh weight of leaves, proline content was determined. measurement of lipid peroxidation to determine the extent of lipid peroxidation, mda concentration was measured using the method of heath & packer (1968). for this purpose, 0.5 g of table 1. sequence of designed primers for the rt-pcr analysis accession number putative function tm (°c) primer sequence (5´-3´) size of amplicon ay641734.2 manganese superoxide dismutase 60.5 f: cggagggcatatcaaccact r: cacccatccagagcctcgt 121 bp ay563528.1 rrna 18 57.7 f: caacacggggaaacttaccag r: taaccagacaaatcgctccac 178 bp fig. 1. a) total rna extracted from tea leaves and electrophoresed on 1% agarose gel with bands of the 5/8s, 18s, and 28s rrna, indicating the absence of rna degradation and lack of protein and dna contamination in the samples; b) 2% agarose gel of mn-sod gene rt-pcr product in clones 100 and dn. bands 1-4 and 5-8 are related to the 121 bp fragment of mn-sod gene in clones dn and 100, respectively. the annealing temperature 48 fresh leaf tissue was ground with 5 ml of 5% trichloroacetic acid. the extract was centrifuged at 14000 rpm for 15 minutes at room temperature and the supernatant was separated using a micropipette. an equal volume of 20% tca containing 0.5% tba was then added to the supernatant. the mixture was placed in a boiling water bath at 100°c for 30 min. and immediately cooled on ice. then, 2 ml of the supernatant was transferred to a microtube and centrifuged for 5 min at 7500 rpm. mda + tba complex absorbance was read at 532 nm and the non-specific absorption of the pigment was determined at 600 nm and subtracted from absorption at 532 nm. the extinction coefficient of 155 mm-1 cm-1 was used to calculate the concentration of mda. finally, the reaction product of lipid peroxidation (malondialdehyde) was calculated as nm per gram of fresh weight. measurement of chlorophyll and carotenoids pigment measurement was performed based on lichtenthaler (1987). chlorophyll and carotenoid concentrations were obtained in terms of µg/ cm2 of leaf area. results extraction of rna from tea leaves the extracted rna bands can be seen in fig. 1a. 3 bands of rrna, corresponding to 5.8s 18s and 28s, are present in the rna extract, with the width and intensity of the 28s band higher than the other bands. the extracted rna sample was used for cdna synthesis in rt-pcr (fig. 1a). fig. 2. melting curves of rt-pcr product (mn-sod gene) in clones 100 and dn. dna was detected with cyber green. a: melting curve of mn-sod gene related to clone dn in control treatments. b: melting curve of mn-sod gene related to clone dn in 10 days drought treatment. c: melting curve of mn-sod gene related to clone 100 in control treatments. d: melting curve of mn-sod gene related to clone 100 in 10 days drought treatment. e: melting curve of mn-sod gene related to clone dn in 17-day treatment (retrieved by re-watering). f: melting curve of mn-sod gene related to clone 100 in 17-day treatment (retrieved by re-watering). biologica nyssana ● 11 (1) september 2020: 45-54 mohammadi et al. ● a real-time pcr assay for evaluation of drought tolerance in a new tea clone 49 electrophoresis and melting curve of rt-pcr products of mn-sod gene as is shown in fig. 1b and 2, the melting temperature for the 121 bp fragment of mn-sod gene rtpcr product and of the rrna 18s gene are 73.5 °c and 77.5 °c, respectively (fig. 1b, fig. 2). as shown in fig. 3 measurement of mn-sod gene expression was carried out in leaf tissue from 2 clones 100 and dn at 0, 10 and 17 days after treatment. standardization of gene expression data was carried out using 18s rrna. examining gene expression during drought stress and re-irrigation yielded different results. results indicate that the greatest amount of mn-sod expression was observed in clones of dn after undergoing drought conditions for 10 days. significant increases in mrna synthesis of mn-sod gene occurred during this period and expression level decreased significantly by day 17 day, i.e., after the 7 day alleviation of the drought conditions, compared to expression levels after the 10 day exposure to drought conditions in the same clone. in contrast, mn-sod gene expression did not significantly change in clone 100 in any of the 2 treatments (fig. 3). measurement of relative water content and soil moisture content measurement of smc showed that soil moisture was decreased meaningfully in all treatments which were inducted by imposition of drought conditions (fig. 4). the effect of drought on rwc changes in the 100 and dn clones showed that responses were similar in both clones and rwc was reduced meaningfully after 10 days of drought stress. in the recovery treatment, rwc levels increased in both clones. this increase was similar in the 2 clones (fig. 4). proline measurement a comparison of proline content in clones dn and 100 indicates that the responses of the two clones differed in samples under drought and recovery conditions. proline levels increased under drought stress in clone dn, whereas significant increases were not observed in clone 100 compared to control. proline content of clone dn was also reduced significantly after the recovery treatment; however, changes of proline content were again not statistically significant in clone 100 at this point (fig. 5). fig. 3. changes in mrna level of the mn-sod gene in clones 100 and dn in treatments of control (0), 10 days without watering (10) and treatments recovered with reirrigation for 7 days (17). data is average of three replicates ± standard error (se) respectively. different letters indicate significant differences between treatments according to duncan’s test with p <0.05. fig. 4. changes in rwc of clones 100 and dn in treatments of control (0), 10 days without watering (10) and treatments recovered with re-irrigation for 7 days (17). data is average of three replicates ± standard error (se) respectively. different letters indicate significant differences between treatments according to duncan’s test with p <0.05. fig. 5. changes of proline content of clones 100 and dn in treatments of control (0), 10 days without watering (10) and treatments recovered with re-irrigation for 7 days (17). data is average of three replicates ± standard error (se) respectively. different letters indicate significant differences between treatments according to duncan’s test with p <0.05. biologica nyssana ● 11 (1) september 2020: 45-54 mohammadi et al. ● a real-time pcr assay for evaluation of drought tolerance in a new tea clone 50 measurement of lipid peroxidation as seen in fig. 6 changes in malondialdehyde levels in clone 100 are different from those in clone dn under drought stress. mda content does not increase in clone 100 under drought stress, mda levels are very substantially increased in clone dn after 10 days of drought treatment. mda levels in clone dn dropped dramatically in the 7 day recovery period, as assayed on day 17 day of the overall experiment (fig. 6). measurement of photosynthetic pigments measurement of carotenoids data shows that the highest concentrations of carotenoids occurred in clone 100 in the 10 day treatment. changes in carotenoid level in clone dn are substantially lower than in clone 100 (fig. 7). measurement of chlorophyll a measurement of chlorophyll a in the 2 clones showed that in clone dn levels of chlorophyll a were increased after 10 days of drought stress, whereas significant changes were not observed in clone 100. the amount of chlorophyll a in clone dn was significantly greater than in clone 100 after the 10 day drought treatment. by day 17, i.e., after 7 days recovery, the amount of chlorophyll a in clone dn had dropped dramatically and the levels in it and dn were not significantly different (fig. 8). measurement of total chlorophyll measurement of total chlorophyll content from the 2 clones indicated that chlorophyll content in clone 100 did not vary throughout the experiment. although total chlorophyll content appeared higher at day 10 compared to day 0, these differences were not statistically significant. however, there were significant differences in clone dn after the recovery period, i.e. at day 17 compared to day 10 days and the control (fig. 9). discussion many studies have shown that in photosynthetic plants there is a strong relationship between tolerance to oxidative stress induced by environmental stresses and increasing concentrations of antioxidant enzymes (sairam & saxena, 2000; sairam & srivastava, 2001). researchers have shown that the concentrations of antioxidant enzymes double under stress, resulting, in particular, in increased resistance fig. 6. changes of mda content of clones 100 and dn in treatments of control (0), 10 days without watering (10) and treatments recovered with re-irrigation for 7 days (17). data is average of three replicates ± standard error (se) respectively. different letters indicate significant differences between treatments according to duncan’s test and with p <0.05. fig. 7. changes in carotenoid content in clones 100 and dn in treatments of control (0), 10 days without watering (10) and treatments recovered with re-irrigation for 7 days (17). data is average of three replicates ± standard error (se) respectively. different letters indicate significant differences between treatments according to duncan’s test with p <0.05. fig. 8. changes of chlorophyll a content in clones 100 and dn in treatments of control (0), 10 days without watering (10) and treatments recovered with re-irrigation for 7 days (17). data is average of three replicates ± standard error (se) respectively. different letters indicate significant differences between treatments according to duncan’s test with p <0.05. biologica nyssana ● 11 (1) september 2020: 45-54 mohammadi et al. ● a real-time pcr assay for evaluation of drought tolerance in a new tea clone 51 to oxidative stress. drought stress increases the activity of glutathione reductase and sod especially (gamble & burke, 1984; lascano et al., 2001). this is not to say that all cultivars of a particular species are stressed to the same degree by drought conditions. in the present study, a significant increase was observed in mn-sod gene expression in clone dn during 10 days of drought treatment, while no significant increase in the expression of this gene was found in clone 100. this correlated well with the other physiological parameters assayed and reflected clone 100’s greater resistance to drought in the first place. clone dn activates resistant mechanisms against oxidative stress caused by drought by increasing the expression of mn-sod because it was more greatly stressed by the drought conditions. malondialdehyde content rose, also, in only clone dn and not in clone 100. increases in this product represent an increase in lipid peroxidation due to ros production and there is a positive correlation (r2=0.66) between the amount of malondialdehyde and sod gene expression. it can be said, therefore, that sod gene expression in clone dn was increased to mitigate the effects of generated ros. again, neither mda content nor sod gene expression were significantly increased in clone 100, indicating that a lower level of cellular stress was induced by drought conditions in this cultivar. on the other hand, appropriate conditions are provided for the growth of clones with re-watering, which is accompanied by reduced expression of the sod gene in clone dn, indicating a reduction in oxidative stress in this clone. ten days of drought applied to clone 100 did not result in considerable stress responses involving a significant stimulation of the antioxidant defense system. many reports are indicating mn-sod expression and activity increases during stress (shah & nahakpam, 2012). for example, increased resistance to cold stress was correlated with a simultaneous increase in the expression of mn-sod in chlorella ellipsoidea (clare et al., 1984). similarly, transformation of wheat plants with the mn-sod gene from brassica species and its overexpression increased resistance to oxidative stress and aluminum toxicity (gachon et al., 2004) in the transgenic wheat. this method to increase sod activity has also been reported in a study by bowler et al. (1992). they found that transformation of maize chloroplasts with the tobacco mn-sod gene enhanced cold tolerance. also, increased expression of mn-sod in tobacco considerably enhances resistance against oxidative stress. in studies conducted by upadhyaya et al. (2008), performed on 4 tea clones, a decrease in sod activity occurred during 10 days drought stress in clone tv 1 that is resistant to water stress, while an increase in activity was seen in the sensitive clone tv 20. rwc is a very important index of plant water status and dehydration tolerance, and consequently reflects the activity of metabolites in the tissue. rwc is at its highest level in the early stages of leaf development and decreases with increasing leaf dry matter and maturation. this physiological parameter has a direct relationship with water uptake of root and loss of water through respiration (anjum et al., 2011). different cultivars show different levels of rwc, depending on duration of stress and ability of the genotypes to maintain osmotic potential in drought conditions at different stages of development (siddique et al., 2000; allen & ort, 2001). interestingly, a comparison of rwc in clones dn and 100 revealed that rwc was the same in both and was similarly reduced after 10 days of drought stress; whilst re-watering caused an essentially identical increase in rwc in the leaves of both clones. that the decrease in leaf water content happens equally in both clones but causes a different response in these clones strongly indicates that lowered rwc does not necessarily result in oxidative stress. although not receiving adequate moisture is observed in both examined clones, similar water deficits will not necessarily stimulate defense responses in the two clones equally. each clone can activate its defense system tailored to its needs and ability. it will be very interesting to learn what the bases of this difference are. perhaps cytoplasmic proline levels are important here. clone 100 showed much higher basal proline than did clone dn. relationships between the production of free radicals and proline accumulation in plants indicate fig. 9. changes of total chlorophyll content in clones 100 and dn in treatments of control (0), 10 days without watering (10) and treatments recovered with re-irrigation for 7 days (17). data is average of three replicates ± standard error (se) respectively. different letters indicate significant differences between treatments according to duncan’s test with p <0.05. biologica nyssana ● 11 (1) september 2020: 45-54 mohammadi et al. ● a real-time pcr assay for evaluation of drought tolerance in a new tea clone a role for proline in reducing toxicity, a non-enzymatic defense mechanism for removing free radicals (matysik et al., 2002). on the other hand, increases in proline, which reduces the acidity of the cytosol, can be due to various stresses (kurkdjian & guern, 1989). removing excess h+ as a positive effect of proline synthesis results in a reduction of stress. the current study suggests that a change in proline content in tea plant is regulating the level of osmotic potential that ultimately controls the water absorption. the same phenomenon has been previously observed in cotton (parida et al., 2008), rice (pirdashti et al., 2009), maize (valentovic et al., 2006) and in tea (upadhyaya & panda, 2013). our investigation shows that higher drought resistance of clone 100 compared to clone dn is due to its higher proline level. this capability of clone 100 might be more effective in weak or moderate drought and probably less effective in extreme drought conditions. clones dn and 100 differed also in their chlorophyll a profiles throughout the experiment. chlorophyll a levels rose under drought conditions in clone dn but were unchanged in clone 100. similarly, upon relief of drought, chlorophyll a levels (and, in this case, total chlorophyll as well) dropped in clone dn and, again, did not significantly change in clone 100. changes in chlorophyll levels during stress are related to the duration and intensity of the stress (zhang & kirkham, 1996). chapman & barreto (1997) believe that the growth stage, cultivar and environmental conditions may be important factors influencing the amount of chlorophyll pigments under stress. sairam et al. (2000 and 2001) have also stipulated that higher levels of carotenoids and chlorophylls in plants under drought stress conditions can be associated with different resistance genotypes. machado & paulsen (2001) found that rapid physiological changes such as tubular leaves, reduced leaf area, and increased stomatal resistance may be factors in the apparent increase in chlorophylls content, as part of an avoidance mechanism to drought stress. during stressful conditions, plants reduce transpiration levels to prevent water loss through lower leaf surfaces. consequently, the chlorophyll content increases per unit leaf area, despite the decrease in total chlorophyll content. clone dn is a drought resistant cultivar and is considered as a reference strain for evaluating new iranian clones used at tea research institute of iran. perhaps at least a part of the changes in chlorophylls in this cultivar was related to the higher oxidative stress intensity experienced by this clone during the drought conditions. after the 7 day re-watering period the morphological changes mentioned above were reversed but chlorophyll levels nevertheless fell in clone dn to values lower than in control. it is possible that enhanced chlorophyll synthesis could not be upgraded along with these morphological changes and/or not enough time was available for chlorophyll synthesis to reach levels present in control samples. clone 100 displayed better tolerance to drought and changes in chlorophyll content in these clones were minimal. again, it will be very interesting to learn the basis for the lack of apparent oxidative stress in clone 100 as contrasted to the clear oxidative stress experienced by clone dn under conditions of drought. conclusion 10-day drought stress was imposed on two clones of tea plants. this was followed by one week irrigation period (twice a week, according to the usual practice of the research station) to exit from the stress condition. the results showed that 10-day stress caused significant changes in metabolism and activity of antioxidant system in dn clone, while these changes were not observed in clones 100. measurement of membrane lipid peroxidation, as an important physiological indicator to detection oxidative stress intensity and check out the mn-sod gene expression as the first defense enzyme against oxidative stress, showed that during the 10 days of water stress, the clone 100 is not affected, whereas dn clone showed increase in proline, mda and also increased expression of superoxide dismutase. with the re-irrigation of the samples, the expression of superoxide dismutase and the levels of malondialdehyde are also decreased in dn clone similar to clone 100, indicating that this clone has been out of stress. therefore, in terms of some responses to drought stress, clone 100 can be introduced as a more compatible clone for planting in the north region of iran. acknowledgements. the authors acknowledge for the plant material provided by tea research institute of lahijan, iran. references allen, d. j., ort, d. r. 2001: impacts of chilling temperatures on photosynthesis in warm-climate plants. trends in plant science, 6 (1): 36-42. anjum, s. a., xie, x.-y., wang, l., saleem, m. f., man, c., lei, w. 2011: morphological, physiological and biochemical responses of plants to drought stress. african journal of agricultural research, 6 (9): 2026-2032. asada k. 2006: production and scavenging of active oxygen in photosynthesis. plant physiology, 141, 391 ̶ 396. bannister, j. v., bannister, w. h., rotilio, g. 1987: aspects of the structure, function, and applications 52 biologica nyssana ● 11 (1) september 2020: 45-54 mohammadi et al. ● a real-time pcr assay for evaluation of drought tolerance in a new tea clone of superoxide dismutase. critical reviews in biochemistry and molecular biology, 22(2): 111180. barrs h., weatherley p. 1962, a re-examination of the relative turgidity technique for estimating water deficits in leaves. australian journal of biological sciences, 15: 413 ̶ 428. bates l., waldren r., teare i. 1973: rapid determination of free proline for water-stress studies. plant soil, 39: 205 ̶ 207. baum, j. a., scandalios, j. g. 1981: isolation and characterization of the cytosolic and mitochondrial superoxide dismutases of maize. archives of biochemistry and biophysics, 206 (2): 249-264. black c. a. 1965, methods of soil analysis: part i, physical and mineralogical properties. american society of agronomy, madison, wisconsin, 1572 p. bowler, c., montagu, m. v., inze, d. 1992: superoxide dismutase and stress tolerance. annual review of plant biology, 43 (1): 83-116. chapman s.c., barreto h.j. 1997: using a chlorophyll meter to estimate specific leaf nitrogen of tropical maize during vegetative growth. agronomy journal, 89: 557 ̶ 562. clare, d. a., rabinowitch, h. d., fridovich, i. 1984: superoxide dismutase and chilling injury in chlorella ellipsoidea. archives of biochemistry and biophysics, 231 (1): 158-163. faize, m., burgos, l., faize, l., piqueras, a., nicolas, e., barba-espin, g., clemente-moreno m.j., alcobendas r., artlip t., hernandez j.a. 2011: involvement of cytosolic ascorbate peroxidase and cu/zn-superoxide dismutase for improved tolerance against drought stress. journal of experimental botany, 1: 1-15. farooq, m., wahid, a., kobayashi, n., fujita, d., basra, s. 2009: plant drought stress: effects, mechanisms and management. in sustainable agriculture, 29: 153-188. gachon, c., mingam, a., charrier, b. 2004: realtime pcr: what relevance to plant studies? journal of experimental botany, 55 (402): 1445-1454. gamble, p. e., burke, j. j. 1984: effect of water stress on the chloroplast antioxidant system i. alterations in glutathione reductase activity. plant physiology, 76 (3): 615-621. heath r.l., packer l. 1968: photoperoxidation in isolated chloroplasts: i. kinetics and stoichiometry of fatty acid peroxidation. archives of biochemistry and biophysics, 125: 189 ̶ 198. 53 jamal, a., yoo, n. h., yun, s. j. 2006: isoformspecific responses of superoxide dismutase to oxidative stresses and hormones in paraquat-tolerant rehmannia glutinosa. journal of agronomy and crop science, 51 (1): 678-679. kurkdjian, a., guern, j. 1989: intracellular ph: measurement and importance in cell activity. annual review of plant biology, 40 (1): 271-303. lascano, h. r., antonicelli, g. e., luna, c. m., melchiorre, m. n., gómez, l. d., racca, r. w., trippi v. s, casano l. m. 2001: antioxidant system response of different wheat cultivars under drought: field and in vitro studies. functional plant biology, 28 (11): 1095-1102. lichtenthaler h.k. 1987: chlorophylls and carotenoids: pigments of photosynthetic biomembranes. methods in enzymology, 148: 350 ̶ 382. livak k.j., schmittgen t.d. 2001: analysis of relative gene expression data using real-time quantitative pcr and the 2(-delta delta c(t)) method. methods, 25: 402 ̶ 408. machado s., paulsen g.m. 2001: combined effects of drought and high temperature on water relations of wheat and sorghum. plant soil, 233: 179 ̶ 187. matysik j., bhalu a., mohanty p. 2002: molecular mechanism of quenching of reactive oxygen species by proline under water stress in plants. current science, 82: 525 ̶ 532. monakhova, o., chernyad’ev, i. 2004: effects of cytokinin preparations on the stability of the photosynthetic apparatus of two wheat cultivars experiencing water deficiency. applied biochemistry and microbiology, 40 (6): 573-580. mukhopadhyay m, mondal t.k. 2014: the physio-chemical responses of camellia plants to abiotic stresses. journal of plant science and research, 1(1): 105. moody, d. 2001: genomics techniques: an overview of methods for the study of gene expression. journal of animal science, 79 (e-suppl), e128-e135. noctor, g., mhamdi, a., foyer, c. h. 2014: the roles of reactive oxygen metabolism in drought: not so cut and dried. plant physiology, 164 (4): 16361648. palma, j. m., lópez‐huertas, e., corpas, f. j., sandalio, l. m., gómez, m., del río, l. a. 1998: peroxisomal manganese superoxide dismutase: purification and properties of the isozyme from pea leaves. physiologia plantarum, 104 (4): 720-726. biologica nyssana ● 11 (1) september 2020: 45-54 mohammadi et al. ● a real-time pcr assay for evaluation of drought tolerance in a new tea clone parida, a. k., dagaonkar, v. s., phalak, m. s., aurangabadkar, l. p. 2008: differential responses of the enzymes involved in proline biosynthesis and degradation in drought tolerant and sensitive cotton genotypes during drought stress and recovery. acta physiologiae plantarum, 30 (5): 619-627. pirdashti, h., sarvestani, z. t., bahmanyar, m. a. 2009: comparison of physiological responses among four contrast rice cultivars under drought stress conditions. world academy of science., engineering and tech, 49: 52-53. sairam, r., saxena, d. 2000: oxidative stress and antioxidants in wheat genotypes: possible mechanism of water stress tolerance. journal of agronomy and crop science, 184 (1): 55-61. sairam, r., srivastava, g. 2001: water stress tolerance of wheat (triticum aestivum l.): variations in hydrogen peroxide accumulation and antioxidant activity in tolerant and susceptible genotypes. journal of agronomy and crop science, 186 (1): 6370. samis, k., bowley, s., mckersie, b. 2002: pyramiding mn‐superoxide dismutase transgenes to improve persistence and biomass production in alfalfa. journal of experimental botany, 53 (372): 1343-1350. sánchez, f. j., manzanares, m. a., de andrés, e. f., tenorio, j. l., ayerbe, l. 2001: residual transpiration rate, epicuticular wax load and leaf color of pea plants in drought conditions. influence on harvest index and canopy temperature. european journal of agronomy, 15 (1): 57-70. sevilla, f., lopez-gorge, j., gomez, m., & del rio, l. 1980: manganese superoxide dismutase from a higher plant. planta, 150 (2): 153-157. shah, k., nahakpam, s. 2012: heat exposure alters the expression of sod, pod, apx and cat isozymes and mitigates low cadmium toxicity in seedlings of sensitive and tolerant rice cultivars. plant physiology and biochemistry, 57: 106-113. siddique, m., hamid, a., islam, m. 2000: drought stress effects on water relations of wheat. botanical bulletin of academia sinica, 41: 35-39. streller, s., krömer, s., wingsle, g. 1994: isolation and purification of mitochondrial mn-superoxide dismutase from the gymnosperm pinus sylvestris l. plant and cell physiology, 35 (6): 859-867. upadhyaya h., panda s.k., dutta b.k. 2008: variation of physiological and antioxidative responses in tea cultivars subjected to elevated water stress followed by rehydration recovery. acta physiologiae plantarum, 30: 457 ̶ 468. upadhyaya, h., panda, s. k. 2013: abiotic stress responses in tea [camellia sinensis l (o) kuntze]: an overview. reviews in agricultural science, 1: 1-10. valentovic, p., luxova, m., kolarovic, l., gasparikova, o. 2006: effect of osmotic stress on compatible solutes content, membrane stability and water relations in two maize cultivars. plant soil and environment, 52 (4): 184. van breusegem, f., slooten, l., stassart, j.-m., botterman, j., moens, t., van montagu, m., inzé, d. 1999: effects of overproduction of tobacco mnsod in maize chloroplasts on foliar tolerance to cold and oxidative stress. journal of experimental botany, 50 (330): 71-78. vyas, d., kumar, s. 2005: purification and partial characterization of a low temperature responsive mn-sod from tea (camellia sinensis (l.) o. kuntze). biochemical and biophysical research communications, 329 (3): 831-838. zhang, j., kirkham, m. 1996: antioxidant responses to drought in sunflower and sorghum seedlings. new phytologist, 132 (3): 361-373. zhu, j.-k. 2001: plant salt tolerance. trends in plant science, 6 (2): 66-71. 54 biologica nyssana ● 11 (1) september 2020: 45-54 mohammadi et al. ● a real-time pcr assay for evaluation of drought tolerance in a new tea clone microsoft word 0306_vujicic_et_al konacno biologica nyssana 1 (1-2) december 2010: 77-82 vujičić, m. et al. axenically culturing the bryophytes … 77 original article ! axenically culturing the bryophytes: a case study of the moss herzogiella seligeri (brid.) z. iwats. (plagiotheciaceae) milorad vujičić*, aneta sabovljević, marko sabovljević institute of botany and garden jevremovac, faculty of biology, university of belgrade, takovska 43, 11000 belgrade, serbia *e-mail: milorad@bio.bg.ac.rs abstract: vujičić, m., sabovljević, a., sabovljević, m.: axenically culturing the bryophytes: a case study of the moss herzogiella seligeri (brid.) z. iwats. (plagiotheciaceae). biologica nyssana, 1 (1-2), december 2010: 77-82. a moss genus herzogiella, from the pleurocarpous family plagiotheciaceae contains only seven species world wide. it occurs in north, central and south america, europe and asia. in europe, only three species occurred, namely h. seligeri, h. striatella and h. turfacea of which, the last one is threatened. with aim to develop the methodology for protection, conservation and active propagation of h. turfacea, more commonly distributed counterpart, h. seligeri, were taken from the national park fruška gora and axenically culture were established. the study gives overview into the problems of sterilization, in vitro establishing, development, propagation and biology of species, as well as indices applicable to threatened counterpart. key words: herzogiella seligeri, in vitro introduction ! bryophytes (comprising mosses, liverworts, hornworts and alies) are the second largest group of higher plants after flowering plants, with estimated 15,000 species worldwide (h a l l i n g b ä c k and h o d g e t t s , 2000). bryophytes, although the second largest group of terrestrial plants, received much less attention in conservation and protection and in comparison to vascular plants and higher animals much less are known on their biology. they comprise very diverse plant groups (e.g. peatmosses, latern-mosses, leafy liverworts) with quite diverse biological characteristics (i.e. structure, size, ecology etc). although culturing plant tissues and organs under axenic conditions was firstly established and profitably employed in bryophytes, especially mosses (s e r v e t t a z , 1913), bryophytes did not retain for long their rightful place as a highly favored research object; therefore most studies of plant morphogenesis are now being done on vascular plants. besides the problems with bryophyte establishment in axenic culture, it is often problem of material availability, genetic variability of material, disposal of axenic organisms leaving on bryophytes and low level of species biology knowledge (e.g. d u c k e t t et al., 2004). apart from economic considerations of experimental work with bryophytes, many fundamental and applicative physiological, genetical, morphogenetic, ecological and evolutionary, as well as other problems could be studied more easily in bryophytes rather than in vascular plants (s a b o v l j e v i ć et al., 2003). bryophytes are useful objects for the elucidation of comlex biological processes such as apogamy, apospory, stress-induced cellular responses in plants, and the fusion and growth of protoplast, etc (l a l , 1984; c o v e et al., 1997; o l i v e r and w o o d , 1997; s h u m a k e r and d i e t r i c h , 1998; r e s k i , 1998; w o o d et al., 2000; c v e t i ć et al., 2005, 2009; b o g d a n o v i ć et al., 2009; v u j i č i ć et al. 2010). 10th sfses • 17-20 june 2010, vlasina lake1 (1-2) • december 2010: 77-82 biologica nyssana 1 (1-2) december 2010: 77-82 vujičić, m. et al. axenically culturing the bryophytes… 78 besides, axenical cultivation of bryophytes as well as developing of methodology in propagation of bryophytes are significant in rare species conservation both for ex situ and reintroduction (e.g. b a t r a et al., 2003; b i j e l o v i ć et al., 2004; s a b o v l j e v i ć et al., 2005; r o w n t r e e and r a m s a y , 2 0 0 5 ; 2 0 0 9 ; g o n z a l e z et al., 2 0 0 6 ; m a l l o n et al., 2007; r o w n t r e e , 2006; c v e t i ć et al., 2007; b r e z e a n u et al, 2008; c h e n et al., 2009; v u j i č i ć et al., 2009, r o w n t r e e et al., in press). this is especially valuable for the species like bryophytes many of which are dioecious and possibly long-lastingly in sterile condition naturally. axenic culturing of bryophytes seems to be so complicated that many investigators gave up the attempt. however, due to possible interaction with other organisms in non axenic conditions, sterile culturing is necessary for certain experimental procedures. progress in bryophyte tissue culture has not gone as fast as in culture of the cells of vascular plants, and the number of cases achieved still does not satisfy sufficiently the demands of various research fields (f e l i x , 1994). like other members of the bryophyta, the mosses are diverse haploid-dominant plants. mosses did not received a lot attention in chemistry research as a source of newly and/or bioactive compounds (s a b o v l j e v i ć and s a b o v l j e v i ć , 2008). however, the problem for analyzing and/or certain substance production in larger amount is often inadequate axenical material, i.e. impossibility to have clean material in enough amount neither to establish bryophyte monoculture fields. one of solution, even it seems problematic one is to establish in vitro culture, to find the proper developmental conditions and to propagate it for the wanted purpose (s a b o v l j e v i ć et al., 2010) in this study, we have focused to pleurocarpous moss herzogiella seligeri. the aim of the present study was to establish stable in vitro culture of this species and examine its development under axenic conditions. since up to date any plagiothecioid moss were not cultivated axenically, the true challenge was to establish the axenic culture of this moss having in mind its tiny and tender morphology and anatomy. material and methods a moss h. seligeri (bridel) z. iwats. (syn. leskea seligeri bridel, musc. rec. 2(2): 47. 1801; dolichotheca seligeri (bridel) loeske; isopterygium seligeri (bridel) c. jensen; plagiothecium silesianum (weber & mohr) schimper; plagiothecium seligeri (bridel) lindberg; sharpiella seligeri (bridel) z. iwatsuki) has been studied. it is wide spread in contrast to its rare and endangered counterpart h. turfacea (i r e l a n d , 1992). h. seligeri is one of the first invading species on the rotten logs and tree bases of all kind of trees. the capsules are mature at the beginning of summer. this is a species distinctive by its widespreading leaves, appearing in several rows, and its long (2-3.5 mm), arcuate capsules. plants in thin mats, lightto yellowish green, glossy. stems to 30 × 1.5-3 mm, prostrate to ascending, pseudoparaphyllia lacking. leaves widespreading, ovate to ovate-lanceolate, smooth, nondecurrent or 1-3 cells indistinctly decurrent, 12.5 × 0.5-0.9 mm, margins serrulate to serrate; cell walls pitted at leaf base, indistinctly pitted distally, sometimes pits lacking; median cells 30-70 × 5-7 μm; alar cells quadrate to short-rectangular, sometimes rounded to oval and inflated, 17-48 × 1226 μm. sexual condition autoicous. seta light brown to red, 1.5-2.5 cm. capsule light brown to reddish brown, inclined, 2-3.5 × 0.5-0.8 mm, cylindric, strongly arcuate, when dry contracted below mouth; operculum conic, 0.4-0.6 mm. spores 12-22 μm. the materials for axenic culture were collected in fruška gora mt in march 2007 on rotten log and the voucher specimen was deposited in the bryophyte collection of the belgrade university herbarium (beou 4421). after collection, the plants were stored in plastic bags at +4°c, till the begging of the experimental work. in the laboratory conditions, the material were cleaned under dissecting microscope from the visible mechanical impurity. the sporophyte with mature almost ripen but unopened capsules were separated, and the gametophyte tips consisted of the leafy stems of ca. 10 mm longitude were separated carefully, placed in glasses, covered with cheese cloth, and rinsed with tap water for 30 minutes. sporophytes and gametophyte parts were then disinfected for 5 minutes with 3, 5, 7, 10, 13% or 15% solution of sodium hypochlorite (commercial bleach, naocl). finally, they were rinsed three times in sterile deionised water. as a basal medium for establishment of in vitro culture, we used murashige and skoog (1962) (ms) medium containing murashige and skoog mineral salts and vitamins, 100 mg/l inositol, 0.70% (w/v) agar (torlak purified, belgrade), and 3% sucrose and bcd medium (see s a b o v l j e v i ć et al. 2009 for the media details). once, the establishment was done, and the plants produced, the in vitro developed plant segments (tips and protonema pieces) were used for further developmental experiments. biologica nyssana 1 (1-2) december 2010: 77-82 vujičić, m. et al. axenically culturing the bryophytes … 79 in order to observe the influence of sucrose and/or mineral salts on the morphogenesis of this species, the following medium composition combination were tested: ms1: half strength of ms mineral salts, sugar free; ms2: half strength of ms mineral salts, 1.5% sucrose; ms3: half strength of ms mineral salts, 3% sucrose; ms4: ms mineral salts, sugar free; ms5: ms mineral salts, 1.5% sucrose; ms6: ms mineral salts, 3% sucrose; bcd1: bcd mineral salts, 1.5% sucrose; bcd2: bcd mineral salts, 3% sucrose; bcd3: bcd mineral salts, sugar free; the ph of the media was adjusted to 5.8 before autoclaving at 114°c for 25 minutes. the temperature and light duration varied in combined with sets of media: combination c1: 16/8 hours of light to darkness, at 25 ± 2°c. combination c2: 8/16 hours of light to darkness, at 20 ± 2°c. combination c3: 16/8 hours of light to darkness, at 20 ± 2°c. combination c4: 16/8 hours of light to darkness, at 18 ± 2°c. light was supplied by cool-white fluorescent tubes at a photon fluency rate of 47 μmol/m2s. cultures were subcultured for a period of 4-6 weeks. for analysis of condition set influence to development 10mm long apical segments (gametophyte), spores or protonema were transferred to various nutrient media. for each medium composition combined with light conditions, 40 transplants of h. seligeri were cultivated. the influence of tested environmental condition was quantified after 30 days by visually estimation of the plant habitat marked as the best those of appearance like in nature. results and discussion the attempts to establish the axenic culture from gametophytes i.e. 10mm plant tips failed since the concentration for surface sterilizations killed the plant material or was not effective enough to kill the xenic organisms on the plants and not to harm the plants at the same time. so, even there where the plants survive the bleach surface sterilization and transferred to the mineral salts, it was overgrown quickly with fungi, algae and bacterias. the try outs to leave it until transferred plantlets overgrow the xenic organisms, for the purpose of the use of newly grown tips, remained useless (note that the survival does not mean axenic as well). there is rather low probability percentage of good bleach surface sterilization of the moss tips, without harming plants, for establishment in vitro culture (e.g. s a b o v l j e v i ć et al., 2003) surface sterilization of the sporophytes was more successful since we choose the almost mature but unopened capsules and did the sterilization in various concentration of bleech for 5 minutes like for the gametophytes. the advantage of this process was that we did not need the capsules material itself (so we could harmed it lethally) but the spores from inside that should remain viable. once, the surface of sporophytes was sterilized, the capsules were opened in sterile conditions and the spores were taken out with sterile needle to the mineral salt containing media. the success of this way starting culture concerning sterilization of start plant material was achieved with 100% at 10% bleach for 5 minutes. in higher concentration the sterilization percentage remain high but the bleach started to harm the spores quantified by spore germination slightly decrease. 0 2 4 6 8 10 12 14 16 -10 0 10 20 30 40 50 60 70 80 90 100 110 s ur vi va l ( % ) bleach concentration (%) gametophyte sporophyte fig 1. percentage of surviving of gametophyte and sporophyte parts after bleach treatment. the best bleach concentration for surface sterilizing of h. seligeri was 10%. the percentage of tested propagules, both of gametophyte tips and sporophytes survival, decreased with concentration increase (fig.1). however, the bleach could not offer proper sterilization since propagules contained hardly disposal contamination afterwards with fungi, algae or bacteria, or the high concentration is lethal in high percentage (above 10%). the bleach concentration under 10% are functional since the propagules survived in high percentage, but not 100% of used material was axenical. bleach concentrations above 7% for five minutes exposure biologica nyssana 1 (1-2) december 2010: 77-82 vujičić, m. et al. axenically culturing the bryophytes… 80 kill most of the propagules and are not appropriate for this moss gametophytes in vitro culture establishment. figs. 2. and 3. a detail of herzogiella seligeri gemetophyte developed in in vitro condition spores were germinated on ms medium enriched with sucrose (ms3). after releasing from the capsules, spores germinated in relatively high percentage (up to 100%). however, on the ms medium enriched with sucrose they remain in the phase of primary protonema. the subculturing to fresh medium with sucrose was not a signal for plantlets to pass to the next developmental stage. the protonema is not spreading far from spore in any media tested. the variation of light-length and temperature condition in h. seligeri cultures did not show significantly different behavior (figs. 2 and 3). a set of various combination of light length, temperature and mineral salts were tested to achieve the bud induction and gametophyte development. it can be concluded that in the condition when medium contain the sugar (ms2, ms3, ms5, ms6, bcd1, bcd2) the spore germination is stimulated, but the gametophyte development stopped at protonemal eventually caulonemal stage. schoefield (1981) stated that in most bryophytes spores germinate 7-30 days after exposure of spores to good conditions. in our case, it was quicker when media contained sucrose (7-10 days) than the spore germination on sucrose free media. interestingly, difference in gametophyte development was achieved when sucrose was put by (ms1, ms4). on bcd sugar free (bcd3), the bud formation was noticed after a month. bopp (1952) explained that in native conditions protonema have to achieved the certain size which then produce enough amount of kinetin-like growth regulators released in substrate. this is a trigger for bud induction or passing from filamentous to meristemal growth. buds developed rapidly into a stem which again branched and continue growing achieving full size and normal leaf shapes of natural plants but not the plant shape (figs. 4 and 5). a rather very humid air condition of the growth-dishes favors elongation and growth. figs. 4. and 5. herzogiella seligeri gemetophyte developed in in vitro condition the 10mm shoot and branch tips were used further for subculturing into new media combined with four combination of controlled conditions of day length and temperature. biologica nyssana 1 (1-2) december 2010: 77-82 vujičić, m. et al. axenically culturing the bryophytes … 81 the best developed and the most similar to the plants developed in nature were grown on ms1 and bcd3 at temperature of 18°c or 20±2°c, at both day length. in the temperature of 25±2°c the plants produced slightly smaller, shorter, tinny, fragile and unbranched shoots, often developing contamination of blue-green algae. spore germination was not effected by the day length and it was similar in all temperatures. when the plantlets tips transferred to new media, they produced the secondary protonema, not far from the plantlets, developing shot on them which spread further and branching depending on conditions of growth (fig. 5). axenically culturing h. seligeri showed that different developmental stage of this moss species can be stimulated or stopped by various combination of mineral nutrition, light and temperature. the different growth condition should be taken into account for different herzogiella counterpart species conservation and propagation. secondarily, the problems of contamination with blue-green algae can appear but they rather do not harm the plantlets like green algae, bacteria and fungi do. references batra, a., binding, h., rasmussen, s., rudolph, h. & waetzig, g. h. 2003. efficient regeneration of sphagnum fallax from isolated protoplasts. in vitro cellular & developmental biology. plant 39(2): 147-150. bijelović, a., sabovljević, m., grubišić, d. & konjević, r. 2004. phytohormone influence on the morphogenesis of two mosses (bryum argenteum hedw. and atrichum undulatum (hedw.) p. beuav.). israel journal of plant sciences 52(1): 31-36. bogdanović, m., sabovljević, m., sabovljević, a., grubišicć, d. 2009. the influence of gypsiferous substrata on bryophyte growth: are there obligatory gypsophilous bryophytes? botanica serbica 33 (1): 75-82. bopp, m. 1952. entwicklungsphysiologische untersuchungen an laubmoosprotonemen. zeitschrift für botanik 40: 119-152. brezeanu, a., cogălniceanu, g. & mihai, r. 2008. ultrastructural characterization of the in vitro gametophyte of bucegia romanica radian a rare liverwort. romanian journal of biology – plant biology 53(2): 49-61. chen, y.-y., lou, y.x., guo, s.-l. & cao, t. 2009. successful tissue culture of the medicinal moss rhodobryum giganteum and factor influencing proliferation of its protonemata. annales botanici fennici 46: 516-524. cove, d. j., knight, c. d. & lamparter, t. 1997. mosses as model systems. trends in plant science 2: 99–105. cvetić, t., sabovljević, m., sabovljević, a. & grubišić, d. 2005. in vitro culture and apogamy – alternative pathway in life cycle of the moss amblystegium serpens (amblystegiaceae). archives of biological sciences 57(3): 267-272. cvetić, t., sabovljević, a., sabovljević, m. & grubišić, d. 2007. development of the moss pogonatum urnigerum (hedw.) p. beauv. under in vitro culture conditions. archives of biological sciences 59(1): 57-61. cvetić, t., sabovljević, a., bogdanović-pristov, j., sabovljević, m. 2009. effects of day length on photosynthetic pigments and antioxidative metabolism of in vitro cultured moss atrichum undulatum (hedw.) p. beauv. (bryophyta). botanica serbica 33 (1): 83-88. duckett, j.g., fletcher, r., matcham, h.w., read, j.t., russell, a.j. & pressel, s. 2004. in vitro cultivation of bryophytes; practicalities, progress, problems and promise. journal of bryology 26: 3-20. felix, h. 1994. calli, cell, and plantlet suspension cultures of bryophytes. candollea 49: 141-158. gonzález, m. l., mallón, r., reinoso, j. & rodríguez-oubiña, j. 2006. in vitro micropropagation and long-term conservation of the endangered moss splachnum ampullaceum hedw. biologia plantarum 50: 339–345. hallingbäck, t. & hodgetts, n. 2000. mosses, liverworts and hornworts; status survey and conservation action plan for bryophytes. iucn/ssc bryophyte specialist groupoxford ireland, r.r. 1992. synopsis of the genus herzogiella for north america. lindbergia 17: 111-115. lal, m. 1984. the culture of bryophytes including apogamy, apospoary, parthenogenesis and protoplasts. in: dyer, a. f. & duckett, j. g. (eds.) the experimental biology of bryophytes. academic press, london, pp. 97–115. mallon, r., barros, p., luzardo, a., gonzalez, m. l. 2007. encapsulation of moss buds: an efficient method for the in vitro conservation and regeneration of the endangered moss splachnum ampullaceum. plant cell tissue and organ culture 88: 41–49. murashige, t. & skoog, f. 1962. a revised medium for rapid growth and bioassays with tobacco tissue culture . physiologia plantarum 15: 473497. biologica nyssana 1 (1-2) december 2010: 77-82 vujičić, m. et al. axenically culturing the bryophytes… 82 oliver, m. j. & wood, a. j. 1997. desiccation tolerance inmosses. in: koval, t., (ed.) stressinducible processes in higher eucariotic cells. plenum publishing corporation, new york, pp. 1–26. reski, r. 1998. physcomitrella and arabidopsis— the david and goliath of reverse genetics. trends in plant science 3: 209–210. rowntree, j. k. 2006. development of novel methods for the initiation of in vitro bryophytes culture for conservation. plant cell tissue organ cult. 87:191-201. rowntree, j. k. & ramsay, m. m. 2005. ex situ conservation of bryophytes: progress and potential of a pilot project. bol. soc. esp. briol. 26-27:17-22. rowntree, j. k. & ramsay m. m. 2009. how bryophytes came out of the cold: successful cryopreservation of threatened species. biodiversity and conservation 18: 1413-1420. rowntree jk, pressel s, ramsay mm, sabovljevic a sabovljevic m. in press. in vitro conservation of european bryophytes. in vitro cellular and developmental biology plant sabovljević, a. & sabovljević, m. 2008. bryophytes, a source of bioactive and new compounds. in: govil, j. n. (ed.) phytopharmacology and therapeutic values iv, the series "recent progress in medicinal plants". studium press, houston, texas, usa. pp. 9-25. sabovljević, a., sabovljević, m., grubišić, d. & konjević, r. 2005. the effect of sugars on development of two moss species (bryum argenteum and atrichum undulatum) during in vitro culture. belgian journal of botany 138(1): 79-84. sabovljević, a., sabovljević, m. & jocković, n. (2009). in vitro culture and secondary metabolite isolation in bryophytes. in: jain, s. mohan; saxena, praveen k. (eds) protocols for in vitro cultures and secondary metabolite analysis of aromatic and medicinal plants. methods in molecular biology . humana press pp. 117-128. sabovljević, m., bijelović, a. & dragićević, i. 2003. in vitro culture of mosses: aloina aloides (k.f. schultz) kindb., brachythecium velutinum (hedw.) b.s.&g., ceratodon purpureus (hedw.) brid., eurhynchium praelongum (hedw.) b.s.&g. and grimmia pulvinata (hedw.) sm. turkish journal of botany 27: 441–446. sabovljević, a., soković, m., glamočlija, j., ćirić, a., vujičić, m., pejin, b., sabovljević, m. (2010): comparison of extract bio-activities of in situ and in vitro grown selected bryophyte species. african journal of microbiology research 4(9): 808-812 schofield, w. b. 1981. ecological significance of morphological characters in the moss gametophyte. bryologist 84: 149-165. schumaker, k. s. & dietrich, m. a. 1998. hormone-induced signaling during moss development. annu. rev. plant physiol. plant mol. biol. 49: 501–523. servettaz, c. 1913. reserches experimentales sur le development et la nutrition des mousses en milieux sterilise. ann. sci. nat., bot. biol. veg. 17: 111–223. vujičić, m., sabovljević, a. & sabovljević, m. 2009. axenically culturing the bryophytes: a case study of the moss dicranum scoparium hedw. (dicranaceae, bryophyta). botanica serbica 33(2): 137-140. vujičić, m., cvetić, t., sabovljević, a., sabovljević, m. 2010. axenically culturing the bryophytes: a case study of the liverwort marchantia polymorpha l. ssp. ruderalis bischl. & boisselier (marchantiophyta, marchantiaceae). kragujevac journal of science 32: 73-81. wood, a. j., oliver, m. j., cove, d. j. 2000 new frontiers in bryology and lichenology. bryophytes as model systems. bryologist 103: 128–133. yaman et al., 2020, biologica nyssana 11(1) 11 (1) september 2020: 23-29 doi: 10.5281/zenodo.4060285 the endemic plants in bartın (turkey), and their conservation status original article barbaros yaman bartın university faculty of forestry department of forest botany, turkey yamanbar@gmail.com (corresponding author) zafer kaya bartın university faculty of forestry department of forest botany, turkey zkaya23@hotmail.com bilge tunçkol bartın university ulus vocational school department of forestry and forest products program, turkey bilgetunckol@gmail.com halil barış özel bartın university faculty of forestry department of silviculture, turkey halilbarisozel@gmail.com received: february 04, 2020 revised: april 01, 2020 accepted: april 14, 2020 abstract: bartın province located in the western black sea region of turkey has been selected as a floristic research unit. bartın, previously a district of zonguldak, has earned a provincial status in 1991. compared to other provinces of turkey, the flora and vegetation studies in bartın has been started quite late in detail since the second half of the 1990s. the present study aims to determine endemic taxa in bartın province and to evaluate their conservation status on the basis of iucn, cites and bern criteria. turkey has numerous laws, regulations, and programs on biodiversity conservation, but there are many deficiencies and problems in the implementation of these guidelines. our study was the first report on the endemic taxa and their conservation status in bartın province of turkey. we have determined that 36 of turkey’s endemic taxa are also found in bartın’s flora. we state that 3 of bartın’s endemic plant taxa are endangered (en), 2 is critical (cr), 1 is vulnerable (vu), 7 are near threatened (nt) and the others are least concern (lc) categories. key words: endemic taxa, flora, vegetation, conservation status apstract: endemične biljke bartina, turska, i njihov conservacijski status provincija bartın, koja se nalazi u zapadnom crnomorskom regionu turske, izabrana je za florističku istraživačku jedinicu. bartın, prethodno okrug zonguldak, stekao je status provincije 1991. godine. u poređenju sa drugim provincijama turske, detaljnije studije flore i vegetacije u bartınu započete su prilično kasno, od druge polovine 1990-ih. cilj ove studije je bio utvrđivanje endemičnih taksona u provinciji bartın i procena njihovog statusa u zaštiti na osnovu iucn, cites i bern kriterijuma. turska ima brojne zakone, propise i programe o očuvanju biodiverziteta, ali postoje mnogi nedostaci i problemi u njihovoj primeni. naša studija predstavlja prvi izveštaj o endemičnim taksonima i njihovom statusu očuvanosti u provinciji bartın u turskoj. odredili smo da se 36 turskih endemičnih taksona nalazi u flori bartına. utvrđeno je da su na području barrtina 3 endemična biljna taksona ugrožena (en), 2 kritično ugrožen (cr), 1 ranjiv (vu), 7 su skoro ugroženi (nt), a ostali su u kategoriji poslednje brige (lc). ključne reči: endemični taksoni, flora, vegetacija, conservacijski status introduction in terms of biogeography, turkey is at the crossroads of three different flora region within holoarctic ecozone (ketenoğlu et al., 2014) and three biodiversity hotspots (caucasus, irano-anatolian, and mediterranean) (şekercioğlu et al., 2011). because of these and other traits related to its geomorphological and climatic characteristics, turkey has one of the richest floras of the temperate zone in the world (özhatay et al., 2003). ekim (2014) stated that the number of native vascular plant species in turkey is 9753, and 3035 of them (31.12%) is endemic to the country. bartın, previously a district of zonguldak located in the western black sea region of turkey, earned a provincial status in 1991. flora and vegetation studies in bartın province has been started in detail since the second half of the 1990s. the first studies in this regard were carried out by yatkın (1996), başaran (1999), sarıbaş et al., (2002) and sarıbaş et al., © 2020 yaman et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 23 (2007). after these small-scale studies carried out in the restricted areas in bartın, the first flora list for the whole province was published by kaya & basaran (2006), and later first biotope maps for coastal habitats between amasra and inkum in bartın were constituted by nayim (2010). indeed, kaya & basaran (2006) have stated that there are 672 different plant taxa belonging to 368 genera under 97 families in bartın, and the authors stated that 7 of them (1.04 %) were endemic for turkey. afterward, within the scope of bartın’s biodiversity project, kaya & yaman (2017) have worked on the flora of the whole province in detail. according to kaya & yaman (2017), 35 of turkey’s endemic taxa are also found in bartın’s flora, and 9 of them are geophyte taxa. recently, kure mountains national park (bartın section) has been also examined in terms of its flora and vegetation by tunçkol & aksoy (2018). however, in 2017, a study related to the distribution of endemic taxa locations in turkey’s flora according to provinces has shown that any endemic taxon location doesn’t present in bartın (şenkul & kaya, 2017). although some studies on the province’s partial flora, this review by the authors may be due to the lack of a comprehensive article published on bartın flora available until then. the red-list criteria and conservation status of plant species in the world have been determined by different organizations such as iucn, cites, and bern. based on the iucn criteria, the plant species of turkey have been evaluated by the turkish association for the conservation of nature & van centennial university (ekim et al., 2000). the present study aims to determine the endemic taxa in bartın, and to evaluate their conservation status on the basis of iucn, cites and bern criteria. material and methods bartın province determined as a floristic research unit is divided into grids on the map (fig. 1). during the year 2016, 2017 and 2018, we went to the grids representing different habitats to collect plant specimens every week. the photographs of the plants were also taken during the field work. the specimens were pressed and dried between the folds of old newspapers, and the dried specimens are mounted on herbarium sheets of standard size (41x29 cm). the taxa were identified on the basis of flora of turkey and the east aegean islands (davis (ed.), 1965-1982). the nomenclature was harmonized 24 fig. 1. the map of bartın province located on the western black sea region of turkey. biologica nyssana ● 11 (1) september 2020: 23-29 yaman et al. ● the endemic plants in bartin (turkey), and their conservation status 25 family latin name/locality, altitude, legators cites iucn cupressaceae juniperus oxycedrus f. yaltirikiana avci & ziel. a4 bartın: hatipler coast,10 m., z.kaya & b.yaman * pinaceae abies nordmanniana subsp. equi-trojani (asch. & sint. ex boiss.) coode & cullen a4 bartın:ulus,uluyayla plateau, 1000 m., z.kaya & b.yaman nt amaryllidaceae allium kastambulense kollmann a4 bartın: kurucuşile coast, 20 m., z.kaya & b.yaman nt amaryllidaceae allium olympicum boiss. a4 bartın: ulus, drahna valley, 500-950 m., z.kaya & b.yaman lc apiaceae astrantia maxima subsp. haradjianii (grintz.) rech.f. a4 bartın: ulus, uluyayla, gendarme cemetery, 1500 m, z.kaya & b.yaman lc apiaceae ferulago platycarpa boiss. & balansa a4 bartın: kaynarca, slopes across the municipal park, 30 m., z.kaya & b.yaman lc apiaceae seseli resinosum freyn & sint. a4 bartın: ulus, ulukaya waterfall, 310 m., z.kaya & b.yaman vu araceae arum hygrophilum subsp. euxinum (r.r.mill) alpınar a4 bartın: kurucaşile, kapusu coast, 10 m., z.kaya & b.yaman lc asparagaceae bellevalia clusiana griseb. a4 bartın: güzelcehisar, the field edge on the way to the castle ruins, 70 m.,z.kaya & b.yaman lc asteraceae centaurea cadmea subsp. pontica köse & ocak a4 bartın: ulus, ulukaya waterfall, 310 m., z.kaya & b.yaman lc asteraceae centaurea inexpectata wagenitz a4 bartın: uluyayla plateau, 970 m., z.kaya & b.yaman lc asteraceae centaurea kilaea boiss. a4 bartın: mugada, coast dune, 5 m., z.kaya & b.yaman en asteraceae helichrysum arenarium subsp. aucheri (boiss.) davis & cupicha a4 bartın: ulus, uluyayla plateau, 970 m., z.kaya & b.yaman lc asteraceae inula helenium subsp. orgyalis (boiss.) grierson a4 bartın: arıt, zoni plateau, 841 m., z.kaya & b.yaman nt asteraceae turanecio hypochionaeus (boiss.) hamzaoğlu * a4 bartın: güzelcehisar, lava columns, 10 m., z.kaya & b.yaman cr boraginaceae onosma armena dc. a4 bartın: arıt, zoni plateau, roadside, 800m., z.kaya & b.yaman lc boraginaceae onosma intertexta hub.-mor. a4 bartın: ulus, drahna valley, 770 m., z.kaya & b.yaman nt brassicaceae alyssum pateri nyár. subsp. pateri a4 bartın: ulus, border between bartın and karabük, 1305 m.,z.kaya & b.yaman lc brassicaceae aubrieta canescens subsp. canescens a4 bartın: ulus, drahna, in front of kemerli cave, 850 m., z.kaya & b.yaman lc brassicaceae hesperis bicuspidata (willd.) poir. a4 bartın: arıt, çöpbey,yapıkayası hill, 600 m., z.kaya & b.yaman lc campanulaceae campanula grandis fisch. & c.a.mey. subsp. grandis a4 bartın: ulus,ulukaya village, 350 m., z.kaya & b.yaman lc caprifoliaceae cephalaria paphlagonica bobrov. a4 bartın: kurucaşile, kapısuyu village, 80 m., b.tunçkol nt caryophyllaceae minuartia mesogitana subsp. flaccida mcneill a4 bartın: arıt, roadside to zoni plateau, 864 m., z.kaya & b.yaman en celastraceae euonymus latifolius subsp. cauconis coode & cullen a4 bartın: from ulus to arıt, near to şahin village, 775 m., z.kaya & b.yaman nt fabaceae astragalus bartinense aytaç, tunçkol & n. aksoy a4 bartin: ulus, küre mountains national park, above abdurrahman village, 550 m, tunçkol cr iridaceae crocus ancyrensis (herb.) maw a4 bartın: ulus, uluyayla plateau, near to inonu cave, 950 m., z.kaya & b.yaman lc table 1. endemic taxa in bartın province and their conservation status. biologica nyssana ● 11 (1) september 2020: 23-29 yaman et al. ● the endemic plants in bartin (turkey), and their conservation status 26 plants have strong adaptation capabilities to environmental changes, anthropogenic global warming and climate change have a strong effect on plant life cycles and species’ interactions (settele et al., 2014). in spite of worldwide climate crisis, turkey has not a comprehensive conservation program for its own endemic plants yet. the most of endemic plant species in turkey has been threatened by biodiversitydamaging activities such as coal-fired power plant, dam construction, stone and marble quarry, gold mining, clearing grounds for fields, overgrazing, reform of barren lands, urbanization, tourism, wild fires, and afforestation without biodiversity base (bulut & yılmaz, 2010, şekercioğlu et al., 2011). in terms of conservation importance of each grid square and its threatened endemic plant taxa number in turkey, a4 grid square has high conservation importance (türe & böcük, 2010), and bartın province is on this grid square. recently also in bartın as well as many provinces in turkey, a coal-fired power plant in amasra district and a stone and marble quarry in ulus district have been planned by some private enterprises (atmiş, 2016, atmiş & günsen, 2017, yaman, 2019). in the province, the potential threats to biodiversity can result in the extinction of endemic plant species as well as habitat loss, habitat fragmentation and habitat degradation. in addition, intense tourism pressure on the extremely limited and exclusive coastal habitat of centaurea kilaea (en) and turanecio hypochionaeus (cr), which are bartın’s two of five endangered species, can result in the loss of these two species and their habitats. other endangered plant species in bartın are minuartia mesogitana subsp. flaccida and corydalis caubiologica nyssana ● 11 (1) september 2020: 23-29 yaman et al. ● the endemic plants in bartin (turkey), and their conservation status with ngbb electronic herbarium database in turkey (http://bizimbitkiler.org.tr) and the plant list database (http://www.theplantlist.org/). after taxonomic identification, they were labeled and stored in the herbarium of bartın faculty of forestry at the foundation stage. results and discussion bartın province which is determined as a floristic research area is located in the western black sea region of turkey, and mainly it shows bio-geographically the characteristics of euro-siberian region; however, some plant species belonging to the irano-turanian and mediterranean regions occur in the province. according to kaya & yaman (2017) and our recent field studies, the number of plant taxa in bartın province is approximately 1000. 36 of the turkey’s endemic taxa are also found in bartın’s flora, and 9 of them are geophyte taxa. the most of endemic taxa in bartın is euro-siberian element (21 taxa), 7 of them are irano-turanian element, and the others are mediterranean (1 taxon) or common elements (6 taxa) of two or three flora regions. we state that 3 of bartın’s endemic plant taxa are endangered (en), 2 critically endangered (cr), 1 vulnerable (vu), 7 near threatened (nt) and most of the other least concern categories (lc). the endemic taxa of bartın province and their conservation status and the photographs of some of them are given tab. 1 and fig. 2 respectively. today there is a concept on a biodiversity crisis worldwide related to global warming and climate change. it is known that more than 30.000 species are threatened with extinction (url-1). although family latin name/locality, altitude, legators cites iucn iridaceae crocus bolensis (ruksans) ruksans a4 bartın:uluyayla plateau, near to marble quarry, 970 m., z.kaya & b.yaman * iridaceae iris kerneriana asch. & sint. ex baker a4 bartın: ulus, border between bartın and karabük, 1298 m., z.kaya & b.yaman lc lamiaceae phlomis russeliana (sims.) lag. ex benth. a4 bartın: ulus, sarıçiçek hill, 1500 m., z.kaya & b.yama lc lamiaceae sideritis dichotoma huter a4 bartın: ulus, ulukaya waterfall, 310 m., z.kaya & b.yaman lc orchidaceae dactylorhiza nieschalkiorum h.baumann & künkele a4 bartın: ulus, sarıçiçek hill, 1500 m., z.kaya & b.yaman + lc orobanchaceae melampyrum arvense var. elatius boiss. a4 bartın: ulus, near to çerçi village, 560m., z.kaya & b.yaman nt papaveraceae corydalis caucasica subsp. abantensis lidén a4 bartın: arıt, zoni plateau, 900 m., z.kaya & b.yaman en plantaginaceae digitalis lamarckii ivanina a4 bartın: ulus, kavakseydibaşı, 910m., z.kaya & b.yaman lc ranunculaceae delphinium fissum subsp. anatolicum chowdhuri & p.h.davis a4 bartın: ulus, kumluca, road side after kızıllar, 493m., z.kaya & b.yaman lc rubiaceae asperula pestalozzae boiss a4 bartın: ulus, near to abdurrahman village, 710 m., z.kaya & b.yaman lc 27 biologica nyssana ● 11 (1) september 2020: 23-29 yaman et al. ● the endemic plants in bartin (turkey), and their conservation status fig. 2. some of threatened endemic taxa in bartın, turkey, a. seseli resinosum, b. centaurea kilaea, c. turanecio hypochionaeus, d. corydalis caucasica subsp. abantensis, e. minuartia mesogitana subsp. flaccida casica subsp. abantensis. one of vulnerable species in the province, seseli resinosum, is threatened by the planned stone and marble quarries in ulus district. the project in a high plateau (uluyayla) of the district, also threats the habitats of endemic and nonendemic geophyte species such as crocus ancyrensis and galanthus elwesii, respectively as well as crocus bolensis which is a relatively common endemic species (ruksans, 2017). in addition, dactylorhiza nieschalkiorum, which is not in any threat category based on iucn, might be under the risk of illegal plant collection due to its commercial ornamental value, thus d. nieschalkiorum is on the cites appendix ii (url-2). however, in bartın, there is not any endemic species included in bern convention appendix i (url-3). environmental impact assessment (eia) has been applied legally in the industrial projects in turkey since 1993. however, only 1.58 percent of all eia decisions (67040) were negative (url-4). although turkey has numerous laws, reg28 ulations, and programs on biodiversity conservation, there are many deficiencies and problems in the implementation of these guidelines (kaya & raynal, 2001). in practice, although non-governmental organizations oppose, these rules are often stretched by central and local governments with the idea of economic development and profit (atmiş & günsen, 2017, atmiş, 2018). conclusion our investigation was the first report on the endemic taxa and their conservation status in bartın province of turkey. based on a long-term conservation program, in situ conservation systems should be established urgently for turkey’s plant taxa in the cr, en and vu categories of iucn. in addition, while developing the provincial conservation programs for plant species, critical (cr), endangered (en) and vulnerable (vu) endemic species in the provinces should be considered primarily. biodiversity-damaging activities cited above must not be allowed in habitats having critical (cr), endangered (en) and vulnerable (vu) endemic species in bartın province. references atmiş, e. 2016: yerelde dikkate değer bir örnek: bartın platformu’nun termiksiz yaşam mücadelesi. in: yıldırım, d., haspolat, e. (eds.), değişen karadeniz’i anlamak, 501-522, phoenix, ankara. atmiş, e., günsen, b. 2017: türkiye’de orman yıkımına karşı mücadelelerin analizi. in: uydacı, m. (ed.), turkish studies from different perspectives, 315-336, athens institute for education and research, athens. atmiş, e. 2018: a critical review of the (potentially) negative impacts of current protected area policies on the nature conservation of forests in turkey. land use policy, 70: 675-684. başaran, s. 1999: kirazlık (bartın) barajı florası. phd thesis. zkü fen bilimleri enstitüsü. zonguldak. bulut, z., yılmaz, h. 2010: the current situation of threatened endemic flora in turkey: kemaliye (erzincan) case. pakistan journal of botany, 42(2): 711-719. davis, p.h. (ed.) 1965-1982: flora of turkey and east aegean islands, i-ix. university press, edinburg. ekim, t., koyuncu, m., vural, m., duman, h., aytaç, z., adıgüzel, n. 2000: red data book of turkish plants (pteridophyta and spermatophyta). turkish association for the conservation of nature & van centennial university, barışcan ofset, ankara. 246 p. ekim, t. 2014: damarlı bitkiler. in: güner, a., ekim, t. (eds.), resimli türkiye florası (illustrated flora of turkey), 1: 159-162, ali nihat gökyiğit vakfı, flora araştırmaları derneği ve türkiye i̇ş bankası kültür yayınları, i̇stanbul. kaya, z., raynal, d.j. 2001: biodiversity and conservation of turkish forests. biological conservation, 97: 131-141. kaya, z., başaran, s. 2006. bartın florasına katkılar. gazi üniversitesi orman fakültesi dergisi, 6(1): 40-62. kaya, z., yaman, b. 2017: bartın i̇linin karasal ve i̇ç su ekosistemleri biyolojik çeşitlilik envanter ve i̇zleme projesi (flora bölümü). orman ve su i̇şleri bakanlığı doğa koruma ve milli parklar (dkmp) 10. bölge müdürlüğü bartın i̇l şube müdürlüğü. ankara. 526 p. ketenoğlu, o., vural, m., kurt, l., körüklü, t. 2014: vejetasyon. in: güner, a., ekim, t. (eds.), 2019: resimli türkiye florası (illustrated flora of turkey), 1:163-224, ali nihat gökyiğit vakfı, flora araştırmaları derneği ve türkiye i̇ş bankası kültür yayınları, i̇stanbul. özhatay, n., byfield, a., atay, s. 2003: türkiye’nin önemli bitki alanları. wwf türkiye (doğal hayatı koruma vakfı). i̇stanbul. 88 p. nayim, s.y. 2010: amasra-i̇nkum (bartın) arasında yer alan önemli biyotopların haritalanması. phd thesis. i̇stanbul üniversitesi fen bilimleri enstitüsü. i̇stanbul. ruksans, j. 2017: the world of crocuses. latvian academy of sciences. latvia. 568 p. sarıbaş, m., kaya, z., başaran s., yaman, b. 2002: batı karadeniz bölgesi’nde doğal olarak yetişebilen bitkilerden peyzaj uygulamalarında kullanılabilecek bitkilerin saptanması. in: yahyaoğlu, z. (ed.), 2. ulusal karadeniz ormancılık kongresi, 520-537, kafkas üniversitesi artvin orman fakültesi, artvin. sarıbaş, m., kaya, z., başaran, s., yaman, b., sabaz, m. 2007: the use of some natural plant species from the western black sea region of turkey for landscape design. fresenius environmental bulletin 16(2): 193-205. şekercioğlu, ç. h., anderson, s., akçay, e., bilgin, r., can, ö.e., semiz, g., tavşanoğlu, ç., yokeş, m.b., soyumert, a., kahraman, i̇., sağlam, i̇.k., yücel, m., dalfes, h.n. 2011: turkey’s globally important biodiversity in crisis. biological conservation, 144: 2752–2769. biologica nyssana ● 11 (1) september 2020: 23-29 yaman et al. ● the endemic plants in bartin (turkey), and their conservation status 29 settele, j., scholes, r., betts, r, et al., 2014: terrestrial and inland water systems. in: field, c.b., barros, v.r., dokken, d.j. et al., (eds.), climate change: impacts, adaptation, and vulnerability. part a: global and sectoral aspects, contribution of working group ii to the fifth assessment report of the intergovernmental panel on climate change, 271–359, cambridge university press, cambridge. şenkul, ç., kaya, s. 2017: geographical distribution of endemic plants of turkey. türk coğrafya dergisi, 69: 109-120. tunçkol, b., aksoy, n. 2018: flora of küre mountains national park (bartın section). journal of forestry, 14(2): 80-113. tunçkol, b., aytaç, z., aksoy, n. and fişne, a., 2020. astragalus bartinense (fabaceae), a new species from turkey. acta botanica croatica, 79(2): 131-136. türe, c., böcük, h. 2010: distribution patterns of threatened endemic plants in turkey: a quantitative approach for conservation. journal for nature conservation, 18: 296–303. url-1. https://www.iucnredlist.org/ [accession date: 30 december 2019]. url-2. http://checklist.cites.org/ [accession date: 21 january 2020]. url-3. https://www.coe.int/en/web/conventions/ full-list/-/conventions/treaty/104 [accession date: 21 january 2020]. url-4. https://ced.csb.gov.tr/ [accession date: 21 january 2020]. yaman, b. 2019: bartın’ın gözbebeği uluyayla mermer ocaklarına feda edilebilir mi? orman ve av, 97(5): 10-12. yatkın, h. 1996: amasra yöresi floristik kompozisyonu. msc thesis, zkü fen bilimleri enstitüs. zonguldak. biologica nyssana ● 11 (1) september 2020: 23-29 yaman et al. ● the endemic plants in bartin (turkey), and their conservation status anticancer compounds from medicinal plants biologica nyssana 2 (2)  december 2011: 00-00 ljupković r.b. et al..  removal cu(ii) ions from water... 69 original article antimicrobial activity of the three commercial drug’s essential oils: chamomillae flos, calendulae flos and millefolii herba zorica stojanović-radić university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia e-mail: zstojanovicradic@yahoo.com abstract: stojanović-radić, z.: antimicrobial activity of the three commercial drug’s essential oils: chamomillae flos, calendulae flos and millefolii herba. biologica nyssana, 3 (2), december 2012: 69-76. commercial herbal drugs of the three plants belonging to family asteraceae were used for the extraction of the essential oils (hydrodistillation method). since various factors during the processing of the plant material can affect several characteristics of essential oils such as yield, chemical composition and, thus, biological activities of essential oils, comparison of the antimicrobial activity of the herein isolated oils was done with avaiable literature sources dealing with this subject. in order to evaluate antimicrobial potential of the isolated essential oils, a broth microdilution method was employed. the results pointed to very high activity of the tested oils, in all cases much stronger in comparison to the previous results. as a conclusion, the adverse effects of plant material processing did not affect its antimicrobial potential, while the obtained high antimicrobial activity can be explained by sinergistic action of the herein used solvent with essential oils. key words: achillea millefolium, antimicrobial activity, calendula officinalis, chamomilla recutita, herbal drug introduction essential oils are complex mixtures of compounds, synthetized in plant tissues as secondary metabolites. the role of essential oil in plant is multiple: defence of pathogens and herbovores, attraction of insects, inhibition of other plant's seed germination, formation of protective layer which reduces transpiration and formation of specific microclimate. due to their lipophylic character, essential oils can interact with microbial membranes and achieve significant antimicrobial effect. for this reason, during the last several decades, much attention has been devoted to plant essential oils, especially to their antimicrobial activity against various pathogenic and nonpathogenic species. during the research of some plant's medicinal properties, very important indicator of antimicrobial potential is ethnopharmacological use for the treatment of infective diseases. owing to this, many studies have been focused to ethnopharmacological plants and their antimicrobial activity, as a novel source of antimicrobial compounds. in serbia, plants belonging to family asteraceae are very significant medicinal plants and are used extensively in both traditional medicine and current phytotherapy. among them, very commonly used ones are chamomile, yarrow and calendule. yarrow (achillea millefolium) is an aromatic, perennial herbaceous plant, widely distributed in serbia. healing properties of yarrow are well known from the ancient times and it is used in both official and alternative medicine. according to ph. eur. iv, biological source of the yarrow herbal drug is strictly the species achillea millefolium. however, some authors (b o ž i n et al., 2008) stated that achillea millefolium, in wider sence, represents a cytogenetic and chemically polymorphous aggregate 3 (2) • december 2012: 69-76 biologica nyssana 3 (2)  december 2012: 69-76 stojanović-radić z.  antimicrobial activity of.. 70 of 12 species, characterized by a well defined morphological, anatomical and caryological features, while the existence of the naturally occurring hybrids increases the number of the representatives in the aggregate. herbal drug used in medicinal purposes is the above-ground part of the plant in bloom (millefolii herba). rarely, some parts can be used as individual drugs – inflorescence (millefolii flos) or leaves (millefolii folium) (s a r i ć , 1989). the main utilization is connected to the treatment of the various gastrointestinal disorders owing to its carminative, holagogue and spasmolytic effects. also, it can be used to treat hemorrhoids, hypertension, thrombosis, inflammations and respiratory infections (s a r i ć , 1989; k o j i ć et al., 1998; t a j i k et al., 2008; s a n t ’ a n n a et al., 2009). applied externally, yarrow can heal inflammations of skin and mucosal surfaces; it promotes epitelization, heals the wounds and stops the bleeding process by constricting the blood vessels. essential oil of this plant yields from 0.2-1% and can contain up to 50% of chamazulenes (r o s s , 2003). investigations of its chemical composition showed that it is very variable, depending on factors such as geographical origin, duration of inflorescence and level of polyploidy – the oil has characteristic blue color only in the case of its isolation from tetraploid specimens (k u b e l k a et al., 1999; r o h l o f f et al., 2000; b o š k o v i ć et al., 2005), and it is connected to the presence of the sesquiterpene chamazulene. literature data on antimicrobial activity of achillea millefolium are relatively poor, since the most number of investigations studied antimicrobial effects of its various extracts (k o j i ć et al., 1998; n a s c i m e n t o et al., 2000; h o l e t z et al., 2002; c a n d a n et al., 2003; s t o j a n o v i ć et al., 2005; t a j i k et al., 2008). only one of these studies investigated antimicrobial potential of the yarrow essential oil (c a n d a n et al., 2003), where moderate antimicrobial activity was detected. calendula officinalis l. is used extensively in ethnopharmacology. the most frequent application of this plant is in the form of tincture or creme for curing the wounds, abscesses and decubitus. it has beneficial effect to gastrointestinal and urogenital disorders, and it is also used for sedative purposes (w i l l f o r t , 1959). the famous wound healing properties of this plant prompted investigations which proved its proliferative effects (c h a n d r a n & k u t t o n , 2008; l e a c h , 2008). marigold contains small amounts of essential oil: 0.06-0.3% (n a g u i b et al., 2005), but due to its significant pharmacological activities, it has been studied well. in the most papers, its main component is α-cadinol (c h a l c h a t et al., 1991, o k o h et al., 2007, g a z i m et al., 2008a, p e t r o v i ć et al., 2010). investigations of marigold’s essential oil antimicrobial activity inhibited growth of bacillus subtilis, escherichia coli, staphylococcus aureus, pseudomonas aeruginosa and candida albicans (j a n s s e n et al., 1986). also, it has been shown that it possess potent activity against candida albicans and staphylococcus aureus clinical isolates (g a z i m et al., 2008b; f i t et al., 2009). chamomilla recutita is an annual, perennial plant, native in southeastern europe, but today widely distributed and can be found in north africa, asia, america and new zealand (p i r z a d et al., 2006). chamomille is used in therapy since an ancient times (о w l i a et al., 2007). owing to its medicinal properties, it is included as a drug in pharmacopeia of 26 countries (p i r z a d et al., 2006; о w l i a et al., 2007; s h i k o v et al., 2008). this plant possesses anti-inflammatory, anesthetic, antiseptic, sedative, carminative, antidiarrhoeic and vulnerary properties (p i r z a d et al., 2006; m i l l e r , 2009). scientifically investigated and confirmed pharmacological properties of chamomile are multiple antioxidant (a s g a r y et al., 2002; o w l i a et al., 2007; r o m e i l a h , 2009; a y o u g h i et al., 2011), аntiulcer (k a r b a l a y d o s t & n o o r a f s h a n , 2009), antiinflammatory (p r e s i b e l l a et al., 2006; m c k a y & b l u m b e r g , 2006), vulnerary (m c k a y & b l u m b e r g , 2006; m a r t i n s et al., 2009), anticoagulant, antitumor, antigenotoxic, hypocholesterolemic, antispasmolitic and sedative (m c k a y & b l u m b e r g , 2006). the most pharmacologically active compounds are those found in chamomile essential oil – chamazulene, αbisabolol, bisabolol oxides, farnesene and cyclic esters (o r a v et al., 2001; m a r t i n s et al., 2009), but flavonoids, coumarins and pyrones are also significantly active compounds, found in chamomille (g r g e š i n a et al., 1995; p i r z a d et al., 2006). antimicrobial activity of chamomile is confirmed against 25 different gram positive and gram negative bacterial strains, as well as against 20 lysteria monocytogenes strains (l i s b a l h i n et al., 1998). the same study showed antifungal activity against aspergillus niger, a. ochraceus and fusarium moniliforme. study of o w l i a et al. (2007) showed antistreptococcal activity of chamomile essential oil, which was also active against mycobacterium tuberculosis, salmonella typhymurium and staphylococcus aureus (p i r z a d et al., 2006). beside antibacterial activity, it was confirmed that chamomile essential oil possess antiviral activity against polio and herpes viruses (mcka y & b l u m b e r g , 2006). biologica nyssana 3 (2)  december 2012: 69-76 stojanović-radić z.  antimicrobial activity of.. 71 since most of the literature data about antimicrobial activity of these plants is based on unprecise method (disc diffusion) or the data are relatively old, the main goal of this paper was to investigate antimicrobial properties of these three plants, but using more precise microdilution method and to compare the obtained results with the previous ones on this subject. owing to the fact that commercial plant material (for medicinal purposes) can be found at the markets nowdays, there is high possibility that it possess different properties than the same material collected by botanical experts. the processing after the collection such as drying, time of grounding and storage period before the isolation are very important since they can significantly affect chemical composition of essential oil and, thus, antimicrobial and other biological activities. in the light of these facts, this investigation used commercial plant material which had a lot of potential to differe when compared to personally collected material by relevant experts. the paper provides updated and more precise data on these essential oil's antimicrobial activity, as well as information whether usage of commercial material can significantly affect expected beneficial properties. materials and methods plant material plant material used for isolation of the essential oils was puchased in the form of monocomponent herbal drug from the following commercial sources: jeligor, svrljig (yarrow), montes, leskovac (chamomille) and „josif pančić” institute, belgrade (marigold). isolation of the essential oil air-dried, to constant weight, the commercial sample of carlinae radix drug (three batches of 100-150 g) were subjected to hydrodistillation with ca. 1 l of distilled water for 3.5 h using the original clevenger-type apparatus (c l e v e n g e r , 1928). the obtained oils were separated by extraction with freshly distilled diethyl ether and dried over anhydrous magnesium sulphate. the solvent was evaporated under a gentle stream of nitrogen at room temperature in order to exclude any loss of the essential oil and immediately analyzed. when the oil yields were determined, after the bulk of ether was removed under a stream of n2, the residue was exposed to vacuum at room temperature for a short period to eliminate the solvent completely. the pure oil was then measured on an analytical balance and multiple gravimetric measurements were taken during 24 h to ensure that all of the solvent had evaporated. microorganisms and culture conditions antimicrobial activity assays were performed against seven american type culture collection (atcc) strains: gram positive staphylococcus aureus 6538, gram negative escherichia coli 8739, e. coli 25922, proteus vulgaris atcc 8427, pseudomonas aeruginosa atcc 9027, klebsiella pneumoniae 10031 and the yeast candida albicans atcc 10231. bacterial strains were maintained on nutrient agar (na), while c. albicans was maintained on sabouraud dextrose agar (sda) in the culture collection of the microbiology laboratory, department of biology and ecology, faculty of science and mathematics, university of niš. determination of mic and mbc/mfc (minimal inhibitory/microbicidal) concentrations antimicrobial activity determination was performed by a microdilution method as described previously (s t o j a n o v i ć r a d i ć et al., 2012). briefly, overnight cultures of microorganisms were used for the preparation of suspension. final size of the bacterial inoculum was 5 x 10 5 cfu ml -1 . stock solutions of the essential oil were prepared in 50% aqueous ethanol, whose final concentration never exceeded 5% (v/v) in a well. dilution series were prepared in 96-well microtitre plates in the concentration range from 0.001-50 µl ml -1 (essential oil). after attaining the right dilutions, the inoculums were added to all wells. the plates were incubated for 24 h at 37 ºc (bacteria) and 48 h at 30 ºc (yeast). bacterial growth was determined by adding 20 μl of 0.5% triphenyl tetrazolium chloride (ttc) aqueous solution. one inoculated well was included to allow control of the broth suitability for organism growth. one non-inoculated well, free of antimicrobial agents, was also included to ensure medium sterility. positive controls were tetracycline and nystatine, while the solvent for the oil dilution (ethanol) was used as negative control. minimal inhibitory concentration (mic) was defined as the lowest concentration of the oil inhibiting visible biologica nyssana 3 (2)  december 2012: 69-76 stojanović-radić z.  antimicrobial activity of.. 72 growth (red colored pellet on the bottom of wells after the addition of ttc), while the minimal microbicidal (bactericidal and fungicidal) concentration (mbc/mfc) was defined as the lowest oil concentration killing 99.9% of bacterial/fungal cells. to determine mbc/mfc, the broth was taken from each well without visible growth and inoculated in mueller hinton agar (mha) for 24 h at 37 ºc and in sabouraud dextrose agar (sda) for 48 h at 30 ºc in the case of the tested yeast. experiments were done in quintuplicate. results and discussion essential oils were isolated and yields, together with features are presented in the table 1. the highest yield of essential oil was in the case of yarrow, which classifies this plant into the category of aromatic plants. considering literature data about content of essential oils in the investigated plants, the only one which had different amount is chamomile. in the present work, it yielded only 0.1%, which is lower than the up to now determined range from 0.2-1.9%. this can be explained by material processing before purchasing and distillation, where evaporation of the oil during long storage could contribute to the herein obtained lower yield. the results of antimicrobial activity determination showed activity of the three essential oils in the range from 0.09-50.00 µl ml -1 . the most active essential oil was the one isolated from milefolii herba drug, while the essential oil from calendulae flos exhibited the lowest activity among the tested ones. negative control (solvent = ethanol) showed no activity against any of the tested microbial strains. considering activity in comparison to the reference antibiotics (positive controls), all tested oils showed lower activity than the antibiotic (figures 1-3). considering essential oil of chamomilla recutita, the results of microdilution method revealed very significant activity, which was higher than those reported in previous studies on this subject. the range of the active concentrations was from 0.10-1.73 µl ml -1 (table 2, figure 1). among the tested microbial species, candida albicans showed the highest sensitivity, with inhibited growth at only 0.10 µl ml -1 , while concentration of 0.32 µl ml -1 showed fungicidal effect. on the other hand, the most resistant strain was escherichia coli (atcc 8739), where active concentrations were mic=1.73 µl ml -1 and mbc=2.60 µl ml -1 . table 1. yield and features of the essential oils isolated from commercial material of herbal drugs belonging to three species from family asteraceae herbal drug plant species stated as the only one component of the herbal drug features of isolated essential oil yield of isolated essential oil range of yields according to literature data chamomilae flos chamomila recutita liquid, specific blue color, intensive pleasant aroma 0. 100% 0.20-1.90% milefolii herba achillea millefolium liquid, specific blue color, intensive pleasant aroma 0. 430% 0.20-1.00% calendulae flos calendula officinalis liquid, colorless, intensive pleasant aroma 0. 063% 0.06-0.30% biologica nyssana 3 (2)  december 2012: 69-76 stojanović-radić z.  antimicrobial activity of.. 73 previous investigations on chamomile antimicrobial activity were numerous (l i s b a l h i n et al., 1998; a l i s m a i l & t a l a l , 2003; p i r z a d et al., 2006; o w l i a et al., 2007; s h i k o v et al., 2008; p e r e i r a et al., 2008) and it is considered as a plant with moderate antimicrobial activity. it possesses activity against both gram positive and gram negative strains, as well as against fungi (r o m e i l a h , 2009). essential oil isolated from this plant species showed antibacterial activity at concentration of 25 mg ml -1 against bacillus subtilis, staphylococcus aureus, streptococcus mutans and streptococcus salivarius as well as against candida albicans (a g g a g & y o u s e f , 1972; b e r r y , 1995). in the study of l i s b a l c h i n et al. (1998), it has been reported that this oil has effect against 25 different bacterial species and 20 strains of lysteria monocytogenes. the same study revealed significant antifungal activity against aspergillus niger, a. ochraceus and fusarium culmorum. different streptococcal species streptococcus pyogenes, s. mutans, s. salivarius, s. faecalis and s. sanguis were inhibited by chamomile essential oil in the concentration range from 0.10 – 4.0 µl ml -1 (o w l i a et al., 2007), which is similar to the active concentration range in the present work. the mechanism of chamomile essential oil’s activity was investigated against fungal species, where it was reported that it increases permeability of cytoplasmic membrane, which leads to osmotic pressure disturbance and death of the cell as a consequence (t o l o u e e et al., 2010). however, due to different methods used in the mentioned investigations (disc diffusion in most cases), as well as different solvents used (dimethyl sulfoxide), data from the present paper cannot be compared properly to the previously obtained results (a g g a g & y o u s e f , 1972; b e r r y , 1995; l i s b a l h i n et al., 1998). in some of the existing papers, α-bisabolol is considered as the main carrier of the activity (o w l i a et al., 2007; r o m e i l a h , 2009), which is mentioned in some studies as antifungal compound (p a u l i , 2006; t o l o u e e et al., 2010). it inhibits the synthesis of ergosterol, which would explain the highest activity against fungal organism obtained in the present investigation. results of calendulae flos essential oil’s antimicrobial activity testing showed very high effect against all tested microbial strains. obtained active concentration were in the range from 0.0918.7 µl ml -1 (table 2, figure 2). this essential oil exhibited the highest activity against klebsiella pneumoniae and pseudomonas aeruginosa, while escherichia coli 8739 showed the highest resistance, where higher concentration of 18.7 µl ml 1 was necessary for inhibition of its growth. yeast candida albicans showed very significant sensitivity to this oil, which points to the potential of this plant in the treatment of fungal infections. figure 1. antimicrobial activity of chamomillae flos essential oil compared to the reference antibiotic (µg ml -1 ) тable 2. minimal inhibitory and bactericidal concentrations of essential oils from commercial milefolii herba, calendulae flos and chamomillae flos herbal drugs (µl ml -1 ) bacterial/fungal species атcc number milefolii herba calendulae flos chamomillae flos ab mic mbc mic mbc mic mbc mic staphylococcus aureus 6538 0.29 0.78 0.14 0.39 0.16 0.16 0.39 escherichia coli 25922 0.78 3.12 3.12 6.25 0.12 0.32 1.56 escherichia coli 8739 0.65 1.56 18.7 50.0 1.73 2.60 1.56 klebsiella pneumoniae 10031 0.32 12.50 0.09 0.19 0.13 2.60 0.78 pseudomonas aeruginosa 9027 0.09 0.39 0.09 0.19 0.13 0.32 1.56 proteus vulgaris 8427 0.32 3.12 0.10 0.78 0.10 1.30 1.56 candida albicans 10231 0.12 0.39 0.19 0.19 0.10 0.32 6.25 abreference antibiotic (tetracycline and nystatin), given in µg ml -1 biologica nyssana 3 (2)  december 2012: 69-76 stojanović-radić z.  antimicrobial activity of.. 74 marigold is mentioned in the literature data as a plant with high antimicrobial potential with reported activity against gram positive, gram negative and fungal species (j a n s s e n et al., 1986; i a u k et al., 2003). studies dealing with investigation of antimicrobial effect of marigold’s essential oil isolated from flowers (j a n s s e n et al. 1986; g a z i m et al., 2008b; f i t et al., 2009) reported its activity against bacillus subtilis, escherichia coli, staphylococcus aureus, pseudomonas aeruginosa and candida albicans (j a n s s e n et al. 1986). also, this essential oil showed extraordinary effect against 35 different isolates from skin lesions, with average zone of inhibition of 21.3 mm (at concentration of 30 µl/disc), which was the second strongest activity among ten tested essential oils (aloe vera, rattle, fir, savory, marigold, coconut, eucalyptus, lavender, mint and pine), isolated from medicinal plants (f i t et al., 2009). study on antimicrobial activity of marigold’s essential oil against 23 clinical isolates of candida albicans showed intensive antifungal activity, comparable or sometimes even several times higher than nystatin (g a z i m et al., 2008b). all reported studies pointed to very high antimicrobial potential of marigold, where we must highlight that the lack of results obtained by microdilution method makes the comparison with the present ones very difficult. certainly, according to the previous results, the herein obtained activity was expected. previous studies on chemical composition of this essential oil reported the presence of many compounds already reported as antimicrobial such as monoterpenes αthujone, α-pinene, limonene or 1,8-cineole. therefore, we can suppose that the obtained activity might be a result of their synergistic effect. figure 2. antimicrobial activity of calendulae flos essential oil compared to the reference antibiotic (µg ml -1 ) essential oil isolated from commercial drug of yarrow showed unexpected, very high activity, showing antimicrobial effect against all tested strains. inhibitory concentrations were in the range from 0.09-0.78 µl ml -1 , while concentrations from 0.39-12.50 µl ml -1 exhibited microbicidal effect (table 1, figure 3). this oil showed the highest activity against bacterial species pseudomonas aeruginosa (mic=0.09 µl ml -1 , mbc=0.39 µl ml -1 ), followed by candida albicans with mic at 0.12 µl ml -1 and mbc at 0.39 µl ml -1 . the highest tolerance to the essential oil presence showed both species of escherichia coli, where e. coli 25923 was more resistant and showed sensitivity at 0.78 µl ml -1 (mic) and 3.12 µl ml -1 (mbc). previous investigation of this plant essential oil’s antimicrobial activity (c a n d a n et al., 2003; b o ž i n et al., 2008) pointed to moderate effect, while different extracts in most cases showed no activity. comparison of the present results with the previous one obtained by the same antimicrobial testing method (c a n d a n et al., 2003) showed extremely higher activity of the herein tested essential oil. the oil from the mentioned investigation showed antimicrobial activity in the range from 4.50-72.00 mg ml -1 , while the oil in the present study was active at concentrations from 0.08-0.60 mg ml -1 , which can be explained by eventual qualitative differences of the compared essential oils, as well as by different solvents used. c a n d a n et al. (2003) used 0.5% tween 80 for solution and dispersion of the essential oil, while the present study used ethanol as solvent. beside this, the authors of the previous study added solvent directly into the medium and then performed the dilution of essential oil. in the present study, due to figure 3. antimicrobial activity of milefolii herba essential oil compared to the reference antibiotic (µg ml -1 ) biologica nyssana 3 (2)  december 2012: 69-76 stojanović-radić z.  antimicrobial activity of.. 75 very weak solubility, the oil was first dissolved in 50% ethanol. after that, dilutions were made in the same solvent, which was followed by adding of these solutions into the wells of the microtiter plate in order to achieve appropriate concentrations. owing to the low solubility of the yarrow essential oil, it is possible that the previous study did not have completely dissolved oil, which affected its activity due to lower final concentration of the oil in the wells. also, better activity obtained in this study might be connected to the synergistic activity with ethanol, which does not affect the growth of bacteria in the final concentration, but might increase the permeability of the bacterial membrane and facilitate the passage of the active compounds to its target place. conclusion as a final conclusion, we can state that commercial plant material does not have significantly altered biological properties as a consequence of the plant material processing. the only difference was yield of chamomile essential oil, which can be related to evaporation of oil during a longer period of storage. antimicrobial activity of the tested essential oils was, in all three cases very significant and in the range or even lower than the previously investigated essential oil. higher activity obtained in the present investigation is probably due to more precise and quantitative method used herein (microdilution method), while used solvent might contribute by synergistic action with essential oils. synergism of these mixtures might be very promising and future studies should be focused on this object of investigation. references aggag, m.e., yousef, r.t. 1972. study on antimicrobial activity of chamomile oil. planta medica, 22(2): 140-151. al-ismail, k.m., talal, a. 2003. a study of the effect of water and alcohol extracts of some plants as antioxidants and antimicrobial on longterm storage of anhydrous butter fat. dirasat: agricultural sciences, 30(3): 330–337. asgary, s., naderi, g., bashardoost, n., etminan, z. 2002. antioxidant effect of the essential oil and extract of matricaria chamomilla l. on isolated rat hepatocytes. journal of medicinal plants, 1(1): 69-76. ayoughi, f., barzegar, m., sahari, m.a., naghdibadi, h. 2011. chemical compositions of essential oils of artemisia dracunculus l. and endemic matricaria chamomilla l. and an evaluation of their antioxidative effects. journal of agriculture, science and technology, 13(1): 79-88. berry, m. 1995. the chamomiles. pharmaceutical journal, 254: 191-193. bošković, z., radulović, n., stojanović, g. 2005. essential oil composition of four achillea species from the balkans and its chemotaxonomic significance. chemistry of natural compounds, 41(6): 674–678. božin, b. mimica-dukić, n., bogavac, m., suvajdžić, lj., simin, n., samojlik, i., couladis, m. 2008. chemical composition, antioxidant and antibacterial properties of achillea collina becker ex heimerl s.l. and a. pannonica scheele essential oils. molecules, 13(13): 2058-2068. candan, f., unlu, m., tepe, b., daferera, d., polissiou, m., sökmen, a., akpulat, h.a. 2003. antioxidant and antimicrobial activity of the essential oil and methanol extracts of achillea millefolium subsp. millefolium afan. (asteraceae). journal of ethnopharmacology, 87(2-3): 215–220. chalchat, j.c., garry, r.p., michet, a. 1991. chemical composition of essential oil of calendula officinalis l. (pot marigold). flavour and fragrance journal, 6(3): 189–192. chandran, p.k., kutton, r. 2008. effect of calendula officinalis flower extract on acute phase proteins, antioxidant defense mechanism and granuloma formation during thermal burns. journal of clinical biochemistry and nutrition, 43(2): 58-64. clevenger, j.p. 1928. content of essential oil in plants. american perfumer and essential oil review, 23: 467-503. fit, i.n., rapuntean, g., rapuntean, s., chirila, f., nadas, g.c. 2009. antibacterial effect of essential vegetal extracts on staphylococcus aureus compared to antibiotics. notulae botanicae horti agrobotanici cluj-napoca, 37(2): 117-123. gazim, z.c., rezende, c.m., fraga, s.r., filho, b.p.d., nakamura, c.v., cortez, d.a.g. 2008a. analysis of the essential oils from calendula officinalis growing in brazil using three different extraction procedures. brazilian journal of medical and biological research, 44(3): 391395. gazim, z.c., rezende, c.m., fraga, s.r., svidzinski, t.i.e., cortez, d.a.g. 2008b. antifungal activity of the essential oil from calendula officinalis growing in brazil. brazilian journal of microbiology, 39(3): 61-63. grgešina, d., mandić, m.l., karuza, lj., klapec, t., bočkinac, d. 1995. chemical composition of different parts of matricaria chamomilla. biologica nyssana 3 (2)  december 2012: 69-76 stojanović-radić z.  antimicrobial activity of.. 76 prehrambeno tehnološka i biotehnološka revija, 33(2-3): 111-113. holetz, f.b., pessini, g.l., sanches, n.r., cortez, d.a.g., nakamura, c.v., dias filho, b.p. 2002. screening of some plants used in the brazilian folk medicine for the treatment of infectious diseases. memórias do instituto oswaldo cruz, 97(7): 1027-1031. iauk, l., lo-bue, a.m., milazzo, i., rapisarda, a., blandino, g. 2003. antibacterial activity of medicinal plant extracts against periodontopathic bacteria. phytotherapy research, 17(6): 599-604. janssen, a.m., chin, n.l.j., scheffer, j.j.c., svendsen, a.b. 1986. screening for antimicrobial activity of some essential oils by the agar overlay technique. pharmaceutisch weekblad, 8:289–292. karbalay-doust, s., noorafshan, a. 2009. antriulcerogenic effects of matricaria chamomilla extract in experimental gastric ulcer in mice. iranian journal of medical sciences, 34(3): 198-203. kojić, m., stamenković, v., jovanović, d. 1998. lekovite biljke jugoistočne srbije, zavod za udžbenike i nastavna sredstva, beograd. kubelka, w., kastner, u., glasl, s., saukel, j., jurenitsch, j. 1999. chemotaxonomic relevance of sesquiterpenes within the achillea millefolium group. biochemical systematics and ecology 27(4): 437–444. leach, m.j. 2008. calendula officinalis and wound healing: a systematic review. wounds, 20(8): 17. lis-balchin, m., deans, s.g., eaglesham, e. 1998. relationship between bioactivity and chemical composition of commercial essential oils. flavour and fragrance journal 13(2): 98 –104. martins, m.d., marques, m.m., bussadori, s.k., martins, m.a., pavesi, v.c., mesquita-ferrari, r.a., fernandes, k.p. 2009. comparative analysis between chamomilla recutita and corticosteroids on wound healing. an in vitro and in vivo study. phytotherapy research, 23(2): 274-278. mckay, d.l., blumberg, j.b. 2006. a review of the bioactivity and potential health benefits of chamomile tea (matricaria recutita l.). phytotherapy research, 20(7): 519–530. naguib, n.y., khalil, m.y., el sherbeny, s.e. 2005. a comparative study on the productivity and chemical constituents of various sources and species of calendula plants as affected by two foliar fertilizers. journal of applied sciences research, 1(2): 176-189. nascimento, g.g.f., lacatelli, j., freitas, p.c., silva, g.l. 2000. antibacterial activity of plant extracts and phytochemicals on antibioticresistant bacteria. brazilian journal of microbiology, 31(4): 886-891. okoh, o.o., sadimenko, a.a., afolayan, a.j. 2007. the effects of age on the yield and composition of the essential oils of calendula officinalis. journal of applied sciences,7(23): 3806-3810. orav, a., kailas, t., ivask, k. 2001. volatile constituents of matricaria chamomilla l. from estonia. proceedings of estonian academy of sciences: chemistry, 50: 39-45. owlia, p., rasooli, i., hhorieh, s. 2007. antistreptococcal and antioxidant activity of essential oil from matricaria chamomilla l. research journal of biological sciences, 2(2): 155-160. pauli, a. 2006. α-bisabolol from chamomile-a specific ergosterol biosynthesis inhibitor? international journal of aromatherapy, 16(1): 21–25. pereira, n.p., cunico, m.m., miguel, o.c., miguel, m.d. 2008. promising new oil derived from seeds of chamomilla recutita (l.) rauschert produced in southern brazil. journal of the american oil chemists society, 85: 493-494. petrović, l., lepojević, z., sovilj, v. 2010. composition of essential oil obtained from tubular, head and ligulate flowers of calendula officinalis l. by steam distillation of plant material and co2 extracts. journal of essential oil research, 22(2): 143-146. pirzad, a., alyari, h., shakiba, m.r., zehtabsalmasi, s., mohammedi, a. 2006. essential oil content and composition of german chamomile (matricaria chamomilla l.) at different irrigation regimes. journal of agronomy, 5(3): 451-455. presibella, m.m., villas-bôas, l.b., belletti, k.m.s., santos, c.a.m. weffort-santos, a.m. 2006. comparison of chemical constituents of chamomilla recutita (l.) rauschert essential oil and its antichemotactic activity. brazilian archive of biology and technoogy, 49: 717-724. rohloff, j., skagen, e.d., steen, a.h., iversen, t.h. 2000. production of yarrow (achillea millefolium l.) in norway: essential oil content and quality. journal of agricultural and food chemistry 48(12): 6205–6209. romeilah, r.m. 2009. anticancer and antioxidant activities of matricaria chamomilla l. and marjorana hortensis essential oils. research journal of medicine and medical sciences, 4(2): 332-339. ross, j. 2003. combining western herbs and chinese medicine: principles, practice & materia medica. "achillea", greenfields press, 165-181. biologica nyssana 3 (2)  december 2012: 69-76 stojanović-radić z.  antimicrobial activity of.. 77 sant'anna, j.r., franco, c.c., miyamoto, c.t., cunico, m.m., miguel, o.g., côcco, l.c., yamamoto, c.i., junior, c.c., de castro-prado, m.a. 2009. genotoxicity of achillea millefolium essential oil in diploid cells of aspergillus nidulans. phytotherapy research, 23(2): 231235. sarić, m. (1989): lekovite biljke sr srbije, sanu, beograd. shikov, a.n., pozharitskaya, o.n., makarov, v.g. kvetnaya, a.s. 2008. antibacterial activity of chamomilla recutita oil extract against helicobacter pylori. phytotherapy research, 22(2): 252–253. stojanović, g., radulović, n., hashimoto, t., palić, r.. 2005. in vitro antimicrobial activity of extracts of four achillea species: the composition of achillea clavennae l. (asteraceae) extract. journal of ethnopharmacology, 101(1-3): 185190. stojanović-radić, z., čomić, lj., radulović, n., blagojević, p., miltojević, a., mihajilov-krstev, t., rajković, j. 2012. commercial carlinae radix herbal drug: botanical identity, chemical composition and antimicrobial properties. pharmaceutical biology, 50(8): 933-940. tajik, h., jalali, f.s.s., sobhani, a., zadeh, m.s., shahbazi, y. 2008. in vitro assessment of antimicrobial efficacy of alcoholic extract of achillea millefolium in comparison with penicillin derivatives. journal of animal and veterinary advances, 7(4): 508-511. tolouee, m., alinezhad, s., saberi, r., eslamifar, a., zad, s.j., jaimand, k., taeb, j., rezaee, m.b., kawachi, m., shams-ghahfarokhi, m., razzaghi-abyaneh, m. 2010. effect of matricaria chamomilla l. flower essential oil on the growth and ultrastructure of aspergillus niger van tieghem. international journal of food microbiology, 139(3):127-33. willfort, r. 1959. gesunheit durch heilkrauter. rudolf trauner verlag lintz, germany. lošić et al. 2020, biologica nyssana 11(1) 11 (1) september 2020: 55-58 doi: 10.5281/zenodo.4060300 total phenol content and antioxidant potential of different brassica oleracea varieties original article ajna lošić laboratory for plant physiology, faculty of science, university of sarajevo, sarajevo, bosnia and herzegovina ajna.losic@gmail.com adisa parić laboratory for plant physiology, faculty of science, university of sarajevo, sarajevo, bosnia and herzegovina adisacausevic@hotmail.com arnela demir laboratory for plant physiology, faculty of science, university of sarajevo, sarajevo, bosnia and herzegovina arnela_s1@yahoo.com erna karalija laboratory for plant physiology, faculty of science, university of sarajevo, sarajevo, bosnia and herzegovina erna.karalija@gmail.com (corresponding author) received: february 20, 2020 revised: march 24, 2020 accepted: march 28, 2020 abstract: varieties of brassica oleracea are commonly used in human nutrition. beside nutritive role it is suggested that diets reach in cabbage play important role in disease prevention. the aim of this study was to determine total phenol content and the antioxidant potential of different brassica oleracea varieties (broccoli, brussel sprouts, cauliflower, green cabbage and red cabbage). the highest content of total phenols was found in red cabbage and the lowest in broccoli. red cabbage had the highest ability to remove free radicals, while the lowest radical scavenging activity was recorded for broccoli. strong correlation between phenolic content and antioxidant activity suggests the importance of phenolics in health benefits of cabbages. the study identified differences between different varieties suggesting different nutritional advantages, providing useful information in selection of right cabbage variety as diet supplement in disease prevention. key words: brussel sprouts, broccoli, cauliflower, dpph radical scavenging, green cabbage, phenols, red cabbage apstract: ukupan sadržaj fenola i antioksidativni potencijal različitih varijeteta brassica oleracea različiti varijeteti vrste brassica oleracea često se koriste u ishrani ljudi. pored hranljive uloge, smatra se da ishrana bogata kupusom ima važnu ulogu u prevenciji bolesti. cilj ovog istraživanja bio je da se utvrdi ukupan sadržaj fenola i antioksidativni potencijal različitih varijeteta vrste brassica oleracea (brokoli, kelj pupčar, karfiol, zeleni kupus i crveni kupus). najveći sadržaj ukupnih fenola utvrđen je u crvenom kupusu, a najmanji u brokoliju. crveni kupus je imao najveću sposobnost uklanjanja slobodnih radikala, dok je najmanja aktivnost uklanjanja radikala zabeležena kod brokolija. izražena korelacija između sadržaja fenola i antioksidativne aktivnosti ukazuje na značaj fenola u zdravstvenim prednostima kupusa. studija je identifikovala razlike između različitih varijeteta koje sugerišu različite nutritivne prednosti, pružajući korisne informacije u izboru prave sorte kupusa kao dodatak ishrani u prevenciji bolesti. ključne reči: kelj pupčar, brokoli, karfiol, dpph uklanjanje radikala, zeleni kupus, fenoli, crveni kupus introduction brassicaceae (cruciferae) or the cabbage family includes many economically important, edible and industrial types of oilseeds, vegetables, spices and fodder. this family is also known to comprise more than 120 wild species, some of which are very important for agriculture e.g. sinapsis arvensis and raphanus sativus (warwick, 2011). the long history of plant selection has resulted that this crop group is highly variable in use, appearance and phytochemistry. brassica oleracea is a good example with several important varieties: broccoli, cauliflower, brussel sprouts, cabbage, kale (bjorkman et al., 2011). oxidative stress has been identified as a major cause of the development and progression of several diseases. supplementation with exogenous antioxidant or enhancement of endogenous antioxidant defence of the body is a promising way of counteracting the reactive oxygen species (ros) side effects that cause oxidative damage. plants have long been a source of exogenous antioxidants. two-thirds of © 2020 lošić et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 55 the worlds’ plant species are thought to be medically significant, and almost all of them have excellent antioxidant potential. plants have effective, complex enzymatic and non-enzymatic antioxidant defence systems to avoid the toxic effects of free radicals and non-enzymatic antioxidant system including secondary metabolites as antioxidants (kasote et al., 2015). phenolic compounds from plants represent the largest group of secondary plant metabolites found in fruits, vegetables and teas. they are characterized by antioxidant, anti-inflammatory, anticancerogenic and other biological properties, and can protect plants from oxidative stress and some diseases (ahmed et al., 2017). over the last few decades, vegetables from the brassicaceae family have come under scientific attention because of numerous epidemiological studies that provide evidence that diets rich in these vegetables are associated with a reduces risk of several types of cancer and other chronic diseases. because of that, these vegetables are considered promising sources of metabolites for cancer prevention, and mechanism of anticancerogenic activity is mostly associated with glucosinolate degradation products (šamec et al., 2019). the aim of this study was to determine the content of total phenols and antioxidant potential of selected brassica oleracea varieties (broccoli, cauliflower, brussel sprouts, green cabbage, and red cabbage). material and methods plant material five mostly consumed brassica oleracea varieties were selected for the analysis: b. oleracea var. capitata (green cabbage) – fresh leaves; b. oleracea var. capitata f. rubra (red cabbage, bosnia and herzegovina) – fresh leaves; b. oleracea var. italica (broccoli) – broccoli flowers (frozen by rapid freezing technique); b. oleracea var. botrytis (cauliflower) – fresh flowers of cauliflower; b. oleracea var. gemmifera (brussel sprouts) – axillary buds were used (frozen by rapid freezing technique). extract preparation all samples were air-dried and extraction was performed by maceration with 80% ethanol. prepared macerate was incubated in the dark at 4 °c for 24 hours. after incubation, macerate was centrifuged at 5000 rpm for 30 minutes, and supernatant was collected for the analysis. extract yield was determined for all samples. analysis of total phenol content analysis of phenol content in ethanol extracts of different b. oleracea varieties was performed using folin ciocalteu reagent (fcr), by diluting 20 ml of extract with 1580 ml water and adding 100 ml of fcr, followed by addition of 300 ml 7.5% na2co3 after 3 min of incubation (wolfe et al., 2003). total phenols were quantified based on the direction of the calibration curve of gallic acid. the obtained values are expressed as mg of gallic acid equivalents per gram of dry material (mg ga/gdw). analysis of antioxidant capacity the dpph (2,2 – diphenyl – 1 – picrylhydrazyl) method was used to determine the antioxidant activity of the tested extracts (mishra et al., 2012). a stock solution of dpph was prepared by weighing 50 mg of dpph and dissolving in 100 ml of 96% ethanol. a working dpph was prepared by diluting 5 ml of stock solution with 96% ethanol to the 50 ml mark and by adjusting dpph absorbance reading to 1.123 at 517 nm (lambda 25; perkin elmer). extract testing was performed by mixing 100 ml of extract with diluted dpph and final absorbance reading was recorded after 30 minutes against 96% ethanol (blank). free radical scavenging activity was calculated by the formula: aa% antioxidant capacity; a(t0) – absorbance at 0 seconds; a(60) – absorbance after 60 seconds. naringenin was used as a standard in concentration of 1 mg/ml. statistical analysis the data were expressed as means of three independent replicates ± standard deviation. all data were statistically evaluated by analysis of variance with the anova test (p< 0.05 significance level), and the means were analyzed by regression analysis by newman keuls test. pearson’s correlation was used to assess relationships between the variables. all statistical testing was performed using statistica 10 software (copyright© statsoft, inc. 19842011). results and discussion differences in extract yield for different b. oleracea cultivars were recorded. the highest yield was noted for green cabbage (21.5%), followed by red cabbage, cauliflower and sprouts, and the lowest yield was registered for broccoli (2.6%) (tab. 1). the differences in extract yield for the analyzed b. oleracea varieties can be attributed to the availability of the components that are extracted from the sample, which in itself depends on the analyzed variety (hsu et al., 2006). also, the effectiveness of the solvent 56 biologica nyssana ● 11 (1) september 2020: 55-58 lošić et al. ● total phenol content and antioxidant potential of different brassica oleracea varieties 57 biologica nyssana ● 11 (1) september 2020: 55-58 lošić et al. ● total phenol content and antioxidant potential of different brassica oleracea varieties used for extraction to dissolve endogenous components has a significant effect on the yield of the extracts (siddhuraju & becker, 2003). other factors that can affect yield of extract solvent polarity, extraction time and temperature as well as the chemical nature of the sample itself (sun & ho, 2005). the choice of extraction solvent was selected according to targeted components (phenolics). the analysis of total phenolic content showed high concentrations in red cabbage, while broccoli contained only small amounts of phenolics (tab. 1). compared to other extraction solvents, 80% ethanol is less effective and methanol extracts are richer in phenolics as previously recorded by jaiswal et al. (2011). high phenolic content in red cabbage is probably result of high sinapic acid content as previously recorded in other studies (mattila & hellström, 2007). analysis of dpph radical scavenging activity by ethanol extracts of different varieties of b. oleracea are shown in tab. 1. the antioxidant capacity of the tested extracts ranged from 86.63% to 7.14% for red cabbage and broccoli, respectively. total phenolic content strongly correlated with dpph scavenging activity (r2 0,9755; p<0,05). high antioxidant capacity of red cabbage has been previously recorded by several authors (podsędek et al., 2006; isabelle et al., 2010; rokayya et al., 2013; liang et al., 2019). miller et al. (2000) studied the antioxidant effects of different vegetables and noticed a striking difference between red and green cabbage, suggesting a strong role of red cabbage pigment in antioxidant activity. oxidative stress is one of major causes for pathophysiology of several cardiovascular diseases (lahera et al., 2007). dietary phenolic antioxidants play significant role in counteracting cardiovascular diseases (chiu et al., 2018) and intake of red cabbage is beneficial for human health, as suggested by our study as well. conclusion presented study evaluated different brassica oleracea varieties for their phenolic content and antioxidative capacity. among the tested varieties, red cabbage had the highest phenolic content and significant antioxidant capacity, suggesting the importance of red cabbage in human nutrition. references ahmed, e., arshad, m., khan, m.z., amjad, h.s., sadaf, h.m., riaz, i., sabir, s., ahmad, n., 2017: secondary metabolites and their multidimensional prospective in plant life. journal of pharmacognosy and phytochemistry, 6 (2): 205-214. björkman, m., klingen, i., birch, a.n., bones, a.m., bruce, t.j., johansen, t.j., meadow r., mølmann, j., seljåsen, r., smart, l.e., stewart, d. 2011: phytochemicals of brassicaceae in plant protection and human health-influences of climate, environment and agronomic practice. phytochemistry, 72 (7): 538-556. hsu, b., coupar, i.m., ng, k. 2006: antioxidant activity of hot water extract from the fruit of the doum palm, hyphaene thebaica. food chemistry, 98 (2): 317-328. table 1. extract yield, total phenol content and antioxidant capacity of different brassica oleracea varieties sample extract yield (%) total phenol content (mg ga/g dw) antioxidant capacity (%) red cabbage b. oleracea var. capitata f. rubra 12.00 b ± 0.10 27.97a ± 3.23 86.63a ± 1.97 green cabbage b. oleracea var. capitata 21.50 a ± 0.12 7.89bc ± 0.88 19.88c ± 0.60 broccoli b. oleracea var. italica 2.60 d ± 0.09 3.41d ± 0.73 7.14d ± 0.30 brussel sprouts b. oleracea var. gemmifera 9.30 c ± 0.09 6.78c ± 0.67 25.76b ± 0.26 cauliflower b. oleracea var. botrytis 11.20 bc ± 0.11 8.78b ± 0.90 23.49b ± 1.28 standard (naringenin) 90.00a ± 3.89 data expressed as means ± standard deviation. means of samples not sharing the same letter in one column are significantly different (p < 0.05); ga equivalents of gallic acid; dw dry weight. 58 isabelle, m., lee, b.l., lim, m.t., koh, w.p., huang, d., ong, c.n. 2010: antioxidant activity and profiles of common vegetables in singapore. food chemistry, 120 (4): 993-1003. jaiswal, a.k., rajauria, g., abu-ghannam, n., gupta, s. 2011: phenolic composition, antioxidant capacity and antibacterial activity of selected irish brassica vegetables. natural product communications, 6 (9): 1299-1304. kasote, d.m., katyare, s.s., hegde, m.v., bae, h. 2015: significance of antioxidant potential of plants and its relevance to therapeutic applications. international journal of biological sciences, 11 (8): 982–991. lahera, v., goicoechea, m., garcia de vinuesa, s., miana, m., heras, n.d.l., cachofeiro, v., luno, j. 2007: endothelial dysfunction, oxidative stress and inflammation in atherosclerosis: beneficial effects of statins. current medicinal chemistry, 14 (2): 243-248. liang, y., li, y., zhang, l., liu, x. 2019: phytochemicals and antioxidant activity in four varieties of head cabbages commonly consumed in china. food production, processing and nutrition, 1 (1): 3. mattila, p., hellstrōm, j. 2007: phenolic acids in potatoes, vegetables, and some of their products. journal of food composition and analysis, 20 (3): 152-160. miller, h.e., rigelhof, f., marquart, l., prakash, a., kanter, m. 2000: antioxidant content of whole grain breakfast cereals, fruits and vegetables. journal of the american college of nutrition, 19 (3): 312-319. mishra, k., ojha, h., chaudhury, n.k. 2012: estimation of antiradical properties of antioxidans using dpph assay: a critical review and results. food chemistry, 130 (4): 1036-1043. podsȩdek, a., sosnowska, d., redzynia, m., anders, b. 2006: antioxidant capacity and content of brassica oleracea dietary antioxidants. international journal of food science and technology, 41 (1): 4958. rokayya, s., li, c.j., zhao, y., li, y., sun, c.h. 2013: cabbage (brassica oleracea l. var. capitata) phytochemicals with antioxidant and antiinflammatory potential. asian pacific journal of cancer prevention, 14 (11): 6657-6662. siddhuraju, p., becker, k. 2003: antioxidant properties of various solvent extracts of total phenolic constituents from three different agroclimatic origins of drumstick tree (moringa oleifera lam.) leaves. journal of agricultural and food chemistry, 51 (8): 2144-2155. sun, t., ho, c.t. 2005: antioxidant activities of buckwheat extracts. food chemistry, 90 (4): 743749. šamec, d., urlić, b., salopek-sondi, b. 2019: kale (brassica oleracea var. acephala) as a superfood: review of the scientific evidence behind the statement. critical reviews in food science and nutrition, 59 (15): 2411-2422. warwick, s.i. 2011: brassicaceae in agriculture. in: schmidt, r., bancroft, i. (eds.), genetics and genomics of the brassicaceae, 33-65, springer, new york, usa. wolfe, k., wu, x., liu, r.h. 2003: antioxidant activity of apple peels. journal of agricultural and food chemistry, 51 (3): 609-614. biologica nyssana ● 11 (1) september 2020: 55-58 lošić et al. ● total phenol content and antioxidant potential of different brassica oleracea varieties anticancer compounds from medicinal plants biologica nyssana 2 (2)  december 2011: 00-00 ljupković r.b. et al..  removal cu(ii) ions from water... 91 original article basidiomycetes of temska village area (eastern serbia, mt stara planina) dušan sadiković 1*, eleonora čapelja 2 , marko dašić 3 1 mycological society of niš, somborska 81 a/9, 18000 niš, serbia 2 university of novi sad, faculty of siences, department of biology and ecology, trg dositeja obradovica 3, 21000 novi sad, serbia 3 svetosavska 38, 19370 boljevac, serbia *e-mail: dusan.sadikovic@gmail.com abstract: sadiković, d., čapelja, e., dašić, m.: basidiomycetes of temska village area (eastern serbia, mt stara planina). biologica nyssana, 3 (2), december 2012: 91-96. as a result of mycological research, a total of 110 species belonging to 45 genera, 27 families were recorded in the temska village area. the examination of ecological-trophic structure showed that the most numerous were the mycorrhizal fungi. of all identified species 19 are protected by the national law and 16 are included in the preliminary national red list. key words: endangered, macrofungi , red list. introduction the village of temska (43° 15' 38" n, 22° 32' 39" e) is situated in the south-western foothill of the mountain stara planina, at an altitude of about 500 m (fig. 1). it is surrounded by the temska gorge, krušavica, temačko brdo, kulišta and ravna hills. the area has a temperate continental climate. mean annual temperature is 9°c, with the amountof precipitation ranging from 500mm to 1000mm per year (d u c i ć et al., 2005). alluvial deposits can be found in this area. rendzina soil type was developed on marlstone and limestone geological ground (a n đ e l k o v i ć 1958). although an overview of mycromycete fungi for stara planina mt. was given by i v a n c e v i c & b e r o n j a (2004), this is the first paper focusing on basidiomycetes of the area surrounding temska village. materials and methods fungal specimens were being collected in the period between 2010. and 2012. in spring, summer and autumn. endangered and protected species were not collected and were identified on the spot. a large proportion of fungal basidiocarps were collected at the altitudes between 500 and 1100 m in the oak forest belt (quercus cerris l., q. frainetto ten., q. petraea (mattuschka) liebl., q. pubescens willd.), also inhabited by some other wood species such as betula pendula roth, fraxinus excelsior l., f. ornus l., crataegus monogyna jacq., acer campestre l., a. pseudoplatanus l., a. tataricum l., etc. also a number of species was collected from a belt of coniferous forest, consisting mainly of planted pinus nigra j.f.arnold trees. the fungi were identified on the basis of macroscopic and microscopic morphological characteristics and specific chemical reactions of fruiting bodies according to specific identification keys: (b o ž a c 1978; f o h t 1986; b r e i t e n b a c h & k r ä n z l i n 1995; m a c a et al., 1995; m c k n i g h t et al., 1998; j o r d a n 2004; u z e l a c 2009). author citations for each taxon are abbreviated according to the index fungorum (2013). 3 (2) • decemer 2012: 91-96 biologica nyssana 2 (2)  december 2011: 91-96 sadiković d. et al.  basidiomycetes of temska village area... 92 table 1. list of mycromycete fungi with preferred habitat type and substrate taxa habitat life form agaricales agaricaceae agaricus bitorquis (quél.) sacc. edge of broadleaf forest saprophytic agaricus campestris var. campestris l. meadow saprophytic agaricus macrosporus (f.h. moller & jul. schaeff.) pilat meadow saprophytic agarius nivescens (f.h. møller) f.h. møller meadow saprophytic agaricus moelleri wasser. broadleaf forest saprophytic agaricus silvaticus schaeff. edge of broadleaf forest saprophytic lepiota castanea quél. mixed broadleaf forest saprophytic macrolepiota procera (scop.) singer meadow; edge of the forest saprophytic macrolepiota rhacodes (vittad.) singer meadow; edge of the forest saprophytic amanitaceae amanita caesarea (scop.) pers. edge of broadleaf forest mycorrhizal amanita fulva (schaeff.) fr. broadleaf forest mycorrhizal amanita lividopallescens (secr. ex boud.) kühner & romagn. mixed broadleaf forest mycorrhizal amanita pantherina (dc.) krombh. broadleaf forest mycorrhizal amanita phalloides (vaill. ex fr.) broadleaf forest mycorrhizal amanita rubescens pers. mixed broadleaf forest mycorrhizal amanita vaginata (bull.) fr. broadleaf forest mycorrhizal amanita vaginata var. alba (sacc.) romagn. broadleaf forest mycorrhizal amanita virosa (fr.) bertill mixed forest mycorrhizal coprinaceae coprinus niveus (pers.) meadow; tilled fields saprophytic coprinus comatus (o.f. müll.) pers. tilled fields saprophytic figure 1. geographical position of investigated area based on google earth satellite image biologica nyssana 3 (2)  december 2012: 91-96 sadiković d. et al.  basidiomycetes of temska village area.. 93 taxa habitat life form coprinus micaceus (bull.) fr. tilled fields saprophytic panaeolus acuminatus (schaeff.) edge of the forest saprophytic hygrophoraceae hygrocybe conica (schaeff.) quél. meadow; edge of the forest saprophytic marasmiaceae marasmius oreades (bolton) fr. meadow; edge of the forest saprophytic marasmius rotula (scop.) fr. meadow saprophytic marasmius torquescens quél. broadleaf forest saprophytic omphalotus olearius (dc.) singer broadleaf forest saprophytic mycenaceae mycena renati quél. mixed forest saprophytic mycena vitilis (fr.) quél. mixed forest saprophytic pleurotaceae pleurotus eryngii (dc.) quél. broadleaf forest parasitic pleurotus ostreatus (jacq.) p. kumm. broadleaf forest parasitic/ saprophytic pluteaceae pluteus cervinus (schaeff.) p. kumm. edge of broadleaf forest saprophytic pluteus salicinus (pers.) p. kumm. broadleaf forest saprophytic schizophyllaceae schizophyllum commune fr. mixed forest parasitic strophariaceae pholiota highlandensis quadr. & lunghini meadow saprophytic tricholomataceae clitocybe odora (bull.:fr.) kummer broadleaf forest saprophytic leucopaxillus giganteus (quél.) singer broadleaf forest saprophytic oudemansiella radicata (relhan) singer mixed broadleaf forest saprophytic rugosomyces ionides (bull.) bon broadleaf forest saprophytic tricholoma fulvum (fr.) bigeard & h. guill mixed broadleaf forest mycorrhizal auriculariales auriculariaceae auricularia auricula–judae (bull.) quél. mixed broadleaf forest saprophytic auricularia mesenterica (dicks.) pers. edge of the forest saprophytic boletales boletaceae boletus aestivalis (paulet) fr. broadleaf forest mycorrhizal boletus aereus secr. mixed broadleaf forest mycorrhizal boletus chrysenteron (bull.) edge of broadleaf forest mycorrhizal boletus calopus pers. broadleaf forest mycorrhizal boletus edulis (bull.) broadleaf forest mycorrhizal boletus luridus (schaeff.) meadow;edge of the forest; mycorrhizal boletus luridiformis (rostk.) meadow; edge of the forest; mycorrhizal boletus rubellus (krombh.) broadleaf forest mycorrhizal boletus satanas rostk. edge of broadleaf forest mycorrhizal boletus subtomentosus j.f. gmel. broadleaf forest mycorrhizal leccinum carpini (r. schulz) m.m. moser ex d.a. reid mixed broadleaf forest mycorrhizal leccinum scabrum (bull.) gray mixed broadleaf forest mycorrhizal leccinum variicolor watling mixed broadleaf forest mycorrhizal xerocomus porosporus (imler ex bon & g. moreno) contu mixed forest mycorrhizal biologica nyssana 3 (2)  december 2012: 91-96 sadiković d. et al.  basidiomycetes of temska village area... 94 taxa habitat life form xerocomus chrysenteron (bull.) quélet. mixed forest mycorrhizal suillaceae suillus granulatus (l.) roussel coniferous forest mycorrhizal suillus luteus (l.) roussel coniferous forest mycorrhizal cantharellales cantharellaceae craterellus cornucopioides (pers.) broadleaf forest saprophytic cantharellus cibarius fr. broadleaf forest saprophytic cantharellus cinereus (pers.) fr. broadleaf forest saprophytic dacrymycetales dacrymycetaceae ditiola peziziformis (lév.) d.a. reid broadleaf forest saprophytic lycoperdales lycoperdonaceae calvatia excipuliformis (scop.) perdeck mixed broadleaf forest saprophytic lycoperdon echinatum schumach. broadleaf forest saprophytic lycoperdon mammiforme pers. mixed broadleaf forest saprophytic lycoperdon perlatum pers. mixed broadleaf forest saprophytic geastrales geastraceae geastrum fornicatum (huds.) hook. mixed broadleaf forest saprophytic phallales gomphaceae ramaria eumorpha (p. karst.) corner mixed broadleaf forest mycorrhizal ramaria fennica (p. karst.) ricken mixed broadleaf forest mycorrhizal phallaceae phallus hadriani vent. edge of broadleaf forest saprophytic polyporales fomitopsidaceae postia stiptica (pers.) jülich mixed forest saprophytic ganodermataceae ganoderma lucidum (curtis) p. karst broadleaf forest parasitic/ saprophytic ganoderma resinaceum boud. broadleaf forest parasitic / saprophytic polyporaceae daedalea quercina (l.) pers. broadleaf forest saprophytic fomes fomentarius (l.) fr. broadleaf forest parasitic polyporus badius jungh. broadleaf forest saprophytic polyporus leptocephalus (jacq.) fr. broadleaf forest parasitic/ saprophytic polyporus squamosus (huds.) fr. broadleaf forest parasitic/ saprophytic trametes gibbosa (pers.) fr. mixed broadleaf forest saprophytic trametes hirsuta (wulfen) lloyd broadleaf forest saprophytic trametes versicolor (l.) lloyd broadleaf forest saprophytic flammulina velutipes (curtis) singer broadleaf forest saprophytic russulales auriscalpiaceae auriscalpium vulgare gray. coniferous forest saprophytic russulaceae lactarius aurantiofulvus j. blum ex bon meadow;edge of the forest mycorrhizal biologica nyssana 3 (2)  december 2012: 91-96 sadiković d. et al.  basidiomycetes of temska village area.. 95 results the total of 110 basidiomycetes were identified during the mycological investigation of the area surrounding temska village. these species of fungi belong to 45 genera, 27 families and 11 orders and are listed in the table 1 (table 1. list of mycromycete fungi with preferred habitat type and substrate). discussion taxonomic analysis mycological survey of the area surrounding the village temska has resulted in a total of 110 fungi species belonging to 45 genera, 27 families and 11 orders. the families with the highest species diversity are: russulaceae (24 species or 21.82 % of total species number), boletaceae (15 species or 16.64 %), agaricaceae (9 species or 8.19 %) and amanitaceae (9 species or 8.19 %). regarding the species diversity, the richest genera are: russula (16 species), boletus (10 species) and lactarius (8 species). myc 48% sap 43% par 4% par/sap 5% figure 2. the proportion of groups in fungal species (myc mycorrhizal fungi, sap saprophytic fungi, par/sap fungi with parasitic and/or saprophytic ecology, par – parasitic fungi) ecological-trophic structure four trophic groups of fungi were found in the plant communities of the temska village area. analysis of the data presented showed that taxa habitat life form lactarius deliciosus (l.) gray broadleaf forest mycorrhizal lactarius evosmus kuhner & romagnesi mixed broadleaf forest mycorrhizal lactarius fulvissimus (romagn.) broadleaf forest mycorrhizal lactarius piperatus (l.) pers. broadleaf forest mycorrhizal lactarius tabidus fr. edge of broadleaf forest mycorrhizal lactarius vellereus (fr.) fr. broadleaf forest mycorrhizal lactarius zonarius (bull.) meadow; edge of the forest mycorrhizal russula aeruginea lindbl. ex fr. edge of broadleaf forest mycorrhizal russula chloroides (krombholz) bresadola broadleaf forest mycorrhizal russula cyanoxantha (schaeff.) fr. broadleaf forest mycorrhizal russula emetica (schaeff.) pers. broadleaf forest mycorrhizal russula fellea (fr.) fr. broadleaf forest mycorrhizal russula fragilis var. fallax (schaeff.) massee edge of broadleaf forest mycorrhizal russula grisea fr. broadleaf forest mycorrhizal russula heterophylla (fr.) fr. broadleaf forest mycorrhizal russula lepida fr. broadleaf forest mycorrhizal russula mairei (singer) broadleaf forest mycorrhizal russula melliolens quél. broadleaf forest mycorrhizal russula nigricans fr. broadleaf forest mycorrhizal russula sanguinea fr. broadleaf forest mycorrhizal russula turci bres. broadleaf forest mycorrhizal russula vesca fr. broadleaf forest mycorrhizal russula virescens (schaeff.) fr. broadleaf forest mycorrhizal stereaceae stereum hirsutum (willd.) pers. mixed forest parasitic thelephorales bankeraceae sarcodon imbricatus (l.) p. karst. broadleaf forest mycorrhizal biologica nyssana 3 (2)  december 2012: 91-96 sadiković d. et al.  basidiomycetes of temska village area... 96 mycorrhizal fungi represent the highest number of species (48 % of total species number), followed by saprophytic fungi (43 %), fungi with parasitic and/or saprophytic ecology (5 %) and parasitic fungi (4 %) (fig. 2). protected and endangered fungal species 16 of the species recorded in this area are included in the preliminary national red list (ivancevic 1998) : agaricus macrosporous, amanita cesarea, hygrocybe conica, mycena renati, pluteus salicinus, leucopaxillus giganteus, boletus aereus, boletus satanas, xerocomus porosporus, cantharellus cinereus, geastrum fornicatum, phallus hadriani, ganoderma resinaceum, polyporus badius, lactarius zonarius, sarcodon imbracatus. by the nature conservation law (2009) and regulation on the proclamation and protection of strictly protected and protected wild species of plants, animals and fungi (2010), 4 of recorded species are marked as strictly protected: leucopaxillus giganteus, boletus satanas, geastrum fornicatum and phallus hadriani. the following 10 species are listed as protected: amanita cesarea, marasmius oreades, boletus areus, b. edulis, craterellus cornucopioides, cantharellus cibarius, c. cinereus, lactarius deliciosus, russula cyanoxanta, r. virescens (ivancevic et al., 2012). conclusion fungi are essential parts of ecosystems and should be conserved alongside their habitats (ing 1993). in order to establish the actual fungal diversity in the temska area, more extensive, purposeful and systematic mycological surveys are needed. in the course of these studies, numerous new records both for stara planina mountain and the temska area are expected. acknowledgments. the authors want to express their gratitude to branko jotić for the determination of forest plant species in habitats from which fungal specimens were collected. references anđelković, м. (1958). geološki sastav i tektonika jugozapadnih padina stare planine. beograd : naucno delo. božac, romano. 1978: gljive naših krajeva. grafički zavod hrvatske. breitenbach, j., kränzlin, f. 1995: pilze der schweiz. band 4. verlag mykologia, luzern. 371p. dučić, v., radovanović, m., milovanović, b. 2005: temperature variability on the area of stara planlna in the instrumental period. glasnik srpskog geografskog društva 85 (2): 23–28. focht, i. 1986. ključ za gljive: ilustrirani uvod u gljivarstvo. naprijed, zagreb. index fungorum. http://www.indexfungorum.org/names/names.asp [accessed january 2013] ing, b. 1993: towards a red list of endangered european macrofungi. in: pegler, d. n., l.boddy, b. ing & p. m. kirk (eds.) fungi of europe: investigation, recording and conservation. the royal botanic gardens, kew. 322p. ivančević, b. 1998: a preliminary red list of the macromycetes of yugoslavia. conservation of fungi in europe. università degli studi, siena. pp. 57-61. ivančević, b., beronja, j. 2004: first records of macromycetes from the serbian side of stara planina mts (balkan range). mycologia balcanica vol. 1(1): 15-19. ivančević, b., matavulj, m., vukojević, j., karaman, m. 2012: fungi in the legislation of the republic of serbia. zbornik matice srpske za prirodne nauke (123): 51–64. jordan, m. 2004: the encyclopedia of fungi: of britain and europe. frances fincoln ltd., london. josifović, m., (eds)., 1970-1976: flora sr srbije, iix. sanu. beograd. rikardo, m., ileš, m., unković, s. 1995: kako da raspoznate pečurke: jestive gljive i njihovi otrovni dvojnici. evro, beograd. mcknight, k., mcknight v. 1998: a field guide to mushrooms: north america. houghton mifflin harcourt, boston. milanović, a. 2010: klima stare planine. zbornik radova geografskog instituta “jovan cvijić”, sanu, 1-136. uzelac, b. 2009: gljive srbije i zapadnog balkana. bgv logik, beograd. jovanović et al 2020, biologica nyssana 11(2) 11 (2) december 2020: 139-147 doi: 10.5281/zenodo.4393973 optimizing conditions for mgmt promoter methylation status analysis in glioblastoma ffpe samples original article nikola m. jovanović university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia nikolajov90@gmail.com (corresponding autor) vesna nikolov university of niš, faculty of medicine, clinic of neurosurgery, clinical center, 18000 niš, serbia v.novak@yahoo.com nataša vidović university of niš, faculty of medicine, pathology and pathological anatomy center, 18000 niš, serbia vidovic.patologija@gmail.com jelena vitorović university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia jelena.rajkovic@gmail.com svetlana tošić university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia tosicsvetlana59@yahoo.com vladimir j. cvetković university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia biovlada@yahoo.com tatjana mitrović university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia tatjanamitrovic3@gmail.com tatjana jevtović-stoimenov university of niš, faculty of medicine, department of biochemistry, 18000 niš, serbia tjevtovic@yahoo.com received: may 14, 2020 revised: september 15, 2020 accepted: november 30, 2020 abstract: the methylation status of the mgmt promoter represents the valuable prognostic and predictive marker in glioblastoma (gbm) patients undergoing treatment with alkylating agents such as temozolomide. although formalinfixed and paraffin-embedded tissue (ffpe) signifies the most commonly used source for tissue-based molecular testing, its use in methylation-specific polymerase chain reaction (msp) analysis manifests certain limitations due to low dna integrity. our study aimed to identify the optimal mgmt promoter msp reaction conditions concerning the utilization of bisulfite-converted ffpe-derived template dna. several optimizing reactions were conducted and subjected to imagej software analysis. as a result, 4u of hotstartaq and 125 ng of template dna were specified as necessary for successful msp reactions. the confirmation of optimization success was obtained through comparison of semi-quantitative values of dna methylation levels between reference fresh frozen tissue and corresponding ffpe sample obtained from the same gbm patient. key words: glioblastoma, ffpe, methylation, mgmt, msp, optimization, taq polymerase apstract: optimizacija uslova msp reakcije za promotorni region mgmt gena kod ffpe uzoraka glioblastoma status metilacije promotornog regiona mgmt gena predstavlja jedan od najvažnijih prognostičkih i prediktivnih markera pacijenata obolelih od glioblastoma (gbm) podvrgnutih lečenju alikirajućim agensima poput temozolomida (tmz). iako uzorci tkiva fiksiranih u formalinu i ukalupljenih u parafinske blokove (ffpe) predstavljaju najuobičajeniji izvor dnk materijala prilikom molekularno-bioloških analiza, njihova upotreba pri metodi lančane reakcije polimeraze specifične za metilaciju (msp) ispoljava određena ograničenja usled niskog integriteta dnk. cilj ovog rada predstavljalo je definisanje optimalnih uslova msp reakcije za mgmt promotorni region prilikom korišćenja bisulfitno-konvertovane dnk izolovane iz ffpe uzoraka kao matrice za msp reakciju. izvedeno je nekoliko reakcija optimizacije, a dobijeni rezultati su obrađeni u imagej programu. kao rezultat, hotstartaq polimeraza u količini od 4u i dnk matrica u količini od 125 ng izdvojene su kao neophodne za uspešnu msp reakciju. potvrda uspešnosti reakcija optimizacije dobijena je upoređivanjem semi-kvantitativnih vrednosti nivoa metilacije dnk između referentnog sveže-zamrznutog uzorka (ff) i odgovarajućeg ffpe uzorka poreklom od istog gbm pacijenta. ključne reči: glioblastomi, ffpe, metilacija, mgmt, msp, optimizacija, taq polimeraza © 2020 jovanovic et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 139 introduction glioblastoma (gbm) refers to the highly infiltrative type of gliomas, malignant brain tumors. owing to great morphological and genetic heterogeneity, it is characterized by extremely low five-year survival rates – only 5%, and an annual incidence of 5.26 per 100,000 people (soomro et al., 2017). although it is widely accepted that gliomas originate from normal glial cells, increasing evidence supports the role of various cell types such as glial or neural precursors and stem cells as their possible origin (chen et al., 2017). recent findings 140 in the molecular biology of glioma have led to the novel, improved system of classification which was established by the world health organization (who) in 2016 (louis et al., 2016). namely, the shortcomings of histopathology based classification were exceeded through the incorporation of molecular diagnostic criteria testing for isocitrate dehydrogenase (idh) mutation, chromosome 1p/19q deletion, and histone mutations. in 2010, the cancer genome atlas (tcga) classification of gbms had identified 4 subtypes of glioblastomas based upon gene expression profiles and genomic clustering – proneural, neural, classical, and mesenchymal subtypes (verhaak et al., 2010). the methylation of the cytosine-phosphate-guanine (cpg) islands in the promoter region of o6-methyl guanine-dna methyltransferase gene (mgmt) represents one of the most common epigenetic alterations in gbm. it is present in both primary and secondary gbm in 42% and 79% patients respectively (soomro et al., 2017). the mgmt enzyme is an excision repair enzyme that removes alkyl adducts from the o6position of the guanine. thus, it protects normal cells from carcinogens by repairing double-strand breaks and base mispairing which leads to apoptosis and cell death. during that process it is being irreversibly deactivated, which is why mgmt is referred to as “suicidal” enzyme (thon et al., 2013; soomro et al., 2017). regarding the mgmt role of counteracting the activity of alkylating agents, its inactivation through epigenetic silencing was recognized as an important and clinically relevant factor in gbm patients undergoing treatment with alkylating agents such as temozolomide (tmz). the positive methylation status of the mgmt promoter represents a strong and independent predictive factor of favorable survival in gbm. the median survival was significantly longer in patients with a methylated mgmt promoter (21.7 months) in comparison with the patients lacking methylation (12.7 months). additionally, a high frequency of mgmt promoter methylation was documented in long-term gbm survivors undergoing tmz treatment (thon et al., 2011; thon et al., 2013). given the fact that methylated cytosine in cpg islands exhibits the same base-pairing interactions as unmethylated cytosine, the evaluation of methylation status using conventional hybridization-based methods, i.e. microarrays and pcr, is not suitable (holmes et al., 2014). to address that, frommer et al. have designed a protocol that utilizes bisulfite– induced modification of genomic dna resulting in the conversion of unmethylated cytosine to uracil, while 5-methylcytosines remain intact (frommer et al., 1992). such bisulfite-converted dna could be subsequently analyzed via pcr in order to acquire desired epigenetic information. with the numerous commercially available kits and several technological advances, current bisulfite-treatment protocols are more convenient and user friendly in comparison with the original 16 hours protocol (holmes et al., 2014). however, the success of bisulfite-conversion may vary considerably depending on the quality of dna samples and the choice of tissue samples (tournier et al., 2012). there are strong suggestions for avoiding formalin-fixed and paraffin-embedded tissues (ffpe) samples for bisulfite conversion and following methylation-specific polymerase chain reaction (msp). these are supported by evidence that the use of ffpe induces non-reproducible bisulfite conversion leading to unreliable and inconsistent results for methylation levels (tournier et al., 2012). in contrast with ffpe, the use of fresh frozen tissue (ff) brings reproducible and satisfactory results, owing to the process of cryopreservation which provides the adequate dna preservation. given that the ffpe samples consist of degraded dna generally less than 300 bp, the main challenge with managing ffpe samples is to provide an efficient cell lysis which releases dna of sufficient quality and quantity for further analysis (holmes et al., 2014). what makes it even more difficult, formalin-fixation induces the formation of dna-protein crosslinks, which often could not be completely removed by common lysis protocols. nevertheless, as ffpe samples are widely available, this type of tissue sample is the most commonly used source for tissue-based molecular testing (dietrich et al., 2013). alongside the low pricing of long term storage, ffpe is often the only available material for retrospective studies and the most important material for standard routine diagnostics in the era of personalized medicine (dietrich et al., 2013). therefore, several commercially available kits for ffpe dna isolation and bisulfite conversion were developed to ensure results as reliable as possible (holmes et al., 2014; de ruijter et al., 2015; ludgate et al., 2017; kint et al., 2018). this study aimed to define optimal pcr conditions for evaluation of mgmt promoter methylation status in the ffpe sample obtained from gbm patient, considering the recommendations from the previous studies (dietrich et al., 2013). also, to investigate the validity of the results, they were compared with mgmt promoter methylation status obtained from the ff sample of the same gbm patient. material and methods patient and tumor specimens both ff and ffpe tissue tumor specimens were collected from the gbm patient (male, 63 years old) operated on at the neurosurgery clinic (the biologica nyssana ● 11 (2) december 2020: 199-147 jovanović et al. ● optimizing conditions for mgmt promoter methylation status analysis in glioblastoma ffpe samples 141 biologica nyssana ● 11 (2) december 2020: 199-147 jovanović et al. ● optimizing conditions for mgmt promoter methylation status analysis in glioblastoma ffpe samples clinical centre of niš, serbia) in 2013. the patient underwent total resection of the tumor and had a karnofsky score of ≥80%. the diagnosis of glioblastoma who grade iv was confirmed by an expert neuropathologist (n.v and m.k). the written informed consent of study participation was obtained from the gbm patient. the ethics committee of the faculty of medicine, niš, serbia, approved the informed consent form and study protocol (01-211310). dna isolation and bisulfite conversion extraction of genomic dna was performed using qiaamp® dna mini kit (qiagen, hilden, germany) from 25 mg of ff sample and qiaamp dna ffpe tissue kit (qiagen, hilden, germany, catalogue no. 56404) from 8 freshly cut sections with a thickness of 10 µm from ffpe sample. a total of 2 µg of genomic dna was modified by sodium bisulfite using epitect® bisulfite kit (qiagen, hilden, germany) for the ff dna sample and epitect plus ffpe bisulfite kit (qiagen, hilden, germany, catalogue no. 59144) for ffpe dna sample. biospec–nano uv–vis spectrophotometer (shimadzu, kyoto, japan) was utilized for the determination of quantity and quality of isolated dna and bisulfite converted samples. isolated dna samples were inspected for degradation and visualized by running the dna samples on 2% agarose gel. methylation-specific polymerase chain reaction (msp) all of the msp reactions were carried out in a total volume of 20 µl containing 0.2 μm dntp mix, 1 × pcr buffer with 1.5 mm mgcl2 (qiagen, hilden, germany) and 10 pm of appropriate forward and reverse primer (tab. 1). amplification reactions were performed in a mastercycler gradient (eppendorf) using the following program: 95 °c for 15 min, then 35 cycles of 95 °c for 50 s, 59 °c for 50 s and 72 °c for 50 s, and a final extension at 72 °c for 10 min. msp optimization reactions with four different amounts of bisulfite-converted template dna (31.25 ng, 62.5 ng, 125 ng, and 250 ng) in combination with 1u and 4u hotstartaq dna polymerase (qiagen, hilden, germany, catalogue no. 203203) were conducted according to suggestions from the previous study (dietrich et al., 2013). following the determination of template dna optimal concentration, another set of optimization reactions was designed regarding the investigation of the optimal concentration of hotstartaq polymerase for successful mgmt msp reactions using ffpe dna isolates. therefore, msp reactions with 1u, 2u, and 4u of hotstartaq polymerase and unmethylated set of mgmt primers were conducted simultaneously with msp reaction including template dna obtained from the ff sample and 1u of hotstartaq polymerase. for comparison of methylation level evaluation between ffpe and ff dna isolates, another set of msp reactions was carried out using both sets of mgmt primers and ffpe and ff isolates as dna templates, followed by agarose gel electrophoresis. msp reactions using ffpederived bisulfite-converted template dna were conducted in duplicate. analysis of methylation data after each optimization, msp gel images were subjected to imagej software analysis (national institute of health, bethesda, md, usa) with the aim of measuring the fluorescence intensity of methylated (m) and unmethylated (u) msp bands. m/u intensity ratio values represent the common approach of semi-quantitative evaluation of the mgmt promoter methylation level (christians et al., 2012). results ff and ffpe dna isolates concentrations and purity of genomic dna isolates and bisulfite-converted dna samples are presented in tab. 2. gene primer sequence (5’-3’) amplicon size (bp) mgmt unmethylated (u) f: tttgtgttttgatgtttgtaggtttttgt r: aactccacactcttccaaaaacaaaaca 93 mgmt methylated (m) f: tttcgacgttcgtaggttttcgc r: gcactcttccgaaaacgaaacg 81 table 1. primer sequences and amplicon size total yield of dna (ng) od 260/280 yield of bisulfite-converted dna per single conversion (ng) ffpe dna isolate (from 8 sections with a thickness of 10 µm) 4,537.5 1.94 2,265.8 ff dna isolate (50 mg of tissue) 35,209.2 1.87 2,094.4 table 2. quantity and quality of isolated dna and bisulfite converted samples (biospec–nano uv–vis spectrophotometer) 142 total yield of isolated dna from ffpe sample was 4,537.5 ng and 35,209.2 ng from ff sample, with od 260/280 values of 1.94 and 1.87, respectively. recorded yield of bisulfite-converted dna per single conversion was 2,265.8 ng for ffpe and 2,094.4 ng for ff dna sample. agarose gel images representing the integrity of isolated dna are shown in fig. 1. agarose gel images of ff dna samples displayed that most of the dna fragments have migrated conjointly with the largest fragments of the dna ladder marker (~9000 bp) thereby forming a noticeable band. in contrast, highly degraded ffpe dna samples were presented on agarose gels as characteristic large smears originating from dna fragments of various sizes, mostly shorter than 1,264 bp. mgmt msp template dna concentration optimization in ffpe samples agarose gel image of msp optimization of optimal template dna quantity is presented in fig. 2. with the clear difference in fluorescence intensity of both methylated and unmethylated mgmt msp products for 1u and 4u of hotstartaq polymerase, the most appropriate m and u msp products were recorded for 125 ng quantity of template dna and 4u of hotstartaq polymerase. biologica nyssana ● 11 (2) december 2020: 199-147 jovanović et al. ● optimizing conditions for mgmt promoter methylation status analysis in glioblastoma ffpe samples fig. 1. agarose gel electrophoresis images of isolated dna samples (~400ng) a) high-quality dna isolates obtained from ff samples (4000-9000 bp fragments). lane 1 – marker λ dna-bst eii (new england biolabs® inc. #n3014s), lane 2ff dna sample (first dna elution), lane 3ff dna sample (second dna elution) b) low-quality ffpe dna samples with highly degraded dna fragments: lane 1 – marker λ dnabst eii, lanes 2,3,4 ffpe dna samples. fig. 2. msp optimization for ffpe dna isolates a) agarose gel image b) legend 143 biologica nyssana ● 11 (2) december 2020: 199-147 jovanović et al. ● optimizing conditions for mgmt promoter methylation status analysis in glioblastoma ffpe samples fig. 3. results of msp optimization regarding defining the optimal amount of hotstartaq polymerase. a) original agarose gel image with a legend on the right, presenting ffpe unmethylated mgmt promoter msp products with 1u, 2u and 4u of hotstartaq polymerase alongside with ff unmethylated mgmt msp with 1u of hotstartaq polymerase product as a positive control. b) imagej software processed image with fluorescence intensity analysis of specific unmethylated mgmt msp product bands, with corresponding chart representing their relative values. c) imagej fluorescence intensity analysis of primer-dimer presence in msp reactions. 144 biologica nyssana ● 11 (2) december 2020: 199-147 jovanović et al. ● optimizing conditions for mgmt promoter methylation status analysis in glioblastoma ffpe samples mgmt msp hotstartaq polymerase concentration optimization in ffpe samples regarding further determination of the optimal amount of hot start taq polymerase, agarose gel image was acquired following msp optimization and processed with imagej software analysis (fig. 3). fluorescence intensity values acquired using the imagej software have confirmed the 4u of hotstartaq polymerase as an optimal amount for successful msp mgmt reaction in ffpe samples. among tested concentrations, the highest fluorescence intensity value of unmethylated msp products was recorded for 4u of hotstartaq polymerase. it was equivalent to 57,43% of the recorded fluorescence intensity value of the reference ff dna mgmt msp product. furthermore, the fluorescence intensity value of primer-dimers was the lowest for the 4u concentration, being even lower than the one recorded for the reference ff dna template msp 37.16% of the reference value. finally, only for the 4u hotstartaq polymerase concentration was documented that the mgmt product/primer-dimer fluorescence intensity ratio exceeds 100% of the reference value (113.12 %), suggesting optimal reaction conditions (tab. 3 and fig. 4). mgmt methylation assesment in ffpe and ff sample figure 5 (a) shows the agarose gel image of mgmt msp reactions which included bisulfite-converted template dna of the same patient originating from both ffpe and ff samples, alongside with randomly chosen ffpe sample from another gbm patient. m/u ratio values of ff and ffpe samples were acquired via imagej software analysis fig. 5 (b). as a result of imagej software analysis, both the ffpe and ff samples were assessed as strongly methylated semi-quantitative category of mgmt promoter methylation (m/u ratio <1). the observed difference in m/u ratio values was 8.47% (tab. 4). discussion results from our study have confirmed the great difference in dna fragmentation level between ff and ffpe dna isolates (fig. 1). in contrast with the ff sample, the ffpe sample from our study was in great portion consisted of dna fragments shorter than ~1,000 bp, which were forming a smear and lacking a distinguishable band on the agarose gel. this observation is consistent with previously described ffpe dna isolate properties originating from the tissue preparation and fixation process. namely, it was shown that mechanical stress resulting from cross-linking during the fixation process, the concentration of formalin, ph, and salt, the temperature, and tissue type have a great impact on the quality of the ffpe-derived dna (ludgate et al., 2017). among such factors, the deparaffinization was emphasized as the crucial process which reduces the quality of the isolated dna (sengüven et al., total amount of hotstartaq polymerase units in msp reaction 1u 2u 4u fluorescence intensity of unmethylated mgmt mps product compared to ff refference (%) 48.27 34.67 57.43 fluorescence intensity of primer-dimers compared to ff refference (%) 200.60 219.85 37.16 product / primer-dimer intensity ratio compared to ff refference (%) 49.10 36.30 113.12 table 3. fluorescence intensity values of 3 different concentrations of hotstartaq polymerase obtained using imagej software analysis fig. 4. mgmt product / primer-dimer fluorescence intensity ratios detected for 125 ng of template dna and 3 different concentrations of hotstartaq polymerase in ffpe and reference ff dna bisulfiteconverted samples 145 2014). given the fact that bisulfite conversion further degrades dna, the challenges which arise in dna methylation analysis are no surprising (patterson et al., 2011). as a consequence, msp reaction conditions concerning methylation analysis may significantly differ in case of using the ffpe instead of the ff bisulfite-converted sample as template dna. considering the significance of ffpe tissues as a valuable dna source for mgmt methylation analysis in gbm patients, as well as for clinical and cancer research in general, our main goal was to reveal the optimal mgmt promoter methylation msp reaction conditions which could bring the results as valid as those involving ff-derived dna. the most significant msp reaction parameters – taq polymerase and template dna concentrations were selected considering the findings of improved pcr performance in ffpe tissue samples presented by dietrich et al. (2013). primarily, our study tested 4 concentrations of template dna (31.25 ng, 62.5 ng, 125 ng, and 250 ng) and 2 concentrations of hotstarttaq polymerase (1u and 4u) for the capability of alleviating pcr inhibition and amplifying methylated mgmt promoter (81 bp) and unmethylated mgmt promoter (93 bp) msp products. in concordance with previous findings, this optimization reaction revealed the 4u of hotstarttaq polymerase, alongside with 125 ng of template dna as optimal concentrations for both methylated and unmethylated msp products. as seen in fig. 2, the gradual increase of template dna amount enhances the msp product intensity, both in 1u and 4u reaction subgroups. this overcoming of pcr inhibition occurs through increasing the probability of the presence of template molecules of proper length and integrity. as a result, higher template concentrations and 1u concentration of hotstartaq polymerase emerged as one of the possible solutions for optimal amplification conditions. furthermore, a 4-fold increase of the hotstarttaq polymerase clearly showed the successful amplification in replicates containing low amounts of template dna. the listed observations were suggesting the optimal amount of 125 ng and 4u of hotstarttaq polymerase in both methylated and unmethylated mgmt products. however, the presence of additional primer-dimer bands on agarose gel could lead to misinterpretation of methylation level analysis. to address that issue and further elucidate optimal msp conditions, another optimization reaction was conducted. given the relatively short length of the mgmt promoter msp products, this optimization introduced the 2u concentration of hotstarttaq polymerase as a possible solution for economizing enzyme consumption. for that purpose, optimization reaction was performed including 3 concentrations of hotstartaq polymerase (1u, 2u, and 4u), 125 ng amount of template dna, and unmethylated mgmt promoter primer set (fig. biologica nyssana ● 11 (2) december 2020: 199-147 jovanović et al. ● optimizing conditions for mgmt promoter methylation status analysis in glioblastoma ffpe samples fig. 5. differences in mgmt promoter methylation assessment between ff and ffpe samples: a) agarose gel image presenting methylated and unmethylated mgmt promoter msp products with the corresponding legend on the right side. lanes 3 and 4 methylated mgmt promoter msp products involving ffpe samples of the previously listed gbm patient as template dna; lanes 8 and 9 unmethylated mgmt promoter msp products involving ffpe samples of the previously listed gbm patient as template dna; lanes 12 and 13 reference ff sample mgmt promoter msp products (methylated and unmethylated); lanes 5,6,10,11 mgmt promoter msp products (methylated and unmethylated) assessed with ffpe bisulfite-converted sample of different gbm patient. b) fluorescence intensity levels of methylated (m) and unmethylated (u) msp bands obtained using imagej software. products and ffpe-derived template dna. although the above-mentioned pcr conditions refer to the amplification of specific mgmt promoter pcr products using the end-point pcr method, they could also be taken into consideration while performing ms-qpcr reactions. however, it should be noted that due to the small sample size and a relatively small number of measurements, the presented results were not additionally supported by some form of statistical analysis. acknowledgement. this study was supported by the ministry of education, science and technological development of the republic of serbia (contract number: 451-03-68/2020-14/200124). references chen, r., smith-cohn, m., cohen, a.l., colman, h. 2017: glioma subclassifications and their clinical significance. neurotherapeutics, 14: 284297. christians, a., hartmann, c., benner, a., meyer, j., von deimling, a., weller, m., wick, w., weiler, m. 2012: prognostic value of three different methods of mgmt promoter methylation analysis in a prospective trial on newly diagnosed glioblastoma. plos one, 7(3): e33449. de ruijter, t.c., de hoon, j.p., slaats, j., de vries, b., janssen, m.j., van wezel, t., aarts, m.j., van engeland, m., tjan-heijnen, v.c., van neste, l., veeck, j. 2015: formalin-fixed, paraffinembedded (ffpe) tissueepigenomics using infinium humanmethylation450beadchip assays. models and techniques, 95: 833-842. dietrich, d., uhl, b., sailer, v., holmes, e.e., jung, m., meller, s., kristiansen, g. 2013: improved pcr performance using template dna from formalin-fixed and paraffin-embedded tissues by overcoming pcr inhibition. plos one, 8(10): e77771. esteller, m., garcia-foncillas, j., andion, e., goodman, s.n., hidalgo, o.f., vanaclocha, v., baylin, s.b., herman, j.g. 2000: inactivation of the dna-repair gene mgmt and the clinical response of gliomas to alkylating agents. the new england journal of medicine, 343(19): 1350-1354. frommer, m., mcdonald, l.e., millar, d.s., collis, c.m., watt, f., grigg, g.w., molloy, p.l., paul, c.l. 1992: a genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual dna strands. proceedings of the national academy of sciences of the united states of america, 89(5): 1827-1831. holmes, e.e., jung, m., meller, s., leisse, a., 146 biologica nyssana ● 11 (2) december 2020: 199-147 jovanović et al. ● optimizing conditions for mgmt promoter methylation status analysis in glioblastoma ffpe samples 3). ff dna bisulfite-converted sample served as a control msp reaction. despite successful amplification of the specific product in 1u and 2u replicates, an imagej software analysis showed the significant presence of primer-dimer bands in 1u and 2u, but not in 4u replicate, confirming the 4u concentration of hotstartaq polymerase as the optimal one (fig. 4). since the original study concerning mgmt promoter methylation in gbm patients, there were several common approaches of methylation status assessment (esteller et a., 2000). in order of improving the qualitative assessment results obtained by the end-point pcr method, the semiquantitative approach which utilizes additional imagej software analysis of gel images was proposed (dietrich et al., 2013). thus, through the measuring of fluorescence intensity ratios of methylated and unmethylated mgmt promoter product bands, patients could be sorted into three groups – unmethylated (m/u ratio =0), with weak promoter methylation (m/u ratio between 0 and 1) and strongly methylated (m/u ratio >1). although this approach is widely replaced by qpcr methods of methylation status assessment, for this study it presents a valuable method for validation of optimizing msp reaction results. by comparing m/u ratio values with those of ff samples, ffpe mgmt promoter msp reaction conditions could be tested. for that purpose, the final set of mgmt promoter msp reactions in our study have included both reference ff and ffpe dna isolates, performed in duplicate with previously defined pcr conditions (4u hotstarttaq polymerase and 125 ng of template dna). indeed, m/u ratios obtained for corresponding ff and ffpe samples in our study were very similar, differing only in 8.47 percent (tab. 4). in that manner both of the samples were assessed as strongly methylated (tab. 4), although the absolute intensity values were almost 3-fold different (fig. 5b). this observation suggests that the optimizing conditions for mgmt promoter msp using ffpe-derived dna samples presented in this study were properly defined. conclusion the results acquired in our study were in line with previous findings concerning the use of ffpe tissue in msp reactions. thus, gel electrophoresis analysis of the ff and ffpe dna isolates confirmed the high level of degradation in ffpe isolates. based on several optimizing reactions, 4u hotstartaq polymerase and 125 ng of template dna could be singled out with high certainty as suitable concentrations for successful msp reaction concerning methylated and unmethylated mgmt promoter amplification biologica nyssana ● 11 (2) december 2020: 199-147 jovanović et al. ● optimizing conditions for mgmt promoter methylation status analysis in glioblastoma ffpe samples sailer, v., zech, j., mengdehl, m., garbe, l.a., uhl, b., kristiansen, g., dietrich, d. 2014: performance evaluation of kits for bisulfiteconversion of dna from tissues, cell lines, ffpe tissues, aspirates, lavages, effusions, plasma, serum, and urine. plos one, 9(4): e93933. kint, s., de spiegelaere, w., de kesel, j., vandekerckhove, l., van criekinge, w. 2018: evaluation of bisulfite kits for dna methylation profiling in terms of dna fragmentation and dna recovery using digital pcr. plos one, 13(6): e0199091. louis, d.n., perry, a., reifenberger, g., von deimling, a., figarella-branger, d., cavenee, w.k., ohgaki, h., wiestler, o.d., kleihues, p., ellison, d.w. 2016: the 2016 world health organization classification of tumors of the central nervous system: a summary. acta neuropathologica, 131(6): 803-820. ludgate, j.l., wright, j., stockwell, p.a., morison, i.m., eccles, m.r., chatterjee, a. 2017: a streamlined method for analysing genome-wide dna methylation patterns from low amounts of ffpe dna. bmc medical genomics, 10(1): 54. patterson, k., molloy, l., qu, w., clark, s. 2011: dna methylation: bisulphite modification and analysis. journal of visualized experiments, (56): 3170. sengüven, b., baris, e., oygur, t., berktas, m. 2014: comparison of methods for the extraction of dna from formalin-fixed, paraffin-embedded archival tissues. international journal of medical sciences, 11(5): 494-499. soomro, s.h., ting, l.r., qing, y.y., ren, m. 2017: molecular biology of glioblastoma: classification and mutational locations. the journal of the pakistan medical association, 67(9): 14101414. thon, n., eigenbrod, s., grasbon-frodl, e.m., lutz, j., kreth, s., popperl, g., belka, c., kretzschmar, h.a., tonn, j.c., kreth, f.w. 2011: predominant influence of mgmt methylation in non-resectable glioblastoma after radiotherapy plus temozolomide. journal of neurology, neurosurgery, and psychiatry, 82(4): 441–446. thon, n., kreth, s., kreth, f.w. 2013: personalized treatment strategies in glioblastoma: mgmt promoter methylation status. onco targets and therapy, 6: 1363-72. tournier, b., chapusot, c., courcet, e., martin, l., lepage, c., faivre, j., piard, f. 2012: why do results conflict regarding the prognostic value of the methylation status in colon cancers? the role of the preservation method. bmc cancer, 12: 12. verhaak, r.g., hoadley, k.a., purdom, e., wang, v., qi, y., wilkerson, m.d., miller, c.d., ding, l., golub, t., mesirov, j.p., alexe, g., lawrence, m, o’kelly, m.,temayo, p., weir, b.a., gabriel, s., winckler, w., gupta, s., jakkula, l., feiler, h.s., hodgson, j.g., james, c.d., sarkaria, j.n., brennan, c., kahn, a., spellman, p.t., wilson, r.k., speed, t.p., gray, j.w., meyerson, m., getz, g., perou, c.m., hayes, dn.; cancer genome atlas research network. 2010: integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in pdgfra, idh1, egfr, and nf1. cancer cell, 17(1): 98-110. 147 anticancer compounds from medicinal plants biologica nyssana 5 (1)  september 2014: 1-10 kamberović, j. et al.  marshland vegetation of the order phragmitetalia … 1 original article received: 26 june 2014 revised: 28 august 2014 accepted: 10 september 2014 marshland vegetation of the order phragmitetalia on shores of mine pit lakes in north-eastern bosnia and herzegovina jasmina kamberović 1* , senka barudanović 2 , ermin mašić 2 , anita dedić 3 1 university of tuzla, faculty of natural sciences and mathematics, department of biology, univerzitetska 4, 75 000 tuzla, bosnia and herzegovina 2 university of sarajevo, faculty of natural sciences and mathematics, department of biology, ulica zmaja od bosne 33-35, 71 000 sarajevo, bosnia and herzegovina 3 university of mostar, faculty of science and education, department of biology, matice hrvatske bb, 88 000 mostar, bosnia and herzegovina * e-mail: jasmina.kamberovic@untz.ba abstract: kamberović, j., barudanović, s., mašić, e., dedić, a.: marshland vegetation of the order phragmitetalia on shores of mine pit lakes in north-eastern bosnia and herzegovina. biologica nyssana, 5 (1), septemeber 2014: 1-10. marshland vegetation of the order phragmitetalia w. koch 1926 on the four mine pit lakes in the wider area of tuzla was investigated during 2008. in total, two plant communities were noted. on the lakes suhodanj, mušićko and šićki brod association typhetum latifoliae g. lang 1973 were recorded, within which was noted 33 plant species. on the shores of lakes ramićko and šićki brod the dominant association was phragmitetum australis schmale 1939, within which were determined 32 plant species. this paper analyzes the life forms and bioindicator values of plant species. the research results indicate a successful process of colonization by macrophyte species on absolutely degraded habitats such as mine pit lakes. key words: bioindicators, biodiversity, colonization, mine pit lakes, vegetation, wetlands apstrakt: kamberović, j., barudanović, s., mašić, e., dedić, a.: močvarna vegetacija reda phragmitetalia na obalama kopovskih jezera u severoistočnoj bosni i hercegovini.. biologica nyssana, 5 (1), septemebar 2014: 1-10. močvarna vegetacija reda phragmitetalia w. koch 26 istraživana je u toku 2008. godine na obalama četiri kopovska jezera šireg područja tuzle. na istraživanom području određene su dve biljne zajednice. na jezerima suhodanj, mušićko i šićki brod prisutna je zajednica typhetum latifoliae g. lang 1973, u sklopu koje je zabeleženo prisustvo 33 biljne vrste. na obalama jezera ramićko i šićki brod dominantna je zajednica phragmitetum australis schmale 1939, gde su određene 32 biljne vrste. u radu su analizirane životne forme, bioindikatorske vrednosti i socijabilnost biljnih vrsta. rezultati istraživanja upućuju na uspešan proces kolonizacije močvarnih biljnih vrsta na apsolutno degradiranim staništima, kao što su kopovska jezera. key words: bioindikatori, biodiverzitet, kolonizacija, kopovska jezera, vegetacija, vlažna staništa 5 (1) • september 2014: 1-10 biologica nyssana 5 (1)  september 2014: 1-10 kamberović, j. et al.  marshland vegetation of the order phragmitetalia … 2 introduction plant communities of the order phragmitetalia represent clearly defined zone of emersal vegetation on shores of lakes, ponds and river valleys (r a n đ e l o v i ć et al., 2007; p o l i ć , 2006). they develop on wetlands, gley soils, often peaty soils, with intensive reduction processes (r e d ž i ć et al., 2009). in bosnia and herzegovina they are spread on the area of hutovo blato, bardača, ždralovac, velika and mala tišina near bosanski šamac, some parts of the lake modrac and are fragmented around many small lakes (r e d ž i ć et al., eds., 2008). studies of plant communities of the class phragmitetea in bosnia and herzegovina have been known for a sub-mediterranean area (j a s p r i c a et al., 2003), where in hutovo blato 11 plant communities from three vegetation alliances were determined. in addition to the above mentioned natural habitats, emergent hydrophytes in progradation processes also inhabit shores of anthropogenicallyformed lakes. examples are numerous pit lakes, created in the process of surface coal mining. pit lakes have the potential to conserve wetland biodiversity (b a r u d a n o v i ć & k a m b e r o v i ć , 2008), being overgrown by vegetation of submerged hydrophytes (k a m b e r o v i ć & b a r u d a n o v i ć , 2012) and weed vegetation (b a r u d a n o v i ć & k a m b e r o v i ć , 2011). since the community structure of emergent hydrophytes on the shores of pit lakes in tuzla region is insufficiently known, this paper aims to (i) determine the structure of communities from the order phragmitetalia at artificially formed habitats, (ii) compare these communities with the structure of communities at natural habitats and (iii) assess the ecological characteristics of the habitat on the basis of ecological indexes. study area the research on marshland vegetation was carried out in 2008 in northeastern bosnia and herzegovina on the shores of four pit lakes: suhodanj lake (su, fig. 1), mušićko lake (mj, fig. 2), ramićko lake (rj, fig. 3) and šićki brod lake (šb, fig. 4). lakes were formed during the period from 1982-1987 and each have their own water regime. the lakes suhodanj and ramićko were created by damming the flow of surface water with the tailings material on a partly degraded terrain. the lakes mušićko and šićki brod were formed in the final crater after the cessation of surface coal mining. basic characteristics of the figure 1. lake suhodanj (su) figure 2. lake mušićko (mj) figure 3. lake ramićko (rj) figure 4. lake šićki brod (šb) biologica nyssana 5 (1)  september 2014: 1-10 kamberović, j. et al.  marshland vegetation of the order phragmitetalia … 3 table 1. the basic characteristics of the researched lakes (k a m b e r o v i ć , 2010) table 2. the physical and chemical parameters of water quality of the studied lakes in august 2008 indicators su šb mj rj water temperature ( 0 c) 26 25.5 24 26 turbidity (ntu) 1.5 1.3 2.5 3.5 ph 8.1 8.2 7.8 8.3 total alkalinity (mg caco3l -1 ) 157 147 287 172 carbonate hardness (°dh) 8.8 8.3 16.1 9.7 noncarbonated hardness (°dh) 11.2 8.3 40.3 0.1 total hardness (°dh) 20.2 26.5 56.4 9.8 dissolved oxygen (mgl -1 ) 10 8.7 10.2 8.5 oxygen saturation (%) 116 109 127 104 kmno4 consumption (mgl -1 ) 10.5 14.2 12.6 17.2 total nitrogen (mgl -1 ) 0.18 0.02 1.46 0.18 ammonium (mgl -1 ) 0.16 0.02 0.59 0.16 nitrite (mgl -1 ) 0.018 0.004 0.424 0.004 nitrate (mgl -1 ) 0 0 0.45 0.02 calcium (mgl -1 ) 44.1 122 222.4 31.1 magnesium (mgl -1 ) 60.2 40.7 110 23.7 electrical conductivity (µs/cm) 535 682 1562 280 studied lakes are given in tab. 1. the research area belongs to the temperate continental climate region. the average temperature varies from 9 0 c to 10.6 0 c, and the annual precipitation ranges from 830 l/m 2 to 1.150 l/m 2 (s m a j i ć , 2007). material and methods phytocenological relevés of coastal vegetation were carried out using the zűrich–montpellier school in three vegetation seasons (b r a u n b l a n q u e t , 1964). phytocenological relevés from the spring season with the designation of the lake are additionally marked with the letter "a", the summer season with the letter "b" and the autumn season with the letter "c". determination of plants is done on the basis of herbarium materials according to the relevant floras (t u t i n e t a l ., eds., 19641993, d o m a c , 2002, javorka & c s a p o d y , 1979). the cover of taxa was determined using the numerical scale according to westhoff van der maarel (k o j i ć e t a l ., 1997), and syntaxonomic affiliation of associations according to l a k u š i ć e t a l . (1977). life forms of plant species are taken from o b e r d o r f e r (1979). sociability of plant species and environmental factors (temperature, humidity, soil acidity, nitrification, light, continentality and salinity) are given according to ellenberg (b o r h i d i , 1993). the research of physical/chemical properties of water was done on the field and in the laboratory. the water temperature and the ph of water were lakes suhodanj (su šićki brod (šb) mušići (mj) ramići (rj) year of origin 1985 1987 1982 1983 elevation (m) 293 207 357 378 geographic coordinates 44°23`37``n 44°31`42``n 44°23`45``n 44°25`06``n 18°38`41``e 18°34`44``e 18°30`24``e 18°25`29``e lake's surface (ha) 2.5 20.8 5.53 6.2 max. lake's depth (m) 18 33 6 18 geological basis marl sandy marl marl and serpentine biologica nyssana 5 (1)  september 2014: 1-10 kamberović, j. et al.  marshland vegetation of the order phragmitetalia … 4 measured on the field, while other physical/chemical parameters (turbidity, total alkalinity, carbonate hardness, noncarbonated hardness, total hardness, dissolved oxygen, oxygen saturation, kmno4 consumption, total nitrogen, ammonium, nitrite, nitrate, calcium, magnesium, electrical conductivity) were determined in the laboratory of the institute for chemical engineering in tuzla, by the standard procedure of the american health organization (apha, awwa & wef, 1995). in the statistical analysis we used complete linkage clustering method in primer 6 (c l a r k e & g o r l e y , 2006). the diversity of assemblages was quantified with the shannon-wiener diversity index by the use of the biodiversity pro version 2 software packages (m c a l e e c e et al., 1997). results and discussion physico-chemical analysis of water the water of all four researched lakes has a weak alkaline reaction (ph 7.8-8.3). most pit lakes in the world have an acid reaction of water (n i x d o r f et al., 2005). v e l a g i ć h a b u l et al. (2005) concluded that neutralisation and acidification in mining lakes of the tuzla area are to date producing a cirumneutral water quality. investigated lakes except lake mušićko have sufficient oxygen regime and generally low concentrations of nutrients. lake mušićko has the highest level of water hardness, high calcium and magnesium concentrations, high value of electrical conductivity and the highest value of total nitrogen (tab. 2). previous studies (k a m b e r o v i c & b a r u d a n o v i ć , 2012) found that the lakes suhodanj and šićki brod are characterized by oligo/ß-mesosaprobic status of water with submerged vegetation of charophyta. lakes mušićko and ramićko have ß-mesosaprobic water status and are partially overgrown with vegetation of the alliance magnopotamion. marshland vegetation marshland vegetation of the order phragmitetalia w. koch 1926 in the study area is represented with the alliance phragmition australis w. koch 1926 and two plant association: typhetum latifoliae g. lang 1973 and phragmitetum australis schmale 1939. the community typhetum latifoliae g. lang 1973 occurs in habitats with shallow water (up to 0.5 meters deep) at the lakes suhodanj, mušićko and šićki brod (tab. 3). it is most developed at the lake mušićko and has a dominant role in the appearance of the lake scenery. besides broadleaf cattail (typha latifolia), in this community also occur other semiaquatic plants: mentha aquatica, lycopus europaeus, alisma plantago-aquatica, typha angustifolia and juncus effusus. this community is a very widespread community of shallow parts of water pools with a quiet eutrophic water (s t a n č i ć , 2010). mostly it represents the transition between the water vegetation and the vegetation of reed beds. communities of cattail are resistant for a longer period of drying, salinization and water pollution (p o l i ć , 2006). the community phragmitetum australis schmale 1939 occurs on some more drained terrain compared to the previous community and it was identified at two lakes. on the lake šićki brod it grows over the northwestern lake shore and has a significant role in progradation processes, while at ramićko lake occurs in the form of a narrow zone on the west shore. the floristic composition is dominated by common reed phragmites australis, while all other species are represented with clearly low covering degree (tab. 4). determination of plant communities was confirmed by cluster analysis. clearly separated two clusters indicate differentiation in two plant communities, while the subgroups are formed in relation to the research sites (fig. 5). the floristic composition of the association changes over the year. relevés in the spring season have lower number of species compared to the summer and autumn relevés when terrestrial plant species occure. in cluster analysis, in addition to the original, for comparison purposes we included also results of other similar research. from the dendrogram is evident that the community of reed beds on the lake šićki brod is most similar to stands in serbia and croatia (l a z i ć et al., 2005, p o l i ć , 2006, s t a n č i ć , 2010) with which it shares only three common characteristic plant species of the order phragmitetalia (phragmites australis, mentha aquatica and lycopus europaeus). the community of reed beds on ramićko lake is most similar to the stands in the sub-mediterranean part of bosnia and herzegovina (j a s p r i c a et al., 2003) with which it has four plant species in common (phragmites australis, scirpus sylvaticus, potentilla reptans and calystegia sepium). the communities of cattail at pit lakes in comparison with other studies do not show a great similarity, except for those on the lake šićki brod. differences in the structure of the examined communities are pronounced because of the participation of some characteristic plant species at the localities in serbia, croatia and herzegovina (e.g. glyceria maxima, acorus biologica nyssana 5 (1)  september 2014: 1-10 kamberović, j. et al.  marshland vegetation of the order phragmitetalia … 5 calamus, scirpus lacustris) that are absent at pit lakes. table 3. association typhetum latifoliae g. lang 1973 in littoral of studied pit lakes (the cover of taxa is given according to westhoff van der maarel) association typhetum latifoliae g. lang 1973 relevé number 1 2 3 4 5 6 7 8 9 the locality mja mjb mjc sua sub suc šba šbb šbc d e g re e o f p re se n c e area of relevé (m 2 ) 50 50 50 50 50 50 30 30 30 density (%) 60 85 85 80 95 90 90 95 95 char. ass. typha latifolia l. 8 9 9 8 8 8 8 8 8 v typha angustifolia l. 3 3 3 7 7 7 iv phragmites australis (cav.) trin. ex steud. 5 5 5 ii char. alliance phragmition, char. order phragmitetalia & char. class mentha aquatica l. 3 3 3 2 2 3 iv lycopus europaeus l. 3 3 3 2 3 3 iv alisma plantago-aquatica l. 3 3 3 2 3 3 iv sparganium erectum l. 3 3 3 ii carex vulpina l. 3 3 2 ii lythrum salicaria l. 2 2 2 ii phalaris arundinacea l. 2 3 3 ii epilobium palustre l. 2 3 ii companions cyperus fuscus l. 2 3 3 2 2 2 2 2 v ranunculus repens l. 2 2 3 2 iii juncus effusus l. 3 3 3 ii lysimachia vulgaris l. 3 2 2 ii inula britannica l. 2 2 3 ii eupatorium cannabinum l. 2 3 3 ii elymus repens (l.) gould 2 3 3 ii potentilla reptans l. 2 2 2 ii plantago major l. 2 2 2 ii equisetum telmateia ehrh. 2 3 3 ii erigeron annuus (l.) pers. 2 2 2 ii tussilago farfara l. 2 2 2 ii artemisia vulgaris l. 2 2 2 ii polygonum persicaria l. 2 2 2 ii rumex sanguineus l. 2 3 ii poa pratensis l. 2 2 ii prunella vulgaris l. 2 2 ii bidens tripartita l. 2 2 ii polygonum lapathifolium l. 3 3 ii carex hirta l. 3 i mentha pulegium l. 2 i lycopus exaltatus l. 2 i biologica nyssana 5 (1)  september 2014: 1-10 kamberović, j. et al.  marshland vegetation of the order phragmitetalia … 6 table 4. association phragmitetum australis schmale 1939 in littoral of studied pit lakes (the cover of taxa is given according to westhoff van der maarel) association phragmitetum australis schmale 1939 relevé number 1 2 3 4 5 6 the locality šba šbb šbc rja rjb rjc d e g re e o f p re se n c e area of relevé (m 2 ) 30 30 30 50 50 50 density (%) 80 90 90 100 100 100 char. ass. phragmites australis (cav.) trin. ex steud. 9 9 9 9 9 9 v typha latifolia l. 3 3 3 iii char. alliance phragmition, char. order phragmitetalia & char. class lycopus europeus l. 2 2 2 2 2 2 v alisma plantago-aquatica l. 2 2 2 2 2 2 v epilobium palustre l. 2 2 2 3 iv mentha aquatica l. 2 2 2 iii sparganium erectum l. 2 2 2 iii scirpus sylvaticus l. 2 2 2 iii lythrum salicaria l. 3 3 ii companions ranunculus repens l. 3 2 2 3 3 3 v poa pratensis l. 2 2 3 iii salix purpurea l. 2 2 2 iii equisetum palustre l. 2 2 2 2 iv elymus repens (l.) gould 5 5 5 iii potentilla reptans l. 3 3 3 iii agrostis stolonifera l. 3 3 3 iii calystegia sepium /l./r.br. 5 5 5 iii plantago altissima l. 2 2 2 iii tanacetum vulgare l. 2 2 ii conyza canadensis (l.) cronquist 2 2 ii cyperus fuscus l. 2 2 ii eupatorium cannabinum l. 3 3 ii mentha pulegium l. 3 2 ii bidens tripartita l. 3 3 ii polygonum mite schrank 3 3 ii polygonum lapathifolium l. 2 2 ii echinocystis lobata (michx.) torr. & a.gray 5 5 ii atriplex patula srn. 3 3 ii trifolium repens l. 3 3 ii sonchus arvensis l. 2 3 ii pastinaca sativa l. 2 2 ii verbena officinalis l. 2 2 ii biologica nyssana 5 (1)  september 2014: 1-10 kamberović, j. et al.  marshland vegetation of the order phragmitetalia … 7 figure 5. dendrogram of the original (mj, su, šb and rj) and campared relevés (s t a n č i ć , 2010, p o l i ć , 2006, j a s p r i c a et al., 2003, l a z i ć 2006), complete linkage cluster analysis. p.a. phragmitetum australis; t.l. typhetum latifoliae. biodiversity of associations as part of the order phragmitetalia at the researched sites, a total of 50 plant species were determined. the value of shannon -wiener diversity index ranges from 2.10 to 3.15 (fig. 6). within the community typhetum latifoliae were recorded 33 plant species, and the largest number of species was observed on the lake mušićko (19). in the community phragmitetum australis were determined 32 plant species. here we observed largest deviations in biodiversity with a small number of species at the lake šićki brod (13), which is probably caused by unfavorable pedological conditions (sandy soil and poor water regime). shannon index results mja typh.lat. mjb typh.lat. m.j. typh.lat. su.a typh.lat. su.b typh.lat. su.c typh.lat. š.b.a-typh.lat. š.b.b typh.lat. š.b.c-typh.lat. š.b.a -phr. aus. š.b.b phr. aus.. š.b.c phr. aus. r.j.a phr. aus. r.j.b phr. aus. r.j.c phr. aus. v a lu e sample 3,1 3,2 3,3 2,4 2,5 2,6 2,7 2,8 2,9 2,10 2,11 2,12 2,13 3,14 3,15 0 1 2 3 4 0 5 10 15 figure 6. the values of shannon wiener diversity index biologica nyssana 5 (1)  september 2014: 1-10 kamberović, j. et al.  marshland vegetation of the order phragmitetalia … 8 figure 7. ecological analysis of the studied association (t-temperature, w-humidity, r-soil acidity, n-nitrification, l-light, k-continentality, s-salinity) ecological analysis of associations ecological indexes of plant species from the analyzed communities indicate that stands appear on alkaline (r=6.3-6.8), non-salinated (s=0.5-0.7), well exposed to sunlight or shaded occasionally (l=7.17.6), briefly flooded (w=7.5-8.7) and mesotrophic soils (n=5-6.7). habitat requirements about heat are moderate (t=5.1-5.8), and are characterized by a large proportion of sub-oceanic species which find their main areal in the central europe (k=4-4.6) (fig. 7). comparing ecological indexes of the studied communities with the stands in the "labudovo okno", all values are in close ranges, except for the humidity index, which was lower for the pit lakes habitats (w = 7.5 8.7), indicating a less favorable hydro regime. biological spectrum and sociability of plant species in the studied communities at the localities of the lakes suhodanj and šićki brod the most dominant life form are hydrophytes (up to 46.15%), while hemicryptophytes are dominant at other sites (up to 44.8%) (fig. 8). compared to the stands of the order phragmitetalia in serbia (p o l i ć , 2006) and croatia (s t a n č i ć , 2010), where the dominance in the stands is achieved by hydrophytes, hemicryptophytes and geophytes, in the communities on pit lake shores the share therophytes is not negligible (7.69 25.92%). this phenomenon makes the community's response to frequent changes in water levels and to periods of drought during the summer months. figure 8. the biological spectrum of the studied association (t therophytes, g geophytes, ch chamaephytes, h hemicriptophytes, w hydrophytes, pn phanerophytes) biologica nyssana 5 (1)  september 2014: 1-10 kamberović, j. et al.  marshland vegetation of the order phragmitetalia … 9 figure 9. the social behavior types spectrum of the studied association (ac invasive species, rc ruderal competitors, w weed species, dc pioneer species of secondary successions, g associated plant species, c competitors, s sensitive species) the spectrum of sociability of the plant species indicates the state of the natural vegetation progradation. analysis of the spectrum of plant species sociability indicates that the most frequent are pioneering elements of secondary successions (dt = 23.07 55%) and associated plant species (g=18,18-30,76%). a lower frequency have the competitor plant species (c = 7.69 27.23) and they are most numerous in the communities on the lake šićki brod (fig. 9). weed plant species are also present (plantago major, bidens tripartita, artemisia vulgaris, atriplex patula, verbena officinalis and sonchus arvensis), and from invasive species there were identified three plant species (echinocystis lobata, conyza canadensis and erigeron annuus). r a n d j e l o v i c et al. (2007) emphasize the eurivalence of the community of reed beds for many environmental factors, therefore it is very often penetrated by elements of meadow and weed vegetation. on the shores of pit lakes this phenomenon is very evident, due to close contact of these communities with trampled and ruderal habitats in landfills of tailings, leading to the appearance of invasive plant species. progradation process in natural habitats the communities of reed beds and cattail play a crucial role for the stability and the climax of wetland ecosystems, while in the degraded habitats such as pit lakes they are crucial for the restoration and progradation. vegetation of cattail often grow over anthropogenic habitats of eutrophic character that had been under water for a long period of time (s t a n č i ć , 2010). among the researched localities it was most developed at the lake mušićko, which has a minimum depth, a small slope of the southern shore and water of lower quality. at the lakes suhodanj and šićki brod this community grows over a very small area (up to 50 m 2 ). the factors that prevent its spread are probably variations of water levels and the dominance of communities of the class bidentetea tripartitae (b a r u d a n o v i ć & k a m b e r o v i ć , 2011) on the lake suhodanj and a large slope of the littoral zone on the lake šićki brod. the vegetation of reed beds is more eurivalent for the moisture factor. therefore, at the lakes with large slope of the littoral zone (šićki brod and ramićko lakes), where there are not achieved conditions for communities of cattail, reed beds grow over the coastal region and tolerate longer periods of dryness. frequent fluctuations of water levels throughout the year and steep banks of pit lakes allow colonization of emersed hydrophytes in fragments only on the flat shores. therefore the progradation processes in degraded habitats in terms of establishing wetland ecosystems are possible only in lakes with lower slopes of the shore and more or less stable water levels. conclusion on the shores of four pit lakes within the order phragmitetalia there were determined 50 plant species within two plant communities. in phytocenological releves thirteen species characteristic of the association, alliance, order or class were identified. sociability spectrum of plant species, along with the domination by species of pioneering elements of secondary succession, indicates the state of natural progradation processes. bearing in mind that pit lakes in the initial stages of biologica nyssana 5 (1)  september 2014: 1-10 kamberović, j. et al.  marshland vegetation of the order phragmitetalia … 10 formation are absolutely degraded surfaces, the results of research point to the successful colonization of wetland plant species, whose natural progradation is ongoing. references apha, awwa & wef, 1995: standard methods for the examination of water and wastewater. 19 th edition, washington. barudanović, s., kamberović, j. 2011: weed vegetation on the shores of artificial reservoirs of surface mining pits in the area of tuzla. herbologia. 12 (3):1-14. barudanović, s., kamberović, j. 2008: potencijali turizma i okoliša bosne i hercegovine restauracija napuštenih površinskih kopova. zbornik radova međunarodne konferencije „zaštićena područja u funkciji održivog razvoja“, bihać, fram ziral, 497 – 507. borhidi, a., 1993: social behaviour types of the hungarian flora, its naturalness and relative ecological indicator values. janus pannonius tudom. kiadv. pecs. braun-blanquet, j. 1964: pflanzensociologie. 2 aufl. in ellenberg, h. 1986: vegetetation mitteleuropes mit den alpen in okologischer sicht. verlag eugen ulmer, stuttgart. clarke, k.r., gorley, r.n. 2006: primer v6: user manual/tutorial. primer-e, plymouth. domac, r. 2002: flora hrvatske priručnik za određivanje bilja, ii izdanje. školska knjiga, zagreb. jasprica n., carić, m., batistić, m. 2003: the marshland vegetation (phragmitomagnocaricetea, isoeto-nanojuncetea) and hydrology in the hutovo blato natural park (neretva river delta, bosnia and herzegovina). phyton. 42 (2): 281-294. javorka, s., csapody, v. 1979: ikonographie der flora des sudostlichen metelleurope. gustav fisher verlag, germany. kamberović, j., barudanović, s. 2012: algae and macrophytes of mine pit lakes in the wider area of tuzla, bosnia and herzegovina. natura croatica, 21 (1): 101-118. kamberović, j. 2010: antropogena močvarna staništa kao konzervacijski potencijal područja tuzle. magistarski rad. prirodno-matematički fakultet. univerzitet u sarajevu. kremer, b.p. 2005: steinbachs groser pflanzenfuhrer. eugen ulmer kg, stuttgart. kojić,m., popović, r., karadžić, b. 1997: vaskularne biljke srbije kao indikatori staništa. institut za istraživanja u poljoprivredi “srbija”. institut za biološka istraživanja “siniša stanković”. beograd. lakušić, r., pavlović, d., abadžić, s., grgić, p. 1977: prodromus biljnih zajednica bosne i hercegovine. godišnjak biološkog instituta univerziteta u sarajevu, 30. lazić, d., škorić, m., stojanović, s., knežević, a., nikolić, lj. 2005: syntaxonomic review of the vegetation of the jegrička watercourses. savremena poljoprivreda, 54 (3-4): 269-274. mcaleece, n., lambshead p.j.d., paterson g.l.j. 1997: biodiversity pro. the natural history museum, london. nixdorf, b., lessmann, d. & deneke, r. 2005: mining lakes in a disturbed landscape: application of the ec water framework directive and future management strategies. ecological engineering 24: 67–73. oberdorfer, e. 1983: pflanzensoziologishe excursions flora. verlag eugen ulmer, stuttgart. polić, d. 2006: florističko-fitocenološko proučavanje labudovog okna. biblioteka academia. zadužbina adrejević, beograd. smajić, s. 2005: klimatske karakteristike tuzle. zbornik radova, svezak geografija. prirodno matematički fakultet u tuzli, god ii, (2) tuzla. ranđelović, v., zlatković b., matejić, j. 2007: močvarna vegetacija reda phragmitetalia u jugoistočnoj srbiji. proceeding of the 9th symposium on flora of southeastern serbia and neighbouring regions, niš, 2007. redžić, s., barudanović, s., radević, m. (ed.) 2008: bosna i hercegovina – zemlja raznolikosti, pregled i stanje biološke i pejzažne raznolikosti bosne i hercegovine, prvi izvještaj bih za cbd, bemust, sarajevo. redžić, s., barudanović, s. trakić, s., kulijer, d. 2009. reedbeds, tall sedges and vegetation of phragmito-magnocaricetea in: biodiversity working group 2009: habitat interpretation sheets natura 2000 habitat types occurring along the sava river. final technical document, no. bwg-2008-01:44. stančić, z. 2010: marshland vegetation of the class phragmito-magnocaricetea in northwest croatia (krapina river valley). biologia, 65 (1): 39-53. tutin, t. g., heywood, v. h., burges, n. a., valentine d. h., walters, s.m. & webb, d.a. /ed./ 1964-1993: flora europaea 1-5, cambridge university press, cambridge. velagić-habul, e., bašagić, m. & omanović, e. 2005: uticaj jalovišta površinskih kopova uglja na kvalitet vode tekućica. radovi poljoprivrednog fakulteta univerziteta u sarajevu, l. 56. biologica nyssana 5 (1)  september 2014: 1-10 kamberović, j. et al.  marshland vegetation of the order phragmitetalia … 11 boskov et al. 2022, biologica nyssana 13(2) 13 (2) december 2022: 119-128 doi: 10.5281/zenodo.7437250 quality and stability of topical formulation based on the extract of black locust flower (robiniae pseudoacaciae flos) original article ivana a. boskov faculty of technology, university of niš, bulevar oslobođenja 124, 16000 leskovac, serbia ivannab.92@gmail.com (corresponding author) ivana m. savić gajić faculty of technology, university of niš, bulevar oslobođenja 124, 16000 leskovac, serbia ivan m. savić faculty of technology, university of niš, bulevar oslobođenja 124, 16000 leskovac, serbia received: june 30, 2022 revised: october 15, 2022 accepted: october 22, 2022 abstract: black locust flowers are the source of a significant amount of antioxidants that can be useful against free radicals formed on the skin after the environmental factors. having in mind these facts, the ultrasound-assisted extraction of antioxidants from plant materials was previously developed. the aim of this study was to prepare a topical formulation based on the extract of black locust flowers. the extract enriched with antioxidants was prepared using the ultrasound-assisted extraction using 60% (v/v) ethanol at 60 °c and a liquidto-solid ratio of 10 ml/g for 30 min. the two topical formulations in the form of o/w emulsion at different concentrations of the extract (1% and 2%) were prepared by a hot-hot emulsification process. the product quality was estimated and concluded that the emulsion is white in the semi-solid state. the formulations can have adequate application properties due to their present ingredients, which do not leave a greasy film on the skin. the ph value of the prepared formulations was according to the allowed range for the application to the skin. the proper choice of ingredients enabled the achievement of satisfactory formulation stability. the formulations can be microbiologically safe to use for human purposes. a better antimicrobial activity against the investigated microorganism strains showed the formulation with higher content of the extract so it can be selected as an adequate for further analysis. key words: black locust flowers, extract, topical formulation, quality control, stability apstrakt: kvalitet i stabilnost topikalne formulacije na bazi ekstrakata cvetova bagrema (robiniae pseudoacaciae flos) cvetovi bagrema su izvor značajnih količina antioksidanasa koji mogu biti korisni za hvatanje slobodnih radikala nastalih na koži nakon dejstva spoljašnjih faktora. imajući u vidu ove činjenice, ultrazvučna ekstrakcija antioksidanasa iz biljnog materijala je prethodno razvijena. cilj ove studije je bio da se pripremi topikalna formulacija na bazi ekstrakta cvetova bagrema. ekstrakt obogaćen antioksidansima je pripremljen primenom ultrazvučne ekstrakcije korišćenjem 60% (v/v) etanola na 60 °c i pri odnosu tečnosti i čvrste materije 10 ml/g u toku 30 min. dve topikalne formulacije u obliku u/v emulzije pri različitim koncentracijama ekstrakta (1% i 2%) pripremljene su toplo-toplo emulzifikacionim procesom. kvalitet proizvoda je procenjen i zaključeno je da su emulzije bele polučvrste konzistencije. formulacije mogu da imaju adekvatna aplikativna svojstva zbog prisutnih sastojaka, s obzirom da ne ostavljaju mastan film na koži. ph vrednost pripremljenih formulacija bio je u skladu sa dozvoljenim opsegom za primenu na koži. pravilan izbor sastojaka omogućile su postizanje zadovoljavajuće stabilnosti formulacije. formulacije mogu da budu mikrobiološki bezbedne za ljudsku upotrebu. bolju antimirkobnu aktivnost na analiziranim sojevima mikroorganizmima ispoljila je formulacija sa većim sadržajem ekstrakta, tako da za dalju analizu može biti pogodna. ključne reči: cvetovi bagrema, ekstrakt, topikalna formulacija, kontrola kvaliteta, stabilnost introduction the use of medicinal plants and their extracts in pharmaceutical and cosmetic industries increases due to the desire for a healthier lifestyle and youthful appearance. medicinal plants contain bioactive compounds, which have positive effects on human health (veiga et al., 2020). among the bioactive © 2022 boskov et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 119 compounds, polyphenols retaken a significant place. this group of compounds, also known as secondary metabolites of plants, shows antioxidant (de lima cherubim et al., 2020), antimicrobial, anticancer (dzah et al., 2020), anti-inflammatory, anticollagenase, and antielastase activities (madan & nanda, 2018). according to these properties and the ability to pass through the stratum corneum and penetrate the epidermis and dermis, they are suitable for topical application (zillich et al., 2015). they are topically applied for care (guimarães et al., 2021) and photoprotection of skin (farjadmand et al., 2021), as well as for the prevention of skin cancer (souto et al., 2019). to demonstrate their certain biological activities, polyphenolic compounds have to release from the formulation. the formulations should be chemically, physically, and microbiologically stable. to ensure good migration of polyphenolic compounds into the skin, their precipitation in the vehicle of the formulation should not occur. polyphenolic compounds precipitate rapidly because of their poor solubility in water (figueroa-robles et al., 2020). the solubilization of polyphenolic compounds can be improved and their precipitation can be prevented by the addition of surfactants, such as labrasol and tween (jee et al., 2019) or block copolymers (shin et al., 2018) in the formulation. emulsions are the most suitable type of vehicle for topical formulations since they have adequate solubilization capacity for lipophilic and hydrophilic ingredients (yang et al., 2020). the lower content of the oily phase in the emulsions makes it easier to release polyphenolic compounds and a higher rate of their penetration through the skin (zillich et al., 2013). this type of emulsion has a lower viscosity, which allows better diffusion of polyphenols within the emulsions. skin hyperhydration can be achieved using formulations with a high content of water (o/w emulsion) that cause the higher permeability of polyphenols through the skin. the addition of aqueous and oily plant extracts does not disturb the structure of the emulsion itself (smaoui et al., 2012), but it may affect its rheological properties. due to the dilution effect and the interaction of polyphenols with protein or polysaccharide emulsifiers (li et al., 2019), the viscosity is increased by the addition of the extract (leite et al., 2019). the interactions of polyphenols with nonionic or anionic surfactants, commonly used in emulsions, are rarely described. on the other hand, polyphenols have also surfactant properties (katsouli et al., 2017). the oxidative and storage stability of emulsions can be improved in the presence of polyphenols (choulitoudi et al., 2021). there are dermo-cosmetics formulations with polyphenolic compounds (quercetin, resveratrol, gallic acid, rutin, curcumin, apigenin, etc.) and 120 biologica nyssana ● 13 (2) december 2022: 119-128 boskov et al. ● quality and stability of topical formulation based on the extract of black locust flower (robiniae pseudoacaciae flos) extracts enriched with these compounds (grape extract, liquorice root extract, aloe vera extract, calendula extract, lilac extract, lavender extract, etc.) in the market. due to the growing market demand for dermo-cosmetic products recognized as safe, curative, and with synergistic or multifunctional effects, there is a continual need to find new sources of polyphenolic compounds. according to our previous studies, the ethanol extract of black locust flowers is an excellent source of polyphenolic compounds with expressed antioxidant activity (savic gajic et al., 2019). the aim of this study was to prepare the topical formulation with the extract of black locust flowers enriched with antioxidants. based on the properties of the topical formulation and its physico-chemical stability, the product quality was estimated. materials and methods in this study, sabowax sx and sabonal c1618 (cetearyl alcohol) (sabo s.p.a., italy), white beeswax (ceraalba, extra pure, centrohem, srbija), triethanolamine (care basf, germany), phenoxyethanol sa (schülke & mayr gmbh, germany), mineral oil (techno-chem d.o.o., serbia), glycerol (85%), carbomer, trichloroacetic acid (sigma-aldrich, serbia), and distilled water (aqua purificata) was used. ethanol extract of black locust flowers was obtained by ultrasound-assisted extraction (savic gajic et al., 2019). preparation of topical formulation the o/w topical formulations with and without (base) black locust flower extract were prepared according to tab. 1. the oily and water phases were prepared separately by heating in enameled paten via water bath at a temperature of 70±1 °c. the topical formulation was prepared by the hot-hot emulsification process. this process involves the addition of the oily phase into the water phase gradually with stirring using a laboratory propeller mixer at 800 rpm and constant temperature. after the emulsification process, a stirring of the phases was continued at 500 rpm until the temperature was below 40 °c. phenoxyethanol sa was added as a preservative after cooling the sample. triethanolamine was added to adjust the ph of the formulation. the prepared formulations were stored at room temperature (22±2 °c) in a plastic polypropylene container. the base was prepared in the same way but without the addition of the black locust flower extract. formulation quality assay organoleptic properties (appearance, color, homogeneity, and separation of phases) of the base biologica nyssana ● 13 (2) december 2022: 119-128 boskov et al. ● quality and stability of topical formulation based on the extract of black locust flower (robiniae pseudoacaciae flos) 121 and topical formulations with black locust flower extract (f1 and f2) were assessed by visual observation (ph. jug. iv, 1984). determination of emulsion type was achieved by electrical conductivity measuring (stojiljković et al., 2013). the measurement of electrical conductivity was performed by direct immersion of the conductometer electrode (cdm 230, radiometer, copenhagen, denmark) into the creams samples at room temperature (22±2 °c). before starting the work, the measuring electrode was calibrated with 0.01 mol/lkcl solution. to measure the ph value of the samples, a potentiometric method was used (stojiljković et al., 2013). a glass electrode of ph meter (hi 9321, hanna instruments, lisbon, portugal) was directly immersed in the samples at room temperature (22±2 °c). the device was calibrated with standard ph buffers of 4.0 and 7.0. the viscosity of prepared formulations was determined using a viscosimeter (visco basic plus, fungilab inc., usa) with an sp-r5 spindle, at a 12 rpm rotation speed (maia filho et al., 2012). physical stability of the formulations centrifugation assay was applied to assess the physical stability of the prepared samples on the gravity effect according to the previously described method with slight modification (juttulapa et al., 2017). precisely 4 g of the samples were weighed into a 10 ml plastic test cuvette and centrifuged twice at 3000 rpm for 15 min. a laboratory centrifuge (lc 320, tehtnica, železniki, slovenia) was used. the measurements were performed at room temperature (22±2 °c) 24 h after preparation. the samples were observed visually to notice eventual changes (phase separation). cyclic temperature stress test or accelerated aging test of formulations was realized through 6 cycles. each cycle included the following steps: 1) the samples were placed in the freezer at -10 °c for 24 h; 2) the samples were removed from the freezer and left at room temperature (22±2 °c) for 24 h; 3) the samples were placed in thermostat oven (sutjeska, yugoslavia) at the temperature of 40 °c for 24 h; 4) the samples were removed from the thermostat oven and left at room temperature (22±2 °c) for 24 h. at the same time, one sample was left under the isothermal condition at room temperature (22±2 °c), in a fridge at +4 °c, and in a thermostat oven at +40 °c. after the third and sixth cycles, the ph value and electrical conductivity were measured in each sample. if the product is stable, the appearance, color, and consistency should not change. a long-term aging test or shelf test was applied to estimate the physical stability of the prepared formulations based on the changes in organoleptic properties, ph values, and electrical conductivity. the samples were stored in primary packaging (plastic containers) at room temperature for 3 months, and the tests were performed 7, 30, 60, and 90 days after their preparation. the chemical stability of the extract in pure form and after its incorporation into the formulations (f1 and f2) was assessed based on the change in total antioxidant content (tac) over time. the tac in the black locust flower extract and formulations was determined by the folin-ciocalteu method (savic gajic et al., 2019). the analyzed samples ingredients base topical formulation f1 f2 oily phase white beeswax 1 1 1 mineral oil 6 6 6 sabowax sx 5 5 5 sabonal c1618 (cetearyl alcohol) 6.5 6.5 6.5 trichloroacetic acid 7 7 7 triethanolamine 0.7 0.7 0.7 phenoxyethanol sa 0.2 0.2 0.2 water phase water 68.1 67.1 66.1 carbomer 0.5 0.5 0.5 glycerin 5 5 5 black locust flower extract 1 2 table 1. composition of topical formulations expressed in the percentage (f1-topical formulation with 1% of the extract; f2–topical formulation with 2% of the extract) 122 biologica nyssana ● 13 (2) december 2022: 119-128 boskov et al. ● quality and stability of topical formulation based on the extract of black locust flower (robiniae pseudoacaciae flos) were exposed to the influence of lower and higher temperatures (-18 °c, +4 °c, and +22 °c), as well as daylight/darkness for 3 months. sampling was performed after 7, 30, 60, and 90 days, whereby the tac expressed as milligrams of gallic acid equivalent per 100 g of dry weight (mg gae/100 g) was determined for each sample. the absorbance of the samples was measured at 740 nm after incubation at room temperature (+22 °c) for 30 min. varian cary-100 uv-vis spectrophotometer (malgrave, victoria, australia) and 1×1 cm quartz cuvettes were applied for scanning the samples. microbiological safety of formulations the method is based on the isolation and identification of bacteria, yeasts, and molds present in the sample (https://www.paragraf.rs/ propisi/pravilnik-uslovima-pogledu-zdravstveneispravnosti-predmeta-opste-upotrebe.html).the samples were seeded on a nutrient medium and incubated under optimal conditions. antimicrobial activity antimicrobial activity of black locust flower extract, the base, and formulations was examined against gram-positive (staphylococcus aureus atcc 6538, streptococcus pneumoniae atcc 49619) and gram-negative bacteria (escherichia coli atcc 8739, proteus mirabilis atcc 25933, klebsiella pneumoniae atcc 10031), as well as on fungi (candida albicans atcc 10231) using disk diffusion method. the samples were dissolved in dimethyl sulfoxide (dmso) which was used as a negative control. the antibiotic medium-1 nutrient medium was used for the growth of bacteria, while sabouraud 4% dextrose agar nutrient medium was used for fungal growth. an adequate amount of nutrient medium was dissolved in purified water and then sterilized at 121 °c in an autoclave for 15 min. the sterilized medium was cooled to 40–45 °c and an appropriate concentration of microorganisms’ suspension was added to it. the nutrient medium (15 ml) was poured into petri dishes with a diameter of 90 mm. the paper discs with a diameter of 6 mm, soaked with 30 μl of extract (1 mg/ml) and formulations (0.1 g/ml) were arranged on a cooled and solid surface. gentamicin was used as a positive control. bacteria were incubated at 37 °c for 18–24 h and fungi at 25 °c for 24–48 h under anaerobic conditions. after the incubation, the inhibition zones were measured in millimeters. based on the inhibition zones, the antimicrobial activity of the samples was estimated. statistical analysis all obtained data were presented as mean value±standard deviation. all statistical analyzes were performed using the originpro9 software (originlab corporation, northampton, usa), with a significance level of p<0.05. results and discussion the o/w emulsion formulations were prepared by a technological procedure in the laboratory conditions. the oily phase consisted of white beeswax, mineral oil, sabowsax sx, cetearyl alcohol (sabonal c1618), trichloroacetic acid (tca), triethanolamine, and phenoxyethanol sa. white beeswax is a mixture of esters of higher fatty acid and higher fatty alcohols, free fatty acids and alcohols, carotenoids, aromatic and mineral substances. due to its regenerative, emollient, and soothing properties, white beeswax is suitable for dry skin and winter skincare routines. mineral oil is the odorless, colorless, and tasteless oily liquid of mineral origin. this oil is a mixture of saturated hydrocarbons, which due to their size cannot penetrate the skin and therefore do not close the skin pores. it is not carcinogenic and does not cause allergic reactions on the skin. it can form a protective layer on the skin that prevents transdermal water loss, making skin soft and smooth. white beeswax and mineral oil, in addition to achieving an occlusive effect, are also important for regulating the consistency of the developed formulation (baumann, 2011). sabowax sx is an anionic self-emulsifying o/w base and presents a mixture of cetearyl alcohol, fatty alcohols, and sodium alkyl sulfate. in topical formulations, it is used in concentrations of 4–7% and can improve the stability and consistency of the preparation. cetearyl alcohol (sabonal c1618) is a fatty alcohol used as an emulsifier in the formulations, improving the viscosity of the product. it has a soothing effect on the skin, making it smooth and soft. in the formulations, tca can be used in different concentrations depending on the purpose. it has a significant role in smoothing fine surface wrinkles, removing surface stains, and correcting pigment problems. in the developed formulations, triethanolamine was used as a ph adjusting agent. depending on the desired ph value, it can be used in the concentration of 0.1–1%. due to good antimicrobial properties and low toxicity, phenoxyethanol sa was used as a preservative for the developed formulation. if the formulation contains only one preservative, the maximum permissible concentration is 0.4%, while for a mixture of preservatives, the allowable value is 0.8%. unlike the oily phase, the water phase consisted of demineralized water, carbomer, and glycerin. prepared ethanol extract of black locust flower in the concentration of 1% and 2% was added to the water 123 biologica nyssana ● 13 (2) december 2022: 119-128 boskov et al. ● quality and stability of topical formulation based on the extract of black locust flower (robiniae pseudoacaciae flos) al., 2013). the electrical conductivity for the base, f1, and f2 of 78, 86, and 91 μs/cm, respectively, indicate that the prepared formulations are o/w emulsion. the physiological ph value of skin ranges from 4 to 7 (lambers et al., 2006), while the average value is 5.5. topical formulations should have a ph value close to this range. the ph values of the base, f1, and f2 were 6.41, 6.38, and 6.35, respectively. the addition of the extract to the base led to a slight decrease in the ph value (akhtar et al., 2011). the viscosity of the formulation is important for handling and storage. based on this parameter, the stability of the product is possible to evaluate. the measured values for the viscosity of base, f1, and f2 of 13.522, 13.261, and 12.789 mpa·s, respectively, indicated that the formulations were semi-solid consistent. the addition of extract to the base led to a decrease in viscosity. physical stability of formulations the prepared formulations were physically stable because there was no phase separation after the action of mechanical stress (centrifugation assay). shelflife assessment is a bottleneck during designing a new product. therefore, it is necessary to lead an „accelerated agingˮ test. applying this test in a short time interval can provide information about the physical stability of the product. in that case, it is necessary to observe parameters (ph value, electrical conductivity, stratification rate, flocculation, etc.) with rapid changes in values under the influence of some external factors. temperature is one of phase. the extract was obtained by ultrasoundassisted extraction and characterized in our previous study (savic gajic et al., 2019). the tac in the extract was 3.12 gae/100 g d.w. among the identified individual polyphenolic compounds, rutin was the most abundant compound (56.9 mg/100 g d.w.). the ic50 value of the extract was 120.5 µg/ ml, which makes it suitable for the development of dermo-cosmetic formulations. the water used in the formulations was toxins and microbes free. carbomer found wide application in the topical products as a safe substance with extremely low irritant and sensitizing potential. in the developed formulations, a carbomer was used to stabilize the emulsion. it should be in the concentration range of 0.1-0.5% to use as an emulsion stabilizer. glycerol as humectant was added to the aqueous phase of the emulsion. it is known that glycerol forms an invisible film on the skin which binds water molecules from the air and thus hydrates the skin. formulation quality assay the prepared formulations were a semi-solid consistency, homogeneous, white, and with a characteristic smell. the adequate selection of oily phase ingredients enabled the samples to be stable 24 h after preparation. determination of electrical conductivity is one of the most reliable methods for the determination of emulsion type. according to the literature data, the electrical conductivity higher than 50 μs/cm indicates o/w emulsion, while values less than 1 μs/cm indicates w/o emulsion (jiang et sample storage conditions parameters ph electrical conductivity (ms/cm) f1 cycle iii 6.39 85 cycle vi 6.34 86 f2 cycle iii 6.36 90 cycle vi 6.33 90 base cycle iii 6.41 77 cycle vi 6.39 78 comparative f1 +4 °c 6.38 86 room temperature 6.38 87 +40 °c 6.22 88 comparative f2 +4 °c 6.35 91 room temperature 6.36 92 +40 °c 6.19 91 comparative base +4 °c 6.39 76 room temperature 6.38 78 +40 °c 6.23 79 table 2. ph and electrical conductivity values of the formulations after the cyclic temperature stress test 124 biologica nyssana ● 13 (2) december 2022: 119-128 boskov et al. ● quality and stability of topical formulation based on the extract of black locust flower (robiniae pseudoacaciae flos) the most commonly used factors for accelerated testing of product stability. in the cyclic temperature stress test, the sample was alternately exposed to higher and lower temperatures, which can cause destabilizing phenomena (stratification, flocculation, and coalescence). in tab. 2, the measured values of ph and electrical conductivity for the samples after iii and iv cycles are presented. the comparative formulations were stored isothermally at +4 °c, room temperature (+22 °c), and +40 °c. the base, f1, and f2 had a slightly lower ph value after cycle vi compared to cycle iii. in comparative samples of the formulation was observed a slight decrease of ph value at +40 °c. the high temperature probably destabilized the formulation, but it did not affect the overall product quality because the ph value was still around 6.0. this value is acceptable since it is close to physiological ph and does not irritate the skin. the changes in electrical conductivity of samples were insignificant under all examined conditions. after using the cyclic temperature stress test, the prepared formulations were relatively stable, because the significant changes in the ph value and electrical conductivity were not noticed. also, the changes in the organoleptic properties were not observed. in addition to the accelerated aging tests (centrifugation assay and cyclic temperature stress test), a long-term aging test was also applied for assessing the physical stability of the prepared formulations. the samples were stored at room temperature after preparation for 3 months. the ph value and electrical conductivity were measured after 7, 30, 60, and 90 days of formulation preparation (tab. 3) the ph values of the formulations were decreased but in the range of acceptable limits. the decrease in the ph value was less than 10% for the formulations stored at room temperature for 90 days. these results indicated that the formulations are physically stable. unlike the ph value, the electrical conductivity was increased in all samples during the time. after the storage, the samples were without visible changes in the organoleptic properties. this behavior is the result of good physic-chemical properties and longterm stability of the formulations. table 3. the change of ph and electrical conductivity values of formulations during storage sample ph electrical conductivity (ms/cm) day 7 day 30 day 60 day 90 day 7 day 30 day 60 day 90 f1 6.34 6.19 6.02 5.90 87 88 89 90 f2 6.31 6.16 5.96 5.86 91 93 95 96 base 6.38 6.22 6.06 5.99 79 81 83 85 chemical stability of formulations polyphenols in their structure contain one or more benzene rings to which are attached at least two hydroxyl groups. these groups are highly reactive and susceptible to epimerization, autooxidation, esterification, alkylation, carboxymethylation and dealkylation reaction, chelate formation, and other reactions. these reactions result in a decrease in the stability or loss of the pharmacological activity of polyphenols (garcia et al., 2016). among the environmental factors, the effect of temperature and light were studied on the stability of polyphenols. to investigate the effect of temperature on stability, black locust flower extract was stored at -18 °c, +4 °c, and room temperature for 90 days. at -18 °c, a significant change in the tac (less than 2%) was not observed. this slight decrease is most likely due to the inhibition of phenoloxidase activity at lower temperatures (wei & zhang, 2008). at +4 °c and room temperature, the decrease in the tac was about 8% until day 90 which was in accordance with available results (tsali & goula, 2018). the effect of temperature on the stability of formulations was carried out in the same way as in the case of the extract. the smallest decrease in the tac was noticed for the samples stored at -18 °c (less than 1%), while this decrease was about 4% at higher temperatures (+4 °c and room temperature). the formulation can be considered stable since the decrease in the tac was less than 5% compared to the initial value (ich q1a (r2)). the satisfactory chemical stability of the formulations is the result of an adequate choice of ingredients, especially those in the oily phase. there is no available literature data about the stability of black locust flower extract. srivastava & gupta (2009) monitored the chemical stability of aqueous and methanol chamomile flower extract at different temperatures (-20, +4, and +25 °c) based on the change in the flavonoid content and obtained similar results. at +4 °c and room temperature, the flavonoid content decreased about 10% during 120 days. the storage of prepared chamomile extracts at -20 °c was not caused a significant reduction in the flavonoid content. certain polyphenolic compounds, such as quercetin (golonka et al., 2020), resveratrol (chen 125 biologica nyssana ● 13 (2) december 2022: 119-128 boskov et al. ● quality and stability of topical formulation based on the extract of black locust flower (robiniae pseudoacaciae flos) et al., 2020), and catechin (yuann et al., 2021), etc. are susceptible to photodegradation. isomerization and polymerization are the most common reactions that occur in the extract under the light effect (deng et al., 2018; latva-mäenpää et al., 2021). the chemical stability of extract and formulations stored in daylight and the dark for 90 days was estimated based on the changes in the tac. after 90 days of storage in daylight and the dark, the change in the tac was about 3% and 2%, respectively. also, the reduction in the tac was about 1.5% in daylight and 1.2% in the dark after 90 days. the formulation remained photostable after the addition of the black locust flower extract. also, it can be concluded that there were no interactions between the extract and other ingredients. microbiological safety of formulations microbiological analysis of the samples showed that the total number of aerobic mesophilic bacteria, yeasts, and mold spores in 1 g of the sample corresponds to the permissible number of microorganisms according to regulations (https:// www.paragraf.rs/propisi/pravilnik-uslovimapogledu-zdravstvene-ispravnosti-predmeta-opsteupotrebe.html). the presence of pathogenic bacteria was not confirmed in 0.1 g of the sample. therefore, it can be concluded that the prepared formulations were microbiologically safe. antimicrobial activity the antimicrobial activity of formulations with black locust flower extract (0.1 g/ml) was tested on appropriate strains of microorganisms. the base and extract were prepared at the concentration of 1 mg/ml and considered the positive controls. the results of antimicrobial activity are shown in tab. 4. both dmso solvent and the base did not show an inhibitory effect against the microorganism strains. gentamicin affected only bacteria, while it did not show an effect on the analyzed fungus. black locust flower extract showed the strongest activity against gram-positive bacteria (s. aureus atcc 6538, s. pneumoniae atcc 49619) and weaker against gram-negative (e. coli atcc 8739, p. mirabilis atcc 25933, k. pneumoniae atcc 10031). it did not show an inhibitory effect against the fungus c. albicans atcc 10231. s. aureus bacterium causes skin infections, such as ulcers, carbuncles, impetigo and cellulitis. this bacterium can also cause serious infections of postoperative wounds and burns. since the extract showed the strongest activity against these strains, it can be used in the treatment of skin infections. rosu et al. (2012) showed that ethanol extract of black locust flower has an effect on gram-positive bacteria at the concentration of 100 mg/ml. black locust flower essential oil has antimicrobial activity against selected food pathogens, such as s. aureus kctc 1621, bacillus subtilis kctc 3569, listeria monocytogenes kctc 3569, e. coli o157:h7, and salmonella enterica atcc 4731 with mic and mbc values of 250-1000 mg/ml (bhalla & bajpai, 2017). conclusions the topical formulations with black locust flower extract were developed in the laboratory conditions. the formulation was free of synthetic fragrances, colors, and aggressive emulsifiers. the prepared formulations were safe to use and their measured ph was about 6 corresponding to the ph value of healthy skin. the viscosity of 13.261 and 12.789 mpa·s for f1 and f2, respectively, indicated that the prepared formulations were semi-solid consistency. the formulations were stable since the significant changes in the ph value, electrical conductivity, organoleptic properties, and tac were not noticed during storage. satisfactory product stability was the result of an adequate choice of ingredients, especially for the oily phase. the optimal conditions microorganisms extract f1 f2 base dmso gentamicin s. aureus atcc 6538 +++ ++ +++ +++ s. pneumoniae atcc 49619 +++ ++ ++ +++ e. coli atcc 8739 ++ + ++ ++ p. mirabilis atcc 25933 ++ + + ++ k. pneumoniae atcc 10031 ++ + c. albicans atcc 10231 table 4. results of antimicrobial activity using disk diffusion method no antimicrobial activity (-), inhibition zone <15 mm. weak antimicrobial activity (+), inhibition zone of 15–16 mm. moderate antimicrobial activity (++), inhibition zone of 17–19 mm. high antimicrobial activity (+++), inhibition zone of 20–22 mm. strong antimicrobial activity (++++), inhibition zone of >23 mm. standard deviation±0,5 mm. biologica nyssana ● 13 (2) december 2022: 119-128 boskov et al. ● quality and stability of topical formulation based on the extract of black locust flower (robiniae pseudoacaciae flos) for product storage were room temperature and a plastic polypropylene container. due to the antioxidant and antimicrobial activity (against s. aureus) of incorporated black locust flower extract, the formulations can be useful against skin diseases caused by the effect of free radicals and infections. also, they can be suitable for daily hydration and softening of the skin due to the presence of emollient (glycerin) and moisturizing ingredients. having in mind that the formulation with higher contents of the extract enabled better antimicrobial activity against s. aureus and e. coli, it can be selected as a better one and subjected to further studies. acknowledgements. this research was funded by the republic of serbia-ministry of education, science and technological development, program for financing scientific research work, number 451-03-68/202214/200133. references akhtar, n., khan, b. a., haji, m., khan, s., ahmad, m., rasool, f., mahmood, t.,rasul, a. 2011: evaluation of various functional skin parameters using a topical cream of calendula officinalis extract. african journal of pharmacy and pharmacology, 5(2): 199-206. baumann, l. 2011: kozmetička dermatologija: načela i praksa. prvo izdanje na hrvatskom jeziku. interpreta usluge d.o.o., zagreb. bhalla, p., bajpai, v.k. 2017: chemical composition and antibacterial action of robinia pseudoacacia l. flower essential oil on membrane permeability of foodborne pathogens. journal of essential oil bearing plants, 20(3): 632-645. chen, s., han, y., jian, l., liao, w., zhang, y., gao, y. 2020: fabrication, characterization, physicochemical stability of zein-chitosan nanocomplex for co-encapsulating curcumin and resveratrol. carbohydrate polymers, 236: 116090. choulitoudi, e., xristou, m., tsimogiannis, d., oreopoulou, v. 2021: the effect of temperature on the phenolic content and oxidative stability of o/w emulsions enriched with natural extracts from satureja thymbra. food chemistry, 349: 129206. de lima cherubim, d.j., buzanello martins, c.v., oliveira fariña, l., da silva de lucca, r.a. 2020: polyphenols as natural antioxidants in cosmetics applications. journal of cosmetic dermatology, 19(1): 33-37. deng, j., yang, h., capanoglu, e., cao, h., xiao, j. 2018: technological aspects and stability of polyphenols. in: polyphenols: properties, recovery, and applications: 295-323, woodhead publishing. dzah, c.s., duan, y., zhang, h., wen, c., zhang, j., chen, g., ma, h. 2020: the effects of ultrasound assisted extraction on yield, antioxidant, anticancer and antimicrobial activity of polyphenol extracts: a review. food bioscience, 35: 100547. farjadmand, f., karimpour-razkenari, e., nabavi, s.m., ardekani, m.r., saeedi, m. 2021: plant polyphenols: natural and potent uv-protective agents for the prevention and treatment of skin disorders. mini reviews in medicinal chemistry, 21(5): 576-585. figueroa-robles, a., antunes-ricardo, m., guajardoflores, d. 2020: encapsulation of phenolic compounds with liposomal improvement in the cosmetic industry. international journal of pharmaceutics, 120125. garcia, d.e., glasser, w.g., pizzi, a., paczkowski, s.p., laborie, m.p. 2016: modification of condensed tannins: from polyphenol chemistry to materials engineering. new journal of chemistry, 40(1): 36-49. golonka, i., wilk, s., musiał, w. 2020: the influence of uv radiation on the degradation of pharmaceutical formulations containing quercetin. molecules, 25(22): 5454. guimarães, i., baptista-silva, s., pintado, m., l oliveira, a. 2021: polyphenols: a promising avenue in therapeutic solutions for wound care. applied sciences, 11(3): 1230. https://www.paragraf.rs/propisi/pravilnik-uslovimapogledu-zdravstvene-ispravnosti-predmeta-opsteupotrebe.html ich q1a (r2) stability testing of new drug substances and drug products, european medicines agency, netherlands. jee, j.p., pangeni, r., jha, s.k., byun, y., park, j.w. 2019: preparation and in vivo evaluation of a topical hydrogel system incorporating highly skinpermeable growth factors, quercetin, and oxygen carriers for enhanced diabetic wound-healing therapy. international journal of nanomedicine, 14: 5449. jiang, j., mei, z., xu, j., sun, d. 2013: effect of inorganic electrolytes on the formation and the stability of water-in-oil (w/o) emulsions. colloids and surfaces a: physicochemical and engineering aspects, 429: 82-90. juttulapa, m., piriyaprasarth, s., takeuchi, h., sriamornsak, p. 2017: effect of high-pressure homogenization on stability of emulsions containing 126 127 biologica nyssana ● 13 (2) december 2022: 119-128 boskov et al. ● quality and stability of topical formulation based on the extract of black locust flower (robiniae pseudoacaciae flos) zein and pectin. asian journal of pharmaceutical sciences, 12(1): 21-27. katsouli, m., polychniatou, v., tzia, c. 2017: influence of surface-active phenolic acids and aqueous phase ratio on w/o nano-emulsions properties; model fitting and prediction of nanoemulsions oxidation stability. journal of food engineering, 214: 40-46. lambers, h., piessens, s., bloem, a., pronk, h., finkel, p. 2006: natural skin surface ph is on average below 5, which is beneficial for its resident flora. international journal of cosmetic science, 28(5): 359-370. latva-mäenpää, h., wufu, r., mulat, d., sarjala, t., saranpää, p., wähälä, k. 2021: stability and photoisomerization of stilbenes isolated from the bark of norway spruce roots. molecules, 26(4): 1036. leite, f.d.g., oshiro júnior, j.a., chiavacci, l.a., chiari-andréo, b.g. 2019: assessment of an antiageing structured cosmetic formulation containing goji berry. brazilian journal of pharmaceutical sciences, 55. li, r., peng, s., zhang, r., dai, t., fu, g., wan, y., liu, c., mcclements, d.j. 2019: formation and characterization of oil-in-water emulsions stabilized by polyphenol-polysaccharide complexes: tannic acid and β-glucan. food research international, 123: 266-275. madan, k., nanda, s. 2018: in-vitro evaluation of antioxidant, anti-elastase, anti-collagenase, anti-hyaluronidase activities of safranal and determination of its sun protection factor in skin photoaging. bioorganic chemistry, 77: 159-167. maia filho, d.c., ramalho, j.b., spinelli, l.s., lucas, e.f. 2012: aging of water-in-crude oil emulsions: effect on water content, droplet size distribution, dynamic viscosity and stability. colloids and surfaces a: physicochemical and engineering aspects, 396: 208-212. ph. jug. iv. 1984: farmakopeja sfrj, pharmacopoea jugoslavica, savezni zavod za zdravstvenu zaštitu. beograd. rosu, a.f., bita, a., calina, d., rosu, l., zlatian, o., calina, v. 2012: synergic antifungal and antibacterial activity of alcoholic extract of the species robinia pseudoacacia l. (fabaceae). european journal of hospital pharmacy: science and practice, 19(2): 216-216. savic gajic, i., savic, i., boskov, i., žerajić, s., markovic, i., gajic, d. 2019: optimization of ultrasound-assisted extraction of phenolic compounds from black locust (robiniae pseudoacaciae) flowers and comparison with conventional methods. antioxidants, 8(8): 248. shin, h.j., beak, h.s., kim, s.i., joo, y.h., choi, j. 2018: development and evaluation of topical formulations for a novel skin whitening agent (ap736) using hansen solubility parameters and peg-pcl polymers. international journal of pharmaceutics, 552(1-2): 251-257. smaoui, s., hlima, h.b., jarraya, r., kamoun, n.g., ellouze, r., damak, m. 2012: cosmetic emulsion from virgin olive oil: formulation and bio-physical evaluation. african journal of biotechnology, 11(40): 9664-9671. souto, e.b., sampaio, a.c., campos, j.r., martins-gomes, c., aires, a., silva, a.m. 2019: polyphenols for skin cancer: chemical properties, structure-related mechanisms of action and new delivery systems. studies in natural products chemistry, 63: 21-42. srivastava, j.k., gupta, s. 2009: extraction, characterization, stability and biological activity of flavonoids isolated from chamomile flowers. molecular and cellular pharmacology, 1(3): 138153. stojiljković, d., arsić, i., kostov, m.t., jovanović, z., tadić, v., ðorđević, s. 2013: investigation of the effects of different emollients on the structure and skin moisturizing potential of the cosmetic creams. acta facultatis medicae naissensis, 30(4): 193-200. tsali, a., goula, a.m. 2018: valorization of grape pomace: encapsulation and storage stability of its phenolic extract. powder technology, 340: 194-207. veiga, m., costa, e.m., silva, s., pintado, m. 2020: impact of plant extracts upon human health: a review. critical reviews in food science and nutrition, 60(5): 873-886. wei, l.i., zhang, y. 2008: the antioxidation effect of pyracantha fortuneana polyphenol in vitro. science and technology of food industry, 29(9): 121–123. yang, s., liu, l., han, j., tang, y. 2020: encapsulating plant ingredients for dermocosmetic application: an updated review of delivery systems and characterization techniques. international journal of cosmetic science, 42(1): 16-28. yuann, j.m.p., lee, s.y., yang, m.j., huang, s.t., cheng, c.w., liang, j.y. 2021: a study of catechin photostability using photolytic processing. processes, 9(2): 293. biologica nyssana ● 13 (2) december 2022: 119-128 boskov et al. ● quality and stability of topical formulation based on the extract of black locust flower (robiniae pseudoacaciae flos) zillich, o.v., schweiggert-weisz, u., hasenkopf, k., eisner, p., kerscher, m. 2013: release and in vitro skin permeation of polyphenols from cosmetic emulsions. international journal of cosmetic science, 35(5): 491-501. zillich, o.v., schweiggert-weisz, u., eisner, p., kerscher, m. 2015: polyphenols as active ingredients for cosmetic products. international journal of cosmetic science, 37(5): 455-464. 128 simić & zlatković 2022, biologica nyssana 13(2) 13 (2) december 2022: 89-107 doi: 10.5281/zenodo.7437214 flora around slavujevac on rujan mountain (se serbia) original article milica simić faculty of applied sciences in niš, university “union nikola tesla”, dušana popovića 22a, belgrade, serbia milican92s@gmail.com milica.stankovic2@pmf.edu.rs (corresponding author) bojan zlatković department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, niš, serbia received: september 15, 2022 revised: october 09, 2022 accepted: october 22, 2022 abstract: this paper presents the results of floristic research conducted in the surrounding of village of slavujevac on rujan mt. in se serbia. the presence of 592 taxa from the group of vascular plants was ascertained in the investigated area, with 566 species and subspecies recorded for the first time. out of a total of 26 species listed in the literature for the flora of the slavujevac area, the presence of 11 species has been confirmed. the presence of 45 plant species was recorded in the category of protected taxa, and 13 in the category of strictly protected taxa. the most common life form in the flora of the studied area is the form of hemicryptophytes present with 40.47% of the total number of recorded taxa. in the phytogeographical sense, the flora of the investigated area is dominated by species of the mediterranean-sumediterranean (31.29%) and eurasian (26.36%) area types, also including several balkan endemic taxa. key words: flora, species diversity, floristic composition, slavujevac, rujan mt., serbia apstrakt: flora okoline slavujevca na rujan planini (jugoistočna srbija) u ovom radu su prikazani rezultati florističkog istraživanja sprovedenog u okolini sela slavujevac na planini rujan u jugoistočnoj srbiji. na istraživanom području konstatovano je prisustvo 592 taksona iz grupe vaskularnih biljaka, pri čemu je prvi put zabeleženo 566 vrsta i podvrsta. od ukupno 26 vrsta navedenih u literaturi za floru ovog područja, potvrđeno je prisustvo 11 vrsta. u kategoriji zaštićenih taksona zabeleženo je prisustvo 45, a u kategoriji strogo zaštićenih 13 biljnih vrsta. najčešća životna forma biljaka u flori istraživanog područja su hemikriptofite prisutne sa 40.47% od ukupnog broja evidentiranih taksona. u fitogeografskom smislu, u flori istraživanog područja dominiraju vrste mediteransko-sumediteranskog (31.29%) i evroazijskog (26.36%) areal tipa, uključujući i veći broj balkanskih endemičnih taksona. ključne reči: flora, specijska raznovrsnost, floristički sastav, slavujevac, rujan planina, srbija introduction the part of the balkan peninsula that is characterized by a high diversity of flora and vegetation primarily refers to its southern part, including the southern parts of serbia and the republic of northern macedonia (nikolić et al., 2007). along the land border of the two mentioned countries, there is the mountain region including rujan mt., whose northern slopes include a specific area covered by our research. weakened climatic as well as florogenetic influences from the aegean mediterranean area reach the southern parts of serbia, including the investigated area, creating a characteristic composition and diversity of flora. the specificity of the richness of the plant world is especially noticeable in the thermophilic and xerophilous habitat types, which dominate the entire surroundings of the village of slaujevac, located in the hilly and mountainous belt of the rujan mt. the first data on the flora around slavujevac come from ranđelović and stamenković (1984), who in the second half of the 20th century presented the first data on the presence of certain plant species in this area. two years later, emphasizing the botanical significance of this area, the same authors published the work “flora i vegetacija rujan planine u jugoistočnoj jugoslaviji” with a large number of important data, some of which relate to the vicinity of slavujevac (ranđelović & stamenković, 1986). thanks to the great contribution of prof. n. ranđelović, d.a. hill and prof. v. ranđelović, a new species of the genus crocus was discovered in the area of this mountain, named crocus rujanensis (ranđelović et al., 1988). mount rujan, with the © 2022 simić & zlatković. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 89 surroundings of slavujevac, as an area of distinct floristic richness, has always been an attractive space for various botanical research, including research for the needs of the conservation of the flora of serbia. the aspect of endangerment and protection of rare species that occur in the area of slavujevac, such as: cachrys cristata, opopanax hispidus and alkanna pulmonaria, is discussed in detail in the “crvena knjiga flore srbije 1” (ranđelović et al., 1983; ranđelović et al., 1999; niketić, 1999). later, in the period from 2007-2018, more data on the flora of the investigated area appear, with some data again referring to newly discovered taxa in the flora of serbia (nikolić et al., 2007; diklić & stevanović, 2012; stevanović & gajić, 2012; buzorović et al., 2013; zlatković & bogosavljević, 2014; bogosavljević & zlatković, 2018; nikolić et al., 2018). the latest data from the researched area appears in 2021, when a new taxon (knautia orientalis) was discovered for the flora of serbia (niketić et al., 2021). although the flora of the rujan mt. has been completely processed (ranđelović & stamenković, 1984), a specific analysis of the flora around slavujevac, for which a small number of specific floristic data is stated, was not done until 2015 (stanković, 2015). also, bearing in mind that more than 30 years have passed since the last thorough research of the flora of rujan mt., the aim of this paper is to present a list of flora, but also to indicate whether there have been some changes in the flora of this area which in geographical and phytogeographical terms. materials and methods field research of flora covered the period from march to september 2014 and 2015, i.e. the period from march to september 2021 and from march to june 2022. the area covered by our research includes parts of the rujan mt. located near the village of slavujevac (amedan, dedovica, lismider,majden, mali orljak,veliki orljak,), as well as several localities that make up its rural areas (dolinska mahala, garska mahala and karadačka mahala). the investigated part of the area covers a total area of about 20 km2. the results of field research were documented with herbarium material that was herbarized, labeled and deposited in the herbarium of the faculty of science in niš (hmn). the collected plant material was identified using the keys of contemporary european monographs, including “flora europea” (tutin et al., ed. 19681980), as well as literature sources reference for the researched area (josifović, ed. 1970-1977, sarić, ed. 1986, 1992). the nomenclature is largely in line with the latest checklists, papers, monographs and databases (tutin et al., ed. 1968 – 1980; https://www. europlusmed.org; http://www.worldfloraonline.org). the affiliation of taxa to the appropriate life form was also determined according to the raunkiaer system (1934), which was supplemented by mueller-dombois, ellenberg (1974), and developed for taxa at the level of serbia by stevanović (1992a). the method of meusel et al. (1965, 1978), meusel, jäger (1992) was used to define the range of types and their adequate classification, which according to stevanović (1992) was modified for the territory of serbia. the legend for abbreviations of life forms and flora elements are shown in tab. 1. results and discussion the presence of 592 species from the group of vascular plants was ascertained in the investigated area. all recorded taxa were classified into 312 genera and 73 families. the number of 566 species was recorded for the first time in the vicinity of slavujevac. out of a total of 26 species listed in the literature for the flora of the slavujevac area, the presence of 11 species was confirmed (alkanna pulmonaria, centaurea benedicta, colchicum doerfleri, crocus olivieri subsp. olivieri, crocus pallasii subsp. pallasii, crocus rujanensis, jurinea polycephala, knautia orientalis, petrorhagia velutina, trifolium cherleri, vinca herbacea). the 90 biologica nyssana ● 13 (2) december 2022: 89-107 simić & zlatković ● flora around slavujevac on rujan mountain (se serbia) table 1. abbreviations of life forms and floral elements of plant taxa ch chamaephytes adv adventitive g geophytes ea eurasian h hemicryptophytes eap eurasian mountain hyd hydrophytes hol holarctic p phanerophytes kosm cosmopolitan par parasitophytes (parasitic or semiparasitic plants) med mediterranean s scandentophytes pont pontic t therophytes se central european table 2. overview of the 10 richest families in the flora around slavujevac biologica nyssana ● 13 (2) december 2022: 89-107 simić & zlatković ● flora around slavujevac on rujan mountain (se serbia) 91 presence of 15 species listed in the literature for a given area has not been confirmed by our field research. the group pteridophyta (ferns and horsetails) includes 3 genera, 3 families and 3 species (0.51% of the total number of species) of the flora of the study area. within the group spermatophyta (seeds), the class coniferopsida (gymnosperms) is represented by 1 genus, 1 family and one species (0.17%). the species seeds from the class dicotyledones are by far the most numerous, comprising representatives of 251 genera, 59 families and 486 species (82%). the class monocotyledones is represented by 57 genera, 10 families and 102 species (17.32%). in relation to the number of species with which they are represented in the flora of the researched area, the following families have the highest species richess: compositae (73 species), leguminosae (67) and gramineae (45). families represented with one genus and one species are: aspleniaceae, athyriaceae, alismataceae, araceae, amaranthaceae, anacardiaceae, asclepiadaceae, corylaceae, cupressaceae, dioscoreaeceae, equisetaceae, globulariaceae, haloragaceae, onagraceae, orobanchaceae, protulaceae, saxifragaceae, thymelaceae, tiliaceae and verbenaceae. the richness of the flora around slavujevac as well as the total flora of hilly and lower mountain region of the southern parts of serbia is associated with the fact that the investigated area has a transitional phtogeographical position, i.e. that it is located in the transitional zone between the mediterranean-submediterranean and pontic-south siberian floristic region (zlatković, 2011). tab. 2 shows an overview of 10 by the number of species family n (number of species) % compositae (asteraceae) 73 12.42 leguminosae (fabaceae) 67 11.39 gramineae (poaceae) 45 7.65 caryophyllaceae 36 6.12 labiatae (lamiaceae) 27 4.59 scrophulariaceae 27 4.59 brassicaceae 26 4.42 rosaceae 23 3.91 umbelliferae (apiaceae) 21 3.57 liliaceae 16 2.72 of the richest families in the flora of the investigated area. from the aspect of species conservation, taxa that enjoy one of the two forms of legal protection at the national level on the basis of the law on nature protection are of importance in the researched area (official gazette of the republic of serbia, no. 36/09). the first group is represented by plants that are on the list of strictly protected wild species of plants, animals and fungi, the collection of which is prohibited on the territory of the whole of serbia, i.e. possible only with the permission of the ministry responsible for nature protection. this group refers to 16 species present in the flora of slavujevac. the second group consists of species that are not critically endangered, and whose collecting is allowed only in limited quantities, i.e. controlled on the basis of their presence on the list of protected wild species of plants, animals and fungi. the mentioned group is significantly larger in relation to the previous one and includes a number of 47 species present in the flora of slavujevac. lists of protected species from the two mentioned categories are shown in tab. 3 and tab 4. table 3. list of strictly protected taxa of flora around slavujevac (official gazette of the republic of serbia, no. 36/09) alkanna pulmonaria griseb. astragalus wilmottianus stoj. campanula scutellata griseb. crocus olivieri gay subsp. olivieri crocus pallasii goldb. subsp. pallasii crocus rujanensis randelovic & d. a. hill gladiolus communis l. subsp. communis groenlandia densa (l.) fourr. himantoglossum caprinum subsp. rumelicumh. baumann & r.lorenz pyrus elaeagrifolia pallas ranunculus illyricus l.(literature data) sideritis montana l. subsp. montana spiranthes spiralis (l.) chevall. tuberaria guttata (l.) fourr. vinca herbacea waldst. et kit. haplophyllum suaveolens (dc.) g. don fill. species belonging to the category of endemic taxa are also present in the study area. data on the affiliation of such a category are presented according to the list of endemic taxa of the flora 92 biologica nyssana ● 13 (2) december 2022: 89-107 simić & zlatković ● flora around slavujevac on rujan mountain (se serbia) of serbia provided by tomović et al. (2014). this group includes 12 plant taxa of the flora around table 4. list of protected plant species of flora around slavujevac (official gazette of the republic of serbia no. 36/09) achillea millefolium l. subsp. millefolium herniaria hirsuta l. acinos hungaricus (simonkai) šilić herniaria incana lam. subsp. incana ajuga laxmannii (l.) bentham hypericum perforatum l. anthyllis vulneraria l. limodorum abortivum (l.) schwarz arum orientale bieb. subsp. orientale linum corymbulosum reichenb. campanula lingulata waldst. & kit. ononis spinosa l. subsp. spinosa campanula phrygia jaub. & spach orchis purpurea hudson centaurea cuneifolia sibth. & sm. subsp. pallida (friv.) hayek orchis simia lam. centaurium erythraea rafin. subsp. erythraea ornithogalum montanum cyr. cephalanthera damasonium (miller) druce prunus spinosa l. cephalanthera rubra (l.) l. c. m. richard rosa canina l. colchicum autumnale l. saxifraga bulbifera l. comandra elegans (rochel ex reichenb.) reichenb. fil. scabiosa fumarioides vis. & pančić cornus mas l. scorzoneroides cichoriacea (ten.) greuter crataegus monogyna jacq. subsp. monogyna sedum caespitosum (cav.) dc. centaurea napulifera rochel subsp. tuberosa (vis.) dostál trifolium cherleri l. cyclamen hederifolium subsp. hederifolium aiton trifolium physodes steven ex bieb. equisetum arvense l. trifolium tenuifolium ten. gagea pusilla (f. w. schmidt) schultes & schultes fil. subsp. pusilla trifolium trichopterum pančić galium flavescens borbás tussilago farfara l. galium verum l. subsp. verum valeriana tuberosa l. geranium macrorrhizum l. viola odorata l. gymnadenia conopsea (l.) r. br. subsp. conopsea eryngium palmatum pančič & vis helianthemum salicifolium (l.) miller table 5. list of endemic taxa in the flora around slavujevac (according to tomović et al., 2014) alkanna pulmonaria griseb. dianthus cruentus griseb. subsp. cruentus armeria rumelica bieb. subsp. rumelica dianthus stenopetalus griseb. astragalus wilmottianus stoj. pulsatilla montana (hoppe) reichenb. subsp. bulgarica rummelspecher campanula scutellata griseb. stachys scardica (griseb.) hayek crocus rujanensis ranđelović & d. a. hill trifolium trichopterum pančić centaurea napulifera rochel subsp. tuberosa (vis.) dostál eryngium palmatum pančić & vis. slavujevac. the list of endemic taxa recorded in the vicinity of slavujevac is shown in tab. 5. 93 biologica nyssana ● 13 (2) december 2022: 89-107 simić & zlatković ● flora around slavujevac on rujan mountain (se serbia) specific floristic data from the literature related to this relatively unexplored area include data from only 9 literature references. it should be emphasized that all 26 taxa listed in the literature for these sites have been critically reviewed. if no evidence has been found for a particular taxon listed in the scientific literature during field research, a free assessment of the correctness of the literature citation or possible correction of the citation is given on the basis of a comprehensive analysis of the taxon distribution. in a small number of cases, a dilemma remained regarding the correctness of the literature citation, with such data marked as suspicious or negative. the published data on the critical taxa are compared with our field observation from this study in tab. 6. table 6. list of taxa represented in the published sources compared to data from this study literature data this study observation alkanna pulmonaria griseb. orljak (niketić, 1999) veliki orljak, lismider, dedovica arabis glabra(l.) bernh.subsp. pseudoturritis(boiss. & heldr.) maire orljak (zlatković et al., 2014) not recorded, probably refers to pseudoturritis turrita aristolochia lutea desf. rujan mt., orljak (bogosavljević and zlatković, 2018) not recorded, presence expected astraglus wilmottianus stoj. orljak (ranđelović and stamenković, 1984., 1986; nikolić et al., 2007) not recorded, possibly locally extinct centaurea benedicta (l.) l. rujan mt., slavujevac (zlatković and bogosavljević, 2014) amedan cnicus bulgaricus p. pan. orljak (ranđelović and stamenković, 1984, 1986) not recorded, presence expected colchicum doerfleri halácsy rujan mt., veliki orljak (niketić i saradnici, 2018) veliki orljak crocus olivieri kitanov & drenkovski subsp. olivieri veliki orljak (ranđelović and stamenković, 1986; ranđelović and ranđelović 1999; ranđelović and hill 1986) amedan, veliki orljak crocus pallasii goldbach subsp.pallasii veliki orljak (ranđelović and stamenković, 1986; ranđelović and ranđelović 1999; ranđelović and hill 1986) veliki orljak crocus rujanensis ranđelović & d. a. hill veliki orljak (randjelovic et al., 1988) amedan, dedovica, veliki orljak goniolimon collinum (griseb.) boiss. rujan mt., orljak (ranđelović and stamenković, 1986) not recorded (buzurović et al., 2013) goniolimon tataricum (l.) boiss. rujan mt., orljak, (ranđelović and stamenković, 1984., 1986; buzurović i et al., 2013) not recorded, possibly locally extinct hippocrepis bifloraspreng. rujan mt., orljak (ranđelović and stamenković, 1984.,1986; ranđelović et al., 1986) probably refers to h. glauca 1984.,1986 jurinea polycephala formánek orljak (zlatković and bogosavljević, 2014) veliki orljak knautia orientalis l. rujan mt., mali orljak (niketić et al., 2021) mali orljak 94 biologica nyssana ● 13 (2) december 2022: 89-107 simić & zlatković ● flora around slavujevac on rujan mountain (se serbia) literature data this study observation minurtia glomerata m. bieb orljak (diklić and stevanović, 2012) not recorded, presence expected moenchia graeca boiss. & heldr. orljak (gajić and stevanović, 2012) not recorded, presence expected petrorhagia velutina (guss.) p. w. ball & heywood rujan mt., slavujevac (zlatković et al., 2011; stevanović and gajić, 2012) lismider, amedan ranunculus illyricus l. rujan mt., orljak (ranđelović and stamenković, 1986) not recorded, presence expected scabiosa fumarioides vis. & pančić rujan mt. (ranđelović and stamenković, 1984) not recorded, presence expected trachynia distachya (l.) link rujan mt., slavujevac (zlatković and bogosavljević, 2014) not recorded, presence expected tremastelma palestinum (l.) janch. orljak (ranđelović and stamenković, 1984) not recorded, cer, đeren (zlatković, pers. comm.) trifolium cherleri l. orljak, (ranđelović and stamenković, 1986, 1978) majden, orljak trifolium trichopterum pančić rujan mt., slavujevac (ranđelović and stamenković, 1984) not recorded, presence expected valerianella costata (steven) betcke rujan mt., slavujevac (zlatković et al., 2011) not recorded, presence expected vinca herbacea waldst. & kit. (ranđelović and stamenković, 1984) veliki orljak, dedovica the most common life form in the flora of the investigated area (fig. 1) is the form of hemicryptophytes (h) present with about 40.47% of taxa. with a slightly smaller share in the total flora follows the group of therophytes (t), about 35.37% of the total number of species. since the two mentioned groups of plants have an almost codominant relationship in the overall ecological spectrum of the flora of slavujevac, it can be said that it has a hemicryptophytic-terophytic character. the life forms of geophytes (g), phanerophytes (p) are also relatively well represented, while other life forms are far less represented in the studied area. hydrophytes (hyd) are the least represented (0.51%). such a small number of hydrophytic species is explained by the lack of habitats suitable for the development of aquatic plants. the flora of slavujevac is characterized by the largest share of hemicryptophytes, which is, in general, a characteristic of the flora of the largest number of areas in the temperate climate zone. however, the flora of slavujevac is characterized by only a 5% lower share of therophytes in relation to the life form of hemicryptophytes. the conspicuous percentage of the mentioned life form in the flora of this area indicates significant climatic zone from the mediterranean submediterranean area, and above all the pronounced dry period during the vegetation season which favors the appearance of annual plants as the best adapted ecological groups of plants to hot and dry habitat conditions. based on the horological analysis of the flora slavujevac area, 8 basic habitat types were singled out. in the area spectrum of the flora of the investigated fig. 1. representation of life forms (%) in the biological spectrum of the flora of slavujevac (ch– chamaephytes; g–geophytes; h–hemicryptophytes; hyd–hydrophytes; p– phanerophytes; t–therophytes) 95 biologica nyssana ● 13 (2) december 2022: 89-107 simić & zlatković ● flora around slavujevac on rujan mountain (se serbia) area (fig. 2), the mediterranean-submediterranean area type (31.29%) and the eurasian area type (26.36%) are dominant. these two area types make up a total of 57.65%, i.e. more than half of the total flora of slavujevac. the explanation for the dominance of the mentioned range types lies in the relatively low altitude of the area, while the emphasis of the mediterranean-submediterranean elements is explained by the strong florogenetic influence from the aegean mediterranean area through the lowland area of macedonia and northern macedonia. a significant percentage of the flora of the investigated area belongs to both the central european (12.92%) and the pontic area type (12.58%). indicating strong anthropogenic influences, the group of cosmopolitan plants is also well represented (10.03%). other range types are represented by a much smaller number of plant taxa. fig. 2. representation of types (%) of slavujevac flora (adv–adventive; ea – eurasian; eap–eurasian mountain; hol–holarctic; kosm–cosmopolitan; med–mediterranean; pont–pontic; se–central european) list of recorded taxa* classis: pteridophyta fam: aspleniaceae 1. asplenium trichomanes l. (ch, kosm) fam: athyriaceae 2. cystopteris fragilis (l.) bernh. (ch, kosm) fam: equisetaceae 3. equisetum arvense l. (g, hol) classis: gymnospermae fam: cupressaceae 4. juniperus oxycedrus l. (p, med) classis: monocotyledones fam: alismataceae 5. alisma plantago-aquatica l. (g, kosm) fam: araceae 6. arum orientale bieb.subsp. orientale (g, se) * class, family, and species are given in alphabetical order. the notation (*) in front of the taxon name indicates data from the literature, and the bibliographic data is given after the taxon name. after the bibliographic data in brackets is an abbreviation for life form and area type fam: cyperaceae 7. carex caryophyllea latourr. (h, ea) 8. carex vulpina l. (h, ea) 9. eleocharis palustris (l.) roemer & schultes (h, kosm) 10. heleocharis palustris l. (h, kosm) 11. holoschoenus vulgaris link (h, ea) 12. scirpus setaceus l. (t, kosm) fam: dioscoreaceae 13. tamus communis l. subsp. communis (g/s, med) fam: gramineae (poaceae) 14. aegilops geniculata roth (t, med) 15. aegilops triuncialis l. subsp. triuncialis (t, med) 16. aira elegantissima schur (t, med) 17. alopecurus rendlei eig. (t, med) 18. anthoxanthum odoratum l. (h, se) 19. avena fatua l. (t, ea) 20. bothriochloa ischaemum (l.) keng (h, ea) 21. briza media l. (h, kosm) 22. bromus arvensis l. (t, ea) 23. bromus commutatus schrader subsp. commutatus (t, se) 24. bromus hordeaceus l. subsp. hordeaceus (t, se) 25. bromus racemosus l. (t, ea) 26. bromus scoparius l. (t, ea) 27. bromus squarrosus l. (t, ea) 28. bromus sterilis l. (t, ea) 29. bromus tectorum l. (t, ea) 30. chrysopogon gryllus (l.) trin. (h, pont) 31. cynodon dactylon (l.) pers. (g, kosm) 32. cynosurus cristatus l. (h, kosm) 33. cynosurus echinatus l. (t, kosm) 34. dactylis glomerata l. (h, kosm) 35. dasypyrum villosum (l.) p. candargy (t, med) 36. echinochloa crus-galli (l.) beauv. (t, kosm) 37. elymus hispidus hispidus (opiz) melderis subsp. hispidus (g, med) 38. festuca valesiaca schleicher ex gaudin (h, pont) 39. holcus lanatus l. (h, kosm) 40. holcus mollis l.subsp.mollis (h, se) 41. hordeum murinum hudson subsp. murinum (t, ea) 96 biologica nyssana ● 13 (2) december 2022: 89-107 simić & zlatković ● flora around slavujevac on rujan mountain (se serbia) 42. koeleria nitidula velen. (h, pont) 43. koeleria simonkai adamović (k. glaucovirens aggr.) (h, pont) 44. leersia oryzoides (l.) sw. (h, kosm) 45. lolium perenne l. (h, kosm) 46. lolium temulentum l. (t, kosm) 47. melica altissima l. (h, pont) 48. phleum montanum montanum c. koch subsp. montanum (h, eap) 49. phleum pratense l. subsp. pretense (h, ea) 50. poa annua l. (t, kosm) 51. poa bulbosa l. (h, ea) 52. poa compressa l.(h, ea) 53. poa nemoralis l. (h, ea) 54. poa pratensis l. (h, hol) 55. sclerochloa dura (l.) beauv. (t, ea) 56. taeniatherum caput-medusae (l.) nevski subsp. asperum (simonkai) melderis (t, med) 57. *trachynia distachya (l.) link (t, med) 58. vulpia muralis (kunth) nees (t, med) fam: iridaceae 59. *crocus olivieri kitanov & drenkovski subsp. olivieri (g, med) 60. *crocus pallasii goldbach subsp. pallasii (g, med) 61. *crocus rujanensis ranđelović & d. a. hill (g, med) 62. gladiolus communis l. subsp. communis (g, med) 63. iris germanica l. (g, adv) 64. iris graminea l. (g, pont) 65. iris suaveolens boiss. & reuter (g, med) fam: juncaceae 66. juncus articulatus l. (g, kosm) 67. juncus bufonius l. (t, kosm) 68. juncus effusus l. (g, kosm) 69. juncus inflexus l. (h, kosm) 70. juncus thomasii ten. (g, eap) 71. luzula campestris (l.) dc. (h, se) 72. luzula forsteri (sm.) dc. subsp. forsteri (h, se) fam: liliaceae 73. allium scorodoprasum l. (g, med) 74. allium sphaerocephalon sphaerocephalon l. subsp. sphaerocephalon(g, med) 75. colchicum autumnale l. (g, se) 76. colchicum doerfleri halacsy (g, med) 77. fritillaria gussichiae (degen & dörfler) rix (g, med) 78. gagea pratensis (pers.) dumort. (g, pont) 79. gagea pusilla (f. w. schmidt) schultes & schultes filsubsp. pusilla (g, pont) 80. muscari comosum (l.) miller (g, med) 81. muscari neglectum guss. ex ten (g, med) 82. ornithogalum comosum l. (g, med) 83. ornithogalum kochii parl. (g, pont) 84. ornithogalum montanum cyr. (h, eap) 85. ornithogalum pyramidale l. (g, pont) 86. polygonatum latifolium (jacq.) desf. (g,se) 87. polygonatum odoratum (miller) druce subsp. odoratum (g, se) 88. scilla autumnalis l. (g, med) fam: orchidaceae 89. cephalanthera damasonium (miller) druce (g, se) 90. cephalanthera rubra (l.) l. c. m. richard (g, se) 91. gymnadenia conopsea (l.) r. br. subsp. conopsea (g, ea) 92. himantoglossum caprinum (biebl) c. koch subsp. rumelicum (g, med) 93. limodorum abortivum (l.) swartz (g/par, med) 94. ophrys scolopax cav. subsp. cornuta (steven) camus (g, med) 95. orchis coriophora l. subsp. fragrans (pollini) sudre (g, med) 96. orchis laxiflora lam. subsp. laxiflora (heuffel) soó (g, med) 97. orchis mascula (l.) l.subsp. signifera (vest) soó (g, se) 98. orchis morio l. subsp. picta (loisel.) arcangeli (g, med) 99. orchis papilionacea l. subsp. grandiflora (boiss.) nels (g, med) 100. orchis purpurea hudson (g, med) 101. orchis simia lam. (g, med) 102. orchis tridentata scop. subsp. commutata (tod.) nyman (g, med) 103. platanthera chlorantha (custer) reichenb. (g, se) 104. spiranthes spiralis (l.) chevall. (g, pont) fam: potamogetonaceae 105. groenlandia densa (l.) fourr. (hyd, ea) 106. potamogeton natans l. (hyd, kosm) classis: dicotyledones 97 biologica nyssana ● 13 (2) december 2022: 89-107 simić & zlatković ● flora around slavujevac on rujan mountain (se serbia) 139. sambucus nigra l. (p, se) fam: caryophyllaceae 140. agrostemma githago l. (t, ea) 141. arenaria serpyllifolia l. (t, ea) 142. cerastium banaticum (rochel) heuffel subsp. banaticum (ch, eap) 143. cerastium brachypetalum subsp. brachypetalum (t, med) 144. cerastium glomeratum thuill. (t, ea) 145. cerastium semidecandrum l. (t, ea) 146. dianthus corymbosus sibth. & sm. (t, med) 147. dianthus cruentus griseb. subsp. cruentus (h, eap) 148. dianthus giganteiformis borbás subsp. pontederae (kerner) soó (h, pont) 149. dianthus gracilis sibth. & sm. (ch, med) 150. dianthus stenopetalus griseb. (h, med) 151. gypsophila muralis l. (t, ea) 152. herniaria glabra l. subsp. glabra (h, ea) 153. herniaria hirsuta l. (t, ea) 154. herniaria incana lam. subsp. incana (t, med) 155. holosteum umbellatum l. subsp. glutinosum (bieb.) nyman (t, med) 156. lychnis flos-cuculi l. subsp. flos-cuculi (h, ea) 157. *minurtia glomerata m. bieb (t, pont) 158. *moenchia graeca boiss. & heldr. (t, med) 159. moenchia mantica (l.) bartl. (t, med) 160. petrorhagia illyrica (l.) p. w. ball & heywood subsp. haynaldiana (janka) p. w. ball & heywood (h, med) 161. petrorhagia prolifera (l.) p. w. ball & heywood (t, med) 162. *petrorhagia velutina (guss.) p. w. ball & heywood (t, med) 163. scleranthus perennis l. subsp. dichotomus (schur) stoj. & stefanov (t, pont) 164. scleranthus polycarpos l. (t, med) 165. silene armeria l. (t, med) 166. silene gallynii heuffel ex reichenb. (t, med) 167. silene latifolia poiret subsp. alba (miller) w. greuter & burdet (t, ea) 168. silene nemoralis waldst. & kit. (h, eap) 169. silene rhodopaea janka (h, med) 170. silene subconica friv. (t, pont) 171. silene vulgaris (moench) garcke (h, ea) fam: aceraceae 107. acer campestre l. (p, se) 108. acer tataricum l. (p, pont) fam: amaranthaceae 109. amaranthus albus l. (t, adv) 110. amaranthus retroflexus l. (t, adv) fam: anacardiaceae 111. cotinus coggygria scop. (p, ea) fam: apocynaceae 112. *vinca herbacea waldst. & kit. (ch, pont) 113. vinca minor l. (ch, adv) fam: aristolochiaceae 114. aristolochia clematitis l. (g, med) 115. *aristolochia lutea desf. (g, med) fam: asclepiadaceae 116. vincetoxicum fuscatum (hornem.) reichenb. fil. (h, med) fam: boraginaceae 117. aegonychon purpurocaeruleum (l.) holub (h, pont) 118. *alkanna pulmonaria griseb. (h, med) 119. anchusa barrelieri (all.) vitman (h, pont) 120. anchusa officinalis l. (h, pont) 121. myosotis stricta link ex roemer & schultes (t, ea) 122. buglossoides arvensis (l.) i. m. johnston subsp. arvense (t, kosm) 123. cynoglossum officinale l. (h, ea) 124. echium vulgare l. (h, ea) 125. myosotis arvensis (l.) hill (t, ea) 126. myosotis ramosissima rochel (t, med) 127. myosotis sicula guss. (t, med) 128. myosotis sparsiflora mikan ex pohl (t, ea) 129. myosotis stricta link ex roemer & schultes (t, ea) fam: campanulaceae 130. campanula lingulata waldst. & kit. (h, med) 131. campanula phrygia jaub. & spach (t, med) 132. campanula rapunculoides l. (h, ea) 133. campanula rapunculus l. (h, med) 134. campanula scutellata griseb. (t, med) 135. jasione heldreichii boiss. & orph. (h, eap) 136. legousia speculum-veneris (l.) chaix (t, med) fam: caprifoliaceae 137. lonicera caprifolium l. (ch/s, se) 138. sambucus ebulus l. (h, ea) biologica nyssana ● 13 (2) december 2022: 89-107 98 simić & zlatković ● flora around slavujevac on rujan mountain (se serbia) 172. spergula arvensis l. (t, kosm) 173. stellaria graminea l. (h, ea) 174. stellaria media (l.) vill. (t, kosm) 175. viscaria vulgaris bernh. (h, se) fam: celastraceae 176. evonymus europaeus l. (p, se) 177. evonymus latifolius (l.) miller (p, se) fam: chenopodiaceae 178. atriplex patula l. (t, ea) 179. chenopodium album l. (t, kosm) fam: cistaceae 180. fumana procumbens (dunal) gren. & godron subsp. procumbens (ch, med) 181. helianthemum ledifolium (l.) miller (t, med) 182. helianthemum nummularium (l.) miller subsp. nummularium (ch, se) 183. helianthemum salicifolium (l.) miller (t, med) 184. tuberaria guttata (l.) fourr. (t, med) fam: compositae (asteraceae) 185. achillea coarctata poiret (h, pont) 186. achillea crithmifolia waldst. & kit. (h, pont) 187. achillea millefolium l. subsp. millefolium (h, hol) 188. anthemis arvensis l. subsp. arvensis(t, ea) 189. arctium lappa l. (h, ea) 190. bellis perennis l. (h, se) 191. carduus acanthoides l. (h, kosm) 192. carduus nutans l. subsp. leiophyllus (petrović) stoj. & stefanov (h, pont) 193. carlina corymbosa l. subsp. graeca (boiss.) nyman (h, med) 194. carthamus creticus l. (t, med) 195. *centaurea benedicta (l.) l. (t, med) 196. centaurea calcitrapa l. subsp. calcitrapa (h, kosm) 197. centaurea cuneifolia sibth. & sm. subsp. pallida (friv.) hayek (h, med) 198. centaurea cyanus l. (t, med) 199. centaurea jacea l. (h, ea) 200. centaurea salonitana vis. (h, med) 201. centaurea stoebe stoebe l. subsp. stobe (h, pont) 202. chondrilla juncea l. (h, ea) 203. cichorium intybus l. (h, kosm) 204. cirsium arvense (l.) scop. (h, ea) 205. cirsium ligulare boiss. (h, eap) 206. cirsium vulgare (savi) ten. (h, kosm) 207. *cnicus bulgaricus p. pan. (t, med) 208. cota tinctoria (l.) j. gay (t, ea) 209. crepis biennis l. (h, se) 210. crepis foetida l. subsp. rhoeadifolia (bieb.) čelak (t, ea) 211. crepis sancta (l.) bornm. (t, med) 212. crepis setosa haller fil. subsp. setose (t, med) 213. crupina vulgaris cass. (t, ea) 214. echinops sphaerocephalus l. (h, med) 215. filago arvensis l. (t, ea) 216. filago minima (sm.) pers. (t, ea) 217. galinsoga parviflora cav. (t, kosm) 218. hypochaeris maculata l. (h, hol) 219. inula britannica l. (h, ea) 220. inula germanica l. (h, ea) 221. inula hirta l. (h, pont) 222. inula oculus-christi l. (h, pont) 223. inula salicina l. subsp. aspera (poiret) hayek (h, ea) 224. jacobaea vulgaris gaertn. (h, ea) 225. *jurinea polycephala formánek (h, pont) 226. lactuca saligna l. (h, ea) 227. laphangium luteoalbum (l.) tzvelev (t, kosm) 228. lapsana communis l. subsp. adenophora (boiss.) rech. fil. (t, ea) 229. leontodon biscutellifolius dc. (h, med) 230. leontodon hispidus l. (h, se) 231. leucanthemum ircutianum dc.(h, ea) 232. matricaria chamomilla l. (t, kosm) 233. onopordum acanthium l. subsp acanthium (t, ea) 234. pilosella bauhinii (schultes) arv.-touv. subsp. thaumasia (peter) soják (h, se) 235. pilosella macrantha f. w. schultz & schultz bip. fratt. (h, ea) 236. ptilostemon afer (jacq.) w. greuter (h, med) 237. scorzonera hispanica l. (h, pont) 238. scorzonera mollis bieb. subsp. mollis (g, pont) 239. scorzoneroides cichoriacea (ten.) greuter (g, med) 240. senecio leucanthemifolius poiret subsp. vernalis (waldst. & kit.) greuter (t, pont) 241. senecio vulgaris l. subsp. vulgaris (t, kosm) simić & zlatković ● flora around slavujevac on rujan mountain (se serbia) biologica nyssana ● 13 (2) december 2022: 89-107 99 242. sonchus arvensis l. (t, ea) 243. sonchus asper (l.) hill subsp. asper (t, hol) 244. tanacetum corymbosum (l.) schultz bip. (h, ea) 245. tanacetum macrophyllum (waldst. & kit.) schultz-bip. (h, eap) 246. tanacetum vulgare l. (h, ea) 247. taraxacum erythrospermum besser (h, ea) 248. taraxacum haussknechtii hausskn. (h, med) 249. taraxacum officinale weber (h, kosm) 250. tragopogon dubius scop. subsp. major (jacq.) vollm. (t, pont) 251. tragopogon pratensis l. subsp. pratensis (h, ea) 252. tripleurospermum inodorum (l.) schultzbip. (t, ea) 253. tussilago farfara l. (g, ea) 254. xanthium strumarium l. subsp. italicum (moretti) d. löve (t, adv) 255. xeranthemum annuum l. (t, med) 256. xeranthemum cylindraceum sm. (t, med) fam: convolvulaceae 257. calystegia sepium (l.) r. br. (t/s, ea) 258. convolvulus arvensis l. (t/s, kosm) 259. convolvulus cantabrica l. (h, med) fam: cornaceae 260. cornus mas l. (p, med) 261. cornus sanguinea l. subsp. australis (c.a. meyer) jáv. (p, se) fam: corylaceae 262. carpinus orientalis miller subsp. orientalis (p, med) fam: crassulaceae 263. sedum caespitosum (cav.) dc. (t, med) 264. sedum hispanicum l. (t, med) 265. sedum rubens l. (t, med) 266. sedum urvillei dc. (ch, med) fam: cruciferae (brassicaceae) 267. alliaria petiolata (bieb.) cavara & grande (t, ea) 268. alyssum alyssoides (l.) l. (t, med) 269. alyssum minutum schlecht. ex dc. (t, pont) 270. alyssum montanum l. subsp. reiseri (velen.) hayek (t, pont) 271. alyssum turkestanicum regel & s c h m a l h . ex regel (t, ea) 272. arabidopsis thaliana (l.) heynh. (t, ea) 273. *arabis glabra (l.) bernh. subsp. pseudoturritis (boiss. & heldr.) maire (t, med) 274. arabis sagittata (bertol.) dc. (h, med) 275. berteroa incana (l.) dc. subsp. stricta (boiss. & heldr.) stoj. & stef. (h, ea) 276. bunias erucago l. (t, med) 277. calepina irregularis (asso) thell. (t, ea) 278. camelina rumelica velen. (t, med) 279. capsella bursa-pastoris (l.) medicus (t, kosm) 280. cardamine bulbifera (l.) crantz (g, se) 281. cardamine hirsuta l. (t, ea) 282. descurainia sophia(l.) webb ex prantl (t, ea) 283. erophila verna verna (l.) chevall. subsp. verna (t, ea) 284. erysimum diffusum ehrh. (h, ea) 285. lepidium campestre (l.) r. br. (t, pont) 286. lepidium ruderale l. (h, ea) 287. raphanus raphanistrum l. (t, adv) 288. rorippa prolifera (heuffel) neilr. (h, se) 289. rorippa pyrenaica (lam.) reichenb. (h, med) 290. rorippa sylvestris (l.) besser subsp. sylvestris (h, se) 291. sisymbrium officinale (l.) scop. (t, ea) 292. thlaspi perfoliatum l. subsp. perfoliatum(t, ea) fam: dipsacaceae 293. dipsacus laciniatus l. (h, ea) 294. knautia arvensis (l.) coulter subsp. rosea (baumg.) soó (h, ea) 295. knautia orientalis l. (t, med) 296. scabiosa argentea l. (h, pont) 297. *scabiosa fumarioides vis. & pančić (h, eap) 298. *tremastelma palestinum (l.) janch. (t, med) fam: euphorbiaceae 299. euphorbia cyparissias l. (h, se) 300. euphorbia esula l. subsp. thommasiniana (bertol.) nyman (h, ea) 301. euphorbia helioscopia l. (t, kosm) 302. euphorbia seguierana necker subsp. niciciana (h, med) fam: fagaceae 303. quercus cerris (k. maly) czecz. (p, med) 304. quercus frainetto ten. (p, med) 305. quercus pedunculiflora k. koch (p, med) biologica nyssana ● 13 (2) december 2022: 89-107 simić & zlatković ● flora around slavujevac on rujan mountain (se serbia) 306. quercus pubescens willd. (p, med) fam: gentianaceae 307. centaurium erythraea rafin. subsp. erythraea (t, ea) 308. centaurea napulifera rochel subsp. tuberosa (vis.) dostál (h, med) 309. centaurium pulchellum (swartz) druce (t, ea) fam: geraniaceae 310. erodium cicutarium (l) l’her. (t, kosm) 311. geranium columbinum l. (t, se) 312. geranium lucidum l. (t, ea) 313. geranium macrorrhizum l. (g, eap) 314. geranium molle molle l. subsp. m o l l e ( t , kosm) 315. geranium sanguineum l. (h, se) fam: globulariaceae 316. globularia bisnagarica l. (h, ea) fam: guttiferae (hypericaceae) 317. hypericum perforatum l. (h, ea) 318. hypericum rumeliacum boiss. (h, med) 319. hypericum tetrapterum fries (h, se) fam:haloragaceae 320. myriophyllum spicatum l. (hyd, hol) fam: labiatae (lamiaceae) 321. acinos arvensis (lam.) dandy subsp. villosus (gaudin) sojak (t, ea) 322. acinos hungaricus (simonkai) šilić (ch, med) 323. ajuga chamaepitys (l.) schreber subsp. chia (schreber) arcangeli (t, ea) 324. ajuga genevensis l. (h, se) 325. ajuga laxmannii (l.) bentham (h, pont) 326. ballota nigra l. subsp. foetida hayek (h, se) 327. clinopodium vulgare l. (h, hol) 328. lamium amplexicaule l. subsp. amplexicaule (t, ea) 329. lamium maculatum l. (h, se) 330. lamium purpureum l. subsp. purpureum (t, ea) 331. marrubium peregrinum l. (h, pont) 332. mentha aquatica l. (h, ea) 333. mentha longifolia (l.) hudson subsp. longifolia (h, ea) 334. nepeta nuda l. subsp. nuda (h, ea) 335. origanum vulgare l. subsp. vulgare (h, ea) 336. prunella laciniata (l.) l. (h, pont) 337. prunella vulgaris l. (h, hol) 338. salvia amplexicaulis lam. (h, med) 339. salvia verticillata l. subsp. verticillata (h, pont) 340. scutellaria columnae all. subsp. columnae (h, se) 341. sideritis montana l. subsp. montana (t, med) 342. stachys annua (l.) l. (h, ea) 343. stachys germanica l. (h, ea) 344. stachys officinalis (l.) trevisan subsp. officinalis (h, se) 345. stachys scardica (griseb.) hayek (h, med) 346. teucrium chamaedrys l. (h, med) 347. teucrium polium l.subsp. capitatum (ch, med) 348. thymus glabrescens willd. (ch, pont) fam: leguminosae (fabaceae) 349. anthyllis vulneraria l. (h, se) 350. astragalus glycyphyllos l. (h, se) 351. astragalus onobrychis l. subsp. onobrychis (ch, pont) 352. *astragalus wilmottianus stoj. chamaecytisus tommasinii (vis.) rothm. (h, pont) 353. chamaecytisus triflorus (lam.) scalicka (ch, eap) 354. colutea arborescens l. (p, med) 355. coronilla scorpioides (l.) koch (t, med) 356. coronilla varia l. (h, se) 357. dorycnium germanicum (gremli) rikli (ch, se) 358. genista ovata waldst. & kit.(ch, pont) 359. *hippocrepis biflora spreng. the species has not been confirmed by field research, therefore, the data only refer to the species hippocrepis glauca (h, med) 360. hippocrepis glauca ten. (h, med) 361. lathyrus hirsutus l. (t, med) 362. lathyrus latifolius l. (h, med) 363. lathyrus laxiflorus (desf.) o. kuntze (h, se) 364. lathyrus pratensis l. (h, ea) 365. lathyrus sativus l. (t, ea) 366. lathyrus tuberosus l. (g, ea) 367. lembotropis nigricans (l.) griseb. (ch, pont) 368. lotus angustissimus l. (t, med) 369. lotus corniculatus l. subsp. corniculatus 100 biologica nyssana ● 13 (2) december 2022: 89-107 simić & zlatković ● flora around slavujevac on rujan mountain (se serbia) (h, ea) 370. medicago arabica (l.) hudson (t, kosm) 371. medicago falcata (l.) hudson subsp. falcate (h, kosm) 372. medicago lupulina (l.) hudson (h, kosm) 373. medicago orbicularis (l.) bartal. (t, ea) 374. medicago rigidula (l.) all. (t, med) 375. medicago sativa l.(h, adv) 376. onobrychis alba (waldst. & kit.) desv. subsp. alba (h, med) 377. ononis spinosa l.subsp. spinosa (ch, se) 378. ornithopus compressus l. (t, med) 379. robinia pseudacacia l. (p, adv) 380. trifolium alpestre l. subsp. alpestre (h, se) 381. trifolium angustifolium l. (t, med) 382. trifolium arvense l. (t, ea) 383. trifolium campestre l. (t, ea) 384. *trifolium cherleri l. (t, med) 385. trifolium echinatum bieb. (t, med) 386. trifolium fragiferum l.subsp. bonannii (h, ea) 387. trifolium glomeratum brot. (t, med) 388. trifolium hirtum all. (t, med) 389. trifolium incarnatum l. subsp. mollineri (balbis ex hornem.) syme (t, med) 390. trifolium montanum l. (h, pont) 391. trifolium nigrescens viv. subsp. nigrescens (t, med) 392. trifolium ochroleucon hudson (h, med) 393. trifolium pallidum waldst. & kit. (h, med) 394. trifolium patens schreber (t, med) 395. trifolium physodes steven ex bieb. (h, med) 396. trifolium pratense l. (h, ea) 397. trifolium purpureum loisel. (t, med) 398. trifolium repens l. (h, kosm) 399. trifolium resupinatum l. (t, med) 400. trifolium retusum l. (t, med) 401. trifolium striatum l. (t, se) 402. trifolium strictum l.(t, med) 403. trifolium sylvaticum gerard sec. c. visioso (t, med) 404. trifolium tenuifolium ten. (t, med) 405. *trifolium trichopterum pančić (t, med) 406. trigonella monspeliaca l. (t, med) 407. vicia cracca l. subsp. incana(h/s, med) 408. vicia grandiflora scop. (t/s, pont) 409. vicia hirsuta(l.) s. f. gray (h/s, ea) 410. vicia lathyroides l. (h/s, med) 411. vicia melanops sibth. & sm. (t/s, med) 412. vicia pannonica pannonica crantz subsp. pannonica (t/s, pont) 413. vicia peregrina l. (t/s, ea) 414. vicia tenuifolia roth subsp. dalmatica (a. kern.) greuter (h/s, med) fam: linaceae 415. linum austriacum l. (h, pont) 416. linum corymbulosum reichenb. (t, med) 417. linum hirsutum l. subsp. hirsutum (t/h, pont) 418. linum tenuifolium l. (h, med) 419. linum trigynum l. (t, med) fam: lythraceae 420. lythrum hyssopifolia l. (t, kosm) 421. lythrum salicaria l. (h, kosm) fam: malvaceae 422. alcea pallida (willd.) waldst. & kit. subsp. pallida (h, pont) 423. alt ha ea can na bi na l. (h, med) 424. hibiscus trionum l. (t, ea) 425. malva neglecta wallr. (h, ea) 426. malva sylvestris l. (h, kosm) fam: oleaceae 427. fraxinus ornus l. (p, med) 428. ligustrum vulgare l. (p, se) fam: onagraceae (oenotheraceae) 429. oenothera biennis l. (h, adv) fam: orobanchaceae 430. orobanche minor sm. (g/par, med) fam: papaveraceae 431. corydalis solida (l.) clairv. (g, se) 432. chelidonium majus l. (t, ea) 433. fumaria petteri reichenb. subsp. thuretii (t, med) 434. fumaria rostellata knaf (t, pont) 435. papaver albiflorum (besser) pacz (t, med) 436. papaver dubium dubium l. subsp. dubium (t, ea) 437. papaver hybridum l. (t, med) 438. papaver rhoeas l. (t, med) fam: plantaginaceae 439. plantago holosteum scop. (h, eap) 440. plantago lanceolata l. (h, kosm) 441. plantago major l. subsp. major (h, ea) 442. plantago media l. (h, ea) 101 biologica nyssana ● 13 (2) december 2022: 89-107 simić & zlatković ● flora around slavujevac on rujan mountain (se serbia) fam: plumbaginaceae 443. armeria rumelica bieb. (h, eap) 444. *goniolimon tataricum (l.) boiss. (h, pont) 445. *goniolimon collinum (griseb.) boiss. (h, pont) fam: polygalaceae 446. polygala comosa schkuhr (h, ea) 447. polygala vulgaris l. (h, se) fam: polygonaceae 448. fallopia convolvulus (l.) å. löve (t/s, kosm) 449. persicaria maculosa s.f.gray (t, kosm) 450. persicaria mitis delarbre (t, ea) 451. polygonum aviculare l. (t, kosm) 452. polygonum persicaria l. (t, ea) 453. rumex acetosa l. (h, hol) 454. rumex acetosella l. subsp. acetosella (h, se) 455. rumex patientia l. (h, ea) 456. rumex pulcher l. subsp. pulcher (h, med) fam:portulacaceae 457. portulaca oleracea l. subsp. oleracea (t, kosm) fam: primulaceae 458. anagallis arvensis l. (t, kosm) 459. cyclamen hederifolium aiton subsp. hederifolium (g, med) 460. lysimachia nummularia l. (h, se) 461. lysimachia punctata l. (h, med) 462. lysimachia vulgaris l. (h, ea) 463. primula veris l. subsp. suaveolens (h, eap) fam: ranunculaceae 464. consolida regalis s.f. gray subsp. paniculata (host) soó (t, pont) 465. helleborus odorus waldst. & kit. subsp. odorus (g, se) 466. nigella arvensis l. subsp. arvensis (t, med) 467. pulsatilla montana (hoppe) reichenb. subsp. bulgarica rummelspecher (g, pont) 468. ranunculus acris l.subsp. acris (h, ea) 469. ranunculus arvensis l. (h, ea) 470. ranunculus ficaria l. subsp. calthifolius (g, se) 471. *ranunculus illyricus l. (g, pont) 472. ranunculus lanuginosus l. (h, se) 473. ranunculus laterifolius dc. (t, ea) 474. ranunculus millefoliatus vahl (g, med) 475. ranunculus psilostachys griseb. (g, med) 476. ranunculus repens l. (h, ea) 477. thalictrum lucidum l. (h, se) 478. thalictrum minus l. subsp. minus (h, ea) fam: rosaceae 479. agrimonia eupatoria lebed. (h, ea) 480. aremonia agrimonoides (l.) dc. subsp. agrimonoides (h, se) 481. crataegus monogyna jacq. subsp. monogyna (p, se) 482. filipendula vulgaris moench (h, ea) 483. fragaria viridis duchesne subsp. viridis (h, ea) 484. geum urbanum l. (h, ea) 485. potentilla chrysantha trev. (h, pont) 486. potentilla detommasii ten. (h, med) 487. potentilla inclinata vill. (h, ea) 488. potentilla laciniosa waldst. & kit. ex nestler (h, pont) 489. potentilla neglecta baumg. (h, ea) 490. potentilla pedata l. (h, med) 491. potentilla recta l. (h, pont) 492. potentilla reptans l.(h, kosm) 493. prunus spinosa l. (p, se) 494. pyrus communis l. (p, se) 495. pyrus elaeagrifolia pallas (p, med) 496. rosa canina l. (p, ea) 497. rosa dumalis bechst. (p, se) 498. rosa gallica l. (p, se) 499. rubus caesius l. (ch, ea) 500. sanguisorba minor scop. subsp. muricata briq. (h, ea) 501. sorbus domestica l. (p, med) fam: rubiaceae 502. asperula arvensis l. (t, med) 503. asperula cynanchica l. (ch, pont) 504. crucianella angustifolia l. (t, med) 505. cruciata glabra (l.) ehrend. (h, ea) 506. cruciata laevipes opiz. (h, se) 507. galium album miller subsp. album (h, se) 508. galium aparine l. (t, ea) 509. galium flavescens borbás (h, med) 510. galium spurium l. (h, se) 511. galium tenuissimum bieb. (t, ea) 102 biologica nyssana ● 13 (2) december 2022: 89-107 simić & zlatković ● flora around slavujevac on rujan mountain (se serbia) 512. galium vernum scop. (h, ea) 513. galium verum l. subsp. verum (h, ea) 514. sherardia arvensis l. (t, kosm) fam: rutaceae 515. dictamnus albus l. (ch, pont) 516. haplophyllum suaveolens (dc.) g. don fill. (h, pont) fam:salicaceae 517. populus alba l. (p, ea) 518. populus nigra l. subsp. nigra (p, ea) 519. salix amplexicaulis bory (p, med) 520. salix fragilis l. (p, ea) fam: santalaceae 521. comandra elegans (rochel ex reichenb.) reichenb. fil. (ch/par, pont) 522. thesium arvense horvátovszky ( h / p a r , pont) fam: saxifragaceae 523. saxifraga bulbifera l. (g, med) fam: scrophulariaceae 524. antirrhinum orontium l. (t, ea) 525. digitalis lanata ehrh. (h, med) 526. euphrasia pectinata ten. (t, pont) 527. euphrasia stricta d. wolff ex j. f. lehm. (t/par, ea) 528. gratiola officinalis l. (h, hol) 529. linaria genistifolia genistifolia (l.) miller subsp. genistifolia (h, pont) 530. linaria pelisseriana (l.) miller (t, med) 531. melampyrum arvense l. (t/par, se) 532. melampyrum cristatum l. (t, se) 533. parentucellia latifolia (l.) caruel (t/par, med) 534. rhinanthus angustifolius c.c. gmelin (t/ par, ea) 535. rhinanthus rumelicus velen. (t/par, eap) 536. verbascum banaticum schrader (h, pont) 537. verbascum blattaria l. (t, ea) 538. verbascum lychnitis l. (h, pont) 539. verbascum macrurum ten. subsp. pannosiforme (stoj.) murb. (h, med) 540. verbascum phlomoides l. (h, pont) 541. verbascum phoeniceum l. subsp. phoeniceum (h, ea) 542. veronica agrestis l. (t, se) 543. veronica anagallis-aquatica l. subsp. anagallisaquatica (h, kosm) 544. veronica austriaca l. subsp. jacquinii (baumg.) maly (h, pont) 545. veronica chamaedrys l. subsp. vindobonensis m. fischer (h, se) 546. veronica hederifolia l. (t, se) 547. veronica persica poiret (t, adv) 548. veronica polita fries (t, ea) 549. veronica serpyllifolia l. (h, hol) 550. veronica verna l. (t, ea) fam:solanaceae 551. datura stramonium l.(h, kosm) 552. lycium europaeum l. (p, adv) 553. solanum nigrum l. subsp. schultesii (opiz) wessely (t, ea) fam: thymelaeaceae 554. thymelaea passerina (l.) cosson & germ. (t, ea) fam: tiliaceae 555. tilia platyphyllos scop. (p, se) fam: ulmaceae 556. ulmus minor miller (p, se) fam: umbelliferae (apiaceae) 557. anthriscus caucalis bieb. (t, se) 558. anthriscus cerefolium (l.) hoffm. (t, med) 559. anthriscus nemorosa (bieb.) sprengel (h, ea) 560. bifora radians bieb. (t, med) 561. caucalis platycarpos l. (t, med) 562. chaerophyllum temulum l. (t, se) 563. conium maculatum l. (h, kosm) 564. daucus carota l.(t, sea) 565. daucus guttatus sibth. & sm. subsp. zahariadii heywood (t, pont) 566. eryngium campestre l. (h, med) 567. eryngium palmatum pančić & vis. (h, med) 568. falcaria vulgaris bernh. (h, ea) 569. ferulago sylvatica (besser) reichenb. (h, med) 570. heracleum sphondylium l. subsp. sibiricum (l.) simonkai (h, ea) 571. oenanthe banatica heuffel (h, se) 572. orlaya grandiflora (l.) hoffm. (t, med) 573. physocaulis nodosus (l.) koch (t, med) 574. scandix pecten-veneris l. (t, ea) 575. tordylium maximum l. (t, med) 576. torilis japonica (houtt.) dc. (t, ea) 577. trinia glauca (l.) dumort. (h, pont) fam: urticaceae 103 biologica nyssana ● 13 (2) december 2022: 89-107 simić & zlatković ● flora around slavujevac on rujan mountain (se serbia) 104 578. urtica dioica l. (h, hol) 579. urtica urens l. (h, hol) fam: valerianaceae 580. valeriana tuberosa l. (g, pont) 581. valerianella carinata loisel. (t, med) 582. *valerianella costata (steven) betcke (t, med) 583. valerianella locusta (l.) laterrade (t, med) 584. valerianella rimosa bast. (t, pont) fam: verbenaceae 585. verbena officinalis l. (h, kosm) fam: violaceae 586. viola alba (ten.) w. becker subsp. dehnhardtii (jord.) nyman (h, med) 587. viola arvensis murray subsp. arvensis (t, ea) 588. viola hirta l. (h, ea) 589. viola jordanii hanry (h, med) 590. viola kitaibeliana schultes (t, med) 591. viola odorata l. (h, se) 592. viola tricolor l. subsp. macedonica (boiss. & heldr.) a. schmidt (h, med) conclusions floristic influences from the southern part of the balkan peninsula strongly influenced the flora of southern and southeastern serbia. for that reason, the flora of rujan mt., including the surroundings of the village of slavujevac is largely specific and diverse and requires more detailed botanical research. the presence of 592 plant species, 312 genera and 73 families of vascular plants was established by recent floristic research in the area of slavujevac. only a small number of taxa are listed in the available literature for the vicinity of slavujevac. of that number, only the presence of 11 taxa was confirmed during our field research. at the same time, arabis glabra subsp. pseudoturritis, goniolimon collinum and hippocrepis biflora are incorrectly listed for the studied area. the species of pteridophyta, as well as gymnosperms, are represented by a poor number of species. taxa from the group dicotyledones are by far the most numerous with about 82% of the total number of flora in the investigated area. taxonomic analysis shows that the families compositae, leguminosae and gramineae are the richest in species and subspecies. analysis of the biological spectrum indicates the dominance of the hemicryptophytes and therophytes in the flora. in the phytogeographical sense, the species of the mediterranean-submediterranean and eurasian area type prevail, including 10 species belonging to the group of balkan endemic taxa. a large presence of plants from the group of endangered taxa was found in the investigated habitats. this area is floristically rich, but it is also exposed to numerous negative anthropogenic factors. it is necessary to educate the local population in order to reduce the rate of destruction of natural habitats in the area. also, it is necessary to valorize rujan mt. by the organizations for nature conservation in order to preserve this floristically rich and diverse mountain for a longer period of time. references bogosavljević, s., zlatković, b. 2018: report on the new floristic data from serbia ii. biologica nyssana, 9 (2): 63-75. buzurović, u., stevanović, v., niketić, m., jakovljević, k., tomović, g. 2013: on the distribution of goniolimon tataricum (plumbaginaceae) in serbia. botanica serbica, 37 (2): 67-172. diklić, n., stevanović, v. 2012: cayophylaceace. in: stevanović, v. (eds.). flora sr srbije,2: 218, srpska akademija nauka i umetnosti. beograd. euro+med 2006+ [continuously updated]: euro+med plantbase the information resource for euro-mediterranean plant diversity. – published at http://www.europlusmed.org josifović, m. (ed.) 1970-1977: flora sr srbije 1-9. srpska akademija nauka i umetnosti, beograd. meusel, h., jäger, e. 1992: vergleichende chorologie der zentraleuropäischen flora 3. karten, literatur, register. gustav fischer, jena, stuttgart, new york. meusel, h., jäger, e., weinert, e. 1965: vergleichende chorologie der zentraleuropäischen flora «1». karten. gustav fischer, jena. meusel, h., jäger, e., weinert, e. 1978: vergleichende chorologie der zentraleuropäischen flora «2». karten. gustav fischer, jena. mueller-dombois, d., ellenberg, h. 1974: aims and methods of vegetation ecology. john wiley & sons, new york. niketić, m. 1999: alkanna pulmonaria griseb. in: stevanović, v. (ed.). crvena knjiga flore srbije 1: 171-172. ministarstvo za životnu sredinu republike srbije, biološki fakultet univerziteta u beogradu, zavod za zaštitu prirode republike srbije. beograd. niketić, m., tomović, g., bokić, b., buzurović, b., duraki, š., djordjević, v., biologica nyssana ● 13 (2) december 2022: 89-107 simić & zlatković ● flora around slavujevac on rujan mountain (se serbia) 105 djurović, s., krivošej, z., lazarević, p., perić, r., prodanović, d., radak, b., rat, m., ranimirović, m., stevanović, v. 2021: material on the annotated checklist of vascular flora of serbia. nomenclatura, taxonomic and floristic notes iii. bulletin of the natural history museum, 14: 97.doi:10.5937/bnhmb2114077n niketić, m., tomović, g., perić, r., zlatković, b., anačkov, g., đorđević, v., jogan, n., radak, b., duraki, š., stanković, m., kuzmanović, n., lakušić, d., stevanović, v. 2018: material on the annotated checklist of vascular flora of serbia. nomenclatural, taxonomic and floristic notes i. bulletin of the natural history museum, 11: 101180. nikolić, lj., stojanov, s., ranđelović, n. 2007: the endemic and relict plants species in the valley of the river pčinja. in: trumić, m. (ed.). ecological truth, 27. zbornik radova, 38. univerzitet u beogradu, tehnički fakultet u boru. sokobanja, 34-39. ranđelović, n., hill, d. a. 1986: dopuna flori sr srbije novim podacima o rasprostranjnju biljnih vrsta. in: sarić, m., diklić, n. (eds.). flora sr srbije. 10: 229. srpska akademija nauka i umetnosti. beograd. ranđelović, n., hill, d. a., stamenković, v., ranđelović, v. 1988: crocus rujanensis spec. nova iz agregata c. sieberi u sr srbiji. naučni skup “minerali, stijene, izumrlii živi svijet bih’’,zbornik referata, zemaljski muzej bosne i hercegovine, sarajevo,325-330. ranđelović, n., ranđelović, v. 1999: crocus pallasii golob. subsp. pallasii. in: stevanović, v. (ed.). crvena knjiga flore srbije 1: 189 190. ministarstvo za životnu sredinu republike srbije, biološki fakultet univerziteta u beogradu, zavod za zaštitu prirode republike srbije. beograd. ranđelović, n., ranđelović, v. 1999: crocus olivieri gay subsp. olivieri. in: stevanović, v. (eds.). crvena knjiga flore srbije 1: 335-336. ministarstvo za životnu sredinu republike srbije, biološki fakultet univerziteta u beogradu, zavod za zaštitu prirode republike srbije. beograd. ranđelović, n., stamenković, v. 1978: trifolium cherleri l. nova vrsta u flori srbije. glasnik prirodnjačkog muzeja u beogradu, serija b, 33: 165-167. ranđelović, n., stamenković, v. 1984: flora i vegetacija rujan planine u jugoistočnoj jugoslaviji. leskovački zbornik, 24: 375-392. ranđelović, n., stamenković, v. 1986: travnjačka flora jugoistočne srbije (ii). leskovački zbornik, 26: 405-410. ranđelović, n., stamenković, v., hill, d., ranđelović, v. 1983: rasprostranjenost biljnih vrsta u jugoistočnoj jugoslaviji. glasnik prirodnjačkog muzeja u beogradu, serija b, 38: 67-72. ranđelović, v., ranđelović, n., zlatković, b. 1999: opopanax hispidus (friv.) griseb. in: stevanović, v. (ed.). crvena knjiga flore srbije 1: 277-228. ministarstvo za životnu sredinu republike srbije, biološki fakultet univerziteta u beogradu, zavod za zaštitu prirode republike srbije. beograd. raunkiaer, c. 1934: the life forms of plants and statistical plant geography; being the collected papers of c. raunkiaer, translated into english by h. g. carter, a. g. transley and miss fausboll, clarendon, london. sarić, m. r. (ed.) 1992: flora srbije 1. srpska akademija nauka i umetnosti, beograd. sarić, m. r., diklić, n. (eds.) 1986: flora sr srbije 10. srpska akademija nauka i umetnosti, beograd. stanković m., 2015: florističke karakteristike staništa okoline slavujevca (ji srbija). master rad. prirodno matematički fakultet. univerzitet u nišu. niš. stevanović, v. (ed.) 1999: crvena knjiga flore srbije 1, iščezli i krajnje ugroženi taksoni. ministarstvo za životnu sredinu republike srbije, biološki fakultet univerziteta u beogradu, zavod za zaštitu prirode republike srbije, beograd. stevanović, v. 1992: floristička podela teritorije srbije sa pregledom viših horiona i odgovarajućih flornih elemenata. in: sarić, m. r. (ed.). flora srbije,1: 47-56, srpska akademija nauka i umetnosti, beograd. stevanović, v. 1992a: klasifikacija životnih formi biljaka u flori srbije. in: sarić, m. r. (ed.). flora srbije,1 (2nd edition): 37-49, srpska akademija nauka i umetnosti, beograd. stevanović, v., gajić, m. 2012: cayophylaceace. in: stevanović, v. (eds.). flora sr srbije, 2: 490491, srpska akademija nauka i umetnosti. beograd. tomović, g., niketić, m., lakušić, d., ranđelović, v., stevanović, v. 2014: balkan endemic plants in central serbia and kosovo regions: distribution patterns, ecological characteristics, and centres of diversity. botanica jurnal of the linnean society, 11: 5–14. tutin, t. g., heywood, v. h., burges, n. a., moore, d. m., valentine, d. h., walters, s. m., biologica nyssana ● 13 (2) december 2022: 89-107 simić & zlatković ● flora around slavujevac on rujan mountain (se serbia) 106 webb, d. a. (eds.) 1968-1980: flora europaea 2-5. university press. cambridge. uotila p. (2011+): chenopodiaceae (pro parte majore). – in: euro+med plantbase the information resource for euro-mediterranean plant diversity. published at http://www.europlusmed.org zlatković, b. 2011: flora i fitogeografska pripadnost doline reke pčinje u jugoistočnoj srbiji. doktorska disertacija. biološki fakultet. univerzitet u beogradu (manuskript). beograd. zlatković, b., bogosavljević, s. 2014: report on the new floristic data from serbia. biologica nyssana, 5 (2): 123-129. zlatković, b., bogosavljević, s., smiljković, n., ranđelović, v. 2014: report on the new and insufficiently studied taxa in the flora of serbia. biologica nyssana, 5 (1): 63-69. zlatković, b., ranđelović v., lakušić, d., stevanović, v. 2011: novelties for the vascular flora of serbia. botanica serbica, 35 (2): 103-110. biologica nyssana ● 13 (2) december 2022: 89-107 simić & zlatković ● flora around slavujevac on rujan mountain (se serbia) 107 appendix appendix 1. selected representatives of the flora of slavujevac on rujan mt. (crocus rujanensis (a), colchicum doerfleri (b), spiranthes spiralis (c), pulsatilla montana subsp. bulgarica (d), himantoglossum caprinum subsp. rumelicum (e), gladioulus communis subsp. communis (f), fritillaria gussichiae (g), iris suaveolens (h), aristolochia lutea (i)). authors of photographs: a-f, milica simić; g-i, bojan zlatković. koljanin et al. 2023, biologica nyssana 14(1) 14 (1) june 2023: 15-29 doi: 10.5281/zenodo.8027089 notes on the distribution and conservation status of some rare plants of wet habitats in bosnia and herzegovina original article dragan koljanin faculty of forestry, university of banja luka, stepe stepanovića 75a, 78 000 banja luka, bosnia and herzegovina draagaan98@gmail.com (corresponding author) jugoslav brujić faculty of forestry, university of banja luka, stepe stepanovića 75a, 78 000 banja luka, bosnia and herzegovina vladimir stupar faculty of forestry, university of banja luka, stepe stepanovića 75a, banja luka 78000, bosnia and herzegovina đorđije milanović faculty of forestry, university of banja luka, stepe stepanovića 75a, banja luka 78000, bosnia and herzegovina received: december 02, 2022 revised: december 21, 2022 accepted: december 22, 2022 abstract: wetland habitats in bosnia and herzegovina are usually small, fragmented and under numerous anthropogenic pressures. therefore species confined to such habitats can be considered being among the most threatened plant species in bosnia and herzegovina. the focus of this article is the distribution pattern and threatened status of five such plant species: achillea ptarmica, carex elongata, carex strigosa, comarum palustre and prunus padus. while these taxa can be common in other parts of europe, in bosnia and herzegovina, they are rare and usually restricted to a small number of localities. wetland flora can be found in the dinaric mountains, where it is confined to the karst fields, mountain plateaus, spring areas and banks of glacial lakes, but as well in the lowlands in the north of the country, occupying the remnants of wetland habitats. published, new and unpublished (herbarium) chorological data for bosnia and herzegovina, description of the habitat, population estimate, threats on habitats and species, and estimation of threatened status based on iucn criteria are given for each studied species. key words: achillea ptarmica, carex elongata, carex strigosa, comarum palustre, prunus padus, distribution, ecology apstrakt: o rasprostranjenju i ugroženosti nekih retkih biljnih vrsta vlažnih staništa bosne i hercegovine močvarna staništa u bosni i hercegovini su obično mala, fragmentirana i pod brojnim antropogenim pritiscima. stoga se vrste ograničene na takva staništa mogu svrstati među najugroženije biljne vrste u bosni i hercegovini. fokus ovog rada su rasprostranjenje i status ugroženosti pet takvih biljnih vrsta: achillea ptarmica, carex elongata, carex strigosa, comarum palustre i prunus padus. iako su ovi taksoni većinom uobičajeni u drugim dijelovima evrope, u bosni i hercegovini oni su retki i obično ograničeni na mali broj lokaliteta. močvarna flora se može naći i na dinarskim planinama, gde je svedena na kraška polja, planinske visoravni, izvorišta i obale glacijalnih jezera, ali i u nizijama na severu zemlje, zauzimajući ostatke močvarnih staništa. za svaku proučavanu vrstu dati su objavljeni, novi i neobjavljeni (herbarijski) horološki podaci za bosnu i hercegovinu, opis staništa, procena populacije, ugroženost staništa i vrsta, te procena ugroženosti na osnovu iucn kriterijuma. ključne reči: achillea ptarmica, carex elongata, carex strigosa, comarum palustre, prunus padus, rasprostranjenost, ekologija vrsta introduction at a broad geographical scale, species distribution is determined by macroclimate, evolutionary and migration history (huntley et al., 1995; knollová & chytrý, 2004; pearson et al., 2004; tsiftsis et al., 2012). the distribution of species is also influenced by relief, soil properties, mesoclimate and microclimate at smaller scales, as well as human activities that can introduce or cause the extinction of species in certain locations. at the border of the distribution area, a species often transits from a continuous to a disjunct distribution pattern. this is caused by the fact that favourable ecological conditions appear in disjunct patches in limited areas because of a special constellation of conditions. at the edge of their range, populations have restricted areas of occurrence; often, they are ecologically © 2023 koljanin et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 15 different and characterized by low abundance in terms of population size (lesica & allendorf, 1995; duffy et al., 2009). furthermore, plant populations that are at the edge of their distribution range are often at their ecological limits because their habitats are of declined suitability, and therefore they are more sensitive to changes in ecological conditions (koljanin et al., 2021). knowledge of distribution patterns and ecology can be considered fundamental for species conservation (heywood & iriondo, 2003). species of wet habitats can be considered to be one of the most threatened vascular plants in bosnia and herzegovina (redžić et al., 2009). due to precipitation regimes in the growing season, with long dry periods, significantly higher temperatures, and dominant types of water-permeable geological substrata (especially limestones), the balkan peninsula is less covered by wetland habitats than most of the central and northern parts of europe. therefore, some species that are common in wetlands of the northern parts of europe are rare or absent in the balkans. peatlands are of greater value in southern regions than those further on the north, as they usually have specifically distinctive fauna and flora, as well as isolated and possibly more genetically divergent populations of circumboreal species than those of northern mires (desrochers et al., 2000). in bosnia and herzegovina, mires occupy about 16,250 ha scattered throughout the country, with most of them more or less degraded in the last 150 years (milanović, 2017a). there are only two larger areas with the accumulation of peat (livanjsko polje and hutovo blato), with an area of around 13,000 ha (milanović, 2017a), while all others occupy small areas, mostly on higher elevations, where a particular constellation of ecological factors ensures the existence of these habitats (barudanović et al., 2019). since these sites are very sensitive to changes and usually have a limited area, any exploitation of these sites could have huge negative consequences for biodiversity. the northern part of bosnia and herzegovina overlaps with the edge of the pannonian plain, which is dominantly characterized by typical plain configuration influenced by slow-flowing and large alluvial rivers; this area was very rich in wetland habitats several centuries ago. due to the unregulated banks of these rivers, the surrounding lowlands were normally seasonally flooded, while rivers often changed their beds, creating conditions necessary for the development of various wetland habitats on the river terraces. the deterioration of wetlands in northern bosnia and herzegovina began with the systematic regulation of the flow of the sava river in the second half of the 18th century and continued with the construction of a reclamation system to preserve agricultural land (mrgić, 2007). it could be assumed that these changes caused the disappearance of many populations and even species confined to wetland habitats. such an example is fritillaria meleagris l. which disappeared from many localities after the construction of the drainage system (koljanin et al., 2021). going from north to south of bosnia and herzegovina, the northern lowlands gradually rise to the mountain chain of the dinaric alps, which is an ecologically very heterogeneous area. in this area, wetland flora is typically limited to karst fields, mountain plateaus and depressions, flattened small river terraces, and fragments alongside mountain brooks and around springs. these habitats are relict remnants of vegetation where some species had found a refugium and have great importance for biodiversity on a national level, as they are inhabited by many plant species that are rare in the country. a great example of such a species is liparis loeselii (l.) rich. which is found only in one locality in balkan peninsula (milanović, 2012). one of the fundamental problems in the protection of plants and habitats is the lack of data on these species. the dinaric part of the country is rich in endemic plant species, while they are absent in northern lowlands (lubarda et al., 2014), which makes it less attractive for botanical studies. this resulted in a lack of data for some species that have main distribution in the northern part of bosnia and herzegovina. on the other hand, some species in the dinaric mountains are confined to small, refugial areas; therefore, some populations went undetected. in this paper, we provide new chorological and ecological data for five species that are most common in wetlands of central european countries but occur very rarely in bosnia and herzegovina: achillea ptarmica l., carex elongata l., carex strigosa huds., comarum palustre l. and prunus padus l. also, we estimated the most appropriate red list category for all of them. materials and methods all chorological data on the occurrence of five target species (achillea ptarmica, carex elongata, carex strigosa, comarum palustre and prunus padus) in bosnia and herzegovina were collected from the available literature. all literature records were georeferenced using topographic maps with a scale of 1:25,000 in qgis software. fieldwork was conducted in the period from 2006 to 2021 in various wetlands in the northern plains of bosnia and herzegovina and in the karst fields, where such habitats are registered or 16 biologica nyssana ● 14 (1) june 2023: 15-29 koljanin et al. ● notes on the distribution and conservation status of some rare plants of wet habitats in bosnia and herzegovina biologica nyssana ● 14 (1) june 2023: 15-29 koljanin et al. ● notes on the distribution and conservation status of some rare plants of wet habitats in bosnia and herzegovina 17 expected, but also in the dinaric alps, at higher elevations, especially around glacial lakes and springs. coordinates were recorded in wgs1984 using a gps device, and digital photographs were also collected in the field. herbarium material was stored in the herbarium of the faculty of forestry of the university of banja luka and in the private collection of đorđije milanović. this material was used for identification mainly following identification keys in flora europaea (ball et al., 1968; webb & rix, 1972; richardson, 1976; chater, 1980) while the nomenclature follows the euro+med (2006) and stupar et al., (2021). target species are listed alphabetically with the following parameters: literature and new chorological data, general distribution and ecology, habitat and species threats and conservation status. published and new records are indicated by: locality name (bolded if it is a new record), wgs1984 coordinates, elevation (if available for literature data), mgrs 10×10 km grid code, plant community/habitat type (if available), soil type (if available), population characteristics (if available), date of collecting, collector and bases of record. maps of species distribution in bosnia and herzegovina are presented on a 10×10 km mgrs grid, using different symbols for published, new and unpublished (herbarium) data. short descriptions of habitats and their stability were given based on field observations. threat status for each species was estimated or reassessed following the iucn criteria and categories (iucn species survival commission, 2012a, 2012b). given that the area of occupancy (aoo) for each of the species under study is estimated to be less than 10 km2, we applied the b2 criterion to each species. additionally, considering that the likelihood of immigration from neighbouring countries is very low, based on the iucn species survival commission (2012b), we decided to maintain the original category from the initial assessment in step one for each species, as noted by the iucn species survival commission (2012a). results and discussion achillea ptarmica l. published and new chorological data achillea ptarmica is a herbaceous perennial plant easily recognizable by linear undivided leaves with serrate margins (fig. 1) and without glandular punctuations on the leaf surface (richardson, 1976). this species was previously reported only from three localities in nevesinjsko polje (sagorski, 1901; ritter-studnička, 1954; beck-mannagetta et al., 1983). new records show that species have a wider distribution in various conditions in bosnia and herzegovina (fig. 2). fig. 1. achillea ptarmica at locality of berek (© d. koljanin) fig. 2. distribution of achillea ptarmica in bosnia and herzegovina this species was recorded in the following localities: • between nevesinje and pustoljani (nevesinjsko polje); coordinates: 18.159896°e, 43.292712°n; elevation: ca 850 m; mgrs 10×10 square: bn69; habitat: wet meadows with sparganium microcarpum; population: numerous individuals; date: 1912; collector: e. sagorski; bases of record: literature (sagorski, 1901; ritter-studnička, 1954; beck-mannagetta et al., 1983), marked from a ritter-studnička, (1954) as probably extinct. • banks of dušila stream (nevesinjsko polje); coordinates: 18.15°e, 43.31667°n; elevation: ca 850 m; mgrs 10×10 square: bp60; habitat: stream banks; date: 08.1955, 09.08.1956; 18 biologica nyssana ● 14 (1) june 2023: 15-29 koljanin et al. ● notes on the distribution and conservation status of some rare plants of wet habitats in bosnia and herzegovina collector: z. devetak, hilda ritter-studnička; bases of record: as f. linearis, in herbarium sara 42214, sara 42215; corresponding literature (ritter-studnička, 1954; beck-mannagetta et al., 1983). • zlatac (nevesinjsko polje); coordinates: 18.141459°e, 43.341539°n; elevation: ca 850 m; mgrs 10×10 square: bp60; habitat: stream banks; population: unknown; date: 1954; collector: z. devetak; bases of record: literature (ritter-studnička, 1954; beck-mannagetta et al., 1983), as f. lineare. • right bank of the river sana, near village pejići (downstream from sanski most); coordinates: 16.724544°e, 44.886894°n; elevation: 147 m; mgrs 10×10 square: xk37; habitat: wet meadows; population: unknown; date: 10.08.1985; collector: č. šilić; bases of record: herbarium sara 50346-50349. • at few microlocalities on slopes of the zimomor peak towards štirinsko lake (zelengora mt.); coordinates: 18.490669°e, 43.373269°n; elevation: 1720 m; mgrs 10×10 square: bp90; habitat: mountain spring communities and calthion meadows; population: estimated about one-two thousand flowering individuals; date: 05.08.2008; collector: đ. milanović; bases of record: new – herbarium djm 180/01-001. • gredina near sajković (livanjsko polje); coordinates: 16.660997°e, 43.982725°n; elevation: 707 m; mgrs 10×10 square: xj37; habitat: seasonally flooded molinia meadows in karst fields on sandy soils; population: several hundred flowering individuals; date: 09.08.2015; collector: đ. milanović; bases of record: new – herbarium djm 180/01-002. • berek (lijevče polje); coordinates: 17.2361511°e, 45,0343937°n; elevation: 96 m; mgrs 10×10 square: xk78; habitat: wet meadows in succession; soil type: pseudogley on tertiary sediments; population: several hundred flowering individuals; date: 26.06.2021; collector: d. koljanin; bases of record: new – herbarium herb. fac. silv. 000368. • elezagići (lijevče polje); coordinates: 17.244907°e, 45.066040°n; elevation: 102 m; mgrs 10×10 square: xk79; habitat: on the edge of wet meadow, alongside the ditch; population: ca 50 adult individuals; date: 13.10.2021; collector: d. koljanin; bases of record: observation. general distribution and ecology achillea ptarmica is distributed in most of europe, reaching france and the british isles in the west, scandinavia in the north, the western bank of the odra river in poland and northern ukraine in the east. the species is relatively common in central europe. in most of southern europe, it is rare, occurring sporadically at isolated localities or is completely absent in some countries (bilz, 2012). it has boreal-subboreal eurasian distribution, reaching the southernmost limit in the balkan peninsula and can be considered a boreal relict in balkan flora (stevanović, 1999). the known populations in the balkan peninsula are rare and distant from each other, thus forming a disjunct distribution pattern. also, this species is rare in montenegro, with only one known locality from durmitor mt. (stevanović, 1999). in croatia, the species is known only from a few localities in the northern part of the country (nikolić, 2010). the species has been recorded in serbia in the past, but recent research has not confirmed its occurrence suggesting that it may be considered extinct at the national level (stevanović, 1999; bilz, 2012). in central europe, the species is noted to occur on non-calcareous and permanently moist soils in open habitats, including calthion palustris wet meadows (most often), molinion caeruleae meadows, deschampsion cespitosae alluvial meadows and other wet habitats, including even ruderal habitats (dudáš et al., 2017; kaplan et al., 2020), scrub formations and wood margins (richardson, 1976). habitat and species threats in bosnia and herzegovina achillea ptarmica inhabits various wet meadows and more/less permanently wet banks of small lowland streams and mountain springs in bosnia and herzegovina. in the northern part of the country, the populations are situated on the border of trifolion pallidi and deschampsion caespitosae mown meadows, under intense pressure from alien and/or ruderal plant species and natural succession at the same time. the further negative impact could result in the disappearance of this species in the northern part of the country. the populations on zelengora mt. and in livanjsko polje are numerous and stable, without observed negative influences. however, they still have limited spatial distribution, where some potential impacts could cause a substantial reduction in their populations. conservation status the global iucn status of achillea ptarmica is least concern (bilz, 2012). in serbia, the species has the category extinct in the wild (stevanović, 1999). šilić (1996) assessed the species as vulnerable in the list of vascular plants (pteridophyta and 19 biologica nyssana ● 14 (1) june 2023: 15-29 koljanin et al. ● notes on the distribution and conservation status of some rare plants of wet habitats in bosnia and herzegovina fraxinetum angustifoliae and genisto elataequercetum roboris; population: unknown; date: 1964; collector: m. glišić; bases of record: literature (glišić, 1964). • jakovica near the village of cerik (orig. cerje); coordinates: 18.522109°e, 44.808918°n; elevation: ca 100 m; mgrs 10×10 square: cq06; habitat: carpino betuli-quercetum roboris forest; population: unknown; date: 1975; collector: p. fukarek; bases of record: literature (fukarek, 1975). • hrastik near the village of donji žabar; coordinates: 18.656429°e, 44.941478°n; elevation: ca 85 m; mgrs 10×10 square: cq17; habitat: carpino betuli-quercetum roboris forest; population: unknown; date: 1975; collector: p. fukarek; bases of record: literature (fukarek, 1975). • burumac (orig. baranac) in the vicinity of bosanski šamac; coordinates: 18.521154°e, 45.004933°n; elevation: ca 95 m; mgrs 10×10 square: cq08; habitat: carpino betuli-quercetum roboris forest; population: unknown; date: 1975; collector: p. fukarek; bases of record: literature (fukarek, 1975). • gromiželj in semberija; coordinates: 19.311810°e, 44.866086°n; elevation: ca 82 m; mgrs 10×10 square: cq66; habitat: alnus glutinosa forest; population: numerous individuals; date: 29.05.2010; collector: đ. milanović, j. brujić, v. stupar and d. nikić; bases of record: literature (petronić et al., 2010; milanović et al., 2011;) herb. djm: 65/04–045. • han kram on sljemeska mt.; coordinates: 18.906667°e, 44.038889°n; elevation: ca 1050 m; mgrs 10×10 square: cp37; habitat: edge of pino-betuletum pubescentis; population: scattered individuals; date: 28.06.2010; collector: đ. milanović, j. brujić and j. travar; bases of record: literature (milanović et al., 2011), herb. djm: 65/04–046. • above donja pećina in bijambare; coordinates: 18.502953°e, 44.093964°n; elevation: ca 925 m; mgrs 10×10 square: cp08; habitat: tall herbs with scirpus sylvaticus on a brook terrace; population: less than ten flowering individuals; date: 13.06.2020; collector: đ. milanović; bases of record: herb. djm: 65/04-047. • podbrdo in podrašničko polje; coordinates: 16.985369°e, 44.457024°n; elevation: 730 m; mgrs 10×10 square: xk52, habitat: well preserved alnus glutinosa forest; soil type: gley on diluvial sediments; population: a dozen tussocks at spermatophyta) for the red book of flora of bosnia and herzegovina. in the red list of flora of the federation of bosnia and herzegovina, the species was also assessed as vulnerable (đug et al., 2013). considering the available data, i.e., area of occupancy (aoo) is less than 10 km2, regional population is severely fragmented, and there is continuing decline in area, extent and/or quality of habitat, we suggest regional conservation status for bosnia and herzegovina: critically endangered cr b2ab(iii). carex elongata l. published and new chorological data carex elongata belongs to the subgenus vignea and the section elongatae (kunth) kük. the species can be recognized by the following set of characteristics: densely caespitose plants often forming tussocks, all spikes (6-12) with female flowers on top and male below, utricles without wings with entire not 2-fid beak and two stigmas, lowest bract shorter than inflorescence (chater, 1980) (fig. 3). the species was known from several localities in bosnia and herzegovina scattered throughout the country (fig. 4): • under koran near pale; coordinates: 18.575645°e, 43.804779°n; elevation: ca 850 m; mgrs 10×10 square: cp05; habitat: wet meadows with scirpus sylvaticus, cardamine pratensis and scutellaria galericulata; population: unknown; date: 03.06.1934 and 03.06.1935; collector: k. malý; bases of record: sara 3932 – sara 3933; corresponding literature record (malý, 1935). remark: the area has been urbanized in the last 50 years, and the occurrence of this species is uncertain today. • gradiška; coordinates: 17.248848°e, 45.135396°n; elevation: ca 95 m; mgrs 10×10 square: xl70; habitat: cynosuretum cristati; population: unknown; date: 1959; collector: j. kovačević; bases of record: literature (kovačević 1959). • berek (lijevče polje); coordinates: 17.242484°e, 45.041149°n; elevation: ca 105 m; mgrs 10×10 square: xk78; habitat: cynosuretum cristati; population: unknown; date: 1959; collector: j. kovačević; bases of record: literature (kovačević 1959); remark: several hundred individuals have been recently confirmed but in monodominant swamp forests of quercus robur (d. koljanin, 16.06.2021. bases of record: observation). • in the vicinity of bosanska dubica; coordinates: 16.932538°e, 45.266012°n; elevation: ca 93 m; mgrs 10×10 square: xl51; habitat: leucojobiologica nyssana ● 14 (1) june 2023: 15-29 koljanin et al. ● notes on the distribution and conservation status of some rare plants of wet habitats in bosnia and herzegovina the edge of the forest; date: 29.05.2021; collector: j. brujić and d. koljanin; bases of record: herb. fac. silv: 000277. • marčete in podrašničko polje; coordinates: 16.961526°e, 44.481430°n; elevation: 740 m; mgrs 10×10 square: xk52, habitat: young alnus glutinosa forest with betula pubescens; soil type: gley on diluvial sediments; population: ca 30 tussocks were counted; date: 29.05.2021; collector: j. brujić and d. koljanin; bases of record: observation. • elezagići in lijevče polje; coordinates: 17.241189°e, 45.068918°n; elevation: 96 m; mgrs 10×10 square: xk79; habitat: swamp forest of quercus robur, in a micro-depression; soil type: pseudogley over tertiary sediments; population: several hundred tussocks; date: 26.06.2021.; collector: d. koljanin; bases of record: herb. fac. silv: 000278. • kočićevo in lijevče polje; coordinates: 17.383162°e, 45.078864°n; elevation: 92 m; mgrs 10×10 square: xk89; habitat: carici elongatae-alnetum glutinosae; soil type: gley on tertiary sediments; population: several dozens of tussocks; date: 07.08.2021; collector: d. koljanin; bases of record: observation. • bardača in lijevče polje; coordinates: 17.397036°e, 45.092282°n; elevation: 91 m; mgrs 10×10 square: xk89; habitat: carici elongatae-alnetum glutinosae; soil type: pseudogley on tertiary sediments; population: couple tussocks; date: 18.08.2021; collector: d. koljanin; bases of record: herb. fac. silv. 000367. • mašići in lijevče polje; coordinates: 17.245689°e, 45.0157962°n; elevation: 107 m; mgrs 10×10 square: xk78; habitat: along a temporary brook in a micro-depression with quercus robur and acer tataricum; soil type: pseudogley on tertiary sediments; population: ca. 30 tussocks; date: 17.04.2022; collector: d. koljanin and j. brujić; bases of record: observation. • posavine in lijevče polje; coordinates: 17.161216°e, 45.13591°n; elevation: 92 m; mgrs 10×10 square: xl70; habitat: leucojofraxinetum angustifoliae; soil type: pseudogley on tertiary sediments; population: several hundred tussocks; date: 08.05.2022; collector: d. koljanin; bases of record: observation. • stanari; coordinates: 17.821632°e, 44.750471°n; elevation: 171 m; mgrs 10×10 square: yk25; habitat: carici elongatae-alnetum glutinosae; soil type: planohistosol and pseudogley on tertiary sediments; population: several hundred tussocks; date: 22.05.2022; collector: v. stupar, j. brujić, đ. milanović & d. koljanin; bases of record: djm: 65/04-048. fig. 3. carex elongata near stanari (© đ. milanović) fig. 4. distribution of carex elongata in bosnia and herzegovina general distribution and ecology carex elongata is widespread in europe and western asia. this species is common in central europe, inhabiting various wetland habitats. the species is often a member of hygrophilous forests where stagnant water is present for a significant period of the year, and it is common in the alnus glutinosa carrs (willner & grabherr, 2007; douda, 2008; borhidi et al., 2012; slezák et al., 2014; hulík & douda, 2017). in addition, the species is also common in narrowleaved ash swamp forests (leucojo-fraxinetum angustifoliae) in balkan peninsula (glavač, 1959; glišić, 1964), monodominant swamp quercus robur forests in hungary (borhidi et al., 2012) 20 koljanin et al. ● notes on the distribution and conservation status of some rare plants of wet habitats in bosnia and herzegovina 21 and poland (sokołowski, 1972) and swamp alnus incana communities in austria (willner & grabherr, 2007). in croatia, the species is distributed mainly in the lowlands in the northern and central parts of the country (nikolić, 2020). in serbia, the species is noted to be common in swamp forests, wet meadows and channel margins from lowlands to mountainous regions (josifović, 1972). still, there are only a few precise data on the distribution and ecology of the species in this country. habitat and species threats in bosnia and herzegovina recent research shows that the species is not as rare throughout northern bosnia and herzegovina as previously considered (milanović et al., 2011). here, this species is a typical forest plant, inhabiting more or less permanently moist parts of various riparian forests, with a development optimum in preserved alnus glutinosa swamp forests and quercus robur swamp forests with +/developed peat layer. in contrast, in other forest types, where water is stagnant for a part of the vegetation season, it is restricted to micro depressions, channel margins or alongside permanent or temporal stream beds. due to largescale hydrological meliorations in the past, habitats suitable for this species are severely restricted, degraded, and fragmented, while flood prevention and changes in water regimes still negatively affect its populations. in the montane region of the dinaric alps, the species is less common, with scattered individuals on stream terraces or at the edges of peatland complexes inhabiting wet tall-herb communities and edges of mixed broadleaved-coniferous forests. various negative impacts on these habitats have been recorded there: trampling in the bijambare nature park, urbanization in koran near pale, and hydrological meliorations in han kram at sljemenska mt. conservation status global and european iucn status of carex elongata has not been estimated yet. the species is not listed in national red lists in most central european countries. in neighbouring croatia, the species is listed as data deficient (nikolić & topić, 2005). šilić (1996) has considered doubtful previously known data, and therefore the species was not included in the list of vascular plants (pteridophyta and spermatophyta) for the red book of flora of bosnia and herzegovina. however, considering the available data, i.e., aoo is less than 10 km2, the regional population is severely fragmented, and there is continuing decline in area, extent and quality of habitat, we suggest regional conservation status for bosnia and herzegovina: critically endangered cr b2ab(iii). carex strigosa huds. published and new chorological data carex strigosa belongs to the section strigosae (fries) (chater, 1980). this species was often overlooked in some parts of its distribution due to low study levels and a small percentage of suitable habitats (trčak & bačič, 2017), which could also be stated for bosnia and herzegovina. from other species of the section, it could be recognized by mainly not pendent, thin and lax female spikes, green utricles with a very short beak and acute female glumes (chater, 1980) (fig. 5). the species has been recorded from only several localities in northern bosnia and herzegovina, but recent records confirm that the species has a somewhat wider distribution in this part of the country (fig. 6): • in the vicinity of bosanska dubica; coordinates: 16.932538°e, 45.266012°n; elevation: ca 93 m; mgrs 10×10 square: xl51; habitat: genisto elatae-quercetum roboris; population: unknown; date: 1964; collector: m. glišić; bases of record: literature (glišić, 1964). note: this record is confirmed, and the species was considered rare (a few dozen individuals) at donja gradina near bosanska dubica in the well-preserved forest of fraxinus angustifolia and quercus robur, 15.05.2015, đ. milanović & v. stupar, djm: 65/04-160. • jakovica near the village of cerik (orig. cerje); coordinates: 18.522109°e, 44.808918°n; elevation: ca 100 m; mgrs 10×10 square: cq06; habitat: carpino betuli-quercetum roboris forest; population: unknown; date: 1975; collector: p. fig. 5. carex strigosa at donja gradina (© đ. milanović) biologica nyssana ● 14 (1) june 2023: 15-29 koljanin et al. ● notes on the distribution and conservation status of some rare plants of wet habitats in bosnia and herzegovina 22 biologica nyssana ● 14 (1) june 2023: 15-29 koljanin et al. ● notes on the distribution and conservation status of some rare plants of wet habitats in bosnia and herzegovina fukarek; bases of record: literature (fukarek, 1975). • lipovica forest near the village of pelagićevo; coordinates: 18.564017°e, 44.886848°n; elevation: ca 80 m; mgrs 10×10 square: cq07; habitat: carpino betuli-quercetum roboris forest; population: unknown; date: 1975; collector: p. fukarek; bases of record: literature (fukarek, 1975). • hrastik near the village of donji žabar; coordinates: 18.656429°e, 44.941478°n; elevation: ca 85 m; mgrs 10×10 square: cq17; habitat: carpino betuli-quercetum roboris forest; population: unknown; date: 1975; collector: p. fukarek; bases of record: literature (fukarek, 1975). • a forest in the village of obudovac; coordinates: 18.578385°e, 44.954521°n; elevation: ca 80 m; mgrs 10×10 square: cq08; habitat: carpino betuli-quercetum roboris forest; population: unknown; date: 1975; collector: p. fukarek; bases of record: literature (fukarek, 1975). • burumac (orig. baranac) in the vicinity of bosanski šamac; coordinates: 18.521154°e, 45.004933°n; elevation: ca 95 m; mgrs 10×10 square: cq08; habitat: carpino betuli-quercetum roboris forest; population: unknown; date: 1975; collector: p. fukarek; bases of record: literature (fukarek, 1975). • dubrave near vrbaška; coordinates: 17.157772°e, 45.137031°n; elevation: 94 m; mgrs 10×10 square: xl70; habitat: leucojo-fraxinetum angustifoliae; soil type: pseudogley over tertiary sediments; population: less than 20 flowering individuals; date: 28.05.2016; collector: đ. milanović; bases of record: observation. • suvopolje near grapska; coordinates: 18.062530°e, 44.789970°n; elevation: ca 130 m; mgrs 10×10 square: bq66; habitat: a degraded forest of white willow (salix alba); soil type: fluvisol over alluvial deposits; population: a dozen individuals scattered in the forest; date: 13.05.2017; collector: đ. milanović & v. stupar; bases of record: djm 65/04-161. • jabučik near kožuhe; coordinates: 18.096060°e, 44.859240°n; elevation: 128 m; mgrs 10×10 square: bq77; habitat: well-preserved alnus glutinosa forest; soil type: eugley river terrace; population: less than 100 individuals scattered in the forest; date: 13.05.2017; collector: đ. milanović & v. stupar; bases of record: observation. • kutlovac near podnovlje; coordinates: 18.142250°e, 44.934760°n; elevation: 116 m; mgrs 10×10 square: bq77; habitat: a wellpreserved grove of white willow; soil type: fluvisol over alluvial deposits; population: a dozen scattered individuals throughout the forest; date: 14.05.2017; collector: đ. milanović & v. stupar; bases of record: observation. • rastoka near podnovlje; coordinates: 18.114401°e, 44.919059°n; elevation: 119 m; mgrs 10×10 square: bq77; habitat: degraded grove of white willow; soil type: fluvisol over alluvial deposits; population: only a few individuals; date: 14.05.2017; collector: đ. milanović & v. stupar; bases of record: observation. general distribution and ecology this subatlantic species is widespread in a large part of western and central europe, the caucasus, and northern iran, and very rare in the iberian peninsula (laskurain et al., 2003; liendo et al., 2016). in central europe, the species is found mainly in lowlands, wet to damp places, nutrient-rich and phneutral soils, in semi-shaded to shaded habitats in the floodplains of larger rivers (kaplan et al., 2020). it usually grows in communities of poplars and tall willows, narrowed-leaved ash, european white elm and pedunculate oak which are often under the impact of flooding (borhidi et al., 2012; chytrý, 2013). in slovenia, this species is noted to grow in communities belonging to the montio-cardaminetea class but more often in lowland riparian forest communities (trčak & bačič, 2017). in croatia, this species is mostly found in the northern part of the country near the sava and drava rivers (nikolić, 2020). the species is also found in serbia in wet and fig. 6. distribution of carex strigosa in bosnia and herzegovina 23 swamp lowland forests (josifović, 1976). habitat and species threats in bosnia and herzegovina in bosnia and herzegovina, the species is restricted to northern lowlands where it occurs in remnants of riparian forests, irregularly scattered in the lower flow of major rivers of the danube catchment. these habitats are rare in bosnia and herzegovina due to intensive changes in the water regime and uncontrolled logging. historically, the species was exposed to similar negative impacts as carex elongata, but it has a wider ecological amplitude, ranging from swamps to meso-hygrophilous forests. invasive species are also a threatening factor. severe habitat changes could lead to species extinction in bosnia and herzegovina. conservation status global and european iucn status of carex strigosa is not assessed. it has different conversation statuses in european countries; nederland – vulnerable (sparrius et al., 2014), czech republic – endangered taxa (grulich, 2012), croatia – data deficient (nikolić & topić, 2005), spain – critically endangered taxa (liendo et al., 2016) etc. considering the available data, i.e., aoo is less than 10 km2, the regional population is severely fragmented, and there is continuing decline in area, extent and habitat quality, we suggest regional conservation status for bosnia and herzegovina: critically endangered cr b2ab(iii). comarum palustre l. published and new chorological data genus comarum is represented by only one species in european flora: comarum palustre. it is similar to the genus potentilla, but it has lateral and filiform style and purple petals (ball et al., 1968) (fig. 7). the species is very rare in bosnia and herzegovina, recorded only from wet refugia around glacial lakes (fig. 8): • banks of kukavičko jezero (kupreško polje); coordinates: 17.331487°e, 43.949120°n; elevation: 1202 m; mgrs 10×10 square: xj86; habitat: banks of the lake; population: numerous individuals; date: 1925.09.1924, 04.07.1955, 19.06.1990; collector: ž. protić, č. šilić, hilda ritter-studnička; bases of record: herbarium sara 17641, sara 17642, sara 0051634; corresponding literature (ritter-studnička, 1954). note: recently confirmed in alnus incana forest around a lake in about hundred flowering individuals, 25.07.2021, observation by đ. milanović, m. hayek, p. haykova & d. dite. • banks of gornje bare (zelengora mt.); coordinates: 18.607463°e, 43.320720°n; elevation: 1515 m; mgrs 10×10 square: cn09; habitat: bank of the lake; population: only one individual; date: 1969; collector: r. lakušić; bases of record: literature (bjelčić et al., 1969); stated by authors as new for bosnia and herzegovina. note: recently (19.07.2016) confirmed population numbers of a few hundred individuals in various sedge stands (carex rostrata, c. limosa, c. vesicaria and c. acuta aggr.) and as very rare in deschampsia cespitosa grasslands around the lake (milanović, 2017b). • banks of kotlaničko lake (zelengora mt.); coordinates: 18.482280°e, 43.362913°n; elevation: 1530 m; mgrs 10×10 square: bp90; habitat: comaro-menyanthetum trifoliatae on the bank of the lake; population: unknown; date: biologica nyssana ● 14 (1) june 2023: 15-29 koljanin et al. ● notes on the distribution and conservation status of some rare plants of wet habitats in bosnia and herzegovina fig. 7. comarum palustre around jugovo jezero (© đ. milanović) fig. 8. distribution of comarum palustre in bosnia and herzegovina 24 1978; collector: r. lakušić; bases of record: literature (lakušić et al., 1978; drešković et al., 2011; redžić et al., 2013). note: the record of comarum palustre around kotlaničko lake has been only indirectly gathered from a report on the vegetation map of bosnia and herzegovina for the period of 1977-1980 (was not available to the authors) as well as from prodromus of plant communities of bosnia and herzegovina (lakušić et al., 1978) where the community of comaromenyanthetum is stated but without an exact record of the species. in the same references (drešković et al., 2011; redžić et al., 2013), the community of comarum-menyanthes was also mentioned for some lakes from treskavica mt., based on the report on the vegetation map of bosnia and herzegovina for 1977, but this report was available for the authors, and this community was not stated there. more than 5000 flowering individuals of this species have been recently confirmed in floating carpets of comarum palustre-menyanthes trifoliata, carex limosa and equisetum fluviatile stands around kotlaničko lake, 18.08.2006 and 21-22.07.2016, đ. milanović, djm: 84/09-001. • crno lake (zelengora mt.); coordinates: 18.583443°e, 43.386275°n; elevation: 1440 m; mgrs 10×10 square: cp00; habitat: phragmites communis stands and floating carpets with comarum palustre; population: several hundred flowering individuals; date: 14.07.2016; collector: đ. milanović; bases of record: literature (milanović, 2017b). • jugovo lake (zelengora mt.); coordinates: 18.530804°e, 43.375774°n; elevation: 1550 m; mgrs 10×10 square: cp00; habitat: carex rostrata stands along banks of the lake, and around a source above the lake; population: a few hundred flowering individuals; date: 21.07.2016; collector: đ. milanović; bases of record: observation. general distribution and ecology comarum palustre has a wide circumboreal distribution, encompassing the temperate to arctic regions of eurasia, north america, and the southern parts of greenland (kaplan et al., 2017). in central europe, the species is found in various habitats, but most commonly on moist, acidic, and nutrient-poor soils, such as fens, transitional mires, wet moss-rich meadows, tall sedge stands, edges of fishponds, and alder carrs, less often also in bog hollows (kaplan et al., 2017). it is also usually found in salix cinerea scrub communities (borhidi et al., 2012; chytrý, 2013). the species is rarely found in southern europe. in croatia, the species can be considered rare. it has been recently confirmed in only one locality, while previous literature records stay unconfirmed (nikolić & topić, 2005). in serbia, the species is known from the eastern part of the country and could also be considered rare (josifović, 1972). in montenegro, the species occurs at three spatially close localities at the durmitor mt. (caković & stešević, 2021), all at high elevations. habitat and species threats in bosnia and herzegovina the species is recorded only in mountain depressions around glacial lakes in bosnia and herzegovina. there it occupies wet habitats on the edge of water bodies of the lakes, often forming floating carpets with other herbaceous plants (kotlaničko lake, jugovo lake, crno lake and gornje bare), while only around kukavičko lake it inhabits alnus incana forests. in spite of the fact that the recorded populations are located far from settlements and sometimes within protected areas (sutjeska national park), some of the recorded populations are exposed to strong current or potential human pressures. due to illegal off-road activities around crno lake, water from springs on the slope above the northeast bank of the lake, which provide permanent moistening of the lake terrace, is drained through the tire tracks, causing dryness during the driest part of the year. the populations around jugovo (borilovačko) lake will be even more affected by the plans for the urbanization of the area surrounding the lake. conservation status global iucn status of comarum palustre is least concern (maiz-tome, 2015). the species is considered critically endangered in croatia (nikolić & topić, 2005). šilić (1996) included the species in the list of vascular plants for the red book of bosnia and herzegovina as vulnerable. in the red list of flora of the federation of bosnia and herzegovina, the species was estimated as vulnerable (đug et al., 2013). considering the available data, i.e., aoo is less than 10 km2, the regional population is severely fragmented, and there is continuing decline in area, extent and habitat quality, we suggest regional conservation status for bosnia and herzegovina: critically endangered cr b2ab(iii). prunus padus l. published and new chorological data prunus padus is sometimes planted tree species in horticulture in bosnia and herzegovina, while its natural populations in the country are very rare (fig. biologica nyssana ● 14 (1) june 2023: 15-29 koljanin et al. ● notes on the distribution and conservation status of some rare plants of wet habitats in bosnia and herzegovina 25 9, fig. 10). only known populations are scattered on the edge of kupreško field, where they were originally considered as potentially escaped from gardens (ritter-studnička, 1958) but later confirmed as autochthonous (ritter-studnička, 1972). recently, the species was also mentioned for northern bosnia (šilić, 2005; stupar et al., 2021), potentially based on findings in some prehistoric archaeological sites (maly, 1904), but without any exact locality or coordinates. moreover, our recent research in the area did not confirm these allegations. furthermore, it should be stated that redžić & golić (1985) noted prunus padus in maoča at konjuh mt. in forest clearings of pinus nigra forests (phytosociological table). neither in the following text (redžić & golić, 1985) nor in subsequent research of the same plots (redžić, 1988) prunus padus was not mentioned again, but prunus mahaleb is regularly noted. therefore, we consider that „prunus padus” is a lapse probably referring to prunus mahaleb which is ecologically suited for such habitats. we considered these lapse as this species is not mentioned in the further text and is replaced by prunus mahaleb in further research on those permanent vegetation plots. prunus padus has been reported from the following localities: • humbat hill near prozor; coordinates: 17.616170°e, 43.816082°n; elevation: ca 770 (orig. 856 m) m; mgrs 10×10 square: yj15; habitat: unknown; population: unknown; date: 21.07.1907; collector: j. stadlmann; bases of record: literature (stadlmann, 1912) note: later indicated as erroneous record after consulting the original herbarium material (ritter-studnička, 1972) misidentification with prunus mahaleb. • near the road on slopes of kupreška vrata towards kupres; coordinates: 17.293807°e, 44.007529°n; elevation: 1200-1300 m; mgrs 10×10 square: xj87; habitat: in forests and scrubs; population: numerous individuals; date: 01.08.1958; collector: l. lažetić; bases of record: herbarium sara 18992-18993, corresponding literature (ritter-studnička, 1958, 1972, 1974). note: recently confirmed throughout the slopes of kupreška vrata and stožer mt. towards kupres, optimally developed in alnus incana forest in several hundred flowering individuals and much more in the vegetative stage as a shrub in the canopy, 30.04.2018, đ. milanović, g. tomović & k. jakovljević, djm: 84/24–001; 13.07.2021, j. brujić & d. koljanin, herb. fac. silv: 000274. • banks of kukavičko lake (kupreško polje); coordinates: 17.331487°e, 43.949120°n; elevation: 1202 m; mgrs 10×10 square: xj86; habitat: banks of the lake; population: scattered individuals; date: 1972; collector: h. ritterstudnička; bases of record: literature (ritterstudnička, 1972, 1974). note: recently confirmed in alnus incana forest around the lake – only a few individuals have been registered, 25.07.2021, đ. milanović, m. hayek, p. haykova & d. dite, djm: 84/24–002. • hrbljina; coordinates: 17.097371°e, 44.005508°n (unprecise); elevation: ca 1300 m; mgrs 10×10 square: xj67; habitat: unknown; population: scattered individuals, rare; date: 1972; collector: n. zubić; bases of record: literature (ritterstudnička, 1972). note: the record is a personal observation of novak zubić, a geographer that at the time was employed at the faculty of natural sciences in sarajevo, and having in mind that the reported locality doesn’t fit in the species ecology, biologica nyssana ● 14 (1) june 2023: 15-29 koljanin et al. ● notes on the distribution and conservation status of some rare plants of wet habitats in bosnia and herzegovina fig. 9. prunus padus in blooming stage on slopes of stožer mt. (© đ. milanović) fig. 10. distribution of prunus padus in bosnia and herzegovina 26 we consider this record as doubtful. • vitorog mt.; coordinates: 17.096455°e, 44.084158°n (unprecise); elevation: ca 1500 m; mgrs 10×10 square: xj68; habitat: unknown; population: scattered individuals, rare; date: 1972; collector: n. zubić; bases of record: literature (ritter-studnička, 1972). note: we consider this record doubtful for the same reasons as in the previous case. later research on the flora and vegetation of this mountain did not confirm this species either (redžić et al., 1984; zubić & topalić-trivunović, 2011). • podbrdo in podrašničko polje; coordinates: 16.985369°e, 44.457024°n; elevation: 730 m; mgrs 10×10 square: xj52; habitat: well preserved alnus glutinosa forest; soil type: eugley over diluvial sediments; population: several flowering individuals; date: 29.05.2021; collector: j. brujić & d. koljanin; bases of record: herb. fac. silv: 000275 & herb. fac. silv: 000276. general distribution and ecology prunus padus is a eurasian species with an almost compact distribution range that is gradually becoming disjunct on its southern border. it is widespread in most parts of europe except the southern areas (webb & rix, 1972; kurtto et al., 2013). in central europe, it typically occurs in closed forests and is a common and diagnostic taxon of alnion incanae alliance, but it can be found in a wide range of habitats (willner & grabherr, 2007; borhidi et al., 2012; chytrý, 2013). this species is common in most central european countries (rhodes & maxted, 2013), including slovenia, and northern and western croatia, which represents the edge of its compact distribution range. in the western balkan, it has a disjunct distribution, occurring mostly at higher altitudes with lower temperatures. it appears that the species is frequent in lowland forests only in colder and more humid climates, while in the balkan peninsula, the species could be considered a glacial relict. habitat and species threats in bosnia and herzegovina the species is found at higher altitudes in moist habitats on silicate substrates. on kupres, species grow in grey alder stands and in the succession of fir-beech-spruce forests. some old individuals are also found along roads. it seems that on kupres, this species does not have a narrow distribution, and it is capable of thriving in different habitats. in sitnica, species is found in only one alnus glutinosa stand, but it is likely to exist in other nearby locations. the main threat for prunus padus populations is land use change. any changes in the hydrological regime of its habitats could also be a threat causing population number and density decline. as prunus padus wood is valuable for wood carving and making lockers (nestby, 2020), there is a potential risk for the older trees. also, as some localities in bosnia and herzegovina are in succession stages, it could be expected that this natural process would be followed by a decreasing number of individuals. conservation status global and european iucn status of prunus padus supsp. padus is the least concern (sparrius et al., 2014), and it is not listed on the national red lists of the majority european countries. the species is not considered native in serbia (josifović, 1972) and, therefore, is not included in the list. species was assessed as vulnerable in a proposal for the red list of vascular flora of bosnia and herzegovina (šilić, 1996). in the red list of flora of the federation of bosnia and herzegovina, the species was also assessed as vulnerable (đug et al., 2013). considering the available data, i.e., aoo is less than 10 km2, the regional population is severely fragmented, and there is continuing decline in area, extent and quality of habitat, we suggest regional conservation status for bosnia and herzegovina: critically endangered cr b2ab(iii). references ball, p.w., pawłowski, b., & walters, s.m. (1968). genus potentilla l. in: tutin t.g., heywood v.h., burges n.a., moore d.m., valentine d.h., walters s.m., webb d.a. (eds.), flora europaea 5 (pp. 26-47). united kingdom, london: cambridge university press. barudanović, s., mašić, e., macanović, a., & hatibović, e. (2019). state of peatland ecosystems in bosnia and herzegovina. fondeco science, 1(1), 48-60. beck-mannagetta, g., malý, k., & bjelčić, ž. (1983). flora bosnae et hercegovinae 4 – sympetalae 4. bosna i hercegovina, sarajevo: zemaljski muzej bosne i hercegovine u sarajevu, prirodnjačko odjeljenje. bilz, m. (2012). achillea ptarmica. in: iucn red list of threatened species 2013: iucn red list of threatened species. version 2013.2. www. iucnredlist.org bjelčić, ž., šilić, č., lakušić, r., kutleša, l., mišić, l., & grgić, p. (1969). neke rijetke i interesantne vrste biljaka sa područja planina maglića, volujka i biologica nyssana ● 14 (1) june 2023: 15-29 koljanin et al. ● notes on the distribution and conservation status of some rare plants of wet habitats in bosnia and herzegovina 27 zelengore. posebna izdanja odjeljenja prirodnih i matematičkih nauka anubih, 3, 91-106. borhidi, a., kevey, b., & lendvai, g. (2012). plant communities of hungary. hungary, budapest: akadémiai kiadó, budapest. caković, d. & stešević, d. (2021). catalogue of vascular flora of montenegro ii. crna gora, podgorica: crnogorska akademija nauka i umjetnosti odjeljenje prirodnih nauka. chater, a.o. (1980). genus carex l. in: tutin t.g., heywood v.h., burges n.a., moore d.m., valentine d.h., walters s.m., webb d.a. (eds.), flora europaea 5 (pp. 290–323). united kingdom, london: cambridge university press. chytrý, m. (ed.). (2013). vegetace české republiky 4: lesní a křovinná vegetace. czech republic, praha: academia. desrochers, a., lavoie, c., pellerin, s., & poulin, m. (2000). bog conservation: a canadian perspective. proceedings of the 11th international peat congress11th international peat congress, 2, 1027–1033. douda, j. (2008). formalized classification of the vegetation of alder carr and floodplain forests in the czech republic. preslia, 80, 199–224. drešković, n., đug, s., stupar, v., hamzić, a., lelo, s., muratović, e., lukić-bilela, l., brujić, j., milanović, đ., & kotrošan, d. (2011). natura 2000 u bosni i hercegovini. bosna i hercegovina, sarajevo: centar za okolišno održivi razvoj. dudáš, m., danihelka, j., & eliáš, p. (2017). achillea ptarmica (asteraceae), a scarce and less known species of the slovak flora. thaiszia-journal of botany, 27(2), 95–109. duffy, k.j., scopece, g., cozzolino, s., fay, m.f., smith, r.j., & stout, j.c. (2009). ecology and genetic diversity of the dense-flowered orchid, neotinea maculata, at the centre and edge of its range. annals of botany, 104, 507 –516. đug, s., muratović, e., drešković, n., boškailo, a., & dudević, s. (2013). crvena lista flore federacije bosne i hercegovine. bosna i hercegovina, sarajevo: eu greenway. euro+med plantbase (the information resource for euro-mediterranean plant diversity, continuously updated). 2006. retrieved from http://ww2.bgbm.org/europlusmed/query.asp fukarek, p. (1975). hrastove šume bosanskog posavlja u prošlosti i sadašnjosti. jugoslavenska akademija znanosti i umjetnosti, posebna izdanja, 2, 371-379. glavač, v. (1959). o šumi poljskog jasena sa kasnim drijemovcem (leucoieto-fraxinetum angustifoliae ass. nov.). šumarski list, 1–3, 39–45. glišić, m. (1964). pregled šumske vegetacije na aluvijum kod bosanske dubice. narodni šumar, 18, 135–142. grulich, v. (2012). red list of vascular plants of the czech republic: 3rd edition. preslia, 84, 631– 645. heywood, v.h. & iriondo, j.m. (2003). plant conservation: old problems, new perspectives. biological conservation, 113(3), 321-335. hulík, j. & douda, j. (2017). germination strategies of two dominant carex species in a swamp alder forest: implications for restoration. biologia, 72(4), 370–377. doi: 10.1515/biolog-2017-0045 huntley, b., berry, p.m., cramer, w., & mcdonald, a.p. (1995). modelling present and potential future ranges of some european higher plants using climate response surfaces. journal of biogeography, 22(6), 967–1001. iucn (international union for conservation of nature) species survival commission. 2012a. guidelines for application of iucn red list criteria at regional and national levels. version 4.0. iucn (international union for conservation of nature) species survival commission. 2012b. iucn red list categories and criteria. version 3.1. second edition. josifović, m. (1972). flora sr srbije iv. beograd: srpska akademija nauka i umetnosti. josifović, m. (1976). flora sr srbije viii. beograd: srpska akademija nauka i umetnosti. kaplan, z., danihelka, j., šumberová, k., chrtek jr.j., rotreklová, o., ekrt, l., štěpánková, j., taraška, v., trávníčk, b., & prančl, j. (2017). distributions of vascular plants in the czech republic. part 5. preslia, 89(4), 333–439. doi: 10.23855/preslia.2020.255 kaplan, z., danihelka, j., ekrt, l., štech, m., řepka, r., chrtek, j., grulich, v., rotreklová, o., dřevojan, p., & šumberová, k. (2020). distributions of vascular plants in the czech republic. part 9. preslia, 92, 55–340. doi: 10.23855/preslia.2017.333 knollová, i. & chytrý, m. (2004). oak-hornbeam forests of the czech republic: geographical and biologica nyssana ● 14 (1) june 2023: 15-29 koljanin et al. ● notes on the distribution and conservation status of some rare plants of wet habitats in bosnia and herzegovina 28 ecological approaches to vegetation classification. preslia, 76, 291–311. koljanin, d., milanović, đ., & stupar, v. (2021). new data on the distribution and threat status of three rare spring geophytes from bosnia and herzegovina. phytologia balcanica, 27(1), 107-114. kurtto, a., sennikov, a.n., & lampinen, r. (eds.). (2013). atlas florae europaeae 16. rosaceae (cydonia to prunus, excl. sorbus). helsinki: the committee for mapping the flora of europe & societas biologica fennica vanamo. lakušić, r., pavlović, d., abadžić, s., & grgić, p. (1978). prodromus biljnih zajednica bosne i hercegovine. godišnjak biološkog instituta univerziteta u sarajevu, 30, 5-88. laskurain, n.a., aldezabal, a., lópez de luzuriaga, a., & olano, j.m. (2003). atlas y libro rojo de la flora vascular amenazada de espana. madrid: dirección general de conservación de la naturaleza. lesica, p. & allendorf, f.w. (1995). when are peripheral populations valuable for conservation? conservation biology, 9(4), 753-760. liendo, d., campos, j.a., biurrun, i., & garcíamijangos, i. (2016). new contributions to the native and alien flora in riparian habitats of the cantabrian watershed (northern spain). lazaroa, 37, 173–182. doi: 10.5209/laza.52852 lubarda, b., stupar, v., milanović, đ., & stevanović, v. (2014). chorological characterization and distribution of the balkan endemic vascular flora in bosnia and herzegovina. botanica serbica, 38(1), 167-184. maiz-tome, l. (2015). comarum palustre. in: iucn red list of threatened species 2013: iucn red list of threatened species. version 2013.2. www.iucnredlist.org malý k. (1935). mitteilungen über die flora von bosnien-hercegovina. glasnik zemaljskog muzeja u bosni i hercegovini, 47, 101–111. milanović, đ., brujić, j., & stupar, v. (2011). new floristic records in the balkans 15: reports 6472. phytologia balcanica, 17(1), 141-144. milanović, đ. (2012). liparis loeselii (l.) rich. – a plant rediscovered in the balkan peninsula. botanica serbica, 36(2), 85-89. milanović, đ. (2017a). bosnia and hercegovina. in: joosten h., tanneberger f., moen a. (eds.), mires and peatlands of europe (pp. 310-318). stuttgard: schweizerbart science publishers. milanović, đ. (2017b). vaskularna flora akvatičnih i vlažnih staništa uz glacijalna jezera u nacionalnom parku sutjeska (republika srpska, bosna i hercegovina). glasnik šumarskog fakulteta univerziteta u banjoj luci, 26, 63-73. doi: 10.7251/ gsf1726075m mrgić, j. (2007). lijevče polje beleške o naseljima i prirodi 15-19. vek. historical review, 5, 171–199. nestby, r.d. (2020). the status of prunus padus l. (bird cherry) in forest communities throughout europe and asia. forests, 11(497), 1-18. nikolić, t. & topić, j. (2005). red book of vascular flora of croatia. republika hrvatska, zagreb: ministry of culture, državni zavod za zaštitu prirode. nikolić, t. (2010). flora croatica database, online: (http://hirc.botanica.hr/fcd). department of botany, faculty of science, univeristy of zagreb (accessed 25.11.2022). nikolić, t. (2020). flora croatica 2 vaskularna flora republike hrvatske. zagreb: alfa d.d. pearson, r.g., dawson, t.p., & liu, c. (2004). modelling species distributions in britain: a hierarchical integration of climate and land‐cover data. ecography, 27(3), 285-298. petronić, s., kadić, j., radošević, d., & panić, g. (2010). floristički diverzitet posebnog područja prirode „gromiželj”. arhiv za tehničke nauke, 3, 156–168. redžić, s., lakušić, r., muratspahić, d., bjelčić, ž., & omerović, s. (1984). struktura i dinamika fitocenoza u ekosistemima cincara i vitoroga. godišnjak biološkog instituta univerziteta u sarajevu, 37, 123–177. redžić, s. & golić, s. (1985). uticaj totalnih sječa na sezonsku dinamiku vegetacije u ekosistemu hrastovo-borovih šuma (querco-pinetum nigrae serpentinicum). biološki institut u sarajevu, 38, 115–129. redžić, s. (1988). šumske fitocenoze i njihova staništa u uslovima totalnih sječa. godišnjak biološkog instituta univerziteta u sarajevu posebno izdanje, 41, 1–260. redžić, s., barudanović, s., & radević, m. (eds.). (2009). bosnia and herzegovina land of diversity. first national report of bosnia and herzegovina for the convention on biological diversity. federal ministry of environment and tourism. biologica nyssana ● 14 (1) june 2023: 15-29 koljanin et al. ● notes on the distribution and conservation status of some rare plants of wet habitats in bosnia and herzegovina 29 redžić, s., trakić, s., & barudanović, s. (2013). patterns of vegetation diversity of grasslands and pastures – crvanj mt. (herzegovina, western balkan). scientific research and essays, 8(39), 1944-1965. rhodes, l. & maxted, n. (2013). prunus padus in: iucn red list of threatened species 2013: iucn red list of threatened species. version 2013.2. www.iucnredlist.org. richardson, i.b.k. (1976). genus achillea l. in: tutin t.g., heywood v.h., burges n.a., moore d.m., valentine d.h., walters s.m., webb d.a. (eds.), flora europaea 4 (pp. 159–165). london: cambridge university press. ritter-studnička, h. (1954). flora i vegetacija kraških polja bosne i hercegovine. godišnjak biološkog instituta univerziteta u sarajevu, 7, 25– 109. ritter-studnička, h. (1958). prilozi za floru bosne i hercegovine iii. godišnjak biološkog instituta univerziteta u sarajevu, 11(1-2), 95-121. ritter-studnička, h. (1972). ist prunus padus l. in bosnien eine autochthone pflanze? bulletin scientifique conseil des academies des sciences et des arts de la rsf de yougoslavie, 17(5–6), 158– 159. ritter-studnička, h. (1974). karstpoljen bosniens und der hercegovina als reliktstandorte und die eigentumlichkeiten ihrer vegetation. botanische jahrbücher für systematik, pflanzengeschichte und pflanzengeographie, 94(2), 139–189. sagorski, e.a. (1901). beitrag zur flora der herzegovina i. mitteilungen des thüringischen botanischen vereins, 16, 33–50. slezák, m., hrivnák, r., & petrášová, a. (2014). numerical classification of alder carr and riparian alder forests in slovakia. phytocoenologia, 44(34), 283–308. doi: 10.1127/0340-269x/2014/00440588 sokołowski, a.w. (1972). zespół carici elongataequercetum – dębniak turzycowy. acta societatis botanicorum poloniae, 41(1), 113–120. sparrius, l.b., odé, b., & beringen, r. (2014). basisrapport rode lijst vaatplanten 2012 volgens nederlandse en iucn-criteria. nijmegen: floron rapport 57. stadlmann, j. (1912). eine botanische reise nach südwest-bosnien und in die nördliche herzegowina. mitteilungen des naturwissenschaftlichen vereines an der universitat wien, 10(3), 29–37. stevanović, v. (ed.). (1999). crvena knjiga flore srbije, 1: iščezli i krajnje ugroženi taksoni. republika srbija, beograd: ministarstvo za životnu sredinu, biološki fakultet univerziteta u beogradu i zavod za zaštitu prirode republike srbije. stupar, v., milanović, đ., & brujić, j. (2021). vaskularna flora republike srpske. banja luka: šumarski fakultet univerziteta u banjoj luci. šilić, č. (1996). spisak biljnih vrsta (pteridophyta i spermatophyta) za „crvenu knjigu” bosne i hercegovine. glasnik zemaljskog muzeja bosne i hercegovine, 31, 323-367. šilić, č. (2005). atlas dendroflore (drveće i grmlje). zagreb: matica hrvatska. trčak, b. & bačič, t. (2017). ozkoklasi šaš (carex strigosa huds.) v sloveniji. hladnikia, 39, 33–43. tsiftsis, s., tsiripidis, i., & papaioannou, a. (2012). ecology of the orchid goodyera repens in its southern distribution limits. plant biosystems, 146(4), 857-866. doi:10.1080/11263504.2011.642 416 webb, d.a. & rix, e.m. (1972). genus prunus l. in: tutin t.g., heywood v.h., burges n.a., moore d.m., valentine d.h., walters s.m., webb d.a. (eds.), flora europaea 2 (pp. 77–80). london: cambridge university press. willner, w. & grabherr, g. (eds.). (2007). die wälder und gebüsche österreichs. heidelberg: spektrum. zubić, g. & topalić-trivunović, lj. (2011). vaskularna flora staništa velikog tetrijeba (tetrao urogallus l.) na području planine vitorog. glasnik šumarskog fakulteta univerziteta u banjoj luci, 15, 29-48. biologica nyssana ● 14 (1) june 2023: 15-29 koljanin et al. ● notes on the distribution and conservation status of some rare plants of wet habitats in bosnia and herzegovina nikolic et al. 2021, biologica nyssana 12(2) 12 (2) december 2021: 159-165 doi: 10.5281/zenodo.5759870 evaluation of ecological awareness and superstition on hermann’s tortoise in eastern and southern serbia original article marko nikolić faculty of sciences and mathematics, university of niš, višegradska 33, 18000 niš, serbia biological society “dr. sava petrović”, višegradska 33, 18000 niš, serbia marko@bddsp.org.rs (corresponding author) dimitrija savić-zdravković faculty of sciences and mathematics, university of niš, višegradska 33, 18000 niš, serbia biological society “dr. sava petrović”, višegradska 33, 18000 niš, serbia jelka crnobrnja-isailović faculty of sciences and mathematics, university of niš, višegradska 33, 18000 niš, serbia institute for biological research “siniša stanković” – institute of national importance for republic of serbia, university of belgrade, despota stefana 142, 11000 belgrade, serbia received: november 01, 2021 revised: november 26, 2021 accepted: december 02, 2021 abstract: folk beliefs, religion and mythology, created through various cultural influences in serbia, shaped today’s attitudes toward wildlife. in this study we evaluated different levels of ecological awareness and superstition extent related to hermann’s tortoise (testudo hermanni) through a systematized face-to-face questionnaire. the study was conducted in two protected and two unprotected areas of southern and eastern serbia. the results confirmed the presence of myths and superstitions amongst locals and existing conflict between the humans and t. hermanni. these results also showed the lack of knowledge about national regulations related to protection of nature and t. hermanni, even in protected areas, with statistically signifficant differences among localities. to reduce this human/wildlife conflict, future conservation measures would have to take into account the views of the local population. key words: testudo hermanni, serbia, ethnozoology, survey, questionnaire, protected areas, human-wildlife conflict apstrakt: procena nivoa ekološke svesti i sujeverja o šumskoj kornjači u istočnoj i južnoj srbiji narodna verovanja, religija i mitologija, nastali pod dejstvom različitih kulturoloških uticaja u srbiji, oblikovali su današnji odnos čoveka prema prirodi. u ovoj studiji procenjivali smo različite nivoe ekološke svesti i sujeverja lokalnog stanovništva o šumskoj kornjači (testudo hermanni) putem anketiranja. studija je sprovedena na dva zaštićena i dva nezaštićena područja južne i istočne srbije. rezultati su potvrdili prisustvo mitova i sujeverja među lokalnim stanovništvom i postojanje sukoba između ljudi i šumske kornjače. ovi rezultati, takođe, pokazuju i nedostatak znanja o nacionalnim propisima koji se odnose na zaštitu prirode i zaštitu šumske kornjače, čak i kod stanovništva u zaštićenim područjima, sa statistički značajnim razlikama među lokalitetima. da bi se smanjio ovaj sukob između ljudi i divljih životinja, buduće mere zaštite bi morale da uzmu u obzir stavove lokalnog stanovništva. ključne reči: testudo hermanni, srbija, etnozoologija, anketa, upitnik, zaštićena područja, sukob između ljudi i divljih životinja introduction as it has been shown in many places worldwide, attitude of the local community regarding importance and conservation of wildlife plays a major role in restricting direct and indirect human impact on habitats and populations of endangered species. local resource users can affect the implementation of conservation measures positively or negatively, therefore being a powerful conservation factor (ramstad et al., 2007; røskaft, et al., 2007; ebua et al., 2011; talukdar & gupta, 2018). local folk beliefs, religion and mythology contribute vastly to the formation of attitudes and opinions of the native local human population (further referred to as locals) towards nature and wildlife. therefore, in recent decades, interdisciplinary fields of study, such as ethnozoology, nature conservation marketing, or social psychology, have made significant contributions to biodiversity conservation (dickman, 2010; alves & souto, 2015; wright et al., 2015). in serbia, various cultural influences shaped today’s attitudes toward wildlife. one of the most serious wildlife threats in this country is illegal collecting for a variety of reasons, major ones being: for pet shops, use in food, folk medicine, or due to superstition and magic rituals (durst & mikuška, 2017; nikolić & golubović, 2017; jovanović et al., © 2021 nikolić et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 159 2020). tolstoy & radenkovic (2001) describe the role of many animal species in local mythology, and eastern hermann’s tortoise (testudo hermanni boettgeri) is one of them. in addition to already known threatening factors such as natural catastrophes, road casualties, habitat fragmentation and loss (including urbanization), a direct human-wildlife conflict is also present (van dijk et al., 2004; bertolero et al., 2011; fernández–chacón et al., 2011; celse et al., 2014; nikolić & crnobrnja-isailović, 2017). nowadays, one of the most common myths about hermann’s tortoise is the belief in the healing properties of tortoise blood, which is believed to help treat serious illnesses (nikolić & crnobrnja-isailović, 2017). according to the red book of reptiles of serbia (tomović et al., 2015), the hermann’s tortoise belongs to national category nt (djordjević & ljubisavljević, 2015) and its’ global iucn red list status is also nt (van dijk et al., 2004). besides, hermann’s tortoise is listed in annexes ii and iv of the habitats directive, in annex ii of the bern convention, and in annex ii of the cites convention. although hermann’s tortoise is protected by both international and national acts, the problem of illegal hunting and trading is still unresolved. tortoises are being collected and transported mainly to european union countries. thus, in 2005, 504 hermann’s tortoises were seized on the border between serbia and croatia, and a similar situation occurred in 2006 with 22 hermann’s and greek tortoises (testudo graeca) (jovanović & ajtić, 2011). in order to solve this problem, it is necessary to understand it. success in reducing negative anthropogenic impact and maintaining long-term sustainable management of natural resources depends a lot on the support of the local community. also, understanding the attitudes of the locals, taking into account their needs, and respecting their opinions should become a priority in creating effective protection and conservation measures (macura et al., 2011). the main aim of this research was to determine and evaluate different levels of ecological awareness and superstition extent occurring among local inhabitants in protected and unprotected areas situated in the southern and eastern regions of serbia. to achieve this goal, we conducted a survey about the awareness of the locals on the existence of protected areas and knowledge of national regulations related to protection of both nature and hermann’s tortoise. finally, we examined the presence of myths and superstitions amongst locals, as well as their perceptions of the costs and benefits of hermann’s tortoise protection. materials and methods study area surveying was conducted in a total of four locations in the municipalities of serbia two in the east and two in the south of the country (fig. 1). two of the four sites were located in protected areas: donji milanovac, on the territory of the “djerdap” national park in eastern serbia, and kunovica, 15 km from the city of niš, on the territory of “sićevačka klisura” nature park in southern serbia (niška banja municipality). the other two sites were not located within the protected areas: kladovo in eastern serbia and leskovac in southern serbia. the presence of hermann’s tortoise populations was confirmed on all 4 localities (golubović et al., 2019; nikolić et al., 2020). sampling fieldwork was carried out from may to october 2016. the survey was conducted for 5 days per 160 biologica nyssana ● 12 (2) december 2021: 159-165 nikolić et al. ● evaluation of ecological awareness and superstition on hermann’s tortoise in eastern and southern serbia fig. 1. map of serbia with the research localities marked as following: 1 – donji milanovac, 2 – kladovo, 3 – niš, 4 – leskovac; 1 44°27’50.48”n, 22° 9’2.63”e; 2 44°36’24.96”n, 22°36’49.19”e; 3 43°19’5.73”n, 21°53’47.89”e; 4 42°59’48.00”n, 21°56’39.03”e 161 locality, and residents were interviewed between 9:00 and 17:00. the research team toured the defined area on foot and, according to the principle of chance, the residents were interviewed face-toface. the conversation with one respondent lasted 15-20 minutes. during one day, 20-25 respondents were interviewed. our total sample included 389 inhabitants (tab. 1). questionnaire the face-to-face questionnaire was designed based on a study by veličković et al. (2015). the questions were divided into three sections. the first section contained questions about the respondents’ awareness of the existence and role of protected areas, and the presence of such areas in their close surroundings. the second section contained questions about superstition on one hand; as well as questions about the presence of myths related to the healing properties of hermann’s tortoise and whether the respondents believed in them, on the other hand. respondents were also able to state whether they were aware of what was believed to be used for these purposes (blood, eggs, legs or something else that was not listed), which diseases are believed to be curable in this way, have they heard of cures, and have they had similar experiences. finally, respondents answered questions about the existence of legal protection for hermann’s tortoise and whether conservation actions should be done to biologica nyssana ● 12 (2) december 2021: 159-165 nikolić et al. ● evaluation of ecological awareness and superstition on hermann’s tortoise in eastern and southern serbia table 1. basic data about respondents locality region protected area number of inhabitants number of respondents average age (%) male respondents (%) donji milanovac east serbia yes 2410 97 49.1 49.5 kladovo east serbia no 8869 93 44.5 55.9 niš (niška banja municipality) south serbia yes 14098 100 48.6 40 leskovac south serbia no 60288 99 49.8 51.5 preserve this species. the questions were formulated in a non-suggestive way to enable to the respondents to give as honest and complete answers as possible. statistical analysis respondents’ answers to superstition questions were presented in the form of categorical variables, and the chi-square (with yates correction for continuity for 2x2 tables) test of the ibm spss statistics ver. 25. software package was used to assess the statistical significance of differences in respondents’ answers. the awareness of the locals about the existence of protected areas in their surroundings and the presence of myths about the healing properties of tortoises was marked with appropriate codes in relation to the attitude towards their claims (1–yes, 2–no, 3–not familiar with the topic). the data was organized using microsoft office 365 excel apps, where graphic figures were also created. results the survey conducted at four localities in serbia included a total of 389 respondents (tab. 1). by analyzing the survey results and applying the chi-square test (tab. 2) we found that there were mild statistically significant differences between the number of superstitious residents of protected areas compared to other respondents: x2(1, n=389) = 4.07, p=0.044. the results have shown that many respondents believe in the myth that blood, eggs or meat of hermann’s tortoise have healing properties (tab. 3). as many as 72.6% of respondents in eastern serbia answered that these myths are present in their environment and that they knew someone who used tortoises for healing purposes. in relation to 55.3% of respondents in southern serbia, significant statistical differences were found: x2(1, n=389)=11.93, p=0.001. statistically significant differences were also found when it comes to the percentage distribution by localities x2(3, n=389) =16.24, p=0.001. most respondents who believed table 2. superstitious respondents in protected and unprotected areas (in percentages) % of respondents df syg. protected area 34.2 1 0.044 non-protected area 24.4 1 162 in the myth of the healing properties of hermann’s tortoise were recorded in donji milanovac (76.3%), and the least in leskovac (50%) (tab. 4). out of a total of 197 respondents living in selected localities within protected areas in southern and eastern serbia, 55.3% of respondents were informed about the existence of protected areas and of the fact that they are located in their immediate vicinity (fig. 2). additionally, 44.7% of respondents outside protected areas were informed according to the survey. the most informed inhabitants were found in donji milanovac (68%, p=0.000), and the least in leskovac (19%, p=0.000). attitudes of the locals that the hermann’s tortoise should be protected and preserved were statistically significantly differentamong localities x2 (3, n =389)=8.71, p=0.033. out of the total number of respondents who agree that hermann’s tortoise should be protected, the smallest share was donji milanovac with 23% (fig. 3), which is located in both eastern serbia and in the protected area. discussion the interaction of the local, indigenous human population with nature is conditioned by various direct or indirect reasons. the direct reasons are the purchase of food, the purchase of medicinal raw materials, or the quest for new natural sites (alves & souto, 2015). as humans have become the dominant species on the planet (ehrlich & ehrlich, 2008), throughout history human civilization has influenced the living world and biodiversity in many ways. in general, the interaction of the locals with nature has always taken place in different ways, and they vary depending on the cultural influence and the environment itself that we observe (alves & souto, 2015). understanding the ecological context for resolving human-wildlife conflicts is not enough; therefore, different socio-economic aspects, cultural influences, attitudes of the actors involved and interactions between them must be considered (salom et al., 2021). our research has shown that 34.2% of the population in protected areas was superstitious (tab. 2), in comparison to 24.4% (p=0.044) of biologica nyssana ● 12 (2) december 2021: 159-165 nikolić et al. ● evaluation of ecological awareness and superstition on hermann’s tortoise in eastern and southern serbia table 3. respondents per region who believe in the healing properties of hermann’s tortoise (in percentages) region % of respondents df syg. east serbia 72.6 1 0.001 south serbia 55.3 1 fig. 2. awareness of the respondents about the existence of protected areas in their environment in relation to whether they live in the territory of the protected area or not fig. 3. respondents who are aware that the hermann’s tortoise should be protected by law (percentage representation of respondents in relation to the total sample) locality ht df syg. pa df syg. donji milanovac 76.3 3 0.001 68.0 3 0.001 kladovo 68.8 3 65.6 3 niš 60.6 3 33.3 3 leskovac 50.0 3 19.0 3 table 4. respondents per locality who believe in the healing properties of hermann’s tortoise (in percentages) (column “ht”) and difference inawareness of the respondents from different localities about the existence of protected areas in their surroundings (column “pa”) 163 biologica nyssana ● 12 (2) december 2021: 159-165 nikolić et al. ● evaluation of ecological awareness and superstition on hermann’s tortoise in eastern and southern serbia superstitious people who live in non-protected areas. the population that inhabits protected areas lives in more rural environment (economically underdeveloped areas far from large settlements), so it can be assumed that this is another reason why myths and folk beliefs are more present there. in the results of the study by nikolić et al. (2019) it was stated that superstition is more present in eastern than in southern serbia. the presence of superstitions related to wild animals also means a greater threat to wildlife (kulišić et al., 1998). our previous research confirmed the assumption that local beliefs and myths can be a powerful cause of animal endangerment in both eastern and southern serbia (nikolić & crnobrnja-isailović, 2017; nikolić et al., 2018). a superstitious human population also could mean a greater impact of myths and folk beliefs on increase of hunting, killing and expelling animals from their natural habitat. in tab. 3, 72.6% (p=0.001) of the respondents who inhabit the protected areas believed that hermann’s tortoise has healing properties. myths about the healing properties of hermann’s tortoise blood, eggs or meat are most present in donji milanovac (76.3%, p=0.001), followed by kladovo (68.8%, p=0.001). also, in southern serbia, the presence of these myths was higher in protected areas, but in a smaller percentage than in the east of the country (tab. 3). one of the warning results of the research is the uninformedness of local inhabitants about the legal regulations on the protection of nature and, particularly, on the protection of the hermann’s tortoise. according to the collected data, 55.3% of the population in protected areas knows about the existence of protection regimes in their environment. although no statistically significant difference was found in relation to the population living outside the protected areas (fig. 2), the worrying fact is that only slightly more than half of the population was informed on this issue. the results from tab. 4 revealed that 68% of the inhabitants of donji milanovac knew about the existence of the national park (x2(3, n=389) = 69.080, p<0.001). thus, almost a third of the respondents did not know that they live within the territory of the largest protected area in serbia, which covers 63,608.45 ha and was placed under protection in 1974 (stanisavljević et al., 2012). the lowest level of awareness in the population from southern serbia was in leskovac–only 19% (p<0.001). there, the inhabitants live far from protected areas, so only a fifth of the total surveyed population was aware of their existence. we recorded large populations of hermann’s tortoise near all four investigated localities (nikolić et al., 2020), so we also know that the residents are in frequent contact with them. but, this study shows more warning results, and that is the attitude of the respondents on the topic of whether hermann’s tortoise should be protected. our research showed that 80.2% of the respondents are aware that hermann’s tortoise should be protected (p=0.033), but a negligible number of respondents knew that this species isalready protected by law (n=7 in total) (službeni glasnik rs, 5/2010, 47/2011, 32/2016 i 98/2016). however, among the localities, the smallest share (23%) belonged again to donji milanovac (fig. 3). as 25.8% (p=0.033) of the inhabitants of donji milanovac were aware that hermann’s tortoise should not be protected (tab. 5), and, at the same time, the presence of folk beliefs and myths was greatest in that locality, this lack of informedness plus superstition suppose to be a direct threat to the populations of this species in the national park. what is also warning are different motives of the respondents when it comes to “protection” of the hermann’s tortoise. an indirect threat to this species can behidden by the motives for protection (ballouard et al., 2020): the motive of caring for hermann’s tortoise should be investigated further, because his “care” for tortoises can be a concern to have enough tortoises for healing, illegal hunting, or for various magical rituals. hermann’s tortoises live near humans, they are harmless, they are slow and relatively small, so people can easily take them from nature to keep as pets (williams, 1999). also, many people find that collecting hermann’s tortoises and carrying them home is safer for the tortoise, than if they are in their natural habitat, because they look vulnerable. this parallel between caring for and endangering tortoises is widespread in children (ballouard et al., 2020). the adequate tool for reducing this type of negative anthropogenic impact in creating successful conservation strategies is education (howe, 2009). education of young people (students and other locals), should become a priority in the sphere of nature conservation, as table 5. the attitude of the respondents per locality that the hermann’s tortoise should be protected and preserved locality % of respondents df syg. donji milanovac 74.2 3 0.033 kladovo 79.6 3 niš 89.9 3 leskovac 77.0 3 well as continued education of the locals as a part of conservation activities in protected areas. the presence of myths and superstitions, as well as residents’ perceptions of the benefits and costs of hermann’s tortoise protection was, according to our knowledge, examined for the first time in the recent scientific literature in serbia. it revealed that the conflict between the humans and this species still cannot be neglected and that, to reduce this conflict, future conservation measures would have to take into account the views of the local population, and even to involve the locals in the planning and implementation of conservation actions. acknowledgements. we are grateful to the rufford small grants foundation for financial support (grants no. 18761-1, 22238-2, 26448-b and 34161-d). we are also indebted to many volunteers – members of biological society “dr. sava petrovic” for participating in the suveying. thanks to phd biljana macura for her help in creating the questionnaire and thanks to phd miloš popović for help in creating maps for the manuscript. jci was also supported the ministry of education, science and technological development of the republic of serbia (contracts nos. 451-03-68/2020-14/200007 and 451-0368/2020-14/200124). references alves, r.r.n., souto, w.m.s. 2015: ethnozoology: a brief introduction. ethnobiology and conservation, 4: 1–13. ballouard, j. m., conord, m., johany, a., jardé, n., caron, s., deleuze, s., bonnet, x. 2020: is popularity a double-edged sword? children want to protect but also harvest tortoises. the journal of environmental education, 51(5): 347-360. bertolero, a., cheylan, m., hailey, a., livoreil, b., willemsen, r. e. 2011: testudo hermanni (gmelin 1789) hermann’s tortoise. conservation biology of freshwater turtles and tortoises: a compilation project of the iucn/ssc tortoise and freshwater turtle specialist group. chelonian research monographs, 5: 059-1. celse, j., catard, a., caron, s., ballouard, j. m., gagno, s., jardé, n., petenian, f. 2014: management guide of populations and habitats of the hermann’s tortoise. life 08 nat/f/000475. arpe paca. 210 p. dickman, a. j. 2010: complexities of conflict: the importance of considering social factors for effectively resolving human–wildlife conflict. animal conservation, 13(5): 458-466. durst, r., mikuška, t. 2017: hunting and bird crime along the adriatic flyway a review of hunting legislation, law enforcement and driving forces. in: sackl p., ferger s. w. (eds.), adriatic flyway – bird conservation on the balkans, 7 – 15, euronatur, radolfzell. đorđević, s., ljubisavlјević, k. 2015: testudo hermanni. in: tomović, l., kalezić, m., džukić, g. (eds): red book of fauna of serbia ii – reptiles, 151– 157, faculty of biology, university of belgrade and institute for nature conservation of serbia. ebua, v. b., agwafo, t. e., fonkwo, s. n. 2011: attitudes and perceptions as threats to wildlife conservation in the bakossi area, south west cameroon. international journal of biodiversity and conservation, 3(12): 631-636. ehrlich, p.r., ehrlich, a.h. 2008: the dominant animal: human evolution and the environment. island press. 440 p. fernández–chacón, a., bertolero, a., amengual, a., tavecchia, g., homar, v., oro, d. 2011: spatial heterogeneity in the effects of climate change on the population dynamics of a mediterranean tortoise. global change biology, 17: 3075–3088. golubović, a., tomović, l., nikolić, m., nikolić, s., anđelković, m., arsovski, d., ivković, v., gvozdenović, s., popović, m. 2019: distribution of hermann’s tortoise across serbia with implications for conservation. archives of biological sciences, 71(3): 509-516. howe, c. 2009: the role of education as a tool for environmental conservation and sustainable development. phd thesis. imperial college london. jovanović, p., ajtić, r. 2011: priručnik za kontrolu prekograničnog prometa i trgovine zaštićenim vrstama. ministarstvo životne sredine, rudarstva i prostornog planiranja. beograd. 146 p. jovanović, s., vukićević, a., rankov, m., ružić, m., moldvai, k. 2020: poslednji let prepoznajte i sprečite stradanje ptica. društvo za zaštitu i proučavanje ptica srbije. novi sad. 31p. kulišić, š., petrović, p. ž., pantelić, n., matlas, m., cvetković, b. 1998: srpski mitološki rečnik. etnografski institut sanu. beograd 328 p. macura, b., zorondo-rodríguez, f., grausatorras, m., demps, k., laval, m., garcia, c. a., reyes-garcía, v. 2011: local community attitudes toward forests outside protected areas in india. impact of legal awareness, trust, and participation. ecology and society, 16(3): 10. nikolić, m., đurđević, a., petković, s., crnobrnja-isailović, j. 2018: narodna verovanja i divlje životinje u srbiji. biological society “dr sava petrović”, niš, serbia. 27 p. 164 biologica nyssana ● 12 (2) december 2021: 159-165 nikolić et al. ● evaluation of ecological awareness and superstition on hermann’s tortoise in eastern and southern serbia 165 biologica nyssana ● 12 (2) december 2021: 159-165 nikolić et al. ● evaluation of ecological awareness and superstition on hermann’s tortoise in eastern and southern serbia nikolić, m., cvetković, j., savić-zdravković, d., conić, j., ilić, m., marković, s., vučković, a., macura, b., crnobrnja-isailović, j. 2019: wildlife conservation and local folklore. 13th symposium on the flora of southeastern serbia and neighboring regions, stara planina mt. june 20-23, 2019. nikolić, m., cvetković, j., stojadinović, d., crnobrnja-isailović, j. 2020: macro-and microhabitat preferences of eastern hermann’s tortoise (testudo hermanni boettgeri). amphibiareptilia, 41(3): 313-322. nikolić, m., crnobrnja-isailović, j. 2017: uticaj lokalnog folklora i kulturnog nasleđa na odnos čoveka prema šumskoj kornjači (testudo hermanni) u srbiji. biološko društvo “dr sava petrović”, niš. 18 p. nikolić, s., golubović, a. 2017: confiscated emys orbicularis l. (1758) dying out in a “temporary” reception facility in serbia: a case study showing the urgency for a regional reptile rescue centre. acta zoologica bulgarica supplementum, 10: 115-120. ramstad, k. m., nelson, n.j., paine, g., beech, d., paul, a., paul, p., allendorf, f. w., daugherty, c. h. 2007: species and cultural conservation in new zealand: maori traditional ecological knowledge of tuatara. conservation biology, 21:455-464. rowcliffe, j.m., de merode e., cowlishaw g. 2004: do wildlife laws work? species protection and the application of a prey choice model to poaching decisions. proceedings of the royal society of london, 271: 2631–2636. røskaft,e., händel, b., bjerke, t., kaltenborn, b. p. 2007: human attitudes towards large carnivores in norway. wildlife biology, 13(2): 172-185. salom, a., suárez, m. e., destefano, c. a., cereghetti, j., vargas, f. h., grande, j. m. 2021: human-wildlife conflicts in the southern yungas: what role do raptors play for local settlers?. animals, 11(5): 1428. službeni glasnik republike srbije (5/2010, 47/2011, 32/2016 i 98/2016): pravilnik o proglašenju i zaštiti strogo zaštićenih divlјih vrsta bilјaka, životinja i glјiva. jp službeni glasnik, beograd. stanisavljević, b., ćosić, n., jelić, i. 2012: vodič kroz biološku i kulturnu raznovrsnost np đerdap. ekološko društvo “endemit”, beograd. talukdar, s., gupta, a. 2018: attitudes towards forest and wildlife, and conservation-oriented traditions, around chakrashila wildlife sanctuary, assam, india. oryx, 52(3): 508-518. tolstoj, s. m., radenković, l. (eds). 2001: slovenska mitologija: enciklopedijski rečnik. zepter book world. tomović lj, kalezić m. džukić g. (eds) 2015: crvena knjiga faune srbije ii – gmizavci. faculty of biology, university of belgrade and institute for nature conservation of serbia. van dijk, p. p., corti, c., mellado, v. p. cheylan, m. 2004: testudo hermanni (errata version published in 2020). the iucn red list of threatened species 2004: e.t21648a176604335. downloaded on 03 september 2021. veličković, v., jović, m., nalić, e., višnjić, a., radulović, o., šagrić, č., ćirić, m. 2015: knowledge, attitudes toward, and acceptability of genetic modification among western balkan university students of life sciences (agree study). journal of the american college of nutrition, 35(2): 150-162. williams, t. 1999: the terrible turtle trade: the pet trade is decimating turtle populations and spreading disease. one veterinarian calls it” the greatest reptile crisis since the demise of the dinosaur”. audubon-new york, 101: 44–51. wright, a. j., veríssimo, d., pilfold, k., parsons, e. c. m., ventre, k., cousins, j.,jefferson, r., koldewey, h., llewellyn, f., mckinley, e. 2015: competitive outreach in the 21st century: why we need conservation marketing. ocean & coastal management, 115: 41-48. microsoft word 0402_stanisavljevic_et_al ispravljeno biologica nyssana 1 (1-2) december 2010: 89-93 stanisavljević, d.m. et al. effects of different drying methods… 89 original article ! effects of different drying methods on the yield and the composition of essential oil from herb mentha longifolia (l.) hudson dragana m. stanisavljević1, sofija m. đorđević2, mihailo s. ristić2, dragan t. veličković1, novica v. ranđelović3 1 college of agriculture and food technology, ćirila and metodija 1, 18400 prokuplje, serbia 2 institute for medicinal plant research "dr josif pančić", tadeuša košćuška 1, 11000 belgrade, serbia 3 university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia * e-mail: draganastanisavljevic72@gmail.com abstract: stanisavljević, d.m., đorđević, s.m., ristić, m.s., veličković, d.t., ranđelović, n.v.: effects of different drying methods on the yield and the composition of essential oil from herb mentha longifolia (l.) hudson. biologica nyssana, 1 (1-2), december 2010: 89-93. this paper discusses the impact of different methods of drying on the content and chemical composition of the essential oil from the herb mentha longifolia (l.) hudson. drying of plant material was carried out naturally in the shade of draughty place, in the laboratory oven at the temperature 45°c and absorptive low temperature condensation drying oven at 35°c (low temperature drying). isolation of essential oil from dried samples in three different ways was conducted by hydrodistillation, whilst chemical analysis was carried out by gc/fid and gc/ms methods. the highest yield of the essential oil was obtained from the herb which was dried at low temperature (1.1%) and the lowest from that dried in the laboratory oven (0.6%). the biggest content of the dominant component of essential oils, piperitone, was recorded in the oil from low temperature dried herb (71.7%), while those isolated from naturally dried drug and in from the laboratory oven contained piperitone in lower concentrations (50.8% and 43.1%, respectively). key words: drying, essential oil, mentha longifolia l., piperiton introduction mentha longifolia (l) hudson, (wild mint, english horsemint) belongs to the genus mentha, lamiaceae family. inhabits wet habitats, the rivers, lakes, ponds and channels, wet meadows, forested terrain, and even drier places along the roads and arable land. it is widespread in serbia except in vojvodina, where it can be found only on fruška gora. in south serbia it is widespread from the regions of lowlands, where it can be found along the roads and on the banks of rivers, to the hilly, near the streams in the community junco-menthetum longifolia. it is widespread throughout the mediterranean, central and northern europe, asia minor, africa (j a n k o v i ć , 1974; s t a m e n k o v i ć , 1995). wild mint is an aromatic and melliferous plant. it is used in the pharmaceutical, tobacco, food industry (in the development of various liqueurs and sweets), and especially in cosmetology. essential oil of english horsemint has pleasant and refreshing odour. according to the pdr exhibits carminative 10th sfses • 17-20 june 2010, vlasina lake1 (1-2) • december 2010: 89-93 biologica nyssana 1 (1-2) december 2010: 89-93 stanisavljević, d.m. et al. effects of different drying methods… 90 and stimulant properties of the gastrointestinal tract. relieves colds, respiratory inflammation, headaches, pain in muscles and joints. internally it is used in the form of infusion, and externally as bath additive (pdr, 2004). there are phytopreparations on the basis of the active ingredients of essential oils of mint (menthol, menthone, pinene, and cineole) in the form of cream, the solution of rubbing (alcohol, oil), syrup, capsule, coated tablet (rote liste, 1997). chemical composition of the essential oil of wild mint herb is very variable depending on the habitat and climate where the species grow. fortyfive constituents were identified in the essential oil of m. longifolia from turkey, with the cis-epoxy piperitone, pulegone and piperitenone oxide as main components, and studied oil exhibits strong antimicrobial activity (g u l l u c e et al., 2007). analysis of essential oil of moroccan m. longifolia showed interesting relative quantities of piperitenone oxide and piperitone oxide (ghoulami et al., 2001). in the essential oil of wild mint from south africa, 31 components were identified. menthone (50.9%), pulegone (19.3%) and 1,8cineole (11.9%) were the main ingredients of the oil (oyedeji and afolayan, 2006). analysis of oil of m. longifolia from italy and israel revealed piperitenone oxide as the main component, while the essential oil from sinai contained 1,8-cineole (28.8%), piperitone oxide (15.4%) and piperitone (13.8%) (m a f f e i , 1988; f l e i s h e r & f l e i s h e r , 1998). there is more then 70% of pulegone in the oil that grows in the desert of jordan (f l e i s h e r & f l e i s h e r , 1991). the dominant components of essential oil of wild mint herb in vojvodina are menthone, isomenthone and 1,8cineole, and the oil exhibits a strong antimicrobial and significant fungicidal effect (m i m i c a et al., 2003). wild mint from croatia contains carvone, piperitenone oxide, limonene and β-caryophyllene as the main ingredients (m a s t e l i ć & j e r k o v i c , 2002). the main components of oil types which were collected on zlatar are cisand transdihidrocarvone (15.9% and 30.6%) (m a t o v i ć & l a v a d i n o v i ć , 1999). piperitone oxide was found as the main component in nine populations of m. longifolia essential oils from greece (k o k k i n i & p a p a g e o r g i o u , 1988). drying is the easiest way of preservation of raw plant material. drying procedures are different and have an impact on the content of active substances in drugs. in the process of drying the plant material moisture content is reduced, but the amount and composition of volatile compounds are changed, too (m o y l e r , 1994). the way of drying has a significant impact on qualitative and quantitative composition of essential oils of aromatic plants. this paper presents the results of the impact of different methods of drying on the yield and the composition of essential oil of m. longifolia herb originating from southern serbia. material and methods plant material plant material was collected in the flowering stage in july 2009 from the municipality of prokuplje, at the rastovnica village, on the mountain pasjača. samples of collected plants are deposited in the herbarium hmd (herbarium moesiacum doljevac) № 233. plants were collected in the morning hours, in dry times and dried in three ways: 15 days in the shade of a draughty place, natural drying (nd), in the laboratory oven at the temperature of 45°c (lod) and in the low temperature absorptive condensation dryer (ntks/60s) at temperature of about 35°c (ltd). the drying air used was of low humidity and a minimally heated. dried plant material was packed in paper bags and kept in a dry and cool place. isolation and determination of the content of the essential oil isolation of the essential oil from dried and cut m. longifolia herb was carried out by water distillation in the clevenger type apparatus, according to procedure ph. jug. iv (ph. jug. iv, 1984). distillation lasted about 2 hours at the boiling temperature. pure essential oils were kept in dark glass ampoules at +4°c. these are colourless liquids, with the characteristic sharp odour. results of determination of the contents of the essential oils in the three studied samples of the herb m. longifolia represent the average value of three comparative analyses and refer to the dry samples. qualitative and quantitative analysis of the essential oils qualitative and quantitative analysis of essential oils was conducted by gc and gc/ms methods. analytical gas chromatography (gc/fid) gc/fid analysis of the oils was carried out on a hp-5890 series ii gc apparatus [hewlettpackard, waldbronn (germany)], equipped with split-splitless injector and automatic liquid sampler (als), attached to hp-5 column (25 m · 0.32 mm, 0.52 µm film thickness) and fitted to flame ionisation detector (fid). carrier gas flow rate (h2) was 1 ml/min, split ratio 1:30, injector temperature biologica nyssana 1 (1-2) december 2010: 89-93 stanisavljević, d.m. et al. effects of different drying methods… 91 table 1 the chemical composition of essential oils of herb m. longifolia dried by different procedures (%) constituents kie kil drying method nd lod ltd α-pinene 932.8 932 0.8 0.6 0.6 sabinene 973.2 969 0.7 0.5 0.4 β-pinene 975.6 974 1.3 0.9 0.8 myrcene 993.0 988 0.7 1.2 0.5 3-octanol 1000.2 988 0.3 0.2 limonene 1029.0 1024 6.3 1.6 2.4 1,8-cineole 1030.9 1026 3.9 3.6 3.5 cis-β-ocimene 1039.6 1032 1.3 0.8 1.2 trans-β-ocimene 1049.2 1044 0.4 linalool 1102.8 1095 0.4 menthone 1154.6 1148 0.6 17.5 iso-menthone 1165.1 1158 8.3 ocimenol 1169.4 n/a 0.3 menthol 1175.3 1167 0.3 cis-isopulegone 1177.1 n/a 0.7 α-terpineol 1193.4 1186 1.1 0.3 0.5 cis-dihydro carvone 1198.2 1191 3.5 0.6 0.3 trans-dihydro carvone 1205.4 1200 2.3 1.1 neoiso-dihydro carveol 1231.8 1226 0.8 cis-3-hexenyl isovalerate 1238.8 1232 0.5 pulegone 1240.7 1233 1.4 carvone 1247.1 1239 20.0 2.9 5.0 piperitone 1257.7 1249 50.8 43.1 71.7 dihydroedulan i 1288.6 1289 0.3 piperitenone oxide 1369.0 1366 2.7 β-bourbonene 1385.1 1387 0.8 β-elemene 1392.9 1389 0.3 trans-caryophyllene 1420.1 1417 4.3 4.1 5.4 α-humulene 1454.1 1452 0.3 γ-muurolene 1482.3 1478 3.1 4.3 3.6 bicyclogermacrene 1497.6 1500 0.4 1.1 0.8 sum of contents % 100.0 100.0 100.0 number of constituents 18 24 20 kie=kovats (retention) index experimentally determined (amdis), kil=kovats (retention) index literature data (a d a m s , 2007), n/a=not available, nd-natural drying , lod-laboratory drying oven, ltd-low temperature drying was 250°c, detector temperature 300°c, while column temperature was linearly programmed from 40-260°c (at rate of 4°/min). solutions of essential oils (~1%) were consecutively injected by als (1 µl, split mode). area percent reports, obtained as result of standard processing of chromatograms, were used as base for the quantification purposes. gas chromatography/mass spectrometry (gc/ms) the same analytical conditions as those mentioned for gc/fid were employed for gc/ms analysis, along with column hp-5ms (30 m · 0.25 mm, 0.25 µm film thickness), using hp g 1800c series ii gcd system [hewlett-packard, palo alto, ca (usa)]. instead of hydrogen, helium was used as carrier gas. transfer line was heated at 260°c. mass spectra were acquired in ei mode (70 ev), in m/z range 40-450. solutions of the essential oils (~1%) were injected by als (200 nl, split mode). the constituents were identified by comparison of their mass spectra to those from wiley275 and nist/nbs libraries, using different search engines. the experimental values for retention indices were determined by the use of calibrated automated mass spectral deconvolution and identification system software (amdis ver.2.1.), compared to those from available literature biologica nyssana 1 (1-2) december 2010: 89-93 stanisavljević, d.m. et al. effects of different drying methods… 92 (adams, 2007), and used as additional tool to approve ms findings. results and disscusion the content of the essential oil from aboveground part of m. longifolia, which has been dried on the three described ways, was variable. the highest yield is obtained from the herb dried at low temperature (1.1%), and then the herb which was dried naturally (0.9%), while the lowest yield was obtained in the laboratory oven (0.6%). chemical composition of essential oils is also variable (tab. 1). twenty-four components were identified in the essential oil obtained from wild mint herb that was dried in the laboratory oven. content of dominant component (piperitone) was 43.1%, along with 1,8-cineole, 3.6%, limonene 1.6%, menthone 17.5%, isomenthone 8.3%, carvone 2.9%, transcaryophyllene 4.1% and γ-muurolene 4.3%. in the essential oil obtained from the naturally dried herb 18 components were identified. content of piperitone was 50.8%, limonene 6.3%, 1,8cineole 3.9%, menthone only 0.6%, carvone 20.0 %, trans-caryophyllene 4.3%, γ-muurolene 3.1%. isomenthone was not recorded. in the essential oil from herb dried in low temperature oven 20 components were identified. content of piperitone was 71.7%, 1,8-cineole 3.5%, limonene 2.4%, carvone 5.0%, trans-caryophyllene 5.4% and γ-muurolene 3.6%. menthone and isomentone were not registered. changes in the concentrations of volatile compounds during drying depend on several factors, such as drying methods and classes of plants. mint belongs to the family lamiaceae, which is known to have storage of essential oil on or near the surface of the leaf (m o y l e r , 1994). pulegone has been proved to be a powerful hepatotoxin, even at low concentrations. it is metabolized by the liver to menthofuran, which is highly reactive metabolite, and it has adverse effect on the liver (c h e n et al., 2001, g o r d o n et al., 1987), and it can also destroy the rat cytochrome p450 (m o o r t h i , 1991). in our tests we can find pulegone only in the oil which was isolated from wild mint herb dried in laboratory oven. conclusion from the results it can be concluded that the highest essential oil content was found in plant material dried at low temperature, then dried naturally, and the lowest content was found in the plant material dried in laboratory oven. the content of the major constituent, piperitone, is reduced in the same order (71.7%, 50.8%. 43.1%). pharmacologically active menthol, cineole, limonene and pinene are the most represented in the oil from the naturally dried herb. so, drying of plant material for isolation of essential oils at low temperatures is could be marked as the best method. simultaneously, draying in the natural way is quite acceptable. pulegone is present only in the oil obtained from the dried herb in a laboratory oven, and from the security aspects this method of drying should be avoided. acknowledgements. the authors wish to thank serbian ministry of science for financial support project № tr 20049. references adams, r. 2007: identification of essential oil components by gas chromatography/mass spectrometry, 4th ed., allured publishing corp., carol stream, il 60188 usa. chen, lj., lebetkin, eh., burka, lt. 2001: metabolism of (r)-(+)-pulegone in f344 rats. drug metabolism and disposition, 29(12): 1567– 1577. farmakopeja sfrj, pharmacopoea jugoslavica, editio quatra (ph. jug. iv), 1984, savezni zavod za zdravstvenu zaštitu, beograd. fleisher, a., fleisher, z. 1991: the essential oils from mentha longifolia growing in sinai and israel aromatic plants of the holy land and the sinai. part iv. journal of essential oil research, 3(1): 57-8. fleisher, z., fleisher, a. 1998: volatile extract of mentha longifolia growing in israel aromatic plants of the holy land and the sinai. part xiii, journal of essential oil research, 10(6): 647-648. ghoulami, s., idrissi, a., fkih-tetouani, s. 2001: phytochemical study of mentha longifolia of morocco, fitoterapia, 72(5): 596-598. gordon, wp., huitric, ac., seth, cl., mcclanahan, rh., nelson, sd. 1987: the metabolism of the abortifacient terpene, (r)-(+)pulegone, to a proximate toxin, menthofuran. drug metabolism and disposition, 15(5): 589–594. gulluce, m., sahin, f., sokmen, m., ozer, h., daferera, d., sokmen, a., polissiou, m., adiguzel, a., ozkan, h. 2007: antimicrobial and antioxidant properties of the essential oils and methanol extract from mentha longifolia l. ssp. longifolia. food chemistry, 103: 1449-1456. janković, m. 1974. rod mentha l. in: josifović, m. (ed.), flora srbije vi: 524-525, srpska akademija nauka i umetnosti, beograd. biologica nyssana 1 (1-2) december 2010: 89-93 stanisavljević, d.m. et al. effects of different drying methods… 93 kokkini, s., papageorgiou, v. 1988: constituents of essential oils from mentha longifolia growing wild in greece. planta medica, 54(1): 59-60. maffei, m. 1988: chemotype of mentha longifolia (l) hudson particularly ridh in piperitenone oxide. flavour and fragrance journal, 3(1): 23-26. mastelić, j., jerković, i. 2002: free and glycosidically bound volatiles of mentha longifolia growing in croatia. chemistry of natural compounds, 38(4): 561-564. matović, m., lavadinović, v. 1999: essential oil composition of mentha longifolia (l) huds. from the mountain zlatar in yugoslavia, journal of essential oil bearing plants, 2(2): 78-81. mimica-dukić, n., bozin, b., soković, m., mihailović, b., matavulj, m. 2003: antimicrobial and antioxidant activites of three mentha species essential oils. planta medica, 69(5): 413-419. moorthy, b. 1991: toxicity and metabolism of r-(+)pulegone in rats: its effects on hepatic cytochrome p450 in vivo and in vitro. journal of the indian institute of science, 71(1): 76-78. moyler, d. 1994: spices-recent advances. in g. charalambous (ed.), spices, herbs and edible fungi. elsevier. amsterdam, 1–70. oyedeji, ao., afolayan, aj. 2006: chemical composition and antibacterial activity of the essential oil isolated from south african mentha longifolia (l.) ssp. capensis (thunb.) briq. journal of essential oil research, 18: 57-59. pdr for herbal medicines, 3rd ed. 2004: thomson pdr at montvale., 283-284. remington, 1995: the science and practice of pharmacy, 19th ed., mack publishing company, easton. rote liste.1997: ecv editio cantor, aulendorf wurtt. shahverdi, ar., rafii, f., tavassoli, f., bagheri, m., attar, f., ghahraman, a., 2004: piperitone from mentha longifolia var. chorodictya rech f. reduces the nitrofurantoin resistance of strains of enterobacteriaceae. phytotherapy research, 18(11): 911-914. stamenković, v. (1995): neškodljive lekovite biljke. i izdanje, solaris, leskovac. williams, ac., barry, bw. 1991: terpenes and the lipid-protein-partitioning theory of skin penetration enhancement. pharmaceutical research, 8:17–24. vitorovic et al. 2021, biologica nyssana 12(2) 12 (2) december 2021: 113-122 doi: 10.5281/zenodo.5759855 antioxidant potential of commercial hemp seed oils and cbd oil original article jelena vitorović faculty of sciences and mathematics, university of niš, višegradska 33, 18000 niš, serbia jelena.rajkovic@pmf.edu.rs (corresponding author) nataša joković faculty of sciences and mathematics, university of niš, višegradska 33, 18000 niš, serbia andrea žabar popović faculty of sciences and mathematics, university of niš, višegradska 33, 18000 niš, serbia received: october 12, 2021 revised: november 24, 2021 accepted: november 25, 2021 abstract: the present study investigates the antioxidant capacity of commercial hemp seed oils, 10% cbd (cannabidiol) enriched hemp seed oil and extra virgin olive oil used for comparison. results indicate a difference in antioxidant activities among tested oils. cbd oil with ic50 of 0.86±0.06 mg/ml was shown to be a better scavenger of hydroxyl radical and dpph (1,1-diphenyl-2-picrylhydrazyl) radical (ic50 value of 5.99±0.34 mg/ml) in comparison with other oils. cbd oil also had the best ability to reduce fe3+ from ferricyanide at a concentration of 20 mg/ml (0.73) and the highest total antioxidant capacity (245±35.16 gae). two commercial fresh hemp seed oils that showed small differences in antioxidant activity had better antioxidant properties than olive oil in three performed assays, while olive oil had the highest total antioxidant capacity. hemp seed oil showed a lower antioxidant potential after a year of storage. key words: cannabis sativa, hemp, cbd, seed oil, antioxidant apstrakt: antioksidativni potencijal komercijalnih ulja iz semena konoplje i cbd ulja cilj studije je ispitivanje antioksidativnog kapaciteta komercijalnih ulja dobijenih iz semena konoplje, ulja iz semena konoplje obogaćenog 10% cbdom (kanabidiol) i ekstra devičanskog maslinovog ulja koje je korišćeno za poređenje. dobijeni rezultati ukazuju na razliku u antioksidativnoj aktivnosti među testiranim uljima. cbd ulje koje ima ic50 vrednost 0.86±0.06 mg/ml se pokazalo kao bolji “hvatač” hidroksilnog radikala i dpph (1,1-difenil2-pikrilhidrazil) radikala (ic50 vrednost 5.99±0.34 mg/ml) u poređenju sa drugim uljima. cbd ulje je takođe imalo najveću sposobnost da redukuje fe3+ u koncentraciji od 20 mg/ml (0.73) i najveći ukupni antioksidativni kapacitet (245±35.16 gae). sveža komercijalna ulja iz semena konoplje koja su pokazala male razlike u antioksidativnoj aktivnosti su imala bolje antioksidativne karakteristike od maslinovog ulja u tri korišćena testa dok je maslinovo ulje imalo najveći ukupni antioksidativni kapacitet. ulje iz semena konoplje je pokazalo slabiji antioksidativni potencijal godinu dana nakon otvaranja. ključne reči: cannabis sativa, konoplja, cbd, ulje semena, antioksidans introduction reactive oxygen species (ros) are highly reactive molecules generated normally in cells during the metabolism of oxygen. they are mainly derived from aerobic respiration during the mitochondrial electron transport but other pathways including the activity of oxidoreductase enzymes and metal catalyzed oxidation can also be involved in their production (birben et al., 2012; ozcan & ogun, 2015). ros include free radicals and radical precursors such as superoxide anion, hydroxyl radical, singlet oxygen and hydrogen peroxide. free radicals are unstable highly reactive species because of possessing unpaired electrons that tend to pair with other molecules. at physiological concentrations, they participate in cell signaling for normal biological processes including cell proliferation, apoptosis, gene expression but when the redox balance is disturbed they can cause tissue damage by the oxidation of cellular components such as membrane lipids, proteins and dnas (salganik, 2001). there is a lot of evidence that ros underly many chronic diseases including cancer, cardiovascular diseases, neurodegenerative diseases, inflammatory diseases etc. (halliwell, 2006; halliwell, 2007; ferguson, 2010). the organism defends itself using endogenous cell antioxidant protection system consisted of non© 2021 vitorović et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 113 enzymatic and enzymatic components (kurutas, 2015). it maintains balance between ros production and removal and thus prevents organism from oxidative stress and the diseases associated with it. antioxidants can also be exogenous, ingested via antioxidant-rich food and supplements and in this way, they can help the antioxidative defense of the cell to cope with free radicals directly by scavenging them (breaking the chain reaction) or indirectly by upregulating the antioxidant defense or inhibiting the ros production. they act as free radicals scavengers, reducing agents, metal chelators and singlet oxygen quenchers (lobo et al., 2010). in this way, they prevent cell damage and consequent diseases (cox et al., 2000; eastwood, 1999). the use of synthetic antioxidants as food preservatives or supplements has been shown to be harmful to health (witschi, 1986). on the other hand, plants are rich source of natural antioxidants such as phenolics, flavonoids, tannins, carotenoids and vitamins with excellent antioxidant properties that have been shown in many studies. therefore, consuming food rich in natural antioxidants is crucial for health and the development of natural products with antioxidants is of great interest to the food industry. the plant seed oils occupy a special place in the food industry due to their nutritional value and high content of unsaturated fatty acids, as well as due to antioxidant compounds which, in addition to contributing to the stability of the oil, also have various pharmacological properties (kriese et al., 2004). various seed oils that have good antioxidant capacity are in use (ramadan & moersel, 2006; smeriglio et al., 2016). among them, special attention is paid to hemp seed oil (hso), the product of the cannabis sativa l. (cannabaceae) plant, due to the specific ratio of fatty acids in its composition. the oil obtained from hemp seeds has high nutritional value and a number of health effects (leizer et al., 2000; callaway, 2004). it contains >80% polyunsaturated fatty acids (pufas) with linoleic acid as dominant, present in concentration of 52-62%, followed with α-linolenic acid in a concentrations ranging from 12-23% (leizer et al., 2000). the linoleic acid (18:2 omega-6) and αlinolenic acid (18:3 omega-3) are present in a favorable ratio of 3:1 found to contribute to the better lipid profiles and have a cardioprotective effect (callaway, 2004; simopoulos, 2008; yang et al., 2016). hso contained also γ-linolenic acid that showed significant anti-inflammatory and immunoregulatory activity (kapoor & huang, 2006). the chemical analysis of the hemp seed oil also showed the presence of tocopherols, phenols, polyphenols and lignanamides which contribute to the stability of the oil and at the same time they are natural antioxidants desirable in nutrition (kriese et al., 2004; yan et al., 2015; andre et al., 2016; smeriglio et al., 2016). tocopherols are significant in the prevention of the oxidation of pufa in oils and have many beneficial health effects including prevention of cardiovascular diseases, cancer, and age-related macular degeneration (kriese et al., 2004). the chemical composition of the hemp seed oil indicates its function in maintaining redox balance and lowering oxidative stress and associated diseases. in vivo studies have shown a beneficial effect of hemp seed oil on oxidative status and reduction of oxidative stress (vitorović et al., 2021). cannabis sativa l. plant is known for the presence of compounds called cannabinoids such as delta-9 tetrahydrocannabinol (thc) and cannabidiol (cbd). unlike thc, the most famous psychoactive cannabinoid, cannabidiol is a non-psychoactive phytocannabinoid obtained from leaves and flowers of the hemp plant that showed many pharmacological activities and therefore it is in the center of interests of the pharmacological industry. however, although preclinical and pilot studies suggest the use of cbd in the treatment of different conditions such as epilepsy, migraine, alzheimer’s disease, chronic pain, inflammatory conditions etc. (friedman & devinsky, 2015; watt and karl, 2017; irving et al., 2018; van dolah et al., 2019), it is not allowed yet as a therapeutic drugs. it is already known that cbd has anti-inflammatory and antioxidant activities (costa et al., 2007; irving et al., 2018; pellati et al., 2018; atalay et al., 2020) which make it a promising agent in the prevention and treatment of illness associated with oxidative stress. cbd showed both, direct antioxidant effects through the impact on the ros generation and components of antioxidative defense and indirect antioxidant effects, reacting with other molecules that are important for redox balance (atalay et al., 2020). the hemp industry become increasingly popular worldwide and many different commercial hemp products can be found in the market. the aim of the present study was to examine the possible differences in the antioxidant potential of commercial hemp seed oils from different manufacturers and to compare the effectiveness with the antioxidant capacity of cbd oil. the antioxidant activity was also compared with the well-known extra virgin olive oil, known to have oleic acid (mufa) as a basic fatty acid in the oil (5583%) and a lot of minor compounds (about 2% of the olive oil weight) including polyphenols, carotenoids, squalene and tocopherols (cicerale et al., 2008; kabaran, 2018). since the hemp seed oil is highly rich in unsaturated fatty acids that are susceptible to oxidation which impairs their stability and function, another goal of the study was to examine how one year of storage affects the antioxidant ability of 114 biologica nyssana ● 12 (2) december 2021: 113-122 vitorović et al. ● antioxidant potential of commercial hemp seed oils and cbd oil 115 the oil. in this case, the same commercial oil was used for the tests after the opening (hso2) and a year later (hso1) and the obtained results were compared. materials and methods oil samples in our study, we used two commercial hemp seed oils (world of hemp, kisac and gramina, novi sad), commercial cbd oil (gramina, novi sad) and extra virgin olive oil (latzimas s.a., crete). hemp seed oils were obtained by cold pressing while cbd oil (cbd enriched hemp seed oil) represents 10% ethanolic extract of cannabidiol dissolved in the cold pressed hemp seed oil. oil samples used in study are listed in the tab. 1. old and new hemp seed oils represent the samples of the same oil analyzed immediately after opening (new hemp seed oil, hso2) and one year after opening (old hemp seed oil, hso1). different oil concentrations in antioxidant assays were prepared in ethanol. bht was used as a positive control in all tests. in vitro antioxidant activities of oils dpph radical scavenging assay dpph assay is using to assess the reduction capacity of the antioxidants toward dpph radical. the method is based on the scavenging of the 1,1-diphenyl-2-picryl-hydrazyl (dpph) radical by the antioxidants which change the initial purple color of dpph solution to pale yellow due to reduction reaction. the assay was performed according to the previously described method with some modifications (minioti & georgiou, 2010). hemp seed oils and olive oil in concentrations of 20, 60, 80, and 120 mg/ml and cbd oil in concentrations of 1, 5, 10, 15 and 20 mg/ml were mixed with 4 ml of dpph solution in ethanol (0.1 mm). the mixtures were placed in the dark for 1 hour at room biologica nyssana ● 12 (2) december 2021: 113-122 vitorović et al. ● antioxidant potential of commercial hemp seed oils and cbd oil temperature. discoloration of the initial purple color of dpph solution indicates the antioxidant potential of oils, which was measured at 517 nm against ethanol blank on uv-vis spectrophotometer (shimadzu, 1650 pc). dpph scavenging activity was calculated from the following formula: dpph scavenging (%)=((abcontrol–absample)/ abcontrol)*100 ic50 which represents the concentration of the oil required to scavenge 50% of dpph was calculated and utilized for easier comparations. the lower ic50 indicate the better antioxidant capacity of oils. oh radical scavenging assay hydroxyl radical scavenging assay was conducted according to the previous study with some modifications (yang et al., 2017). hydroxyl radical was generated in vitro through the fenton reaction model system. the addition of the salicylic acid (trapping agents) served to trap hydroxyl radicals because of its very short half-life leading to produce dihydroxybenzoic acids with absorption at 510 nm. samples were dissolved in ethanol in concentrations of 0.5, 1, 2, 4 and 6 mg/ml for hsos and olive oil and 0.01, 0.05, 0.1, 0.5, 1 and 2 mg/ml for cbd oil. 2 ml of each oil concentration was mixed with 0.6 ml 8 mm aqueous solution of feso4 and 2 ml 3 mm ethanol solution of salicylic acid. the reaction was activated by adding 0.5 ml of 0.6% h2o2, and the mixture was incubated at 37 °c for 30 min. after cooling, mixtures were centrifuged at 2000 rpm for 10 min and absorbances of supernatants were measured at 510 nm. the hydroxyl radical scavenging activity (%) was calculated from the formula: [1 – (aa – ac) /ab] × 100% where: aa represents the absorbance of the whole sample, ab represents mixture without hydrogen peroxide, with 0.5 ml dh20 , ac represents blank without oil with 2 ml of ethanol. ic50 was determined from the plot where % scavenging activity is presented in the function of concentration and utilized for easier comparations. ferric reducing power assay the method was used according to previous studies (baba & malik, 2015; bhatti et al., 2015). 0.2 ml of each oil (concentrations 5, 10, 20, 40 mg/ml for hsos and olive oil and 0.1, 0.5, 1, 10 and 20 mg/ ml for cbd oil) was added to 0.5 ml of 0.2 mm, ph 6.6 phosphate buffer and 0.5 ml of 1% potassium ferricyanide. this mixture was incubated for 20 table 1. oil samples used in antioxidant tests samples abv. manufacturer old hemp seed oil hso1 world of hemp, kisac new hemp seed oil hso2 world of hemp, kisac gramina hemp seed oil hso3 gramina, novi sad extra virgin olive oil evoo latzimas s.a., crete cannabidiol oil cbd gramina, novi sad 116 minutes at 50 °c. then, 0.5 ml of 10% tca was added to the mixture followed by centrifugation at 3000 rpm for 10 minutes. 0.5 ml of supernatant was mixed with 0.5 ml of distilled water and 0.1 ml of ferric chloride (0.1%). after incubation at room temperature for 10 minutes, the absorbances were measured at 700 nm. the reducing power of the oil is represented as a function of concentration. phosphomolybdenum assay for total antioxidant capacity to determine the total antioxidant capacity of the oils, the test with phosphomolybdenum was performed and the gallic acid was used as a standard. the test is based on the reduction of mo (vi) to mo (v) by the antioxidants and formation of a green phosphate/mo (v) complex at acid ph. hemp seed oils and olive oil were tested in different concentrations prepared in ethanol (5, 20, 60 and 120 mg/ml). cbd oil was tested in concentrations: 0.1, 0.5, 1, 5 and 10 mg/ml. in the test, 0.1 ml of each concentration was mixed with a previously prepared reagent composed of 0.6 m sulfuric acid, 28 mm sodium phosphate and 4 mm ammonium molybdate as described previously (chahar et al., 2012). incubation was performed at 95 °c for 90 min. the mixtures were cooled and the absorbances were measured at 695 nm. standard curve of gallic acid prepared in concentrations of 50, 100, 150, 200 and 250 µg/ml in ethanol was used to express the total antioxidant capacity of oil as a µg equivalent of gallic acid (gae). statistical analysis all the data were expressed as means ± standard deviation (sd). the one-way analysis of variance (anova) and tukey’s post hoc multiple comparison test were used for analysis. for p<0.05 differences were considered statistically significant. results and discussion free radical scavenging activity free radicals are very dangerous reactive species capable of damaging biomolecules in cells such as dna, proteins and lipids (salganik, 2001). therefore, the capability to scavenge free radicals is a very important feature of antioxidants. in our study, the capability of oils to scavenge free radicals were tested in dpph and oh scavenging assays (tab. 2, fig. 1 and 2). dpph scavenging activity dpph assay was conducted to estimate the antiradical activity of different oils toward dpph radical. results are presented in tab. 2 and fig. 1. cbd oil showed the best free radical scavenging activity in dpph assay with ic50 of 5.99±0.34 mg/ ml. the percentage of scavenging activity ranged from 32.34% to 99.69 % in the used concentrations of cbd oil (1 to 20 mg/ml). to reach 50% efficiency in scavenging dpph radicals, it is required about ten times higher concentration of evoo and hso1 and about seven times higher concentration of hso2 and hso3 compared to cbd oil. maximum inhibitory activity was noticed in the concentration of 120 mg/ ml for all these oils and fig. 1a shows that hso2 and hso3 showed a similar correlation between the concentration and the scavenging activity. the other two oils were less efficient scavengers until the concentration of 120 mg/ml where all the oils achieved a high degree of inhibition. this can also be seen from the calculated ic50value (tab. 2) which was lower for the hso2 and hso3 oils (39.96±1.01 and 43.09±1.21 respectively) than for hso1 and evoo (61.42±0.81 and 64.03±0.81, respectively). results also indicate a lowering in the antioxidant capacity of the oil after one year of storage. the capacity of scavenging was reduced by 35% in biologica nyssana ● 12 (2) december 2021: 113-122 vitorović et al. ● antioxidant potential of commercial hemp seed oils and cbd oil table 2. antioxidant activities of hemp seed, cbd and extra virgin olive oil antioxidant assays samples hso1 hso2 hso3 evoo cbd bht dpph assay (ic50, mg/ml) 61.42±0.81b 39.96±1.01d 43.09±1.2c 64.03±0.8a 5.99±0.34e 0.06±0.01f phosphomolybdenum assay (gae) 13±1.04b 28.6±2.07b 35.9±0.99b 40.3±3.97b 245±35.2a / hydroxyl radical scavenging assay (ic50, mg/ml) 6.45±0.35b 4.61±0.21c 3.56±0.15d 7.15±0.24a 0.86±0.06e 1.36±0.07e data are presented as mean ± sd of triplicate determinations. values in the same row with different superscript letters are significantly different (p<0.05). bht was used as a positive control 117 biologica nyssana ● 12 (2) december 2021: 113-122 vitorović et al. ● antioxidant potential of commercial hemp seed oils and cbd oil one year and, according to results this reduced oil activity was a little lower than the activity of olive oil to scavenge dpph radical (tab. 2). the most commonly used antioxidant bht had much lower ic50 than all oils (0.06±0.01 mg/ml). however, it is shown that synthetic antioxidants such as bht can cause organ damage and act as tumor promoting compounds in laboratory animals (vuolo & schuessler, 1985; witschi, 1986) and that is the most important reason for using natural antioxidants, safe and effective in reducing oxidative stress. dpph assay as a widely used test for measuring the antiradical ability of bioactive compounds belongs to a group of tests based on both, single electron transfer (sem) and hydrogen atom transfer (hat) mechanisms. studies that investigated in vitro antioxidant activity of seed oils showed their good scavenging activity toward dpph radical indicating that the unsaturated fatty acids, phospholipids, phytosterols and tocopherols are carriers of this activity (luzia et al., 2013; cherbi et al., 2017). in the paper of smeriglio et al. (2016), finola hso also showed antiradical activity and the percentage of inhibition of free radicals was the highest in the case of whole oil followed by lipophilic and hydrophilic fractions. the authors explained this activity by means of the presence of high content of flavonoids in oil that are effective in free radical scavenging and metal chelating activity fig. 1. dpph scavenging activity a) hemp seed oils and extra virgin olive oil, b) cbd oil, c) bht. data are presented as mean ± sd of triplicate determinations of the percentage scavenging of free radicals. bht was used as a positive control fig. 2. hydroxyl free radical scavenging activity presented via ic50 values (mg/ml) for different oils. data are presented as mean ± sd of triplicate determinations. values marked with different letters are significantly different (p<0.05). bht was used as a positive control (kandaswami & middleton, 1994). the weaker dpph radical scavenging activity of olive oil in comparison with hso noticed in our study was also showed in the study of ramadan et al. (2006). they found a negative correlation between radical scavenging activity and amounts of phenolics and tocopherols and a positive correlation with levels of unsaponifiable and phytosterols among oils with the high radical scavenging activity level (coriander seed oil and black cumin seed oil) and concluded that scavenging of dpph was combined action of different compounds in oils. it is most likely that the composition and proportion of the fatty acids and ancillary components from seed oils such are lipophilic flavonoids, tocopherol, or some other bioactive substances, are responsible for the dpph scavenging by hso. in our study, cbd enriched hemp seed oil was also tested, and it was shown that cbd oil is a strong scavenger of dpph radical which is consistent with other studies (hacke et al., 2019; kitamura et al., 2020; petrovici et al., 2021). cbd is a compound with two phenolic groups in the structure and an antioxidant mechanism characteristic of phenol derivates that make it a strong antioxidant. phenol compounds are strong dpph scavengers by hydrogen transfer mechanism (leopoldini et al., 2004; leopoldini et al., 2011). like polyphenols, cbd can eliminate free radicals via electron or hydrogen transfer from the phenol group (borges & da silva, 2017). hydroxyl free radical (oh) scavenging activity hydroxyl radical is the most biologically active and dangerous free radical which can cause serious damage to biomolecules and lead to the development of disease (lipinski, 2011; birben et al., 2012; ozcan & ogun, 2015). therefore, hydroxyl radical scavengers are very important for human’s health. generation of hydroxyl radical in fenton reaction in the presence of hydrogen peroxide and iron ions is a model used in vitro to estimate hydroxyl radical scavenging by the antioxidants. for easier comparison, the ic50was calculated for all tested oils and presented in tab. 2 and fig. 2. all the ic50 values are significantly different except ic50 for cbd and bht indicating their similar hydroxyl radical scavenging ability. ic50 for the cbd oil (0.86±0.06 mg/ml) was the lowest among oils (fig. 2). moreover, all analyzed hemp seed oils had lower ic50 than olive oil indicating that they, together with cbd that was also dissolved in hso, are better oh radical scavengers. good oh radical scavenging activity of cbd oil was previously explained by the presence of two phenolic groups in cannabinoids (borges & da silva, 2017; atalay et al., 2020). additionally, cbd is dissolved in hso rich in pufa that are known as good oh radical scavengers. the high affinity of hydroxyl radical to double bonds of pufa that turn into single bounds is thought to be responsible for the scavenging activity of pufa (lipinski, 2011). hso is rich in pufa (70 to 90% of hso are pufa) and their content is much higher than in olive oil (kabaran, 2018) that can explain its better oh radical scavenging activity. the presence of polyphenolic natural substances such as flavonoids, also contributes to the antioxidant capacity of oil. polyphenols, in addition to their ability to be oxidized, have also the ability to scavenge hydroxyl radicals reducing the double bonds to single ones and thus creating hydroxyl derivatives. this is only the case of polyphenols with available ortho position in the ring. thus, phenol compounds that can be found in olive oil and in hso are also responsible for their ability to scavenge oh radicals (tuck et al., 2001). hso2 and hso3 were shown as excellent scavengers of oh radical especially hso3 with the lowest ic50value (3.56±0.15). the scavenging ability of hso decreased after one year of storage which can be seen through a comparison of ic50 of old and new oil (6.45±0.35 for old hso and 4.61±0.21 for the new hso). this is probably because of the oxidation of the fatty acids in oil which impairs their anti-oh radical function. metal reducing ability of oils reduction of metals by natural compounds is another method used to evaluate the antioxidant ability (capacity) of natural compounds. for that purpose, we performed ferric reducing power assay and phosphomolybdenum assay. ferric reducing power the ability of oils to reduce fe3+ from ferricyanide into fe2+ (ferrous) is mainly characteristic of antioxidants such are phenol derivatives. the reducing power of oils is presented in fig. 3. absorbance obtained at 700 nm is directly proportional to the reducing capacity of the oils. the results showed an increase in reducing potential with increasing concentration of oils. the highest reducing activity showed cbd oil at a concentration of 20 mg/ml (0.73±0.01). hso2 and hso3 showed maximal reducing activity of around 0.5 under the concentration of 40 mg/ml (0.51±0.01 and 0.50±0.01, respectively). concentration of cbd oil of 0.1 mg/ml showed similar reducing power (0.24±0.004) as all other hsos (hso1, hso2 and hso3) in the concentration of 5 mg/ml (0.25±0.01, 0.24±0.003 and 0.22±0.006, respectively) as can be seen in fig. 3a and b. cbd can reduce compounds by donating electrons and also can donate h atom to neutralize free radicals (borges & da silva, 118 biologica nyssana ● 12 (2) december 2021: 113-122 vitorović et al. ● antioxidant potential of commercial hemp seed oils and cbd oil 119 biologica nyssana ● 12 (2) december 2021: 113-122 vitorović et al. ● antioxidant potential of commercial hemp seed oils and cbd oil 2017; atalay et al., 2020). petrovici et al. (2021) suggested that cbd-enriched hemp oil showed high antioxidant activity having the ability to reduce iron, scavenge free radicals and inhibit lipid peroxidation. reducing power of bht was higher compared to all oils and the nearest to cbd oil especially in lower concentrations (fig. 3b). in comparison with olive oil, all three samples of hso in all tested concentrations showed better reducing power (fig. 3a). the results from the reducing power assay showed that the old oil (hso1) had a very similar or even slightly higher reducing power than the new (hso2) in lower concentrations than 40 mg/ml, which can be seen on fig. 3a. a slight decrease in the reducing ability of the hso after one year was noticed in concentration of 40 mg/ml (0.51±0.01 for the new hso and 0.44±0.01 for the old hso). results that showed reducing power of the hsos are of great importance indicating oil potential to limit the generation of free radicals via fenton reaction. the reducing power of hemp seed extracts determined by frap assay was evaluated in the study of irakli et al. (2019). it was also shown that essential oil from the aerial parts of c. sativa showed good reducing power (zengin et al., 2018). cold pressed hso from finola cultivar of cannabis sativa reduced ferric ions, with the reducing power being better in the lipophilic fraction than in the hydrophilic fraction of oil (smeriglio et al., 2016). authors suggested that probably the polyphenols or tocopherol present in different amounts in hso were mainly responsible for these reducing abilities. in the work of petrovici et al. (2021), it was shown that reducing ability of cbd oil was in the line with the ability of phenolic derivatives. total antioxidant capacity the total antioxidant capacity (tac) of oils was estimated by phosphomolybdenum assay. the molybdenum assay serves to predict the antioxidant activity of the sample by measuring the reduction degree of mo (vi) to mo (v) by the oils and formation of green phosphate/mo (v) complex with absorption at 695 nm. tac of each oil was calculated as µg of gallic acid equivalent per mg of oils (gae) using a standard gallic acid curve. in accordance with other antioxidant tests, cbd oil showed the highest total antioxidant capacity (245.00±35.16) with the statistically significant difference in comparison with all other oils (tab. 2). fig. 3. ferric reducing potential of oils a) hemp seed oils (hso) and extra virgin olive oil (evoo), b) cbd oil and bht. data are presented as mean ± sd of triplicate determinations. bht was used as a positive control fig. 4. total antioxidant capacity of hemp seed oils and extra virgin olive oil. data are presented as mean ± sd of triplicate determinations. total antioxidant capacity is expressed as µg of gallic acid equivalent per mg of oil (gae). values marked with different letters are significantly different (p<0.05) the results obtained from phosphomolybdenum assay for other oils than cbd (fig. 4) showed that all the oil samples reduce mo (vi) to mo (v) with higher total antioxidant capacity obtain for evoo (44.3±3.97 gae) in comparison with all three samples of hso (35.9±0.99 gae, 28.6±2.07 gae and 13±1.04 gae for the hso3, hso2 and hso1, respectively). this could be probably explained by the presence of the antioxidants such are phenolic biologica nyssana ● 12 (2) december 2021: 113-122 120 vitorović et al. ● antioxidant potential of commercial hemp seed oils and cbd oil molecules and other minor compounds in olive oil since olive oil in addition to oleic acid, contains polyphenols that are known to be responsible for such antioxidant activities (bendini et al., 2007; lee et al., 2008). total antioxidant activity for old hso was 13±1.04 gae which was more than twice less compared to fresh oil (28.6±2.07 gae) indicating a loss of antioxidant power with storage time. conclusions oils rich in pufa such as hemp seed oil have been the subject of various discussions regarding the impact on diseases associated with oxidative stress. in vivo studies have shown a beneficial effect of hemp oil on oxidative status and reduction of oxidative stress. a part of the antioxidant activities is probably due to the presence of various metabolites and antioxidants such as polyphenols or tocopherols present in the oil. the high amount of unsaturated fatty acids also indicates antioxidant activity because of the reactions that occur at the double bonds. in vitro antioxidant tests showed good antiradical and reduction capabilities of hsos. therefore, the use of hemp seed oil may be a significant contribution to the protection of diseases caused by oxidative stress but there is still a need for more in vivo evidence. two commercial fresh hemp seed oils showed small differences in activity with little advantage for hso3 noticed in the ability to scavenge oh radical and in total antioxidant capacity compared to hso2 which showed better dpph activity. differences are probably due to the small variations in their composition. more in vivo studies are needed to examine their possible differences in the effect. hemp seed oil didn’t completely lose the antioxidant ability a year after opening although the antioxidant capacity was lower in most tests. although the difference between cbd oil and other hemp products is often not clearly known on the market, these results indicate a difference in activity and much better antioxidant power of cbd oil which is the result of its chemical composition. in the ability to neutralize oh radicals, cbd oil has been shown to be a better antiradical scavenger even than the synthetic bht. acknowledgements. this study was supported by the ministry of education, science, and technological development of the republic of serbia (contract number: 451-03-9/2021-14/200124). references andre, c.m., hausman, j-f., guerriero, g. 2016: cannabis sativa: the plant of the thousand and one molecules. frontiers in plant science, 7: 19. atalay, s., jarocka-karpowicz, i., skrzydlewska, e. 2020: antioxidative and anti-inflammatory properties of cannabidiol. antioxidants, 9(1): 21. baba, s.a., malik, s.a. 2015: determination of total phenolic and flavonoid content, antimicrobial and antioxidant activity of a root extract of arisaema jacquemontii blume. journal of taibah university for science, 9(4): 449-454. bendini, a., cerretani, l., carrasco-pancorbo, a., gómez-caravaca, a.m., segura-carretero, a., fernández-gutiérrez, a., lercker, g. 2007: phenolic molecules in virgin olive oils: a survey of their sensory properties, health effects, antioxidant activity and analytical methods. an overview of the last decade alessandra. molecules, 12(8): 16791719. bhatti, m.z., ali, a., ahmad, a., saeed, a., malik, s.a. 2015: antioxidant and phytochemical analysis of ranunculus arvensis l. extracts. bmc research notes, 8(1): 1-8. birben, e., sahiner, u.m., sackesen, c., erzurum, s., kalayci, o. 2012: oxidative stress and antioxidant defense. world allergy organization journal, 5(1): 9-19. borges, r., da silva, a. 2017: cannabidiol as an antioxidant. in: preedy, v. (ed), handbook of cannabis and related pathologies, e122-e130, elsevier. callaway, j. 2004: hempseed as a nutritional resource: an overview. euphytica, 140(1): 65-72. chahar, m.k., kumar, s.d., lokesh, t., manohara, k. 2012: investigation of in-vitro antioxidant activity of mesua ferrea l. seed oil. international journal of pharmaceutical sciences and research, 3(11): 4260. cherbi, r., hamia, c., gourine, n., bombarda, i., saïdi, m., yousfi, m. 2017: lipid classes, fatty acids, tocopherols compositions and antioxidant activity of lawsonia alba seed oils growing in algeria. current nutrition and food science, 13(2): 121-130. cicerale, s., conlan, x.a., sinclair, a.j., keast, r.s. 2008: chemistry and health of olive oil phenolics. critical reviews in food science and nutrition, 49(3): 218-236. costa, b., trovato, a.e., comelli, f., giagnoni, g., colleoni, m. 2007: the non-psychoactive cannabis constituent cannabidiol is an orally effective therapeutic agent in rat chronic inflammatory and neuropathic pain. european journal of pharmacology, 556(1-3): 75-83. cox, b.d., whichelow, m.j., prevost, a.t. 2000: seasonal consumption of salad vegetables and fresh fruit in relation to the development of cardiovascular disease and cancer. public health nutrition, 3(1): 19-29. eastwood, m. 1999: interaction of dietary antioxidants in vivo: how fruit and vegetables prevent disease? qjm: an international journal of medicine, 92(9): 527-530. ferguson, l.r. 2010: chronic inflammation and mutagenesis. mutation research/fundamental and molecular mechanisms of mutagenesis, 690(1-2): 3-11. friedman, d., devinsky, o. 2015: cannabinoids in the treatment of epilepsy. new england journal of medicine, 373(11): 1048-1058. hacke, a.c.m., lima, d., de costa, f., deshmukh, k., li, n., chow, a.m., marques, j.a., pereira, r.p., kerman, k. 2019: probing the antioxidant activity of δ 9-tetrahydrocannabinol and cannabidiol in cannabis sativa extracts. analyst, 144(16): 4952-4961. halliwell, b. 2006: oxidative stress and neurodegeneration: where are we now? journal of neurochemistry, 97(6): 1634-1658. halliwell, b. 2007: oxidative stress and cancer: have we moved forward? biochemical journal, 401(1): 1-11. irakli, m., tsaliki, e., kalivas, a., kleisiaris, f., sarrou, e., cook, c.m. 2019: effect οf genotype and growing year on the nutritional, phytochemical, and antioxidant properties of industrial hemp (cannabis sativa l.) seeds. antioxidants, 8(10): 491. irving, p.m., iqbal, t., nwokolo, c., subramanian, s., bloom, s., prasad, n., hart, a., murray, c., lindsay, j.o., taylor, a. 2018: a randomized, double-blind, placebo-controlled, parallel-group, pilot study of cannabidiol-rich botanical extract in the symptomatic treatment of ulcerative colitis. inflammatory bowel diseases, 24(4): 714-724. kabaran, s. 2018: olive oil: antioxidant compounds and their potential effects over health. in lagouri, v. (ed), functional foods, intechopen. kandaswami, c., middleton, e. 1994: free radical scavenging and antioxidant activity of plant flavonoids. free radicals in diagnostic medicine, 351-376. kapoor, r., huang, y-s. 2006: gamma linolenic acid: an antiinflammatory omega-6 fatty acid. current pharmaceutical biotechnology, 7(6): 531534. kitamura, m., kiba, y., suzuki, r., tomida, n., uwaya, a., isami, f., deng, s. 2020: cannabidiol content and in vitro biological activities of commercial cannabidiol oils and hemp seed oils. medicines, 7(9): 57. kriese, u., schumann, e., weber, w., beyer, m., brühl, l. 2004: oil content, tocopherol composition and fatty acid patterns of the seeds of 51 cannabis sativa l. genotypes. euphytica, 137(3): 339-351. kurutas, e.b. 2015: the importance of antioxidants which play the role in cellular response against oxidative/nitrosative stress: current state. nutrition journal, 15(1): 1-22. lee, o.h., lee, b.y., kim, y.c., shetty, k., kim, yc. 2008: radical scavenging‐linked antioxidant activity of ethanolic extracts of diverse types of extra virgin olive oils. journal of food science and technology, 73(7): c519-c525. leizer, c., ribnicky, d., poulev, a., dushenkov, s., raskin, i. 2000: the composition of hemp seed oil and its potential as an important source of nutrition. journal of nutraceuticals, functional and medical foods, 2(4): 35-53. leopoldini, m., marino, t., russo, n., toscano, m. 2004: antioxidant properties of phenolic compounds: h-atom versus electron transfer mechanism. the journal of physical chemistry a, 108(22): 4916-4922. leopoldini, m., russo, n., toscano, m. 2011: the molecular basis of working mechanism of natural polyphenolic antioxidants. food chemistry, 125(2): 288-306. lipinski, b. 2011: hydroxyl radical and its scavengers in health and disease. oxidative medicine and cellular longevity, 2011. lobo, v., patil, a., phatak, a., chandra, n. 2010: free radicals, antioxidants and functional foods: impact on human health. pharmacognosy reviews, 4(8): 118. luzia, d.m., jorge, n. 2013: bioactive substance contents and antioxidant capacity of the lipid fraction of annona crassiflora mart. seeds. industrial crops and products, 42: 231-235. minioti, k.s., georgiou, c.a. 2010: comparison of different tests used in mapping the greek virgin olive oil production for the determination of its total antioxidant capacity. grasas y aceites, 61(1): 45-51. ozcan, a., ogun, m. 2015: biochemistry of reactive vitorović et al. ● antioxidant potential of commercial hemp seed oils and cbd oil biologica nyssana ● 12 (2) december 2021: 113-122 121 oxygen and nitrogen species. basic principles and clinical significance of oxidative stress, 3: 37-58. pellati, f., borgonetti, v., brighenti, v., biagi, m., benvenuti, s., corsi, l. 2018: cannabis sativa l. and nonpsychoactive cannabinoids: their chemistry and role against oxidative stress, inflammation, and cancer. biomed research international, 2018. petrovici, a.r., simionescu, n., sandu, ai., paraschiv, v., silion, m., pinteala, m. 2021: new insights on hemp oil enriched in cannabidiol: decarboxylation, antioxidant properties and in vitro anticancer effect. antioxidants, 10(5): 738. ramadan, m.f., moersel, j-t. 2006: screening of the antiradical action of vegetable oils. journal of food composition and analysis, 19(8): 838-842. salganik, r.i. 2001: the benefits and hazards of antioxidants: controlling apoptosis and other protective mechanisms in cancer patients and the human population. journal of the american college of nutrition, 20(5): 464s-472s. simopoulos, a.p. 2008: the importance of the omega-6/omega-3 fatty acid ratio in cardiovascular disease and other chronic diseases. experimental biology and medicine, 233(6): 674-688. smeriglio, a., galati, e.m., monforte, m.t., lanuzza, f., d’angelo, v., circosta, c. 2016: polyphenolic compounds and antioxidant activity of cold‐pressed seed oil from finola cultivar of cannabis sativa l. phytotherapy research, 30(8): 1298-1307. tuck, k.l., freeman, m.p., hayball, p.j., stretch, g.l., stupans, i. 2001: the in vivo fate of hydroxytyrosol and tyrosol, antioxidant phenolic constituents of olive oil, after intravenous and oral dosing of labeled compounds to rats. the journal of nutrition, 131(7): 1993-1996. van dolah, h.j., bauer, b.a., mauck, k.f. 2019: clinicians’ guide to cannabidiol and hemp oils. mayo clinic proceedings, elsevier, 1840-1851 p. vitorović, j., joković, n., radulović, n., mihajilov-krstev, t., cvetković, v.j., jovanović, n., mitrović, t., aleksić, a., stanković, n., bernstein, n. 2021: antioxidant activity of hemp (cannabis sativa l.) seed oil in drosophila melanogaster larvae under non-stress and h2o2induced oxidative stress conditions. antioxidants, 10(6): 830. vuolo, l., schuessler, g. 1985: review: putative mutagens and carcinogens in foods. environmental mutagenesis, 7: 577-598. watt, g., karl, t. 2017: in vivo evidence for therapeutic properties of cannabidiol (cbd) for alzheimer’s disease. frontiers in pharmacology, 8: 20. witschi, h. 1986: enhanced tumour development by butylated hydroxytoluene (bht) in the liver, lung and gastro-intestinal tract. food and chemical toxicology, 24(10-11): 1127-1130. yan, x., tang, j., dos santos passos, c., nurisso, a., simoes-pires, c.a., ji, m., lou, h., fan, p. 2015: characterization of lignanamides from hemp (cannabis sativa l.) seed and their antioxidant and acetylcholinesterase inhibitory activities. journal of agricultural and food chemistry, 63(49): 1061110619. yang, l.g., song, z.x., yin, h., wang, y.y., shu, g.f., lu, h.x., wang, s.k., sun, g.j. 2016: low n-6/n-3 pufa ratio improves lipid metabolism, inflammation, oxidative stress and endothelial function in rats using plant oils as n-3 fatty acid source. lipids, 51(1): 49-59. yang, x., zhang, d., song, l-m., xu, q., li, h., xu, h. 2017: chemical profile and antioxidant activity of the oil from peony seeds (paeonia suffruticosa andr.). oxidative medicine and cellular longevity, 2017. zengin, g., menghini, l., di sotto, a., mancinelli, r., sisto, f., carradori, s., cesa, s., fraschetti, c., filippi, a.,angiolella, l. 2018: chromatographic analyses, in vitro biological activities, and cytotoxicity of cannabis sativa l. essential oil: a multidisciplinary study. molecules, 23(12): 3266. vitorović et al. ● antioxidant potential of commercial hemp seed oils and cbd oil biologica nyssana ● 12 (2) december 2021: 113-122 122 vukotić et al. 2022, biologica nyssana 13(1) 13 (1) september 2022: 59-81 doi: 10.5281/zenodo.7117592 aquatic and wetland plant communities of the pusta river (southern serbia): floristic characteristics and syntaxonomic affiliation original article danica vukotić department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, 18 000 niš, serbia faculty of electronic engineering, university of niš, aleksandra medvedeva 14, 18106 niš, serbia danica.vukotic@elfak.ni.ac.rs (corresponding author) dragana jenačković gocić department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, 18 000 niš, serbia dušica p. ilić faculty of electronic engineering, university of niš, aleksandra medvedeva 14, 18106 niš, serbia danijela nikolić department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, 18 000 niš, serbia vladimir ranđelović department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, 18 000 niš, serbia received: july 19, 2022 revised: september 20, 2022 accepted: september 28, 2022 abstract: at the end of summer 2021, phytocenological research was conducted on aquatic and wetland vegetation of the pusta river left tributary of the south morava river. using upgma classification method and the bray-curtis distance on a set of 97 relevés and 44 species, degree of floristic differentiation between studied vegetation plots was quantified. it was established that 7 associations take part in composing vegetation of the pusta river – myriophyllo-potametum soó 1934, potametum nodosi soó (1928) 1960, segal 1964, phragmitetum australis savič 1926, typhetum latifoliae nowiński 1930, glycerio-sparganietum neglecti koch 1926, phalaridetum arundinaceae libbert 1931 and polygono-bidentetum tripartitae (w. koch 1926) lohm. 1950. the plant associations of vegetation class potamogetonetea klika in klika et novák 1941potametum nodosi and myriophyllo-potametum, are floristically the poorest and entirely consist of native plant species. the associations of vegetation class phragmito-magnocaricetea klika in klika et novák 1941 typhetum latifoliae (34 taxa), glycerio-sparganietum neglecti (33 taxa), phragmitetum australis (24 taxa) and phalaridetum arundinaceae (20 taxa) consist of higher number of species, including invasive plants. vegetation class bidentetea tx. et al. ex von rochow 1951 is represented by only one association polygono-bidentetum tripartitae, which consists of 24 taxa, with a share of 16.67% of allochthonous species. collected phytocenological data are going to be good basis for the future research of the vegetation of lotic ecosystems in the southern serbia. key words: aquatic and wetland vegetation, pusta river, upgma classification apstrakt: vodene i močvarne biljne zajednice puste reke (južna srbija): sintaksonomska pripadnost i florističke karakteristike krajem leta 2021. godine, sprovedena su fitocenološka istraživanja vodene i močvarne vegetacije puste reke – leve pritoke južne morave. korišćenjem upgma klasifikacione metode i bray-curtis-ove distance nad setom podataka od 97 fitocenoloških snimaka i 44 vrsta kvantifikovan je stepen florističke diferencijacije između proučavanih sastojina. ustanovljeno je da u izgradnji biljnog pokrivača puste reke učestvuje 7 biljnih zajednica myriophyllopotametum soó 1934, potametum nodosi soó (1928) 1960, segal 1964, phragmitetum australis savič 1926, typhetum latifoliae nowiński 1930, glycerio-sparganietum neglecti koch 1926, phalaridetum arundinaceae libbert 1931 i polygono-bidentetum tripartitae (w. koch 1926) lohm. 1950. asocijacije vegetacijske klase potamogetonetea klika in klika et novák 1941 potametum nodosi i myriophyllo-potametum, su floristički najsiromašnije i u potpunosti izgrađene od autohnotnih biljnih vrsta. u sastav zajednica vegetacijske klase phragmito-magnocaricetea klika in klika et novák 1941 typhetum latifoliae (34 vrste), glycerio-sparganietum neglecti (33 vrste), phragmitetum australis (24 vrste) i phalaridetum arundinaceae (20 vrste) ulazi veći broj vrsta, uključujući i invazivne biljne vrste. vegetacija klase bidentetea tx. et al. ex von rochow 1951 predstavljena je zajednicom polygono-bidentetum tripartitae izgrađenom od 24 vrsta, sa udelom alohtonih vrsta od 16.67%. prikupljeni fitocenološki podaci predstavljaju svojevrsnu osnovu za buduća, intenzivnija istraživanja biljnog pokrivača lotičkih ekosistema južne srbije. ključne reči: vodena i močvarna vegetacija, pusta reka, upgma klasifikacija © 2022 vukotić et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 59 14th symposium on the flora of southeastern serbia and neighboring regions introduction macrophytes are a complex group of plants adapted to the life in water. they include all aquatic vascular plants and aquatic mosses, and some authors also include macroscopic algae (stevanović & janković, 2001). they form aquatic plant communities that, compared to terrestrial communities, consist of a smaller number of species and, in most cases, are dominated by one species. macrophytes can be both submerged and floating, and can be further divided into morphoecological types of plants that are rooted and those that are not rooted, but whose roots float in water, or these types of plants don’t develop roots at all (stevanović & janković, 2001). communities of rooted, floating plants as well as submerged macrophytes belong to the vegetation class potamogetonetea klika in klika et novák 1941, while floating, non-rooted macrophytes of relatively still and nutrient-rich waters, belong syntaxonomically to the vegetation class lemnetea o. de bolòs et masclans 1955 (mucina et al., 2016). aquatic vegetation in serbia has been previously studied in lentic systems (vukov et al., 2003; topuzović et al., 2009; stanković et al., 2009, ranđelović & zlatković, 2010; marković et al., 2015), while in the lotic systems it has been studied only in a few rivers and channels (stojanović et al., 2007; vukov et al., 2008; jenačković et al., 2010; ljevnaić-mašić, 2010; džigurski et al., 2016). wetland vegetation belongs to the class phragmito-magnocaricetea klika in klika et novák 1941 (mucina et al., 2016). in contrast to aquatic vegetation, the structure and physiognomy of phytocenoses in wetlands is determined by emergent macrophytes. this type of vegetation develops in places where water dries out during the summer months but remains close to the ground surface, as well as in habitats that are flooded throughout the year (ranđelović et al., 2007a). wetland vegetation provides refuge for many species, often rare and endangered, that inhabit only habitats where wetland vegetation is present. they are considered very sensitive and one of the most endangered habitats in the world, and their degradation is continuous and uncontrolled (svitok et al., 2011). wetland vegetation in serbia has been studied mainly in the plains of vojvodina (slavnić, 1956; vukićević et al., 1966; babić, 1971; stojanović et al., 1987; butorac & crnčević, 1987; parabućski & butorac, 1994; polić, 2006; stojanović et al., 2007; ljevnaić-mašić, 2010; džigurski et al., 2010; džigurski et al., 2011), while it has been less researched in the southern parts of serbia (katić, 1910; košanin, 1910; mišić et al., 1987; ranđelović, 1978; ranđelović & zlatković, 2010; jenačković et al., 2010; jenačković, 2017; jenačković et al., 2019). aquatic and wetland vegetation show high floristic and ecological similarity, which is why they are often studied together. these types of vegetation on the south morava river and its tributaries have not been sufficiently investigated in terms of their floristic composition and ecological differentiation. wetland vegetation south of the autonomous province of vojvodina was examined near vlaško polje (jovanović, 1958), in the valley of the velika morava river (jovanović, 1965), near kragujevac (veljović, 1967), in the valley of the južna morava river (ranđelović, 1988), in the moravica river (milenović & ranđelović, 2005), in the batušinačke swamps located to the left of the south morava river (ranđelović et al., 2007a), on the banks of the murina river, brestovac (ranđelović et al., 2007b), also on the banks of vlasina lake, cvetkova river, dedina dolina (ranđelović & zlatković, 2010), in the svrljiški and beli timok rivers (jenačković et al., 2010), smilovsko lake, krupac lake, oblačina lake, wetland habitats in surroundings of the cities of bela palanka and prokuplje, as well as near the villages of žitkovac, vrtište, medoševac, lepaja, bresničić, lalinac and levosoje which have been investigated by jenačković (2017). aquatic vegetation was also investigated in the beli and svrljiški timok rivers (jenačković et al., 2010). the classification of aquatic and semi-aquatic vegetation of lakes in serbia was also carried out by laketić (2013), as well as the review of complete aquatic vegetation studied so far in serbia (cvijanović et al., 2018). the main objectives of this paper are to determine: • the diversity of aquatic and wetland phytocenoses of the pusta river, • the degree of floristic differentiation among the communities, • the syntaxonomic affiliation of the registered communities. materials and methods research area phytocenological research was conducted on the aquatic and wetland vegetation of the pusta river along its course from the municipality of bojnik to the municipality of doljevac (fig. 1). the pusta river is located in the southern part of serbia and is a left tributary of the south morava river. the river’s spring is located near the village of donji statovac. it is 71 km long and its catchment is 569 km2. it flows into the south morava river near the village of pukovac (gavrilović & dukić, 2002). phytocenological research of aquatic and wetland communities was conducted during september 2021, 60 biologica nyssana ● 13 (1) september 2022: 59-81 vukotić et al. ● aquatic and wetland plant communities of the pusta river (southern serbia): floristic characteristics and syntaxonomic affiliation 61 biologica nyssana ● 13 (1) september 2022: 59-81 vukotić et al. ● aquatic and wetland plant communities of the pusta river (southern serbia): floristic characteristics and syntaxonomic affiliation as this part of the vegetation season is considered optimal for their sampling (jenačković et al., 2019). phytocenological relevés were recorded in the lower course of the pusta river, from its confluence with the south morava river to the municipality of bojnik, along a river length of about 35 km. the largest number of phytocenological relevés were recorded in the part of the river stream near the villages of pukovac and kočane, as in these localities the phytocenoses of aquatic and wetlands were fully developed. in the part of the river course between the village of pukovac and the municipality of bojnik, the areas with wetland vegetation were drastically reduced. the wetland phytocenoses were represented only by a few smaller patches of vegetation plots that were several kilometers apart. the cleaning of the riverbed significantly destroyed the wetland vegetation in bojnik, which is believed to have developed in the riparian zone. the phytocenological surveys were carried out according to the principle of the swiss-french braun-blanquet school (braun-blanquet, 1964). the areas of the sampled stands had a standard size of 4 m2 for aquatic vegetation and 16 m2 for wetland vegetation. sampling points were georeferenced using the gps device (global positioning system). the plant material collected in the sampled stands was deposited in the herbarium collection “herbarium moesiacum” of the faculty of natural sciences and mathematics (hmn) of the university of niš. the determination was performed with the help of dichotomous identification keys: “flora sr srbije i-x” (josifović, 1970-1980, sarić, 1986, 1992), “флора на нр българияˮ (йорданов, 19631986, велчeв, 1989), and “flora europaea” (tutin et al., 1964-1980). the nomenclature and taxonomy of plant species were standardized according to the euro+medplantbase database (https://www. emplantbase.org). nomenclature of syntaxa, at the level of vegetation classes, orders and alliances was standardized according to mucina et al. (2016). the names of associations, and their syntaxonomic positions, have been adjusted with tzonev (2009), cvijanović (2018) and landucci et al. (2020). statistical analyses the turboveg software package was used to digitize 97 phytocenological relevés recorded during field research (hennekens & schaminée, 2001). classification analysis was performed using the upgma (unweighted pair group method with arithmetic mean) classification method and the bray-curtis similarity index. the value of the “crispness of classification” (botta-dukát et al., 2005) was used to determine the optimal number of clusters. groups of phytocenological relevés belonging to one or more associations, identified by the hierarchical classification analysis as a separate group (cluster), are characterized by diagnostic species. diagnostic species were determined based fig. 1. map of the investigated area on the pusta river, start point (bojnik) and end point (doljevac) 62 on the phi-coefficient, which expresses the degree of taxon fidelity to a particular cluster (chytrý et al., 2002). in addition, virtual standardization of all vegetation groups was performed to eliminate the dependence of the phi-coefficient on the size of the vegetation groups – clusters (tichý & chytrý, 2006). fisher’s accuracy test was used to exclude all phi-coefficient values that were not statistically significant at the significance level p<0.05. species with a phi-coefficient greater than 0.20 were considered as diagnostic species, while species with a phi-coefficient greater than 0.50 were considered as highly diagnostic species. in addition to the classification analysis, where the optimal number of clusters was determined, one more cluster analysis was performed to better understand the floristic similarity between individual phytocenological relevés. second cluster analysis was performed without determining the optimal number of clusters. the dominant and constant species were determined for the vegetation groups identified using the results of the classification analysis. dominant species were determined by calculating the average cover values in relevés within the groups using the cover index (ic) (lausi et al., 1982) and the share in total cover (d%) (surina, 2005). dominant species were determined to be those that were present in at least 10% of the relevés with a cover of at least 25%. constant species were considered those that occurred in at least 40% of the relevés within the clusters. the determination of diagnostic, constant and dominant species within the clusters, as well as the determination of the optimal number of clusters during classification analysis, were performed using the software package juice 7.0 (tichý, 2002, http://www.sci.muni.cz/botany/juice). the software package juice 7.0 was also used to create the biologica nyssana ● 13 (1) september 2022: 59-81 vukotić et al. ● aquatic and wetland plant communities of the pusta river (southern serbia): floristic characteristics and syntaxonomic affiliation combined synoptic table. the combined synoptic table contains information about the floristic composition of the studied communities and the quantitative proportion of species in each of them. results and discussion based on the results of the classification analysis, with determined optimal number of clusters (fig. 2) and the composition of the diagnostic species within the clusters defined (tab. 1), four main vegetation groups (clusters) were distinguished. the results of classification analysis, performed without determining optimal number of clusters, are presented in fig. 3, and illustrate the floristic similarities between individual relevés more clearly. based on the results of these analyses, it can be noticed that cluster ii (fig. 2) is clearly divided into 3 subclusters (fig. 3), and cluster iv (fig. 2) into 2 subclusters (fig. 3), resulting in 7 vegetation groups that participate in the construction of the plant cover of the investigated part of the pusta river. the first cluster (fig. 2) corresponds with relevés of typhetum latifoliae nowiński 1930 (fig. 3), second one (fig. 2), divided into 3 subclusters (fig. 3), unifies relevés of phalaridetum arundinaceae libbert 1931, polygono-bidentetum tripartitae (w. koch 1926) lohm. 1950 and glycerio-sparganietum neglecti koch 1926, third cluster corresponds with relevés of phragmitetum australis savič 1926, and fourth, divided into two subclusters, unifies relevés of potametum nodosi soó (1928) 1960, segal 1964 and myriophyllo-potametum soó 1934. classification analysis showed that the greatest floristic similarity exists between the clusters i and ii, i.e., the communities typhetum latifoliae nowiński 1930 (cluster i) and phalaridetum arundinaceae libbert 1931, polygono-bidentetum tripartitae (w. koch 1926) lohm. 1950 and glycerio-sparganietum fig. 2. dendrogram obtained as a result of the hierarchical upgma classification on bray-curtis resemblance matrix along with determining optimal number of clusters. 63 biologica nyssana ● 13 (1) september 2022: 59-81 vukotić et al. ● aquatic and wetland plant communities of the pusta river (southern serbia): floristic characteristics and syntaxonomic affiliation table 1. the combined synoptic table with the information on frequency and phi-coefficient values (x100) for species occurring in four optimal cluster groups defined by upgma classification analysis. the values for the frequency are represented by the base, while the values for fidelity are given in the form of superscripts species clusters i ii iii iv typha latifolia l. 100 85.6 14 11 . salix alba l. 50 31.3 33 22 . sparganium erectum l. 16 91 69 22 8 lythrum salicaria l. 62 77 42.8 22 . mentha aquatica l. 16 47 42.1 11 . polygonum mite schrank 22 37 29.8 11 . phalaris arundinacea l. 6 28 40 . . tanacetum vulgare l. 3 21 24.7 11 . phragmites communis trin. . . 100100 . urtica dioica l. 25 26 56 37.9 . echinocystis lobata (michx.) torr. & a. gray 3 2 33 46.1 . robinia pseudoacacia l. 6 9 33 37.2 . myriophyllum spicatum l. 6 19 . 92 79.9 potamogeton nodosus poir. 16 28 11 85 60.4 bidens tripartitus l. 47 47 56 . polygonum lapathifolium l. 28 25 22 . salix euxina i. v. belyaeva 31 21 33 . xanthium strumarium l. 28 35 11 . populus sp. 16 9 11 . epilobium hirsutum l. 12 12 11 .. lycopus europaeus l. 9 21 22 rubus caesius l. 6 12 22 . galium aparine l. 6 9 11 . potamogeton crispus l. 3 2 . 15 lemna minor l. 3 7 . 8 calystegia sepium (l.) r. br. 19 23 . . leersia oryzoides (l.) sw. 12 19 . . solanum dulcamara l. 12 19 . . amorpha fruticosa l. 9 9 . . echinochloa crus-galli (l.) p. beauv. 3 9 . . humulus lupulus l. 9 . 11 . rumex conglomeratus murray 3 2 . . lysimachia vulgaris l. 3 2 . . erigeron canadensis l. . 5 11 . scutellaria galericulata l. . 5 . . aristolochia clematitis . 2 . . chenopodium album l. . 2 . . lathyrus latifolius l. . 2 . . neglecti koch 1926 (cluster ii). cluster iv differs the most as this cluster consists of associations potametum nodosi soó (1928) 1960, segal 1964 and myriophyllo-potametum soó 1934 presenting aquatic vegetation (fig. 2). association typhetum latifoliae is characterized by presence of highly diagnostic species typha latifolia (tab. 1). it is floristically rich, includes 34 taxa, and its constant species are lythrum salicaria, salix alba and bidens tripartitus (appendix 1). the stands of this community have been developed in the coastal zone, where t. latifolia has been partly in the water and borders with the stands of myriophyllopotametum and potametum nodosi (fig. 4). on the banks of the river, it forms a dense, impassable cover, and is intersected in some places by the glycerio-sparganietum neglecti. in southern part of serbia, stands of this association were recorded in the batušinačke swamps (ranđelović et al., 2007a), on the banks of the grlište hydro-accumulation (stanković et al., 2009), vlasina lake (ranđelović & zlatković, 2010). in ribinac, the stands in which typha latifolia is the dominant species together with phragmites communis were recorded (ranđelović et al., 2007b). the spectrum of accompanying species is diverse and depends on the habitat. according 64 biologica nyssana ● 13 (1) september 2022: 59-81 vukotić et al. ● aquatic and wetland plant communities of the pusta river (southern serbia): floristic characteristics and syntaxonomic affiliation to šumberová (2011), short-term floodplains are usually much richer in the number of taxa and include not only common wetland plants (e.g. lycopus europaeus l. and lythrum salicaria l.) but also tall sedges (e.g. carex riparia curtis) and species typical for meadow vegetation (e.g. equisetum palustre l., galium palustre l. and symphytum officinale l.). in long-term floodplains, especially in highly eutrophic wetlands, the macrophyte lemna minor l. is common (šumberová, 2011), which is also recorded in the studied stands of the pusta river. it is necessary to stress that high values of phicoefficient for species salix alba (tab. 1) is related with including one relevé characterized by high percentage value of cover of salix alba in database. on the territory investigated, salix alba occurs frequently within stands of the typhetum latifoliae, at its upper border or at the elevated parts of the bank where it occurs mainly with one or two individuals. in some places, one to two individuals of salix alba can be found due to the lack of wetland vegetation. salix alba represents the remnants of once-present floodplain forests that developed near the pusta river. today, the remains of flooded forests in the lower course of the river, are represented only by certain species of the genus salix and populus, some fig. 3. dendrogram obtained as a result of the hierarchical upgma classification on bray-curtis resemblance matrix without identifying optimal number of clusters species clusters i ii iii iv scrophularia nodosa l. . 2 . . berula erecta (huds.) coville . 2 . . stellaria neglecta (lej.) weihe . 2 . . eleocharis palustris (l.) r. br. . 2 . . stachys palustris l. 3 . . . althaea officinalis l. 3 . . . 65 biologica nyssana ● 13 (1) september 2022: 59-81 vukotić et al. ● aquatic and wetland plant communities of the pusta river (southern serbia): floristic characteristics and syntaxonomic affiliation of which extend into the wetland vegetation. the stands of phalaridetum arundinaceae (fig. 4) have been developed on the raised banks of the riverbed, exposed only to occasional floods, after which the surface water stagnates for a short time. the diagnostic species of this association is phalaris arundinacea, while the constant species are mentha aquatica, lythrum salicaria, sparganium erectum, salix alba and xanthium strumarium. it is floristically rich and consists of 20 taxa (appendix 2). this plant community fig. 4. the stands of associations typhetum latifoliae nowiński 1930 (1,2), phalaridetum arundinaceae libbert 1931 (3), phragmitetum australis savič 1926 (4), glycerio-sparganietum neglecti koch 1926 (5) and potametum nodosi soó (1928) 1960, segal 1964 (6) developed in riverbed and on the banks of pusta river, as well. occurs in habitats with pronounced fluctuations of the flood level. the development is conditioned by the hydrological regime of groundwater along the river course (ranđelović & zlatković, 2010). it is present in nutrient rich habitats, on sandy-gravelly to clay substrates. with its strong organic production, this community contributes to the overgrowth of ponds and shallow depressions (panjković, 2005). sometimes it occurs in deeper depressions where shallow (<0.3 m) surface water is retained during most of the growing season (jenačković, 2017). 66 biologica nyssana ● 13 (1) september 2022: 59-81 vukotić et al. ● aquatic and wetland plant communities of the pusta river (southern serbia): floristic characteristics and syntaxonomic affiliation phalarietum arundinaceae is only fragmentarily developed in several localities in southern serbia. according to ranđelović et al. (2007b), the best developed stands are along the šaranica stream near the villages of brestovac, pukovac and bubanj, in the valleys of the vlasina river and murina river. also, stands of this association were recorded on seasonally flooded habitats near the villages of gornje međurovo, levosoje, podvis, vrtište and žitkovac (jenačković et al., 2017). association polygono-bidentetum tripartitae consists of 24 taxa (appendix 3). its dominant species are polygonum lapathifolium and bidens tripartitus (appendix 3). since there are only three vegetation plots, a large number of constant species is noticeable, with a proportion of 16.67% of allochthonous species. this community develops on the banks of the river, where the water occasionally floods the banks, and retreats during the summer months. it is widespread in places along the banks of the pusta river especially where wetland vegetation is absent. also, this community was phytosociological described in svrljiški timok river and beli timok river, where it had a similar species composition (jenačković et al., 2010). similar floristic composition was recorded in nitrophilous stands developed on the shores of artificial lakes placed near the city of tuzla (barudanović & kamberović, 2011). glycerio-sparganietum neglecti is floristically rich association (33 taxa) (appendix 4) and develops on the banks of the river, in the form of narrow belts, some of which extend into the riverbed, and some on slightly elevated banks (fig. 4). its diagnostic and, at the same time, dominant species is sparganium erectum, while the constant species of association are lythrum salicaria and bidens tripartitus. native species have the largest share in the composition of this phytocenosis, but invasive, non-native species such as echinocystis lobata, echinochloa crus-galli, amorpha fruticosa and robinia pseudacacia (lazarević et al., 2012) were also recorded. a few years ago, in the part of the pusta river which flow through the village of pukovac and suffering strong anthropogenic influence via discharge of waste water, the stands of this association were phytocoenological described (jenačković, 2017). stands recorded in svrljiški timok river and beli timok river (jenačković et al., 2010), whose the most common species are sparganium erectum and mentha aquatica, show a remarkable floristical similarity with the stands developed in the pusta river. so far, communities with domination of sparganium erectum have been recorded in the batušinačke swamps (ranđelović et al., 2007a), on suitable habitats near the village of bubanj (ranđelović, 1988), žitkovac, vrtište, prokuplje, gornje međurovo, podvis, rgošte, levosoje (jenačković et al., 2019) and on the banks of vlasina lake (ranđelović & zlatković, 2010) and smilovsko lake (jenačković et al., 2019). the stands of the association glycerio-sparganietum neglecti koch 1926 are frequently involved in the establishment of the plant cover of lowland rivers, not only in our country, but also in the other european countries (gecheva et al., 2013). the dominant species of this community – sparganium erectum, is considered a reliable bioindicator of eutrophic waters (schneider & melzer, 2003), which explains the frequent occurrence of its monodominant communities in the lower reaches of the river, where high concentrations of nutrients, especially nitrogen, accumulate and are maintained as a result of anthropogenic influence. sparganium erectum shows exceptional frequency in the studied rivers of bulgaria, where it occurs in no less than 80 of 223 studied sites, with a frequency of 35% (gecheva et al., 2013). the taxa sparganium erectum and typha latifolia show ecological preferences for wetlands with similar physicochemical properties. they find optimal growing conditions in habitats characterized by relatively deep, slightly acidic water with low electrical conductivity. they prefer habitats where the substrate has the following characteristics: low ph, low values of conductivity values and concentration of carbonates, bicarbonates and potassium (jenačković, 2017). on the area investigated, stands of phragmitetum australis (fig. 4) develops on the highest parts of the riverbanks, often along the road. they have been recorded in only 9 localities in the villages of kočane and pukovac. this community is floristically rich and consists of 24 taxa (appendix 5), and its highly diagnostic species is phragmites communis (tab. 1). in addition to the species phragmites communis, high values of phi-coefficient were established for the species urtica dioica, echinocystis lobata and robinia pseudoacacia. these values probably reflect reduced substrate moisture and increased concentrations of nutrients, primarily nitrogen. abundant presence of individuals of echinocystis lobata and robinia pseudacacia can be explained by their high invasive capacity (lazarević et al., 2012). often, in nature, stands of phragmitetum australis are bordered to the stands of typhetum latifoliae. they usually prefer mineral sand, mineral-organic, as well as organic substrate (pełechaty, 1999). in terms of water depth and degree of soil moisture, they successfully develop on diverse habitat types completely flooded, occasionally flooded and non-flooded. according to jenačković (2017), dominant species of association phragmitetum 67 biologica nyssana ● 13 (1) september 2022: 59-81 vukotić et al. ● aquatic and wetland plant communities of the pusta river (southern serbia): floristic characteristics and syntaxonomic affiliation australis shows affinities to the habitats with high values of substrate ph. stands of phragmitetum australis are widespread in the central balkans. in serbia, they have been recorded in the velika morava river (jovanović, 1958; veljović, 1967), on the slopes of stara planina (mišić et al., 1987), in the south morava basin (ranđelović, 1988), in the toplica river (perišić et al., 2003), on the shores of vlasina lake (ranđelović & zlatković, 2010), smilovsko lake, oblačina lake, near the villages of žitkovac, lalinac, vrtište, rgošte and levosoje, on habitats placed in suburb of the city of bela palanka (jenačković et al., 2019). otherwise, the communities phragmitetum australis, typhetum latifoliae and glycerio-sparganietum neglecti belong to the phytocenoses that most frequently overgrow the wetland habitats of the central balkans (jenačković, 2017). the stands of the myriophyllo-potametum develop in the riverbed itself and form a relatively stable submerged community, consisting mainly of rooted hydrophytes (myriophyllum spicatum and potamogeton crispus) and floating hydrophytes (lemna minor). the association develops on a muddy waterbed at depths of up to 2 m, forming a dense cover. in some parts of the pusta river, the stands of this association form small, separated groups which look like islands composed of plants, while in other parts of the stream they cover the entire water surface from one bank to the another. they prefer slow to moderately slow water flow, but they also tolerate strong water strikes, and are indicators of mesotrophic and eutrophic waters. the association is floristically poor and consists of 3 taxa (appendix 6). myriophyllum spicatum is the highly diagnostic species (tab. 1), while the status of constant species have potamogeton nodosus and potamogeton crispus (appendix 6). study conducted on the aquatic vegetation of rivers in slovenia (germ et al., 2021) have shown that myriophyllum spicatum and potamogeton nodosus are the most common aquatic species in undisturbed aquatic ecosystems, while investigation of slovakian rivers have confirmed that the highest relative mass in rivers has myriophyllum spicatum (hrivnak et al., 2006). the species myriophyllum spicatum, potamogeton crispus and lemna minor have taken part in composing aquatic vegetation of the svrljiški and beli timok rivers (jenačković et al., 2010), river systems and channels in the northern part of serbia (radulović et al., 2010; vukov et al., 2012; džigurski et al., 2016; vukov et al., 2017) as well. otherwise, myriophyllum spicatum and potamogeton nodosus are indicators of eutrophic waters (schneider & melzer, 2003), which explains their occurrence in the lower parts of the river, where high nutrient concentrations accumulate and remain. potametum nodosi develops in the riverbed itself, mainly along the coast, and partly invades coastal vegetation where water flow is slower. in deeper parts of the riverbed, however, the community borders with myriophyllo-potametum. the highly diagnostic species of this community is potamogeton nodosus (tab. 1), and the constant species is myriophyllum spicatum (appendix 7). the community is relatively species-poor and is composed of only 4 taxa (appendix 7). a similar floristic composition have had stands developed in the svrljiški and beli timok rivers (jenačković et al., 2010). floristically, this community is also described in the moravica river, highlighting rare and endangered species (ljevnaić-mašić et al., 2016). the stands noted in the danube-tisa-danube canal in vojvodina, in the northern part of serbia, have had a slightly richer floristic composition compared to the stands recorded in pusta river (džigurski et al., 2016). it is known that both, wetlands and aquatic vegetation, have developed over the years on the once disturbed river. since the vegetation along the entire course of the river has never been described completely so far, but only sporadically, we do not have the possibility to compare the data. what is certain is that this area has been disturbed several times and has changed due to the needs of agriculture. nowadays, the river is surrounded on all sides by fields and arable land, and thus exposed to constant anthropogenic influences. the previously described associations belong to the following vegetation classes, orders and alliances: potamogetonetea klika in klika et novák 1941 potamogetonetalia koch 1926 potamogetonion libbert 1931 myriophyllo-potametum soó 1934 potametum nodosi soó (1928) 1960, segal 1964 phragmito-magnocaricetea klika in klika et novák 1941 pragmitetalia communis koch 1926 phragmition communis koch 1926 phragmitetum australis savič 1926 typhetum latifoliae nowiński 1930 glycerio-sparganietum neglecti koch 1926 phalaridetum arundinaceae libbert 1931 bidentetea tx. et al. ex von rochow 1951 bidentetalia br.-bl. et tx. ex klika et hadač 1944 bidention tripartitae nordhagen ex klika et hadač 1944 polygono-bidentetum tripartitae (w. koch 1926) lohm. 1950 68 biologica nyssana ● 13 (1) september 2022: 59-81 vukotić et al. ● aquatic and wetland plant communities of the pusta river (southern serbia): floristic characteristics and syntaxonomic affiliation conclusions based on the results of the classification analyses and composition of diagnostic species per clusters, 7 associations typhetum latifoliae nowiński 1930, phalaridetum arundinaceae libbert 1931, polygono-bidentetum tripartitae (w. koch 1926) lohm. 1950, glycerio-sparganietum neglecti koch 1926, phragmitetum australis savič 1926, potametum nodosi soó (1928) 1960, segal 1964 and myriophyllo-potametum soó 1934, are described on the territory researched. the stands of typhetum latifoliae nowiński 1930 are abundantly developed in the part of the flow from the confluence of the pusta river to south morava river all the way upstream to the village of pukovac, where they are the most developed and occupies the largest recorded area on the pusta river. the stands of glycerio-sparganietum neglecti koch 1926 were developed in some places along the whole studied part of the river, and they were mainly developed 2-3 km upstream from the village of pukovac. the stands of phalaridetum arundinaceae libbert 1931, polygono-bidentetum tripartitae (w. koch 1926) lohm. 1950, phragmitetum australis savič 1926, diverse in size, occur sporadically. in the village of pukovac and a few kilometers upstream, the aquatic vegetation is more abundantly developed and in some places forms a dense cover that covers the entire water surface. this is one of the first complete research of this kind conducted on the pusta river, and it represents a good basis for future research on the vegetation of lotic ecosystems in southern serbia. acknowledgements. this work has been supported by the ministry of education, science and technological development of republic of serbia (grant no. 451-0368/2022-14/200102 and no. 451-03-68/2022-14/200124). references babić, n. 1971: močvarna i livadska vegetacija koviljskog rita. zbornik matice srpske za prirodne nauke, 41: 19-87. barudanović, s., kamberović, j. 2011: weed vegetation on the shores of artificial reservoirs of surface mining pits in the area of tuzla. herbologia, 12(3): 1-14. botta-dukát, z., chytrý, m., hájková, p., havlová, m. 2005: vegetation of lowland wet meadows along a climatic continentality gradient in central europe. preslia, 77: 89-111. braun-blanquet, j. 1964: pflanzensoziologie, grundzüge der vegetationskunde 3rd ed. springer, berlin. 631 p. butorac, b., crnčević, s. 1987: zajednice acoretoglycerietum slavnić 56 i sparganio-glycerietum fluitantis br.-bl. 25 na području jugozapadnog banata. zbornik matice srpske za prirodne nauke, 72: 169-184. chytrý, m., tichý, l., holt, j., botta-dukát, z. 2002: determination of diagnostic species with statistical fidelity measures. journal of vegetation science, 13: 79-90. chytrý, m., tichý, l. 2018: national vegetation classification of the czech republic: a summary of the approach. phytocoenologia, 48(2): 121–131. cvijanović, d. l., lakušić, d., živković, m., novković, m., anđelković, a., pavlović, d., vukov, d. m., radulović, s. 2018: an overview of aquatic vegetation in serbia. tuexenia, 38: 269–286. džigurski, d., knežević, a., stojanović, s., nikolić, l., ljevnaić-mašić, b. 2010: the vegetation of canal novi sad savino selo. thaiszia journal of botany, 20: 137-145. džigurski, d., knežević, a., nikolić, l., ljevnaićmašić, b. 2011: emergent vegetation in the main canals of the hs dtd in the region of bačka. contemporary agiculture, 60(1-2): 73-79. džigurski, d., nikolić, l., ljevnaić-mašić, b. 2016: vegetation of the hydrochari-lemnetea and potametea classes in the danube-tisza-danube hydrosystem (serbia). contemporary problems of ecology, 9(3): 329–341. gavrilović, l., dukić, d. 2002: reke srbije. zavod za udžbenike i nastavna sredstva, beograd. 218 p. gecheva, g., yurukova, l., cheshmedjiev, s. 2013: patterns of aquatic macrophyte species composition and distribution in bulgarian rivers. turkish journal of botany, 37(1): 99-110. germ, m., janež, v., gaberščik, a., zelnik, i. 2021: diversity of macrophytes and environmental assessment of the ljubljanica river (slovenia). diversity, 13(6): 278. hennekens, s., schaminée, j. 2001: turboveg, a comprehensive data base management system for vegetation data. journal of vegetation science, 12: 589-591. hrivnak, r., otahelova, h., jarolimek, i. 2006: diversity of aquatic macrophytes in relation to environmental factors in the slatina river (slovakia). biologia, 61(4): 413-419. jenačković, d., dimitrijević, d., ranđelović, v. 2010: macrophytic flora and vegetation of the rivers 69 biologica nyssana ● 13 (1) september 2022: 59-81 vukotić et al. ● aquatic and wetland plant communities of the pusta river (southern serbia): floristic characteristics and syntaxonomic affiliation svrljiški and beli timok (eastern serbia). biologica nyssana, 1(1-2): 23-26. jenačković, d. 2017: fitocenološko-ekološka studija močvarne vegetacije (phragmitetea communis r. tx. et preising 1942) centralnog balkana. phd thesis. univerzitet u beogradu, biološki fakultet, beograd. jenačković, d., lakušić, d., zlatković, i., jušković, m., ranđelović, v. 2019: emergent wetland vegetation data recording: does an optimal period exist? applied vegetation science, 22(2): 200-212. josifović, m. (ed.) 1970-1980: flora sr srbije i-x. sanu, beograd. jovanović, r. 1958: tipovi močvarne vegetacije u jasenici. zbornik radova biološkog instituta n. r. srbije, 2(1): 1-36. jovanović, r. 1965: tipologija, ekologija i dinamika močvarne i livadske vegetacije u dolini velike morave. phd thesis. beograd. йорданов, д. (еd.) 1963-1986: флора на нр българия, i-viii. софия: издателство на бан. katić, d. 1910: vlasinska tresava i njezina prošlost. spomenik srpske kraljevske akademije, prvi razred, 50(8): 14-56. košanin, n. 1910: vlasina biljnogeografska studija. glas srpske kraljevske akademije, 81: 86-186. laketić, d. 2013: fitocenološka klasifikacija vegetacije jezerskog tipa u srbiji. phd thesis. univerzitet u beogradu, biološki fakultet, beograd. landucci, f., šumberová, k., tichý, l., hennekens, s., aunina, l., biță‐nicolae, c., borsukevych, l., bobrov, a., čarni, a., bie, e.d., golub, v. 2020: classification of the european marsh vegetation (phragmito‐magnocaricetea) to the association level. applied vegetation science, 23(2): 297-316. lausi, d., gerdol, r., piccoli, f. 1982: syntaxonomy of the ostrya carpinifolia woods in the southern alps (n-italy) based on numerical methods. studia geobotanica, 2: 41-58. lazarević, p., stojanović, v., jelić, i., perić , r., krsteski, b., ajtić, r., sekulić, n., branković, s., sekulić, g., bjedov, v. 2012: preliminarni spisak invazivnih vrsta u republici srbiji sa opštim merama kontrole i suzbijanja kao potpora budućim zakonskim aktima. zaštita prirode, 62(1): 5-31. ljevnaić-mašić, b. 2010: hidrofite osnovne kanalske mreže hidrosistema dtd na području banata. phd thesis. prirodno-matematički fakultet, univerzitet u novom sadu. novi sad. ljevnaić-mašić, b., džigurski, d., nikolić, l. 2016: rare and endangered plant species and associations in the moravica river (serbia). zbornik matice srpske za prirodne nauke, 131: 121-132. marković, g., vićentijević-marković, g., tanasković, s. 2015: first record of water chestnut (trapa natans l., trapaceae, myrtales) in central serbia. journal of central european agriculture, 16(4): 436-444. mccune, b., mefford, m. 2011: pc-ord. multivariate analysis of ecological data. version 6. mjm software. gleneden beach, oregon, united states of america. milenović, v., ranđelović, n. 2005: association sparganio-glycerietum fluitantis br.-bl. in the moravica river valley in eastern serbia. proceeding of the 8th symposium on flora of southeastern serbia and neighbouring regions, 61-65. mišić, v., jovanović-dunjić, r., popović, m., borisavljević, l., antić, m., dinić, a., danon, j., blaženčić, ž. 1987: biljne zajednice i staništa stare planine. beograd: srpska akademija nauka i umetnosti odeljenje prirodno-matematičkih nauka, knjiga dxi, 49: 1-389 mucina, l., bültmann, h., dierßen, k., theurillat, j.p., raus, t., čarni, a., šumberová, k., willner, w., dengler, j., garcía, r.g., chytrý, m. 2016: vegetation of europe: hierarchical floristic classification system of vascular plant, bryophyte, lichen, and algal communities. applied vegetation science, 19(s1): 3-264. panjković, b. 2005: akvatična i semiakvatična vegetacija apatinskog i monoštorskog rita. phd thesis. prirodno-matematički fakultet, univerzitet u novom sadu. novi sad. parabućski, s., butorac, b. 1994: general review of the vegetation along the lower course of the tisa river. thaiszia journal of botany, košice, 4: 99106. pavletić, z. 1968: flora mahovina jugoslavije. institut za botaniku, sveučilište u zagrebu, zagreb. 431 p. pełechaty, m. 1999: the phytosociological characteristic and habitat requirements of the phragmitetum communis (gams, 1927) schmale 1939 phytocoenoses in the lakes of the wielkopolski national park. hydrobiologia 408/409: 327–334. perišić, s., karadžić, b., petković, b. 2003: swamp vegetation of the blace lake (southern serbia). third international balkan botanical congress. sarajevo. polić, d. 2006: florističko-fitocenološko proučavanje labudovog okna. biblioteka academia. zadužbina andrejević, beograd. radulović, s., laketić, d., vukov, d. 2010: a riverside tale: assessment of altered habitat effects on macrophyte assemblage on the river tamiš, serbia. archives of biological sciences, 62(4): 1163-1174. ranđelović, n. 1978: fitocenološko ekološke karakteristike brdskih travnjaka jugoistočne srbije. phd thesis. prirodoslovno-matematički fakultet. zagreb. ranđelović, v. 1988: močvarna vegetacija uz gornji tok južne morave. diplomski rad. prirodnomatematički fakultet, odsek za biologiju., univerzitet u novom sadu. novi sad. ranđelović, v., matejić, j., zlatković, b. 2007a: flora and vegetation of batušinačke swamps near niš. proceeding of the 9th symposium on flora of southeastern serbia and neighbouring regions, 1940. ranđelović, v., zlatković, b., matejić, j. 2007b: swamp vegetation of order phragmitetalia in southeastern serbia. proceeding of the 9th symposium on flora of southeastern serbia and neighbouring regions, 9-18. ranđelović, v., zlatković, b. 2010: flora i vegetacija vlasinske visoravni. prirodnomatematički fakultet, univerzitet u nišu. niš. sarić, m. (ed.) 1986: flora sr srbije, x. beograd: sanu. sarić, m. (ed.) 1992: flora sr srbije, i (2nd edition) beograd: sanu. schneider, s., melzer, a. 2003: the trophic index of macrophytes (tim)—a new tool for indicating the trophic state of running waters. international review of hydrobiology: a journal covering all aspects of limnology and marine biology, 88(1): 49-67. slavnić, ž. 1956: vodena i barska vegetacija vojvodine. zbornik matice srpske za prirodne nauke, 10: 5-72. stanković, ž., borišev, m., simić, s., vučković, m., igić, r., vidović, m., miljanović, b. 2009: macrophytes of the grlište reservoir (serbia): fifteen years after its establishment. archives of biological sciences, 61(2): 267-278. stevanović, b., janković, m. 2001: ekologija biljaka sa osnovama fiziološke ekologije biljaka. nnk international, beograd. 514 p. stojanović, s., butorac, b., vučković, m. 1987: pregled barske i močvarne vegetacije vojvodine. glasnik instituta za botaniku i botaničke bašte univerziteta u beogradu, 21: 41-47. stojanović, s., lazić, d., knežević, a., nikolić, l., škorić, m., kilibarda, p., mišković, m., bugarski, r. 2007: flora i vegetacija osnovne kanalske mreže hs dtd u bačkoj. novi sad: univerzitet u novom sadu, poljoprivredni fakultet i jvp “vode vojvodine”. šumberová k. 2011: typhetum latifoliae nowiński 1930. in: chytrý m. (ed.), vegetace české republiky. 3. vodní a mokřadní vegetace: 401–405, academia, praha. surina, b. 2005: subalpinska in alpinska vegetacija krnskega pogorja v julijskih alpah. scopolia, 57: 1–222. svitok, m., hrivnák, r., oťaheľová, h., dúbravková, d., paľove-balang, p., slobodník, v. 2011: the importance of local and regional factors on the vegetation of created wetlands in central europe. wetlands, 31: 663–674. tichý, l. 2002: juice, software for vegetation classification. journal of vegetation science,13(3): 451-453. tichý, l., chytrý, m. 2006: statistical determination of diagnostic species for site groups of unequal size. journal of vegetation science, 17(6): 809-818. topuzović, m., pavlović, d., ostojić, a. 2009: temporal and spatial distribution of macrophytes in the gruža reservoir. archives of biological sciences, 61(2): 289-296. tutin, t. g., heywood, v. h., burges, n. a., moore, d. m., valentine, d. h., walters, s. m., webb, d. a. (eds.), 1964-1980: flora europaea, i-v. cambridge university press, london. tzonev, r. 2009: syntaxonomy of the natural and semi-natural vegetation of the middle danube plain in bulgaria. biotechnology & biotechnological equipment, 23(s1): 354-359. велчeв, в. (еd.) 1989: флора на нр българия, iх. софия: издателство на бан. veljović, v. 1967: vegetacija okolne kragujevca. glasnik prirodnjačkog muzeja u beogradu, 22 (b): 1-180. vukićević, e., cincović, t., kojić, m. 1966: pregled šumskih i močvarnih fitocenoza mačve. glasnik prirodnjačkog muzeja, 21 (b): 23-36. vukov, d., anačkov, g., igić, r. 2003: rare and protected plants in zasavica river (vojvodina, serbia). viith international symposium interdisciplinary vukotić et al. ● aquatic and wetland plant communities of the pusta river (southern serbia): floristic characteristics and syntaxonomic affiliation biologica nyssana ● 13 (1) september 2022: 59-81 70 regional research–isirr, 43-50. vukov, d., boža, p., igić, r., anačkov, g. 2008: the distribution and the abundance of hydrophytes along the danube river in serbia. central european journal of biology, 3(2): 177-187. vukov, d., igić, r., rućando, m., radulović, s. 2012: diversity of vascular hydrophytes in the zasavica river (serbia) – changes after thirteen years. archives of biological sciences, 64(4): 16071617. vukov, d., ilić, m., ćuk, m., igić, r., janauer, g. 2017: the relationship between habitat factors and aquatic macrophyte assemblages in the danube river in serbia. archives of biological sciences, 69(3): 427-437. vukotić et al. ● aquatic and wetland plant communities of the pusta river (southern serbia): floristic characteristics and syntaxonomic affiliation biologica nyssana ● 13 (1) september 2022: 59-81 71 n um be r of re le vé s 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 ic d % s % l oc al it y k oč an e pu ko va c b oj ni k r ec or di ng da te 08 s ep 12 s ep 16 s ep 25 s ep t e le va ti on (m ) 19 5 20 0 25 1 l at it ud e 43.1136 43.1141 43.1143 43.1149 43.115 43.1152 43.1131 43.113 43.1126 43.1126 43.1894 43.189 43.1885 43.1884 43.1879 43.1877 43.1871 43.1862 43.1855 43.1842 43.1668 43.1673 43.1681 43.1673 43.1662 43.1657 43.1653 43.1639 43.1629 43.1621 43.1611 43.0129 l on gi tu de 21.5029 21.5028 21.503 21.5034 21.5035 21.5035 21.503 21.5031 21.5034 21.5034 21.8434 21.8437 21.8435 21.8437 21.8436 21.844 21.8444 21.8447 21.845 21.8461 21.8559 21.8556 21.8555 21.8554 21.857 21.858 21.8587 21.8618 21.8618 21.8618 21.8627 21.7245 ty ph a la tif ol ia 5 5 5 5 5 5 5 5 5 5 5 5 4 5 5 5 5 5 5 5 5 5 5 5 5 5 4 5 5 5 5 5 99 .3 52 10 0 ly th ru m sa lic ar ia + + + + + + + + + . . + + + . 1 . . . r . r r . r r r . r . . . 11 .8 6. 2 62 .5 sa lix a lb a + . r + + . + + + + . . . . + . + . + + . + . 1 . 1 . . . + . . 11 .5 6 50 b id en s tr ip ar tit us . 1 . r + . . + + + . r + . . . . . r . . r . r . r . . r . r + 8 4. 2 46 .9 sa lix e ux in a . . . + . . . . . . . . . . . . + . . . . . + 1 + + 1 + . . + + 7. 6 4 31 .2 p ol yg on um la pa th ifo liu m . 1 . . + + . . . . + r + . . . . . . . . r . . . . . . r . r . 5. 2 2. 7 28 .1 x an th iu m st ru m ar iu m . . . . + . . . . + 1 . r . + . . . r . . r . r . . . . . . + . 5. 2 2. 7 28 .1 p ol yg on um m ite . + . . + + . . . . . . + . . . . . . . . . . . . . . . r r r . 3. 8 2 21 .9 sp ar ga ni um er ec tu m . . . + + + . . 1 + . . . . . . . . . . . . . . . . . . . . . . 3. 8 2 15 .6 u rt ic a di oi ca . . . . . + . r . . . . r . . . . . . . . r . . . . r r r . . + 3. 5 1. 8 25 c al ys te gi a se pi um . + . . . r . . . + . . + . . . . . . . . r . . . . r . . . . . 3. 1 1. 6 18 .9 m en th a aq ua tic a . . r r . + . + . + . . . . . . . . . . . . . . . . . . . . . . 2. 8 1. 5 15 .6 p op ul us s p. . . . . . . + r . + . . . . r . + . . . . . . . . . . . . . . . 2. 8 1. 5 15 .6 biologica nyssana ● 13 (1) september 2022: 59-81 vukotić et al. ● aquatic and wetland plant communities of the pusta river (southern serbia): floristic characteristics and syntaxonomic affiliation a pp en di x 1. p hy to co en ol og ic al t ab le o f ty ph et um l at ifo lia e n ow iń sk i 1 93 0. i c co ve ra ge in de x ac co rd in g to l au si e t al . (1 98 2) ; d % s ha re in t ot al co ve ra ge a cc or di ng to s ur in a (2 00 5) ; s % sp ec ie s pe rc en ta ge fr eq ue nc y w ith in th e cl us te r a pp en di ce s 72 73 biologica nyssana ● 13 (1) september 2022: 59-81 vukotić et al. ● aquatic and wetland plant communities of the pusta river (southern serbia): floristic characteristics and syntaxonomic affiliation n um be r of re le vé s 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 ic d % s % le er si a or yz oi de s . + . . + . . . . + . . . . . . . . . . . . . . . . . . . r . . 2. 4 1. 3 12 .5 so la nu m du lc am ar a . + . . . + . . . + . . . . . . . . . . . . . . . . r . . . . . 2. 4 1. 3 12 .5 p ot am og et on no do su s + . . . . . . . . . . r . . . . . . . . . . . . r r . r . . . . 2. 1 1. 1 15 .6 e pi lo bi um hi rs ut um . . + . + r . . . . . . . . . . . . . . . . . . . r . . . . . . 2. 1 1. 1 12 .5 a m or ph a fr ut ic os a . . . . . . + + . . . . . . . . . . . r . . . . . . . . . . . . 1. 7 0. 9 9. 4 h um ul us lu pu lu s . . . . . r . . . . . . . . . . . . . . . . . . . . + . . . r . 1. 4 0. 7 9. 4 p ha la ri s ar un di na ce a + . . . . . . . . . . . . . . + . . . . . . . . . . . . . . . . 1. 4 0. 7 6. 3 ly co pu s eu ro pa eu s . . r r . . . . . . . . . . . . . . . . . . . . . r . . . . . . 1 0. 5 9. 4 st ac hy s pa lu st ri s . . . . . 1 . . . . . . . . . . . . . . . . . . . . . . . . . . 1 0. 5 3. 1 r ob in ia ps eu do ac ac ia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . r + . 1 0. 5 6. 3 r um ex co ng lo m er at us + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0. 7 0. 4 3. 1 ly si m ac hi a vu lg ar is . . . . + . . . . . . . . . . . . . . . . . . . . . . . . . . . 0. 7 0. 4 3. 1 ta na ce tu m vu lg ar e . . . . . . . . . . . . + . . . . . . . . . . . . . . . . . . . 0. 7 0. 4 3. 1 m yr io ph yl lu m sp ic at um . . . . . . . . . . . . . . . . . . . . r . . . r . . . . . . . 0. 7 0. 4 6. 3 r ub us c ae si us . . . . . . . . . . . . . . . . . . . . . . . . . . r r . . . . 0. 7 0. 4 6. 3 g al iu m ap ar in e . . . . . r . . . . . . . . . . . . . . . . . . . . r . . . . . 0. 7 0. 4 6. 3 a lth ae a offi ci na lis . . . . . . . . . . . . r . . . . . . . . . . . . . . . . . . . 0. 3 0. 2 3. 1 p ot am og et on cr is pu s . . . . . . . . . . . . . . . . . . . . . r . . . . . . . . . . 0. 3 0. 2 3. 1 e ch in oc hl oa cr us -g al li . . . . . . . . . . . . . . . . . . . . . . . . . . . . r . . . 0. 3 0. 2 3. 1 biologica nyssana ● 13 (1) september 2022: 59-81 vukotić et al. ● aquatic and wetland plant communities of the pusta river (southern serbia): floristic characteristics and syntaxonomic affiliation n um be r of re le vé s 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 ic d % s % le m na m in or . . . . . . . . . . . . . . . . . . . . . . . . . . . . r . . . 0. 3 0. 2 3. 1 e ch in oc ys tis lo ba ta . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . r . 0. 3 0. 2 3. 1 74 75 biologica nyssana ● 13 (1) september 2022: 59-81 vukotić et al. ● aquatic and wetland plant communities of the pusta river (southern serbia): floristic characteristics and syntaxonomic affiliation appendix 2. phytocoenological table of phalaridetum arundinaceae libbert 1931. ic coverage index according to lausi et al. (1982); d% share in total coverage according to surina (2005); s% species percentage frequency within the cluster number of relevés 1 2 3 4 5 6 7 8 ic d% s% locality kočane kosančić recording date 8 sep 12 sep 25 sep elevation (m) 194 194 194 195 195 195 195 222 latitude 43 .1 14 3 43 .1 15 3 43 .1 13 43 .1 12 4 43 .1 89 3 43 .1 88 5 43 .1 87 4 43 .0 78 8 longitude 21 .5 03 21 .5 03 5 21 .5 03 1 21 .5 03 5 21 .8 43 5 21 .8 43 7 21 .8 44 3 21 .7 86 4 phalaris arundinacea 5 5 5 5 5 5 4 5 99 41 100 mentha aquatica r + + 1 + . 2 . 21 8.6 75 lythrum salicaria r + + + + . + . 15 6.3 75 sparganium erectum + + + + . 1 . . 15 6.3 63 salix alba + + + + . . . + 14 5.7 63 xanthium strumarium . . + + + . + . 11 4.6 50 populus sp. + r . . 1 . . . 8.3 3.4 38 leersia oryzoides + . + + . . . . 8.3 3.4 38 typha latifolia + . . . + . + . 8.3 3.4 38 urtica dioica r . + . r . . . 8.3 3.4 38 epilobium hirsutum r . . . + . . . 4.2 1.7 25 polygonum mite + . . . r . . . 4.2 1.7 25 calystegia sepium . . + . . r . . 4.2 1.7 25 tanacetum vulgare . . 1 . . . . . 4.2 1.7 13 bidens tripartitus . . . . . . r + 4.2 1.7 25 salix euxina + . . . . . . . 2.8 1.1 13 amorpha fruticosa . + . . . . . . 2.8 1.1 13 lycopus europaeus . . . . + . . . 2.8 1.1 13 polygonum lapathifolium . . . . + . . . 2.8 1.1 13 myriophyllum spicatum . . . . . . . + 2.8 1.1 13 biologica nyssana ● 13 (1) september 2022: 59-81 vukotić et al. ● aquatic and wetland plant communities of the pusta river (southern serbia): floristic characteristics and syntaxonomic affiliation appendix 3. phytocoenological table of polygono-bidentetum tripartitae (w. koch 1926) lohm. 1950. ic coverage index according to lausi et al. (1982); d% share in total coverage according to surina (2005); s% species percentage frequency within the cluster number of relevés 1 2 3 ic d% s% locality kočane recording date 08 sep elevation (m) 195 latitude 43 .1 14 1 43 .1 14 3 43 .1 12 9 longitude 21 .5 02 8 21 .5 03 21 .5 03 1 polygonum lapathifolium 4 5 4 93 23 100 bidens tripartitus 1 1 4 52 13 100 polygonum mite 2 + + 33 8.4 100 lythrum salicaria 1 + . 19 4.7 67 xanthium strumarium + 1 . 19 4.7 67 leersia oryzoides . 2 . 19 4.7 33 sparganium erectum + + . 15 3.7 67 calystegia sepium + + 15 3.7 67 echinochloa crus-galli r + . 11 2.8 67 erigeron canadensis . r + 11 2.8 67 tanacetum vulgare . r + 11 2.8 67 lysimachia vulgaris 1 . . 11 2.8 33 mentha aquatica + . . 7.4 1.9 33 lycopus europaeus + . . 7.4 1.9 33 aristolochia clematitis + . . 7.4 1.9 33 urtica dioica + . . 7.4 1.9 33 phalaris arundinacea + . . 7.4 1.9 33 solanum dulcamara + . . 7.4 1.9 33 rubus caesius + . . 7.4 1.9 33 amorpha fruticosa . + . 7.4 1.9 33 salix euxina . + . 7.4 1.9 33 chenopodium album . + . 7.4 1.9 33 robinia pseudoacacia . . + 7.4 1.9 33 rumex conglomeratus . . + 7.4 1.9 33 76 77 biologica nyssana ● 13 (1) september 2022: 59-81 vukotić et al. ● aquatic and wetland plant communities of the pusta river (southern serbia): floristic characteristics and syntaxonomic affiliation a pp en di x 4. p hy to co en ol og ic al ta bl e of g ly ce rio -s pa rg an ie tu m n eg le ct i k oc h 19 26 . i c co ve ra ge in de x ac co rd in g to l au si e t a l. (1 98 2) ; d % s ha re in to ta l c ov er ag e ac co rd in g to s ur in a (2 00 5) ; s % sp ec ie s pe rc en ta ge fr eq ue nc y w ith in th e cl us te r n um be r of re le vé s 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 ic d % s % l oc al it y k oč an e pu ko va c donje brijanje bojnik r ec or di ng da te 08 s ep 12 s ep 16 s ep 25 s ep e le va ti on (m ) 19 5 19 6 20 0 21 8 25 1 l at it ud e 43.11356 43.11404 43.11429 43.11311 43.11255 43.11239 43.11222 43.18922 43.18825 43.18822 43.18779 43.18643 43.18616 43.18478 43.16677 43.16702 43.16714 43.16611 43.16495 43.16474 43.16388 43.16378 43.16343 43.16296 43.16218 43.16152 43.16073 43.16055 43.09847 43.0984 43.01257 l on gi tu de 21.50288 21.50282 21.50307 21.50306 21.50336 21.5034 21.50352 21.84363 21.84367 21.84347 21.84394 21.84498 21.84496 21.84558 21.85568 21.85548 21.85567 21.85677 21.86029 21.86074 21.86166 21.86159 21.86172 21.86183 21.86182 21.8619 21.8626 21.86237 21.81782 21.81832 21.72439 sp ar ga ni um er ec tu m 5 5 5 5 5 5 5 5 5 5 5 5 4 5 3 5 5 5 5 5 5 5 5 5 5 5 5 5 5 4 5 98 .6 48 .8 10 0 ly th ru m sa lic ar ia 1 1 + + + + + + . 1 + r + + 2 + r + . r . + r + r r . r . . . 16 .8 8. 3 77 .4 p ol yg on um m ite . + + 1 . . . . . . . . . + . . + . r . . . + . . r . . + 4 + 9. 7 4. 8 35 .5 b id en s tr ip ar tit us . + r . + + . . . . . . . + . . + . + r r . + r r . . . + + + 9 4. 4 48 .4 p ol yg on um la pa th ifo liu m . 1 + + . . + . . . . . . + . . 1 . r r . . + . . . . . + . 1 8. 2 4. 1 35 .5 m en th a aq ua tic a . + . + 1 . . . + + . + + . r r . r . . r . . . . r . . . . . 7. 2 3. 6 38 .7 sa lix a lb a . . + r + . + . . . + . 1 . . . . . . . . . . . . . . + + . . 5. 7 2. 8 25 .8 p ot am og et on no do su s . . . . . + r + . . . + r . . . . r r r r . . . r . r r . . . 5. 4 2. 7 38 .7 sa lix e ux in a . r . . . . . . . . . . 1 . . + . . . . r . . . . + . . + + . 4. 7 2. 3 22 .6 x an th iu m st ru m ar iu m . . . . . r . . + . . . . + . . r . r . . . . r . r . . . r + 4. 3 2. 1 29 biologica nyssana ● 13 (1) september 2022: 59-81 vukotić et al. ● aquatic and wetland plant communities of the pusta river (southern serbia): floristic characteristics and syntaxonomic affiliation n um be r of re le vé s 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 ic d % s % ly co pu s eu ro pa eu s . + . . . . . . . . . . . . r . . r r . . . . . . r . r . r . 2. 9 1. 4 22 .6 p ha la ri s ar un di na ce a 1 . . . . . . . . + . . + . . . . . . . . . . . . . . . . . . 2. 5 1. 2 9. 7 ty ph a la tif ol ia + . . + . . . . . . . . . . 1 . . . . . . . . . . . . . . . . 2. 5 1. 2 9. 7 u rt ic a di oi ca . . . r . . . r . . . . r . r . . r . . . . . . . r . . . . r 2. 5 1. 2 22 .6 m yr io ph yl lu m sp ic at um . . . . . . . . . . . . . . . . . r . r r . . r r . r r . . . 2. 5 1. 2 22 .6 ta na ce tu m vu lg ar e . . . . . + . . . . . . . . . . . r . . . . r . . r . r . . . 2. 2 1. 1 16 .1 c al ys te gi a se pi um . . . . . . . . . . . r . . r . . r . . . . . . . r . . . r r 2. 2 1. 1 19 .4 r ob in ia ps eu do ac ac ia . . . . . . . . . + . . . . . . . . . r . . . . . . . + . . . 1. 8 0. 9 9. 7 so la nu m du lc am ar a . r . . . . . . . . . . . . + r . . . . . . . . . . . . . . . 1. 4 0. 7 9. 7 a m or ph a fr ut ic os a . . + . + . . . . . . . . . . . . . . . . . . . . . . . . . . 1. 4 0. 7 6. 5 le er si a or yz oi de s . . . + . . . . . . . . . . . . . r . r . . . . . . . . . . . 1. 4 0. 7 9. 7 g al iu m ap ar in e . . . . . . . r . . . . . . . . . r . . . . r . . r . . . . . 1. 4 0. 7 12 .9 r ub us c ae si us . . . . . . . . . . . r . . . . . . r r . r . . . . . . . . . 1. 4 0. 7 12 .9 e pi lo bi um hi rs ut um . . . . . . . . . . . . . . . . . . . r . r . . . r . . . . . 1. 1 0. 5 9. 7 le m na m in or . . . . . . . . . . . . . . . . . . . r . . . r . . . . . r . 1. 1 0. 5 9. 7 e ch in oc hl oa cr us -g al li . . r . . . . . . . . . . . . . . . . . . . . . . . r . . . . 0. 7 0. 4 6. 5 p ot am og et on cr is pu s . . . . . . . + . . . . . . . . . . . . . . . . . . . . . . . 0. 7 0. 4 3. 2 sc ut el la ri a ga le ri cu la ta . . . . . . . . . . . . . . . . . r . . . . . r . . . . . . . 0. 7 0. 4 6. 5 sc ro ph ul ar ia no do sa . . . . . . . . . . . . . . . . . r . . . . . . . . . . . . . 0. 4 0. 2 3. 2 78 79 biologica nyssana ● 13 (1) september 2022: 59-81 vukotić et al. ● aquatic and wetland plant communities of the pusta river (southern serbia): floristic characteristics and syntaxonomic affiliationr n um be r of re le vé s 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 ic d % s % b er ul a er ec ta . . . . . . . . . . . . . . . . . . . . r . . . . . . . . . . 0. 4 0. 2 3. 2 st el la ri a ne gl ec ta . . . . . . . . . . . . . . . . . . . . . . . . . r . . . . . 0. 4 0. 2 3. 2 e ch in oc ys tis lo ba ta . . . . . . . . . . . . . . . . . . . . . . . . . r . . . . . 0. 4 0. 2 3. 2 e le oc ha ri s pa lu st ri s . . . . . . . . . . . . . . . . . . . . . . . . . . r . . . . 0. 4 0. 2 3. 2 biologica nyssana ● 13 (1) september 2022: 59-81 vukotić et al. ● aquatic and wetland plant communities of the pusta river (southern serbia): floristic characteristics and syntaxonomic affiliation appendix 5. phytocoenological table of phragmitetum australis savič 1926. ic coverage index according to lausi et al. (1982); d% share in total coverage according to surina (2005); s% species percentage frequency within the cluster number of relevés 1 2 3 4 5 6 7 8 9 ic d% s% locality kočane pukovac recording date 08 sep 12 sep 16 sep elevation (m) 195 200 latitude 43 .1 12 7 43 .1 88 43 .1 85 3 43 .1 84 8 43 .1 64 5 43 .1 63 6 43 .1 63 5 43 .1 63 1 43 .1 63 longitude 21 .5 03 3 21 .8 43 8 21 .8 45 3 21 .8 45 5 21 .8 61 2 21 .8 61 6 21 .8 61 7 21 .8 61 8 21 .8 62 1 phragmites communis 5 5 5 5 5 5 5 5 5 100 56 100 bidens tripartitus + . . . + r r r . 9 5 56 urtica dioica r . r . + r r . . 7 4 56 salix.euxina . . . . + + + . . 7 4 33 robinia pseudoacacia . r + . . . . . + 6 4 33 salix alba + + . . . . . . . 5 3 22 echinocystis lobata . . . . + . . r r 5 3 33 typha latifolia 1 . . . . . . . . 4 2 11 lythrum salicaria + . . . . r . . . 4 2 22 polygonum lapathifolium + . . . . . . r . 4 2 22 sparganium erectum . . . r + . . . . 4 2 22 humulus lupulus + . . . . . . . . 3 1 11 mentha aquatica + . . . . . . . . 3 1 11 polygonum mite . . . . + . . . . 3 1 11 epilobium hirsutum . . . . + . . . . 3 1 11 galium aparine . . . . + . . . . 3 1 11 rubus caesius . . . . . r r . . 3 1 22 lycopus europaeus . . . . . r r . . 3 1 22 populus sp. . r . . . . . . . 1 1 11 potamogeton nodosus . . . r . . . . . 1 1 11 erigeron canadensis . . . . . r . . . 1 1 11 tanacetum vulgare . . . . . r . . . 1 1 11 xanthium strumarium . . . . . . r . . 1 1 11 80 81 biologica nyssana ● 13 (1) september 2022: 59-81 vukotić et al. ● aquatic and wetland plant communities of the pusta river (southern serbia): floristic characteristics and syntaxonomic affiliation appendix 6. phytocoenological table of myriophyllo-potametum soó 1934. ic coverage index according to lausi et al. (1982); d% share in total coverage according to surina (2005); s% species percentage frequency within the cluster number of relevés 1 2 3 4 5 ic d% s% locality kočane pukovac recording date 08 sep 16 sep elevation (m) 195 200 latitude 43 .1 14 6 43 .1 64 5 43 .1 64 43 .1 62 3 43 .1 66 8 longitude 21 .5 03 2 21 .8 61 2 21 .8 61 6 21 .8 61 7 21 .8 55 8 myriophyllum spicatum 5 5 4 5 5 98 69 100 potamogeton crispus 1 . . . 4 24 17 40 potamogeton nodosus + . 2 + . 20 14 60 appendix 7. phytocoenological table of potametum nodosi soó (1928) 1960, segal 1964. ic coverage index according to lausi et al. (1982); d% share in total coverage according to surina (2005); s% species percentage frequency within the cluster number of relevés 1 2 3 4 5 6 7 8 ic d% s% locality pukovac recording date 16 sep elevation (m) 200 latitude 43 .1 66 8 43 .1 66 3 43 .1 65 3 43 .1 64 9 43 .1 64 7 43 .1 61 7 43 .1 60 8 43 .1 60 6 longitude 21 .8 55 8 21 .8 56 7 21 .8 59 1 21 .8 59 8 21 .8 60 9 21 .8 62 2 21 .8 62 6 21 .8 62 7 potamogeton nodosus 5 5 5 4 4 5 5 5 97 63 100 myriophyllum spicatum . 4 1 + 1 1 2 4 44 29 87.5 sparganium erectum . . . 3 . . . . 10 6 12.5 lemna minor . . . . + . . . 3 2 12.5 aladejana, oluduro 2021, biologica nyssana 12(1) 12 (1) september 2021: 79-86 doi: 10.5281/zenodo.5523057 molecular characterization of extended spectrum β-lactamaseproducingenterobacteriaceae from bats (eidolon helvum) faeces in osun state, nigeria original article oluwatoyin modupe aladejana department of biological sciences, microbiology unit, kings university, odeomu, osun state nigeria om.aladejana@kingsuniversity.edu.ng (corresponding author) anthonia olufunke oluduro department of microbiology, faculty of science, obafemi awolowo university, ile-ife, osun state, nigeria aoluduro2003@yahoo.co.uk received: october 23, 2020 revised: april 01, 2021 accepted: april 04, 2021 abstract: enterobacteriaceae from faecal samples of eidolon helvum were isolated and identified biochemically then further confirmed using analytical profile index 20e kit. antibiotic susceptibility testing and extended spectrum b-lactamase (esbl) production using mast disc were carried out. detection of ctx-m gene was done using polymerase chain reaction (pcr) employing appropriate primers. a total of 337 bacterial isolates was recovered from the studied locations consisting of forty-one species belonging to fifteen genera. species of citrobacter, enterobacter, salmonella, klebsiella spp, and escherichia coli were the most abundant and common to the three studied locations, with salmonella spp being the most predominant. a total of 35.9% of the selected isolates was produced elbs and 14.28% of the isolates harbours ctx-m gene. the study concludes that e. helvum in the study areas are reservoirs of enterobacteriaceae and some harboured ctx-m gene which confirmed their pathogenicity with public health consequences. key words: eidolon helvum, enterobacteriaceae, extended spectrum b-lactamase apstract: molekularna karakterizacija enterobacteriaceae iz fecesa slepog miša (eidolon helvum) u osunu, nigerija koje produkuju β-laktamaze proširenog spektra enterobacteriaceae su izolovane iz fekalnih uzoraka eidolon helvum i biohemijski identifikovane korišćenjem api (analytical profile index) 20e kita. korišćenjem mast diskova urađeno je i testiranje osetljivosti na antibiotike i testiranje produkcije β-laktamaze proširenog spektra (esbl). detekcija gena ctx-m je urađena je polimeraznom lančanom reakcijom (pcr) korišćenjem odgovarajućih prajmera. na ispitivanim lokacijama utvrđdeno je ukupno 337 izolata koji pripadaju 41 vrsti iz 15 rodova. vrste citrobacter, enterobacter, salmonella, klebsiella spp, i escherichia coli bile su najzastupljenije i česte za tri ispitivane lokacije, sa najčešćom salmonella spp. ukupno 35.9% odabranih izolata produkovalo je esbl i 14.28% izolata nosilo je gen ctx-m. istraživanje je zaključilo da su e. helvum rezervoari enterobacteriaceae u istraživnim oblastima i da neki nose ctx-m gen koji potvrđuje njihovu patogenost i posledice po javno zdravlje. ključne reči: eidolon helvum, enterobacteriaceae, β-laktamaze proširenog spektra introduction enterobacteriaceae consist of a large number of closely related bacterial species that inhabit the large bowel of man and animals, soil, water, and decaying matter (hassan et al., 2011). these organisms are responsible for various infections including nosocomial or hospital-acquired infections that cause urinary tract and wound infections, pneumonia, meningitis and septicaemia, among others (ruiz et al., 2002). wild animals can serve as sources of various infectious diseases, including the majority of nosocomial or hospital-acquired and illnesses resulting from environmental contamination. emerging infectious diseases are increasingly originating from wildlife. different activities result © 2021 aladejana, oluduro. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 79 into emerging infectious diseases of wildlife and they include:alteration of an ecosystem, transfer of pathogens and vectors by human or other agents as well as advice in microbial genomic studies which reveals more about the infectious agents. the transmission of pathogens from wildlife to other animals and humans could be through direct or indirect contact (rabinowitz et al., 2009). wildlife although not directly exposed to antimicrobial agents can acquire resistance to clinically used antimicrobial agents through contact with the polluted environment, domestic animals and humans. bat is a natural reservoir of deadly viruses, such as rabies, ebola viruses and zoonotic bacterial pathogens, such as escherichia coli and staphylococcus aureus among others. (oluduro, 2012; akobi et al., 2012). bat can easily spread multiple antibiotic resistant enterobacteriaceae due to their versatile feeding and migratory capability. antibiotics are used in the treatment of infections caused by enterobacteriaceae. the active antimicrobial agents available for use against these organisms include; fluoroquinolones, β -lactams and aminoglycosides. resistance to betalactams in enterobacteriaceae is mainly conferred by β-lactamases. these enzymes inactivate betalactam antibiotics by hydrolysis. the most common esbls are shv-, tem-, ctx-m. the ctx-ms are increasingly detected worldwide (paterson & bonomo, 2005; pierre et al., 2020). materials and methods study area the study area were selected cities where bats roost over a human-populated place in osun state, nigeria. these included: ile ife lies between 7°31’14’’ n and 7°31’14’’.7612 n and longitudes 4°32’3.161’’e and 4°32’2.591’’ e coordinates, osogbo (machine tools area, km 8, osogboikirun road) located at 7°50’9.0168’’ n and 4°36’30.0708’’ e and ilesa (oba’s palace area, ilesa) located at 7°37’0’’ n and 4°43’0’’ e (fig. 1). sample collection faecal droppings of bats were collected randomly from various roosting places in osun state, in the locations stated above. the samples were collected between 6.00 a.m and 7.00 a.m according to the method of akobi et al. (2012) and transported to the laboratory immediately in an ice bag for bacteriological analysis. isolation of bacteria the faecal samples collected were immediately streaked on prepared macconkey agar plates and incubated at 37 °c for 24 hours for isolation of enterobacteriaceae. distinct colonies were picked and sub-cultured by successive streaking on freshly prepared macconkey agar plates, incubated at 37 °c for 24 hours and a pure isolates were obtained. these were stored on nutrient agar slant and kept in the refrigerator at 4 °c for further use. all the isolates were identified using cultural, morphological, microscopic examination and biochemical tests following standard procedures and interpreted according to bergey’s manual of systematic bacteriology. isolates were further confirmed using analytical profile index (api) 20e test kit (biomérieux, inc., france). 80 biologica nyssana ● 12 (1) september 2021: 79-86 aladejana, oluduro ● molecular characterisation of extended spectrum β-lactamase-producingenterobacteriaceae from bats faeces... fig. 1. map of osun state and the study areas 81 phenotypic detection of extended spectrum b-lactamase (esbl) production selected multiple antibiotic-resistant enterobacteriaceae isolates from faecal samples of bats were tested for esbl production. this was carried out by using mask disc comprising six antibiotics of ceftazidime (30 µg), ceftazidime plus clavulanic acid (30 µg), cefotaxime (30 µg), cefotaxime plus clavulanic acid (30 µg), cefpodoxime (30 µg), and cefpodoxime plus clavulanic acid (30 µg). the standardised inoculum of the isolates were inoculated aseptically on müeller-hinton agar plates. these discs were firmly placed on the surface of the culture plates aseptically using a sterile forceps and incubated in an inverted position at 37 °c for 24 hours. the diameters of inhibition zones of each antibiotic on the bacterial isolates were measured with a transparent ruler to the nearest millimetre. an increase in zone diameter greater than or equal to 5 mm in the presence of clavulanic acid from any or all the sets of mast id esbl detection disc was considered to indicate the presence of esbl in the tested organisms. detection of resistance gene (ctm gene) in selected esbl-producing enterobacteriaceae isolates by polymerase chain reaction (pcr) for the extraction of dna, randomly selected esbl-producing enterobacteriaceae isolates grown overnight were transferred to eppendorf tube and spun down at 14,000 rpm for 2 minutes. the supernatant was discarded and 600 µl of prewarmed extraction buffer cetyl trimethyl ammonium bromide (ctab) was added to the pellet and incubated at 65 °c for 30 minutes. the sample was removed from the incubator and allowed to cool to room temperature, chloroform was added and mixed by inverting tubes for 15 minutes. after that, the sample was spun at 14,000 rpm for 15 minutes and the supernatant was transferred into a new eppendorf tube, afterwards equal volume of cold isopropanol was added to precipitate the dna. the sample was kept in the freezer for 1hour and later spun at 14,000 rpm for 10 minutes. the supernatant was discarded and the pellet was washed with 70% ethanol. the sample was then air dried for 30 minutes on the bench. the pellet was resuspended in 100 µl of sterile distilled water. the dna concentration of the samples was measured on spectrophotometer at 260 nm and 280 nm and the genomic purity was determined. the genomic purity was between 1.8 –2.0 µl for all the dna samples. ctx-m genes were profiled in the isolates by pcr using the primer ctx-m-f at g t g c a g ya c c a g ta a r g t k at g g c , ctx-m-r-tgggtraartargtsaccagaa ysagcgg (8). pcr was performed in a total volume of 25 μl containing 2.5 μl of both the forward and the reverse of the primers, 12.5 μl master mix, 2.5 μl nuclease free water and 5 μl of the extracted dna (as dna template), then dna amplification was carried out with the thermal cycler. the reaction mixtures were amplified by denaturation for 4 minutes at 95 °c, 30 cycles at 95 °c for 30 seconds, annealing temperature at 55 °c for 60 seconds. the pcr products were electrophoresed using 1.5% agarose gel. it was stained with 1% ethidium bromide and run at 80 v for 2 hours, the electrophoretic products were scanned with uvtransilluminator. data analysis data were analysed using spss software (version 20). proportions and the actual number of esblproducing enterobacteriaceae isolates was used to describe frequency outputs for categorical variables. mean and standard deviation were used to describe continuous variables. results in all, 337 bacterial isolates were recovered from the three locations which includes 142 (42.12%) isolates from obafemi awolowo university, ile ife, 84 (24.93 %) from nigeria machine tools area, osogbo and 111 (32.94 %) from oba’s palace area, ilesa all in osun state, southwest, nigeria. the identified enterobacteriaceae belong to 15 genera comprising 41 different species. the bacterial species identified based on their various biochemical reactions to the api kits include the following: citrobacter freundi, c. koseri, c. diversus, c. werkmanii, c. rodentium; enterobacter aerogenes, e. intermedius, e. cloacae, e. aquatilis e. asburiae, e. sakazakii; proteus vulgaris, p. mirabilis, salmonella arizonae, s. bongori, s. typhi, s. enterica, s. choleraesuis; klebsiella pneumoniae, k. oxytoca, k. ornithinolytica, k. planticola; shigella sonnei, sh. dysentery, sh. flexineris; serratia. liquefaciens, s. marcescens, s. plymutica, s. odorifera; raoultella terrigena, r. ornithinolytica; yersinia frederiksenii, y. aleksiciae; y. mollaretii; escherichia coli, erwinia amylovora, edwardsiella ictaluri, kluyvery ascorbate, providenica rettgeri, and rahnella aquatilis. table 1 shows the distribution of the bacterial isolates in the bats faeces across the three studied locations. for ile-ife location, s. arizonae has the highest frequency of 24 (16.90%) followed by e. coli 17 (11.97%) and the least frequency of 1 (0.70%) were recorded in e. sakazaki, e. hormaecei, k. planticola, k. ornitinolytica, r. aquatilis, r. terribiologica nyssana ● 12 (1) september 2021: 79-86 aladejana, oluduro ● molecular characterisation of extended spectrum β-lactamase-producingenterobacteriaceae from bats faeces... 82 table 1. the percentage of enterobacteriaceae isolates in faeces of eidolon helvum from the three locations bacterial isolates ile-ife(n=142) (%) osogbo (n=84) (%) ilesa (n=111) (%) number of isolates (n=337) (%) citrobacter freundi 3(2.11) 2(2.38) 5(4.51) 10 (2.97) c. koseri 3(2.11) 2(2.38) 3 (2.70) 8 (2.37) c. werkmanii 1(0.70) 1 (0.30) c. rodentium 6(4.23) 3 (0.89) c. farmer 2(1.41) 2 (0.59) c. diversus 4 (3.60) 4(1.19) escherichia coli 17 (11.97) 11(13.10) 8 (7.20) 36 (10.68) enterobacter aerogenes 2 (1.41) 5(5.59) 6 (5.40) 13 (3.86) e. aquatilis 1(1.19) 1 (0.30) e. intermidius 3 (2.11) 2(2.38) 5 (1.48) e. asburiae 12(8.45) 4(2.80) 5 (4.50) 21 (0.21) e. cloacae 2 ( 1.41) 1(1.19) 6 (5.40) 9 (2.67) e. sakazaki 1 (0.70) 2(2.38) 3 (0.89) e. hormaecei 1 (0.70) 1 (0.30) edwardsiella ictaluri 3 (2.11) 3 (0.89) erwinia amylovora 1 (0.90) 1 (0.30) klebsiella pneumonia 10 (7.04) 10 (11.91) 11 (9.91) 31 (9.20) k. planticola 1 (0.70) 3 (2.70) 4(1.19) k. oxytoca 13 (9.16) 9 (10.71) 5 (4.51) 27 (8.01) k. ornitinolytica 1 (0.70) 1 (0.30) kluyvery ascorbate 2(1.41) 1 (1.19) 1 (0.90) 4 (1.19) proteus mirabilis 5(3.04) 1 (1.19) 6 (5.40) 12 (3.56) p. vulgaris 2(1.41) 1(1.19) 3 (2.70) 6 (1.78) providenica rettgeri 2(1.41) 2 (0.60) rahnella aquatilis 1(0.70) 1 (0.30) raoultella terrigena 1 (0.70) 1 (0.30) r. ornithinolytica 3 (2.11) 3 (0.89) serratia plymutica 5 (3.04) 3 (2.70) 8 (2.37) s. odorifera 2 (1.41) 2 (1.80) 4 (1.19) s. liquefaciens 1 (0.70) 1 (1.19) 2 (0.60) s. marcescens 3 (2.11) 2 (1.80) 5 (1.48) salmonella typhi 1 (1.19) 4 (3.60) 5 (1.48) s. enterica 7 (8.33) 5 (4.51) 12 (3.56) s. bongori 2 (1.41) 2 (2.38) 4 (1.19) s. arizonae 24 (16.90) 14(16.67) 12 (10.80) 50 (41.56) s. choleraesuis 1 (1.19) 1 (0.30) shigella flexineris 3 (2.11) 3(3.57) 3 (2.70) 9 (2.67) s. sonnei 4(4.76) 4 (3.60) 8 (2.37) yersinia aleksiciae 1 (0.70) 1(1.19) 2 (0.60) y. mollaretii 1 (0.70) 2 (1.80) 3 (0.89) y. fredrikseni 3 (2.11) 3 (0.89) biologica nyssana ● 12 (1) september 2021: 79-86 aladejana, oluduro ● molecular characterisation of extended spectrum β-lactamase-producingenterobacteriaceae from bats faeces... gena, s. liquefaciens, y. aleksiciae and y. mollaretii. for osogbo location, salmonella arizonae has the highest frequency of 14 (16.67%) followed by escherichia coli 11 (13.10%) and the leat frequency of 1 (1.19%) were recorded in enterobacter aquatilis, e. cloacae, kluyvery ascorbate, proteus mirabilis, p. vulgaris, s. liquefaciens, s. typhi, s. choleraesuis and y. aleksiciae. also for ilesa location. s. arizonae has the highest frequency of 12 (10.80%) followed by k. pneumoniae 11 (9.91%) and the leat frequency of 1 (0.9%) were recorded in erwinia amylovora and kluyvery ascorbate. in total, s. arizonae has the hig-hest frequency of 50 (41.56%), followed by e. coli 36 (10.68 %), k. pneumoniae 31 (9.20 %), k. oxytoca 27 (8.01%). salmonella spp., citrobacter spp., enterobacter spp., klebsiella spp., yersinia spp. and e. coli were most abundant and common to the three locations as depicted in fig. 2. table 2 shows the occurrence of the esbl-producing isolates in selected multiple antibiotic resistant bacterial isolates from bat faecal samples. the esblproduction by selected multiple antibiotic-resistant isolates using mast disc showed that 14 (35.90%) of the 39 selected antibiotic-resistant isolates were positive to esbls production. 83 fig. 2. distribution of bacterial isolates common to the three locations (ile-ife, osogbo and ilesa) fig 3: agarose gel electrophoresis of ctx –m (529 bp) amplicons of the selected multiple antibiotics resistant e coli spp. isolated from faecal samples of eidolon helvum from three locations key lane m –dna ladder (100 bp). lanes 1 to 3 were amplicons from e. coli lanes 4 to 6 were amplicons from enterobacter sakazakii lanes 7 and 10 were amplicons from salmonella arizonae lanes 11 and 12 were amplicons from serretia liquefacience lanes 13 and 14 were amplicons from citrobacter koseri biologica nyssana ● 12 (1) september 2021: 79-86 aladejana, oluduro ● molecular characterisation of extended spectrum β-lactamase-producingenterobacteriaceae from bats faeces... the ctx –m (529 bp) gene was detected in e. coli and e. sakazakii as shown in fig. 3. discussion fifteen different genera comprising 41 different species of enterobacteriaceae were recovered from the faecal samples of bats from the three study locations (ile-ife, osogbo and ilesa) in osun state, nigeria. the high diversity of isolates could be as a result of the ability of bats to travel far distance, which may result with feeding from different habitats. the enterobacteriaceae recovered from the faecal samples of bats in this study were bacterial isolates that are significant in faecal samples of animals (both domestic and wild) across the world (sader et al., 2018). salmonella arizonae has the highest frequency of 50 (41.56%), followed by escherichia coli 36 (10.68 ), k. pneumoniae 31 (9.20%) and k. oxytoca 27 (8.01%). klebsiella pneumoniae had the highest occurrence of 10 (7.04%), 10 (11.91%) and 11 (9.91%) in ileife, osogbo and ilesa, respectively. meanwhile, k. oxytoca had the next high percentage occurrence of 13 (9.16%), 9 (10.71%) and 5 (4.91%) in ileife, osogbo and ilesa location. these percentage occurrences were relatively low for k. pneumoniae (35.8 %) but relatively high for k. oxytoca (0.9%) when compared to the occurrence from wild birds (10). escherichia coli had a prevalence of 17 (11.97%), 11 (13.10%) and 8 (7.20%) in ile-ife, osogbo and ilesa locations, respectively. this percentage occurrence was relatively low compared to 50.5 % occurrence reported from wild birds (carlos et al., 2016). the result of this study can be compared to the record of the presence of e. coli in the faecal samples of bats in ile-ife, nigeria (oluduro, 2012). salmonella spp. and escherichia coli have also been isolated from bat faeces in trinidad according to adesiyun (2009). ten (10) genera were common to the three studied locations while 13 species were common to the three studied locations, 13 species were common to two of the locations and 15 species were present in only one studied location. ile-ife location has the highest diversity of enterobacteriaceae with a total of 34 different bacteria species identified, osogbo has a total of 21 different species while ilesa has a total of 25 different species identified. the results show great diversity of enterobacteriacea in the studied area, although statistical data indicate that there is no significant difference in the occurrence of isolates across the three locations. bats are unique with their ability to fly long distance (calisher et al., 2006). therefore they can migrate from one geographical location to another. this capability makes it easy for them to acquire new microorganism either from the environment, contact with one another or other animals which aid the resistance of their microbial commensals to antimicrobial agents. the antimicrobial resistance among these bacteria leads to ineffective treatment of the infections caused by these organisms. various mechanisms of drug resistance have been attributed with the production of betalactamases which leads to resistance. the increase in the prevalence of esbl-producing isolates globally increases the burden of treating infectious disease especially in developing countries with poor resources and weak health management system. in the recent decade, esbl-producing enterobacteriaceae has resulted in an increase of resistance to β-lactam antibiotics leading to challenges in the management of infections caused by these bacteria (cantòn & coque, 2006). in this study, 35.9% of the selected multiple resistant isolates were positive to (esbls) produc84 s/n isolates name specific isolate frequency 1 escherichia coli a84, b83, c42 3 2 enterobacter sakazakii a3, b29, a120 3 3 salmonella arizonae a5, b6, c15, b32, b44 4 4 serretia liquefacience a140, a69 2 5 citrobacter koseri a105, b3 2 * isolate code isolates from ile-ife: a84, a3, a120, a5, a140, a69 and a105 isolates from osogbo: b83, b29, b6, b32, b44, and b3 isolates from ilesa: c42 and c25 table 2. occurrence of extended-spectrum β-lactamase (esbl) -producing isolates from faecal samples of eidolon helvum from the three locations using mast disc biologica nyssana ● 12 (1) september 2021: 79-86 aladejana, oluduro ● molecular characterisation of extended spectrum β-lactamase-producingenterobacteriaceae from bats faeces... tion. it is an indication that substantial amount of the multiple resistant isolates were esbls producers. the reason may be that the third-generation cephalosporins are commonly in use over a long period in the studied areas and might have been abused, therefore leading to the acquisition of resistance mechanisms by the organisms. the decreased susceptibility of these antimicrobial could also be as a result of the production of esbl and ampc b-lactamase. two of the isolates profiled harboured ctx-m gene (e. coli (b83 from osogbo) and enterobacter sakazakii (a120 from ife)). although the ctx-m family of esbls has been reported in germany since 1989 (doi et al., 2017) it has now spread to different parts of the world. the ctx-m enzyme has been found in e. coli and klebsiella spp., more frequently then in other enterobacteriaceae species (bush, 1989). wildlife are not directly exposed to clinically used antibiotics, but they could have contacted it through environment (sewages and animal manure). resistance could also be gained through feeding on some materials that contain antimicrobial substances in nature, like plants, but this still remain unclear. due to the ability of migratory birds to travel far distance, they can carry antibiotic-resistant bacteria from one continent to another leading to global transmission of bacterial resistance among bats. direct contact with faeces of wildlife and indirect contact through food, water and air is an important risk factor for the dissemination and transmission of pathogens or antimicrobial resistance from animals to both other animals and humans. conclusions currently, no studies have described the prevalence of esbl-producing enterobacteriaceae in faecal samples of eidolon helvum in nigeria, hence the study is novel. although much has not been done on the probable source of contamination, however the result of this study indicates that bat is a source of esbl-producing enterobacteriaceae and hence prevention need to be taken since they roost over human-populated area and release their faeces indiscriminately. acknowledgements. i acknowledge professor oluduro’s laboratory, department of microbiology, obafemi awolowo university, ile-ife and microbiology laboratory, kings university, odeomu, osun state nigeria. references adesiyun, a. a., stewart-johnson, a., thompson, n. n. 2009. isolation of enteric pathogens from bats in trinidad. journal of wildlife disease, 45: 952– 961. akobi, b., aboderin, o., sasaki, t., shittu a. 2012. characterisation of staphylococcus aureus isolates from faecal samples of the straw-coloured fruit bat (eidolon helvum) in obafemi awolowo university (oau), nigeria, 12: 279. bush. k. 1989. classification of beta-lactamases: groups 1, 2a, 2b, and 2b’. antimicrobial agents chemotherapy, 33(3): 264–70. calisher, c.h., childs, j.e., field, h.e., holmes, k.v., schountz, t. 2006. bats: important reservoir hosts of emerging viruses. clinical microbiology reviews, 19(3), 531–545. cantòn, r., coque, t.m. 2006. the ctx-m β-lactamase pandemic. current opinion in microbiology, 9: 466–475. carlos, a. r. m., ingrid, a. p., eliane, m. f., dália dos, p. r., salvatore, s. 2016. frequency of zoonotic bacteria among illegally traded wild birds in rio de janeiro. brazilian journal of microbiology, 47: 4. dierikx, c.m., van duijkeren, e., schoormans, a.h. 2012: occurrence and characteristics of extended-spectrum-beta-lactamaseand ampcproducing clinical isolates derived from companion animals and horses. journal of antimirobial chemotherapy, 67: 1368-74. doi, y., iovleva, a., & bonomo, r. a. 2017: the ecology of extended-spectrum β-lactamases (esbls) in the developed world. journal of travel medicine, 24(suppl_1), s44–s51. hassan, r., el-naggar, w., el-sawy, e., el-mahdy a. 2011. characterisation of some virulence factors associated with enterbacteriaceae isolated from urinary tract infections in mansoura hospitals. egyptian journal of medical microbiology, 20: 2. oluduro, a.o. 2012. antibiotics resistant commensal escherichia coli in faecal droplets from bats and poultry in nigeria. veterinaria italiana, 48(3): 297308. mbehang nguema, p. p., onanga, r., ndong atome, g. r., obague mbeang, j. c., mabika mabika, a., yaro, m., lounnas, m., dumont, y., zohra, z.f., godreuil, s., bretagnolle, f. 2020. characterisation of esbl-producing enterobacteria from fruit bats in an unprotected area of makokou, gabon. microorganisms, 8: 138. paterson, d.l., bonomo, r.a. 2005. extendedspectrum beta-lactamases: a clinical update. clinical microbiology reviews, 18(4): 657–686. rabinowitz, p., scotch, m., conti, l. 2009. human and animal sentinels for shared health risks. 85 biologica nyssana ● 12 (1) september 2021: 79-86 aladejana, oluduro ● molecular characterisation of extended spectrum β-lactamase-producingenterobacteriaceae from bats faeces... veterinaria italiana, 45(1), 23–24. ruiz, j., simon, k., horcajada, j.p., velasco, m, barranco m, roig g, moreno-martínez a, martínez, j.a., jiménez de anta t. et al., 2002. differences in virulence factors among clinical isolates of escherichia coli causing cystitis and pyelonephritis in women and prostatitis in men. journal of clinical microbiological, 40: 4445–4449. sader, h.s., castanheira, m., duncan, l.r., flamm, r.k. 2018. antimicrobial susceptibility of enterobacteriaceae and pseudomonas aeruginosa isolates from united states medical centers stratified by infection type: results from the international network for optimal resistance monitoring (inform) surveillance program, 20152016. diagnostic microbiology infectious diseases, 92(1): 69-74. 86 biologica nyssana ● 12 (1) september 2021: 79-86 aladejana, oluduro ● molecular characterisation of extended spectrum β-lactamase-producingenterobacteriaceae from bats faeces... anticancer compounds from medicinal plants biologica nyssana 3 (2)  december 2012: 61-67 jović j. et al..  influence of solvent on antimicrobial activity... 61 original article influence of solvent on antimicrobial activity of carlinae radix essential oil and decoct jovana jović * , tatjana mihajilov-krstev, andrea žabar, zorica stojanović-radić department of biology and ecology, faculty of science and mathematics, university of niš, višegradska 33, 18000 niš, serbia * e-mail: jovanajović85@gmail.com abstract: jović, j., mihajilov-krstev t., žabar a., stojanović-radić z.: influence of solvent on antimicrobial activity of carlinae radix essential oil and decoct. biologica nyssana, 3 (2), december 2012: 61-67. plants of the family asteraceae are known for their use in ethnopharmacology, available as commercial drugs. in this study, antimicrobial activity of carlinae radix commercial drug’s vinegar decoction and essential oil, dissolved in various solvents (etohethanol, dmsodimethyl sulfoxide and tween 80 polyoxyethylene sorbitan monolaurate) was tested to investigate the effect of solvents on activity and to compare the results with previous researches. the microdilution method was used to determine the minimum inhibitory concentration (mic) and minimum bactericidal concentration (mbc). the results showed that antimicrobial activity of the carlinae radix oil significantly depends on the solvent and that most efficient antimicrobial effect had the essential oil dissolved in 7% ethanol, which points to significant synergistic effect of the oil with this solvent. key words: antimicrobial activity, carlina acanthifolia l. root, synergistic activity, solvent, mic and mbc introduction plants of the family asteraceae are known for their use in ethnopharmacology. essential oil is present in high amounts in these plants, but in traditional medicine much more frequent way of utilisation is using of decoct. carlina acaulis l. (asteraceae) is widely spread herb in east asia and europe. commonly used part of this plant in ethnopharmacology is root, known as carlinae radix. in traditional medicine, well known use of this drug is in the form of tinctures and decoction against urinary tract infections, skin diseases and wound irrigation (t u c a k o v , 1971). it was confirmed that inulin and flavonoids from roots have the antitumor, antiviral, antibacterial, antidiabetic, antioxidant and neuroprotective activity (a l b u l e s c u et al., 2004; c h a n et al., 2010). previous investigations on this commercial drug’s composition, based on morphological and anatomical features of the dried root material, revealed that in serbia, carlinae radix mostly contains roots of c. acanthifolia, instead of c. acaulis (đ o r đ e v i ć et al., 2004; s t o j a n o v i ć r a d i ć , 2011). together with this, chemical analyses determined very similar chemical compositions of the oils from both species, suggesting that adulteration of the drugs would not affect biological activities of commercial drug material. studies of c. acanthifolia essential oil showed yield from 1-2%, while chemical analysis identified 11 compounds of the oil (đ o r đ e v i ć et al., 2005; s t o j a n o v i ć r a d i ć , 2011). carlina 3 (2) • december 2012: 61-67 biologica nyssana 3 (2)  december 2012: 61-67 jović j. et al..  influence of solvent on antimicrobial activity... 62 oxide was a major component (98.9±0.9%), which is known as potent antimicrobial compound (c h a l c h a t et al., 1996; w i c t h l et al., 2002). the essential oil showed inhibitory activity on reference strains of staphylococcus aureus, escherichia coli, pseudomonas aeruginosa, proteus vulgaris, klebsiella pneumoniae and candida albicans (đ o r đ e v i ć et al., 2007; s t o j a n o v i ć r a d i ć et al., 2012). together with essential oil’s antimicrobial activity, s t o j a n o v i ć r a d i ć et al. (2012) investigated antimicrobial properties of different root decocts, where vinegar decoct had the highest inhibitory effect on the bacterial strain s. aureus atcc 6538 in comparison to water and wine decocts (s t o j a n o v i ć r a d i ć et al., 2012). in this study, we tested the antimicrobial activity of vinegar decoct and essential oil isolated from commercial carlinae radix drug against total of eighteen microbial strains. among them, one set was made of atcc (american type culture collection) reference strains, while the second set was comprised of multiresistant clinical isolates from wounds. these strains were chosen in order to explore the effect on common wound pathogens, since this plant is commonly used for wound irrigation. together with this, the tested oil was dissolved in three different solvents (etoh ethanol, dmsodimethylsulfoxide and tween 80 polyoxyethylene sorbitan monolaurate) in order to investigate the influence of solvent to antimicrobial activity and compare the results with previous researches. materials and methods essential oil isolation the essential oil was obtained from 100 g of dried root material of the commercial herbal drug carlinae radix (“jeligor”, svrljig) by hydrodistillation method, using clevenger’ s apparatus (c l e v e n g e r , 1928). the essential oils (1.05–1.60 g per batch) were obtained in the mean yield of 1.05% (w/w). the obtained oils were separated by extraction with freshly distilled diethyl ether and dried over anhydrous magnesium sulphate. the solvent was evaporated under a gentle stream of nitrogen at room temperature in order to exclude any loss of the essential oil and immediately analyzed. when the oil yields were determined, after the bulk of ether was removed under a stream of n2, the residue was exposed to vacuum at room temperature for a short period to eliminate the solvent completely. the pure oil was then measured on an analytical balance and multiple gravimetric measurements were taken during 24 h to ensure that all of the solvent had evaporated. preparation of decoct. decoct was prepared from 2 g of the roots, which were cut into small pieces and then extracted with 100 ml of boiling apple vinegar for 10 min, as described previously (s t o j a n o v i ć r a d i ć et al., 2012). after the plant material was filtered off, the obtained extract (decoct) was used as such in the antimicrobial tests. microorganisms antimicrobial activity assays were performed against eight american type culture collection (atcc) strains: salmonella enteritidis 13076, pseudomonas aeruginosa 3554, enterococcus faecalis 19433, enterobacter aerogenes 13048, proteus mirabilis 12453, clostridium perfringens 19404, klebsiella pneumoniae 10031 and yeast candida albicans 10231. multiresistant bacterial strains were isolates from wounds (clinical isolatesci): enterobacter aerogenes, acinetobacter sp. (2 clinical isolates), proteus mirabilis, escherichia coli, staphylococcus aureus, pseudomonas aeruginosa (3 clinical isolates) and klebsiella oxytoca. bacterial strains were maintained on the nutrient agar and yeast on sabouraud dextrose agar (microbiological laboratory, department of biology and ecology, faculty of science and mathematics, niš). antimicrobial activity testing antimicrobial activity was evaluated by microdilution method as described previously (s t o j a n o v i ć r a d i ć et al., 2010). bacterial strains were transfered on new nutrient agar and candida albicans on sabouraud dextrose agar, and incubated for 18 h at 37°c. overnight strains were used to make suspensions in sterile saline solution (0.9% nacl). standard turbidity was adjusted to 0.5 mcfarland (density of bacterial cells 1.0-1.5 x 10 8 and yeast 1.0-1.5 x 10 7 cfu/ml) and this inoculum size was used to prepare a final colony number of 12 x 10 6 (1-2 x 10 5 for yeast) colony forming units (cfu/ml) in a plate with sterile mueller hinton broth (mhb). carlinae radix essential oil was dissolved in three different solvents: 70% ethanol, dmso and 0.05% tween 80. a serial doubling dilutions of the oil in mueller hinton broth (methodology 1) or in the same solvent (methodology 2) were prepared in 96 well microtiter plate and used for experimental work. biologica nyssana 3 (2)  december 2012: 61-67 jović j. et al..  influence of solvent on antimicrobial activity... 63 methodology 1. microtiter plate wells were filled with 100 µl mhb and 100 µl carlinae radix oil (dissolved in 70% ethanol) were added into the first wells. double dilutions were made by transferring 100 µl of the first dilution in subsequent wells (for each microorganism strain) in a concentration range from 0.03 mg ml -1 to 70 mg ml 1 . antimicrobial activity was tested in 96-well plates, prepared by dispensing 90 µl of mhb and 1 µl of the inoculum into each well. then, 10 µl of the appropriate oil dilutions were transferred to wells and initial concentration of the oil was 7 mg ml -1 . methodology 2. microtiter plates were filled with different solvents (100 µl in each well): 70% ethanol, dmso and 0.05% tween 80. double dilutions were made in solvent by adding carlinae radix oil (100 µl into the first wells), dissolved in the same solvent, and transferring 100 µl from the first in subsequent wells. antimicrobial activity was tested like in methodology 1, with oil concentration range from 0.003 mg ml -1 to 7 mg ml -1 . negative controls were solvents in inoculated broth: 7% ethanol, 10% dmso and 0.005% tween 80. in all tests, positive controls were tetracycline (bacteria) and nystatin (yeast), both in the concentration range from 0.25-512 µg ml -1 . decoct activity was tested in plates with mhb (100 µl). the first wells (for each microorganism strain) were filled with 100 µl of decoct for making a series of double dilutions and inoculated. concentration range of decoct was from 0.024% to 50% (v\v). negative control was commercial apple vinegar in initial concentration of 2% (v\v). after incubation period of 24 h at 37°c, bacterial growth was determined by adding 20 µl of 0.5% ttc (triphenyl tetrazolium chloride) aqueous solution. mic (minimum inhibitory concentration) was read as the lowest concentration of oil at which there was no visible growth and red color. the broth from wells without visible growth (100 µl) was transferred to mha (mueller hinton agar) for 24 h at 37°c. minimum bactericidal concentration (mbc) was defined as lowest oil concentration killing 99.9% of microorganism cells. control of microorganisms’ growth was inoculated broth (without oil), while non-inoculated wells were included to ensure broth sterility. the experiment was performed in triplicate and the mean values are presented. results and discussion the results of broth microdilution assay of carlinae radix essential oil are presented in table 1. in methodology 1, the oil showed mic/mbc activity in the range from 0.055-7.000/1.750-7.000 mg ml -1 . the highest microbistatic and microbicidal effect were manifested against reference strain pseudomonas aeruginosa 3554 (mic/mbc = 0.055/1.750 mg ml -1 ). also, significant activity was obtained against clinical isolates pseudomonas aeruginosa (ci 2), acinetobacter sp. (ci 2) (mic/mbc = 0.219/7.000 mg ml -1 ) and atcc strains enterobacter aerogenes 13048 (mic/mbc = 0.437/1.750 mg ml -1 ) and salmonella enteritidis 13076 (mic/mbc = 0.437-3.500 mg ml-1) (graph 1). clinical isolates klebsiella oxytoca, acinetobacter sp. (ci 1), escherichia coli, pseudomonas aeruginosa (ci 1) and proteus mirabilis were resistant to the highest tested concentration (7.000 mg ml -1 ). graph 1. minimum inhibitory concentrations of carlinae radix essential oil dissolved in ethanol (methodology 1). the bars above the concentration of 7.00 mg ml -1 are placed to represent strains resistant to the highest tested concentration of essential oil. microbial strain legend: 1enterobacter aerogenes (clinical isolate), 2acinetobacter sp. (clinical isolate 1), 3 proteus mirabilis (clinical isolate), 4escherichia coli (clinical isolate), 5staphylococcus aureus (clinical isolate), 6 pseudomonas aeruginosa (clinical isolate 1), 7pseudomonas aeruginosa (clinical isolate 2), 8klebsiella oxitoca (clinical isolate), 9pseudomonas aeruginosa (clinical isolate 3), 10 acinetobacter sp. (clinical isolate 2), 11: salmonella enteritidis 13076, 12pseudomonas aeruginosa 3554, 13 enterococcus faecalis 19433, 14enterobacter aerogenes 13048, 15proteus mirabilis 12453, 16clostridium perfringens 19404, 17klebsiella pneumoniae 10031 and 18 candida albicans 10231. biologica nyssana 3 (2)  december 2012: 61-67 jović j. et al..  influence of solvent on antimicrobial activity... 64 biologica nyssana 3 (2)  december 2012: 61-67 jović j. et al..  influence of solvent on antimicrobial activity... 65 generally, gram-negative bacterial strains possess higher resistance to external agents, which can be attributed to their characteristic membrane structure (beveridge, 1999). the results obtained in the present study for klebsiella pneumoniae 10031 and candida albicans 10231, showed higher resistance in comparison with the previous research (s t o j a n o v i ć r a d i ć et al., 2012). in previous investigation, solvent was absolute alcohol which could lead to synergism and, thus, higher activity of oil. results of microdilution method (methodology 2) where the essential oil was dissolved in ethanol showed inhibitory activity against all tested strains at surprisingly low concentrations, lower than the tested concentration range (mic<0.003 mg ml -1 ), but without bactericidal effect (mbc>7.000 mg ml -1 ) (table 1). the results could be explained by the synergism between the antimicrobial components of the oil and ethanol. this method used the same concentration of the solvent in all tested wells of the microtiter plate (7%) in combinations with different concentrations of the oil (0.003 mg ml -1 to 7 mg ml -1 ). in methodology 1, concentration of 7% ethanol was in the first well only and then subsequently two-fold diluted, so we can conclude that at this concentration, ethanol exhibits efficient synergistic action, probably by increasing permeability of the membrane for the active compounds of the essential oil. it is also very important to mention that this solvent (7% ethanol), tested alone against all model microorganisms did not exhibited any inhibitory effect. in this study, inhibitory effect of the oil dissolved in dmso ranged from 0.437 to 3.500 mg ml -1 (graph 2). h i l l i et al. (1997) tested thirteen oils dissolved in dmso and showed their antimicrobial activity. however, cinnamon oil, which was not dissolved in dmso had higher activity against the tested strains. the results confirmed that higher concentrations of dmso indicate antagonistic effect (h i l l i et al., 1997). 10% dmso and 0.005% tween 80 had no activity against our strains (except for candida albicans and pseudomonas aeruginosa clinical isolate 2), as in the previous research (p r a b u s e e n i v a s a n et al., 2006). increased resistance of microorganisms, compared to mic/mbc of the oil dissolved in ethanol, could be explained by antagonistic action of dmso with carlina oxide (the main component of the oil) or by the lack of membrane permeability alteration effect. graph 2. minimum inhibitory concentrations of carlinae radix essential oil dissolved in dmso (methodology 2) the oil dissolved in tween 80 had relatively low activity against all tested strains of microorganisms (mic = 3.500-7.000 mg ml -1 ) or the strains showed complete resistance. there was not bactericidal activity of oil in these solvents. b a u m a n n et al. (2011) confirmed the stimulatory effect of tween 80 on protein secretion in yeast (pichia pastoris). a source of oleic acid such as tween 80 enhanced subsequent acid survival of probiotic lactobacilli when added to the growth medium (c o r c o r a n et al., 2007). graph 3. minimum inhibitory concentrations of carlinae radix essential oil dissolved in tween 80 (methodology 2). the bars above the concentration of 7.00 are placed to represent strains resistant to the highest tested concentration of essential oil. biologica nyssana 3 (2)  december 2012: 61-67 jović j. et al..  influence of solvent on antimicrobial activity... 66 non-ionic detergents are characterized by their uncharged, hydrophilic head groups, so that could be the reason of low oil activity. antagonistic and synergistic effect is probably reflected by oil’s distribution between two phases, water (broth) and the solvent, which influences the effect of oil components on the microorganisms. bacterial strains also used in this study were multiresistant isolates from wounds. this set was chosen as model since, according to the literature, decoct of carlinae radix is used in traditional medicine for rinsing wounds on skin (k o j i ć et al., 1998). comparison of decoct activity with apple vinegar (control) confirmed that decoct exhibited antimicrobial activity. apple vinegar concentration was generally higher (or equal to) mic/mbc of the control when compared to mic/mbc of decoct. the results showed that decoct caused minimal inhibitory activity at a concentration of 0.7811.562% v\v and minimal bactericidal activity from 0.781-6.250% v\v. the highest resistance showed acinetobacter sp. (ci 1), pseudomonas aeruginosa (ci 3), enterobacter aerogenes 13048 and candida albicans 10231 (table 2). in previous studies, apple vinegar decoct showed higher activity against s. aureus (mic = 0.78%, mbc = 3.12%) when compared to water and wine decoct (s t o j a n o v i ć r a d i ć , 2011). activity can be attributed to the extracted compounds at low ph. graph 4. minimum inhibitory concentrations of carlinae radix decoct. the mic bars of decoct which are at the same values as the mic of vinegar present the lack of decoct activity (strains 1, 2, 3, 4, 5 and 9). table 2. antimicrobial activity of carlinae radix decoct bacterial/fungal strain strain type carlinae radix decoct apple vinegar mic mbc mic mbc (%) (%) (%) (%) enterobacter aerogenes clinical isolate 1.562 1.562 0.063 0.125 acinetobacter sp. clinical isolate 1 1.562 3.125 0.063 0.125 proteus mirabilis clinical isolate 1.562 1.562 0.063 0.125 escherichia coli clinical isolate 1.562 1.562 0.063 0.125 staphylococcus aureus clinical isolate 1.562 1.562 0.063 0.25 pseudomonas aeruginosa clinical isolate 1 0.781 1.562 0.063 0.125 pseudomonas aeruginosa clinical isolate 2 1.562 3.125 0.125 0.25 klebsiella oxitoca clinical isolate 1.562 3.125 0.125 0.25 pseudomonas aeruginosa clinical isolate 3 1.562 3.125 0.063 0.063 acinetobacter sp. clinical isolate 2 0.781 0.781 0.063 0.125 salmonella enteritidis atcc 13076 0.781 6.25 0.125 0.125 pseudomonas aeruginosa atcc 3554 0.781 3.125 0.063 0.063 enterococcus faecalis atcc 19433 1.562 3.125 0.125 0.125 enterobacter aerogenes atcc 13048 1.562 3.125 0.063 0.063 proteus mirabilis atcc 12453 1.562 1.562 0.125 0.125 clostridium perfringens atcc 19404 1.562 1.562 0.125 0.125 klebsiella pneumoniae atcc 10031 1.562 1.562 0.125 0.125 candida albicans atcc 10231 1.562 3.125 0.125 0.125 conclusion based on the obtained results it can be concluded that the antimicrobial activity of the oil carlinae radix depends on the solvent. the most efficient inhibitory effect had the oil dissolved in ethanol at 7% concentration, which can be explained by the effect of the ethanol in this concentration on membrane permeability, which leads to increased transfer of the oil’s active compounds into the target places on/inside the microbial cell. vinegar decoct of carlinae radix exhibited very significant antimicrobial activity, even against all tested multiresistant microbial strains, which justifies its ethnopharmacological utilization as an efficient biologica nyssana 3 (2)  december 2012: 61-67 jović j. et al..  influence of solvent on antimicrobial activity... 67 wound rinsing agent. as a final recommendation, the oil should be used in combination with ethanol (in the form of tinctures) at minimum 7% of the solvent concentration to achieve highly efficient antimicrobial effect. also, since the decoct of carlinae radix shows remarkable antimicrobial properties at very low percentage concentrations, it should be considered as more frequently used wound rinsing agent for the treatment of infected wounds. references albulescu м., alexa n. and cojan c. 2004: calendula officinalis flowers, source of extracts with antioxidant activity. journal of the serbian chemical society, 13(2): 169-176. baumann k., adelantado n. , lang c., mattanovich d. and ferrer p. 2011: protein trafficking, ergosterol biosynthesis and membrane physics impact recombinant protein secretion in pichia pastoris. microbial cell factories, 10:93. beveridge t.j. 1999: structures of gram-negative cell walls and their derived membrane vesicles. journal of bacteriology, 181: 4725–4733. chalchat j.c., đorđević s. and gorunović m. 1996: composition of the essential oil from the root of carlina acaulis l. asteraceae, journal of essential oil research, 8: 577-578. chan y.s., cheng l.n., wu j.h., chan e., kwan y.w., lee s.m.y., leung g.p.h., yu p.h.f. and chan s.w. 2010: a review of the pharmacological effects of arctium lappa (burdock). inflammopharmacology, 110. clevenger j.p. 1928: content of essential oil in plants. american perfumer and essential oil review, 23: 467-503. corcoran b.m., stanton c., fitzgerald g. f. and ross r.p. 2007: growth of probiotic lactobacilli in the presence of oleic acid enhances subsequent survival in gastric juice. microbiology, 153: 291-299. đorđević s, lakušić b, petrović s, niketić m., 2004: morphoanatomical characteristics of carlina acaulis subsp. caulescens and c. acanthifolia subsp. utzka (asteraceae). arh farm, 54, 773–783. đorđević s., petrović s., ristić m. and đoković d. 2005: composition of carlina acanthifolia root essential oil. chemistry of natural compounds, 41(4): 410-412. ðorđević s., lukić s., petrović s., dobrić s., milenković m., vučićević d., žižić s. and kukić j. 2007: antimicrobial, anti-inflammatory, anti-ulcer and antioxidant activities of carlina acanthifolia root essential oil. journal of ethnopharmacology, 109: 458–463. hili p., evans c.s. and veness r.g. 1997: antimicrobial action of essential oils: the effect of dimethylsulphoxide on the activity of cinnamon oil. letters in applied microbiology, 24: 269-275. kojić m., stamenković v. and jovanović d. 1998: lekovite biljke jugoistočne srbije, zavod za udžbenike i nastavna sredstva, beograd. prabuseenivasan s., jayakumar m. and ignacimuthu s. 2006: in vitro antibacterial activity of some plant essential oils. bmc complementary and alternative medicine, 6:39. stojanović-radić z. 2011: biotička aktivnost etarskih ulja odabranih biljaka iz familije asteraceae i mehanizmi njihovog antibakterijskog delovanja u uslovima in vitro. phd thesis. prirodno – matematički fakultet. kragujevac. stojanović-radić z., nešić m., čomić lj., radulović n. 2010: antimicrobial activity and cytotoxicity of commercial rosemary essential oil (rosmarinus officinalis l.). biologica nyssana, 1 (1-2): 83-88. stojanović-radić z., čomić lj., radulović n., blagojević p., mihajilov-krstev t., rajković j. 2012: commercial carlinae radix herbal drug: botanical identity, chemical composition and antimicrobial properties. pharmaceutical biology, 50 (8): 933-40. tucakov j., 1971: lečenje biljem. fitoterapija. izdavačko preduzeće rad: beograd, jugoslavija, 402, 535-537. wichtl m., 2002: teedrogen und phytopharmaca. wissenschaftliche verlagsgesellschaft mbh, stuttgart, germany, pp. 114–115. willfort r., 1959: gesunheit durch heilkrauter. rudolf trauner verlag lintz, germany. http://www.ncbi.nlm.nih.gov/pubmed?term=stojanovi%c4%87-radi%c4%87%20z%5bauthor%5d&cauthor=true&cauthor_uid=22480199 http://www.ncbi.nlm.nih.gov/pubmed?term=%c4%8comi%c4%87%20l%5bauthor%5d&cauthor=true&cauthor_uid=22480199 http://www.ncbi.nlm.nih.gov/pubmed?term=radulovi%c4%87%20n%5bauthor%5d&cauthor=true&cauthor_uid=22480199 http://www.ncbi.nlm.nih.gov/pubmed?term=stojanovi%c4%87-radi%c4%87%20z%5bauthor%5d&cauthor=true&cauthor_uid=22480199 http://www.ncbi.nlm.nih.gov/pubmed?term=%c4%8comi%c4%87%20l%5bauthor%5d&cauthor=true&cauthor_uid=22480199 http://www.ncbi.nlm.nih.gov/pubmed?term=radulovi%c4%87%20n%5bauthor%5d&cauthor=true&cauthor_uid=22480199 http://www.ncbi.nlm.nih.gov/pubmed?term=blagojevi%c4%87%20p%5bauthor%5d&cauthor=true&cauthor_uid=22480199 http://www.ncbi.nlm.nih.gov/pubmed?term=blagojevi%c4%87%20p%5bauthor%5d&cauthor=true&cauthor_uid=22480199 http://www.ncbi.nlm.nih.gov/pubmed?term=mihajilov-krstev%20t%5bauthor%5d&cauthor=true&cauthor_uid=22480199 http://www.ncbi.nlm.nih.gov/pubmed?term=rajkovi%c4%87%20j%5bauthor%5d&cauthor=true&cauthor_uid=22480199 biologica nyssana 3 (2)  december 2012: 61-67 jović j. et al..  influence of solvent on antimicrobial activity... 68 veljić, 2019, biologica nyssana 10(2) 10 (2) december 2019: 87-98 doi: 10.5281/zenodo.3600179 bryology in serbia: from floristics to chemosystematics review article milan veljić university of belgrade, faculty of biology, institute of botany and botanical garden, takovska 43, 11000 belgrade, serbia veljicm@bio.bg.ac.rs (corresponding author) received: october 11, 2019 revised: october 13, 2019 accepted: november 24, 2019 abstract: bryological research in serbia can be divided into several periods and research fields, the first of which being floristic research. an important exploration of bryophytes in serbia started at the end of the 19th century, and with a few discontinuances it continued up today. during the last decade of the 20th century couple of researchers started their work on the exploration of bryophytes. they focused on bryologically important habitats like sources, riverbanks, peat bogs and mountains. a significant number of new species was reported for the country, but the revision of the old records has been done as well, hence today flora of serbia numbers 831 bryophyte taxa in total. progress of the botanical research has brought interdisciplinarity into bryology. as a result, bryology in serbia nowadays spread in different types of bryophyte research. for example, bryophytes are studied to investigate the biological, antimicrobial, antifungal and cytotoxic activity, biotransformation of molecules, phytochemical composition and as model organisms in stress physiology. special emphasis is also given on active conservation of the species. this represents only some directions of what is currently studying on bryophytes in serbia. key words: bryophytes, serbia, history, floristics, phytochemistry, biological activity apstract: kolekcija mahovina beou zanemareno nacionalno istraživačko blago istoriju briologije u srbiji možemo podeliti na nekoliko perioda i pravaca. prvi je pravac florističke (inventarizacione) briologije. naime značajnija briološka istraživanja u srbiji započeta su krajem devetnaestog veka i sa znatnim prekidima traju do danas. u poslednjoj dekadi dvadesetog veka pojavilo se nekoliko istraživača koji su se prevashodno posvetili istraživanju mahovina. istraživanja su usmerena na briološki zanimljiva staništa kao što su vrela, reke, tresetišta, planine. otkriven je značajan broj novih taksona ali izvršena je i revizija nekih ranijih podataka, tako da trenutno brioflora srbije broji 831 takson. kako je opšti način botaničkih istraživanja napredovao, i u briologiji se prešlo na multidisciplinarnost. grupa mlađih istraživača bavi se istraživanjima biološke aktivnosti mahovina, antimikrobne, antifungalne i citotoksične, fitohemijom mahovina, biotransformacijom molekula izolovanih iz mahovina ali i fiziologijom stresa. poseban akcenat je stavljen na aktivnu zaštitu i reintrodukciju. ovo je samo deo onoga što se danas iz briologije radi u srbiji. ključne reči: briofite, srbija, istorija, floristika, fitohemija, biološka aktivnost introduction bryological research in serbia could be divided into three periods of investigation, and several research fields. first period coincide to the onset of the botany in serbia, or with the work of josif pančić and his students. this period is characterized mainly by floristic or inventory studies, with relatively little bryophyte records made. after a brief break in investigation, research slightly intensified during the period from 1920s to 1960s with the work of group of phytocenologists. third, or the modern period, began in the end of 20th century, and is the most fruitful period so far (pantović & sabovljević, 2017). during this period, in addition to extensive floristic research, ecological investigations, the biological activity of moss extracts, their phytochemical analysis, as well as the transformation of molecules isolated from the extracts, were also initiated and successfully realized. at present, a lot of research is © 2019 veljić. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 87 13th symposium on the flora of southeastern serbia and neighboring regions conducting in the field of stress physiology in serbia, as well. a group of researchers is intensively working on the conservation of bryophytes, with an emphasis on active protection and reintroduction of the rare and threatened species. the institute of botany and botanical gardens “jevremovac” has valuable collection of over 260 species of living bryophytes from around the world, of which about 60% are regionally or globally endangered. floristic-ecological research of the bryophytes of serbia as a part of general botanical research in the territory of serbia, research of the bryophyte flora had started in the middle of the 19th century. the first bryophyte records were published in the works of the researchers of that time (grisebach, 1843; pančić, 1859, 1863). more extensive research of bryophyte flora has began at the end of the century when svetozar obradović, đura ilić and lujo adamović started researching bryophytes; they were sending most of the collected material for determination and verification by more experienced botanists (wettstein, 1890; simić, 1892; matouschek, 1899, 1901). simić (1897, 1898) published part of his research on the vranje surrounding in the reports of the vranje gymnasium. schiffner (1897) identified one species from the bornmuller’s herbarium. research results obtained during this period were summarized by jurišić (1900) and simić (1900). in these papers, they included part of their unpublished results as well jurišić cites 209 and simić 145 species. for the next ten years, bryological research was done mostly by katić (1900, 1903, 1906, 1907a, 1907b i 1909) and košanin (1909a, 1909b, 1910). after this period, bryological studies were done by podpera (1922), černjavski (1929, 1932, 1937, 1938), pichler (1931, 1939, 1940), grebenschikov (1943, 1949, 1950), soška (1949), knapp (1944), rudski (1949a, 1949b) and rajevski (1951). pavletić (1955) stated in his work “prodromus of the bryophyte flora of yugoslavia” 374 taxa (57 marchantiophyta, 317 bryophyta) to be present in serbia. since the 1950s, only sporadic bryophye records were published within the general floristic research (gigov & nikolić, 1954; gigov, 1956; slavnić, 1956; pavletić, 1956, 1968; janković & janković 1962; blečić & tatić, 1962; čolić et al., 1963; pavlović, 1951, 1964; mišić & popović, 1960; popović, 1966; filipović, 1966; guelmino, 1970, 1972; erdeši, 1971; babić, 1972; tešić et al., 1979; martinčič, 1980; janković & mišić 1980; jovanović 1980; janković & stevanović, 1981; mišić, 1982; grgić, 1983; gajić, 1983, 1986, 1988, 1989; gajić & karadžić, 1991; petković et al., 1988, 1990, 1991; mišić & panić, 1989). based on these studies, the list of bryophyte flora has been updated by gajić et al. (1991), who cited 415 bryophyte species in serbia (60 marchantiophyta, 355 bryophyta). the third period (or modern period) of bryophyte research began in the last decade of the 20th century when several researchers devoted their research to the bryophytes: ranđelović, 1994; stevanović et al., 1995; veljić et al,. 1996, 2001a, 2001b, 2004, 2006, 2008, 2013, 2016a, 2016b; veljić & marin, 1997; pavić et al., 1998; sabovljević & stevanović, 1999; petković et al., 2000; sabovljević, 1998, 1999, 2000, 2003a, 2003b, 2006; papp & sabovljević, 2001, 2002, 2010; grdović & blaženčić, 2001; sabovljević & cvetić, 2003; pócs et al., 2004; cvetić & sabovljević, 2005, papp & erzberger, 2005; grdović, 2005; grdović & stavretović, 2004, 2006; sabovljević & grdović, 2009; papp & erzberger, 2005, 2007a, 2007b, 2009; erzberger & papp, 2007; papp et al., 2004, 2006, 2012a, 2012b, 2013, 2014a, 2014b, 2014c, 2016a, 2016b, 2016c; ranđelović, zlatković, 2010; pantović & sabovljević, 2013, 2017; pantović et al., 2014; ilić et al., 2015. research was focused on biologically interesting and unexplored habitats such as springs, rivers, peatlands, mountains. this phase of intensive research of the flora and ecology of bryophytes is ongoing, and more recently two ph.d. dissertations focusing on bryophytes in serbia were done: “biogeographic and ecological study of the bryophyte flora of serbia” (pantović, 2018) and “diversity, distribution, differentiation of micro-habitats and the structure of moss communities of fruška gora mountain” (ilić, 2019). in this short period of time, 88 biologica nyssana ● 10 (2) december 2019: 87-98 veljić ● bryology in serbia: from floristics to chemosystematics fig. 1. collection of bryophytes in the beou herbarium. shown are digitized specimens included in the bryo database (photo j. pantović) a significant number of new taxa has been reported for the country, but also some previous data has been revised, so serbia currently numbers 830 bryophyte taxa (1 anthocerotophyta, 142 marchantiophyta, 687 bryophyta). historical data on the distribution and ecology of bryophytes has been collected and digitized into a unique database of bryophytes of serbia, the bryo database (fig. 1). updated lists of liverworts and mosses of serbia have been prepared and are currently in press. protection and conservation of bryophytes bryophytes, like all other living organisms, are exposed to various negative environmental factors. they are threatened similarily as vascular plants, but the attention and concern for their conservation is much lower. among bryophytes, there are taxa that are extinct, in different categories of threat, or rare, which has been confirmed by the increasing number of the regional red lists (sabovljević et al., 2004; marka et al., 2012; hodgetts et al., 2019). recognizing this global problem, a group of associates gathered around marko and aneta sabovljević (bbgb bryophyte biology group belgrade) has developed into the widely recognized group for bryophyte conservation (sabovljević et al., 2009; sabovljević et al., 2014; vujičić et al., 2012). the institute of botany and botanical garden “jevremovac” has an in vitro living collection of over 260 species of bryophytes from around the world, of which about 60% are regionally or globally endangered. nearly 52% biologica nyssana ● 10 (2) december 2019: 87-98 89 of the species in the collection are on the european and regional red lists. special attention is given to the active protection, like reintroduction and population strengthening of the rare and endangered taxa. in vitro propagated and grown plants are being prepared for reintroduction to the potentially suitable habitats. several species such as hendiella heimii and entostodon hungaricus (fig. 2) have been propagated this way and reintroduced to habitats in banat, serbia (sabovljević et al., 2012a, 2012b, 2016, 2018). ecophysiological research of bryophytes in serbia with progress of the botanical research, a multidisciplinary approach has been adopted in bryology as well. a group of young researchers has started working on bryophyte stress physiology (rajčić et al., 2015; vujičić, 2016; vujičić et al., 2016; ćosić et al., 2018). as a part of these studies, a doctoral dissertation entitled “biochemical and ecophysiological properties of selected moss species during salt stress in the axenical conditions” (vujičić, 2016) has been done. monitoring the parameters of morphogenesis revealed that bryophyes and vascular plants respond differently to the short-term and long-term salt stress, indicating different survival strategies with the increased salinity of the microenvironment. the effect of salt stress is also observed by the change in the concentration of tocopherols in mosses (physcomitrella patens, entosthodon hungaricus and henediella heimii) (fig. 3). namely, tocopherol as a non-enzymatic component plays an important role in the moss response to the stress caused by increased nacl concentration. the results of the study show that under long-term stress, the concentration of tocopherols increases on moderately saline media (50-100 mm nacl) in h. heimii. the salt stress affects concentration of proline and the ratio of proline and free amino acids in mosses (p. patens, e. hungaricus and h. heimii). proline as an essential amino acid plays a very important role in protecting the cell from osmotic stress, and it is involved in osmotic adaptation. it was discovered that proline concentration increases with the increase in salt concentration in all tested species during short-term stress, and that the ratio of proline and free amino acids increases with the increase in salt concentration in the medium. long-term salt stress induces a decrease of proline concentration in plants grown on a medium with the high concentration of fig. 2. reintroduction of henediella heimii (marked red) and enthostodon hungaricus (marked yellow), propagated axenically in vitro, acclimatized and finally released to nature on potentially suitable site in banat, serbia! (photo m. sabovljević) veljić ● bryology in serbia: from floristics to chemosystematics 90 salt. during long-term stress, the ratio of proline to free amino acids also increases with the increase of salt concentration in the medium. in addition to the non-enzymatic components, as a response to the salt stress enzyme activity (peroxidase, catalase, and superoxide dismutase) was monitored as well. phytochemical research of the bryophytes together with the ecophysiological studies, phytochemical studies of bryophytes have also been done. using the uhplc/-hesi-ms/ms methods, phenolic compounds were analyzed in physcomitrella patens (vujičić, 2016). in plants exposed to the short-term stress compared to plants exposed to the long-term salt stress, a significantly greater variety of compounds was observed. the predominantly detected compound was p-coumarin acid, which was detected in all treatments except in the control group. qualitative analysis of phenolic compounds in the plants exposed to the long-term stress showed that the number of detected phenolic compound decreases with the increase in salt concentration. in all tested species dominant phenolic compound was p-coumaric acid. the chemical composition has been investigated on selected moss species: rodobrium ontariense, atrichum undulatum, hypnum andoi (pejin et al., 2012a, 2012c). however, there are more studies of the chemical composition of liverworts. several liverwort taxa have been the subject of a doctoral dissertation: “chemical composition and antimicrobial activity of various liverwort extracts of scapania aspera m. & h. bernet, s. nemorea (l.) grolle, porella cordaeana (hub.) moore and p. arboris-vitae (with.) grolle)” (bukvički, 2014). analysis of the volatile compounds of different extracts identified 105 compounds in scapania aspera (bukvicki et al., 2013), 79 in scapania nemorea (bukvički et al., 2014), 84 in porella cordaeana (bukvički et al., 2012a)., and 66 in p. arboris-vitae (tyagi et al., 2013). chemical analysis of the volatile compounds of the extracts revealed the presence of large amounts of sesquiterpene and monoterpenes, and a much lower percentage of hydrocarbons, alcohols, aldehydes and ketones. gc-ms analysis of the hexane solution of methanolic extract of lophocolea bidentata showed two major compounds two sesquiterpene lactones. diplophyllolide (eudesmanic-type of sesquiterpene lactone), an important chemotaxonomic marker for marchantiophyta, and 5-hydroxydiplophyllolide have also been identified. bis-bibenzyls are a significant group of chemical compounds in liverworts. methanolic extract of marchantia polymorpha subsp. ruderalis contains bisbibenzyl marchantin a as a fig. 3. the appearance of the investigated species cultivated for three weeks in culture in vitro on a nutrient medium with the addition of various salt concentrations (photo m. vujičić) fig. 4. structures of seven new bisbibenzyls isolated from the methanol extract of the liverwort lunularia cruciata (novaković et al., 2019). biologica nyssana ● 10 (2) december 2019: 87-98 veljić ● bryology in serbia: from floristics to chemosystematics 91 major component (sabovljevic et al., 2017). seven new bis-bibenzyls were isolated from the methanol extract of the liverwort lunularia cruciata (fig. 4) along with one previously known bibenzyl and five known bisbibenzyls (novaković et al., 2019). these are acyclic bisbibenzyls, perrotetins and cyclic analogues, ricardines. recently, microbial transformation of molecules isolated from mosses has been the subject of the investigation. biotransformation represents an important strategy in the structural modification of natural compounds by microorganisms. biological activity of bryophytes secondary metabolites of bryophytes have important antibacterial and antifungal potential. this has been confirmed by a series of studies on both mosses and liverworts. among mosses, following species were studied: bryum argenteum (sabovljevic et al., 2006), pleurozium schreberi, palustriella commutata, homalothecium philippeanum, anomodon attenuatus, rhytidium rugosum, hylocomium splendens, dicranum scoparium, leucobryum glaucum (veljić et al., 2008), fontinalis antipyretica, hypnum cupressiforme, ctenidium molluscum (veljić et al., 2009), abietinella abietina, neckera crispa, platyhypnidium riparoides, cratoneuron filicinum, campylium protensum (bukvički et al., 2012b) and rhodobryum ontariense (pejin et al., 2012b). in general, all extracts showed stronger antifungal than antibacterial potential. however, significantly higher antimicrobial potential has been shown by secondary metabolites in liverworts. in addition to the species from scapania and porella genera that were already mentioned (bukvički, 2014), ptilidium pulcherrimum was investigated as well (veljić et al., 2010). tem analysis was performed to confirm antimicrobial activity and the mode of action. transmission electron micrographs of salmonella enteritidis (fig. 5) show the effect of the extracts: (a) control cells; (b) bacterial cell after the treatment with methanolic extract and (c) bacterial cell after the treatment of ethanol extract of the liverwort p. arborisvitae (fig. 5). more recently, the cytotoxic potential of various molecules (like terpenes, flavonoids, sterols, coumarins, and interesting macrocyclic compounds bibenzyls and bis-bibenzyls) present mainly in marchantiophyta, have also been investigated. the cytotoxic activity of the newly isolated compounds, as well as the already known ricardin g was investigated. the isolated compounds showed cytotoxic activity against a549 lung cancer cells with ic50 values of 5.0, 5.0 and 2.5 mm. pinguisane-type of compounds isolated from porella cordaeana showed imunomodulatory activity (radulović et al., 2016). this chronological overview gives a partial review of bryological researches in serbia, but also shows endless directions and possibilities of bryology. bryology has large potential and perspective, considering that every new initiative in bryophyte research has given many internationally significant results. at this point, perhaps main problem in the further development of bryology is the lack of interested researchers. acknowledgements. this research was supported by the grant from the ministry of education, science and development of serbia (project no. 173029). references babić, n. 1972: močvarna i livadska vegetacija koviljskog rita. matica srpska, zbornik za prirodne nauke, 41: 19–87. blečić, v., tatić, b. 1962. prilog poznavanju smrčevih šuma golije planine. glasnik prirodnjačkog muzeja u beogradu, b18: 39–47. bukvički, d. 2014: hemijski sastav i antimikrobna aktivnost različitih ekstrakata jetrenjača scapania fig. 5. transmission electron micrographs of untreated and treated salmonella enteritidis cells. (a) untreated cells, (b) methanolic p. arboris-vitae extract, and (c) ethanolic p. arboris-vitae extract (tyagi, 2013) biologica nyssana ● 10 (2) december 2019: 87-98 veljić ● bryology in serbia: from floristics to chemosystematics aspera m. & h. bernet, s. nemorea (l.) grolle, porella cordaeana (hub.) moore i p. arboris-vitae (with.) grolle”. phd thesis. univerzitet u beogradu, biološki fakultet. bukvički, d., gottardi, d., veljić, m., marin, p. d., vannini, l., guerzoni, m. e. 2012a: identification of volatile components of liverwort (porella cordaeana) extracts using gc/ms-spme and their antimicrobial activity. molecules, 17: 6982-6995. bukvički, d., gottardi, d., tyagi, a.k., veljić, m., marin, p.d., vujisić, lj., guerzoni m.e. vannini, l. 2014: scapania nemorea liverwort extracts: investigation on volatile compounds, in vitro antimicrobial activity and control of saccharomyces cerevisiae in fruit juice, lwt – food science and technology, 55: 452-458. bukvički, d., тyagi, a., gottardi, d., veljić, m., janković, s., guerzoni, m.e., marin, p. d. 2013: assessment of the chemical composition and in vitro antimicrobial potential of extracts of the liverwort scapania aspera. natural product communications, 8 (9): 1313-1316. bukvički, d., veljić, m., soković, m., grujić, s., marin, p. d. 2012b: antimicrobial activity of metanol extracts of abietinella abietina, neckera crispa, platyhypnidium riparoides, cratoneuron filicinum and campylium protensum mosses. archive of biological sciences, 64 (3): 911-916. ćosić, m., vujičić, m., sabovljević, m., sabovljević, a. 2018. what do we know about salt stress in bryophytes? plant biosystems, 153 (3): 478-489. černjavski, p. 1929: ein betrag zur kenntnis der geh–lzflora der kalktuffes (travertins) von plevlje und prijepolje. glasnik botaničkog zavoda i bašte univerziteta u beogradu, 1(2): 205-208. černjavski, p. 1932: beitrag zur postglazialen geschichte des blace„sees“ in serbien. bulletin de l‘institut et du jardin botaniques de l‘universite de belgrade, 2: 80–90. černjavski, p. 1937: tablice za određivanje biljaka iz okoline beograda. „lito-štampa“, beograd. černjavski, p. 1938: postglacijalna istorija vlasinskih šuma. izdavačko i književno preduzeće “geca kon”, beograd. čolić, d., mišić, v., popović, m. 1963: fitocenološka analiza visokoplaninske zajednice šleske vrbe i planinske jove (saliceto-alnetum viridis ass. nova) na staroj planini, knjiga 6 (5): 1-43. cvetić, t., sabovljević, m. 2005: a contribution to the bryophyte flora of fruška gora (vojvodina, serbia). phytologia balcanica, 11(1): 35–43. erdeši, j. 1971: fitocenoze šuma jugozapadnog srema. doktorska disertacija. šumsko gazdinstvo sremska mitrovica, sremska mitrovica. erzberger, p., papp, b. 2007: new and noteworthy bryophyte records from montenegro and serbia. willdenowia, 37: 339–351. filipović, d. 1966: limnološka karakteristika izvorskog regiona lisinskog potoka na kopaoniku. arhiv bioloških nauka, 18 (3-4): 325-337. gajić, m. 1983: flora deliblatske peščare. prirodno-matematički fakultet oour institut za biologiju novi sad; šumsko industrijski kombinat „pančevo“ oour specijalni prirodni rezervat „deliblatski pesak“, 475 p. gajić, m. 1986: flora i vegetacija subotičko– horgoške peščare. šumarski fakultet beograd, šumsko gazdinstvo subotica, 495 p. gajić, m. 1988: flora nacionalnog parka tara. šumarski fakultet, beograd; nacionalni park tara, bajina bašta, 288 p. gajić, m. 1989: flora i vegetacija golije i javora. šumarski fakultet, beograd; oour šumarstvo „golija”, ivanjica, 592 p. gajić, m., karadžić, d. 1991: flora ravnog srema sa posebnim osvrtom na obedsku baru. šumarski fakultet beograd, šumsko gazdinstvo sremska mitrovica, 67–70. gajić, m., korać, m., obratov, d. 1991: pregled mahovina u srbiji, zbornik radova sa simpozijuma „nedeljko košanin i botaničke nauke”, 401–407. gigov, a. 1956: analiza polena na nekim tresavama stare planine. arhiv bioloških nauka, 1: 45–57. gigov, a., nikolić. v. 1954: rezultati analize polena na tresavama planine ostrozub. arhiv bioloških nauka, 6(1-2): 99–116. grdović, s. 2005: mahovine šireg područja beograda i njihov bioindikatorski značaj, zadužbina andrejević, beograd, 130 str. grdović, s., blaženčić, ž. 2001: prilog poznavanju mahovina rezervata “zasavica”, monografija “zasavica”, 35–41. grdović, s., stavretović, n. 2004: mahovine kao korov u travnim površinama. acta herbologica, 13(1): 235–242. grdović, s., stevanović, v. 2006: the moss flora in the central urban area of belgrade. archives of biological sciences, 58(1): 55–59. 92 biologica nyssana ● 10 (2) december 2019: 87-98 veljić ● bryology in serbia: from floristics to chemosystematics grebenščikov, o. 1943: prilog poznavanju vegetacije planine koprivnik kod peći. srpska kraljevska akademija, posebna izdanja 136, prirodnjački i matematički spisi 35, ohridski zbornik, 2: 241–267 (1–29). grebenščikov, o. 1949: pregled radova na polju proučavanja mahovina u srbiji, glasnik prirodnjačkog muzeja srpske zemlje, b, 1-2: 315321. grebenščikov, o. 1950: o vegetaciji centralnog dijela stare planine. zbornika radova instituta za ekologiju i biogeografiju, 1: 1–36. grgić, p. 1983: prilog poznavanju mahovina u ekosistemima sa pančićevom omorikom. godišnjak biološkog instituta, 36: 69–72. grisebach, a. 1843: spicilegium florae rumelicae et bitynicae exhibens synopsis plantarum, brunsvigae. guelmino, j. 1970: prva nalazišta mahovine funaria hungarica boros u jugoslaviji, zbornik za prirodne nauke, matica srpska, 37: 176–178. guelmino, j. 1972: nova nalazišta dve malo poznate mahovine kod nas (funaria hungarica boros i tortula velenovsky schiffner). zbornik za prirodne nauke, matica srpska, 42(1): 106–109. hodgetts, n., cálix, m., englefield, e., fettes, n., garcía criado, m. et al. 2019: a miniature world in decline: european red list of mosses, liverworts and hornworts. brussels, belgium: iucn. ilić, m. 2019: diverzitet, distribucija, diferencijacija mikrostaništa i struktura zajednica mahovina fruške gore. phd thesis. univerzitet u novom sadu, prirodno-matematički fakultet, departman za biologiju. ilić, m., vukov, d., rućando, m., ćuk, m., igić, r. 2015: contribution to the bryophyte flora in beech forests of vidlič mountain (serbia). zbornik matice srpske za prirodne nauke, 174: 67–27. janković, m., janković, m. m. 1962: ekološki uslovi vegetacije u vrelu mlave kod žagubice, sa posebnim osvrtom na biljku callitriche verna. arhiv bioloških nauka, 14 (3-4), 157-168. janković, m., mišić, v. 1980: šumska vegetacija i fitocenoze fruške gore. monografije fruške gore. matica srpska, novi sad, 191 p. janković, m., stevanović, v. 1981: prilog poznavanju fitocenoza sa srpskom ramondijom (ramonda serbica panč.) u klisurama severnih ogranaka šarplanine. ekologija, 16(1): 1–34. jovanović b., vukićević, e., avdalović, v. 1983: neke planinske zajednice crne i bele jove u okolini sjenice. zaštita prirode, 36: 49–68. jovanović, b. 1980: šumske fitocenoze i staništa suve planine. glasnik šumarskog fakulteta, serija a šumarstvo, posebno izdanje 55. jurišić, j. ž. 1900: prilog poznavanju mahovina u srbiji. spomenik srpske akademije nauka, 45: 47– 60. katić, d. 1900: drugi prilog flori okoline kragujevca. izveštaj gimnazije kneza miloša u kragujevcu za 1899/1900. godinu, 43–45. katić, d. 1903: treći floristički prilog (iz kragujevačke okoline). izveštaj gimnazije kneza miloša velikog u kragujevcu za 1902/1903, 3–7. katić, d. 1906: beitrag zur mossflora von serbien. hedwigia, 45: 92–99. katić, d. 1907a: nekoliko mahovinskih prinova flori srbije. prosvetni glasnik, 12: 884–888. katić, d. 1907b: sitniji prilozi flori srbije. nastavnik, list profesorskoga društva, 18: 184–191. katić, d. 1909: priložak mahovinskoj flori srbije. nastavnik, 20 (7-8): 285–286. knapp, r. 1944: vegetationsstudien in serbien. halle. košanin, n. 1909a: das vorkommen von polytrichum alpinum l. auf einem hochmoor in serbien. hedwigia, 48: 205–206. košanin, n. 1909b: moose aus dem gebiete des golia-gebirges in südweat-serbien. hedwigia, 48: 207–209. košanin, n. 1910: elementi vlasinske flore (algae, bryophyta, pteridophyta et phanerogamae). prosvetni glasnik beograd, 31 (8-9): 684–700. lakušić, d. 1996: pregled flore kopaonika (jz srbija, jugoslavija). ekologija, 31(2): 1–35. lazarević, p., pantović, j., szurdoki, e., papp, b., sabovljević, m. 2016: distribution, ecology and threat status evaluation of sphagnum species in serbia. wulfenia, 23: 37–51. marinković, p., gajić, m. 1956: o jednom nalazištu sfagnumske tresave u srbiji. šumarstvo, 4–5: 258– 262. marka, j., papp, b., erzberger, p., colacino, c., sabovljević, m. 2012: towards a red list of the albanian bryophytes. studia botanica hungarica, 43: 13-25. martinčić, a. 1980: prispevek k poznavanju mahovne flore jugoslavije ii šar planina. biološki vestnik, 28 (2): 87–102. 93 biologica nyssana ● 10 (2) december 2019: 87-98 veljić ● bryology in serbia: from floristics to chemosystematics matouschek, f. 1899: beitrag zur mooskenntnis von südserbien. verhandlungen der zoologischbotanischen gesellschaft in wien, 49: 386–390. matouschek, f. 1901: bryologisch floristisches aus serbien. allgemeine botanische zeitschrift für systematik, floristik, pflanzengeographie etc., 7 (2): 21–22. mišić, v. 1982: reliktne polidominantne šumske zajednice srbije. matica srpska, 178 p. mišić, v., panić, i. 1989: šumska vegetacija doline studenice. zaštita prirode, 41-42: 33–52. mišić, v., popović, m. 1960: fitocenološka analiza smrčevih šuma kopaonika. zbornik radova biološkog instituta n. r. srbije, 3(5): 1–26. novaković, m., bukvički, d., andjelković, b., ilić tomić, t., veljić, m., tešević, v., asakawa, y. 2019: cytotoxic activity of riccardin and perrottetin derivatives from the liverwort lunularia cruciata, journal of natural products, doi:10.1021/acs. jnatprod.8b00390, in press. pančić, j. 1859: die flora der serpentinberge in mittel-serbien. verhandlungen der zoologischbotanischen gesellschaft in wien, 9: 139–150. pančić, j. 1863: živi pesak i bilje što raste na njemu. glasnik društva srbske slovesnosti, 16: 197–233. pantović, j. 2018: biogeografska i ekološka studija flore briofita srbije. phd thesis. univerzitet u beogradu, biološki fakultet. pantović, j. grdović, s., sabovljević, a., sabovljević, m. 2014: new and interesting bryophyte records for the flora of serbia. archives of biological sciences, 66 (2): 701–704. pantović, j., sabovljević, m. 2013: contribution to the bryophyte flora of mt boranja (west serbia). phytologia balcanica, 19 (1): 23–27. pantović, j., sabovljević, m. 2017: bryophytes of kosovo. phytotaxa, 306 (2): 101–123. pantović, j., sabovljevic, m. s. 2017: overview of bryophyte flora research in serbia with presentation of the serbian bryo database. botanica serbica, 41(2): 153-162. papp, b., alegro, a., erzberger, p., szurdoki, e., šegota, v., sabovljević, m. 2016a: bryophytes of saline areas in the pannonian region of serbia and croatia. studia botanica hungarica, 47 (1): 141– 150. papp, b., erzberger, p. 2005: the bryophyte flora of golija studenica biosphere reserve and some adjacent sites (sw serbia montenegro). studia botanica hungarica, 36: 101–116. papp, b., erzberger, p. 2007a: contributions to the bryophyte flora od western stara planina mts (e serbia). studia botanica hungarica, 38: 95–123 papp, b., erzberger, p. 2007b: new and noteworthy bryophyte records from montenegro and serbia. willendowia, 37: 339–351. papp, b., erzberger, p. 2009: contributions to the bryophyte flora of southeastern serbia: suva planina mts and its surroundings. studia botanica hungarica, 40: 125–142. papp, b., erzberger, p., sabovljević, m. 2004: contributions to the bryophyte flora of kopaonik mts (serbia, serbia-montenegro). studia botanica hungarica, 35: 67–79. papp, b., erzberger, p., sabovljević, m. 2006: contribution to the bryophyte flora of the djerdap national park (e serbia). studia botanica hungarica, 37: 131–144. papp, b., natcheva, r., erzberger, p., sabovljević, m. 2012a: didymodon sicculus, new to bulgaria and serbia and notes on its ecology. nova hedwigia, 95 (1-2): 221–226. papp, b., pantović, j., sabovljević, m., szurdoki, e. 2014a: myurella sibirica, a moss species new to montenegro and serbia: its range extension towards south-eastern europe. cryptogamie, bryologie. 35(3): 1–6. papp, b., pantović, j., szurdoki, e., sabovljević, m. 2014b: interesting and new species for the bryophyte flora of serbia. herzogia, 27(1): 221–225. papp, b., sabovljević, m. 2001: contribution to the knowledge of the bryoflora of the region of petnica (w serbia, yugoslavia). studia botanica hungarica, 32: 107–120 papp, b., sabovljević, m. 2002: the bryophyte flora of tara national park (w serbia, yugoslavia). studia botanica hungarica, 33: 25–39. papp, b., sabovljević, m. 2010: contribution to the bryophyte flora of the vršačke planine mts., serbia. botanica serbica, 34(2): 107–110. papp, b., szurdoki, e., pantović, j., sabovljević, m. 2013: physcomitrium eurystomum and pohlia proligera, new mosses in the bryophyte flora of serbia. archives of biological sciences, 65 (2): 703– 706. papp, b., szurdoki, e., pantović, j., sabovljević, m. 2014c: contributions to the bryophyte flora of the pešter plateau, sw serbia. studia botanica hungarica, 45: 33–47. 94 biologica nyssana ● 10 (2) december 2019: 87-98 veljić ● bryology in serbia: from floristics to chemosystematics papp, b., szurdoki, e., pantović, j., sabovljević, m. 2016b: an insight into the bryophyte flora of the ibar george and its surroundings (central and sw serbia). acta botanica hungarica, 58: 411–423. papp, b., szurdoki, e., pantović, j., sabovljević, m. 2016c: new records of mediterranean-atlantic mosses in the flora of serbia. herzogia, 29:185–189. papp, b., szurdoki, e., sabovljević, m. 2012b: bryophyte flora of lake vlasina and its surroundings (se serbia). studia botanica hungarica, 43: 27–45. pavić, s., sabovljević, m., stevanović, v. 1998: diversity and threat status of the yugoslav bryoflora. lindbergia, 23: 38-44. pavletić, z. 1955: prodromus flore briofita jugoslavije. jugoslavenska akademija znanosti i umetnosti, 578 p. pavletić, z. 1956: prilog poznavanju briofitskog endemizma u flori jugoslavije. acta musei macedonici scientiarium naturalium, 4: 2–33. pavletić, z. 1968: flora mahovina jugoslavije. institut za botaniku sveučilišta u zagrebu, 431 p. pavlović, z. 1951: vegetacija planine zlatibor. srpska akademija nauka, zbornik radova 11, institut za ekologiju i biogeografiju, 2: 115–182. pavlović, z. 1964: borove šume na serpentinima u srbiji. glasnik prirodnjačkog muzeja u beogradu, b 19: 25–65. pejin, b., sabovljević, a., soković, m., glamočlija, j., ćirić, a., vujičić, m., sabovljević, m. 2012a: antimicrobial activity of rhodobryum ontariense. hemijska industrija, 66 (3): 381-384. pejin, b., bianco, a., newmaster, s., sabovljević, m., vujisić, lj., tešević, v., vajs, v., de rosa, s. 2012b: fatty acids of rhodobryum ontariense (bryaceae). natural product research, 26 (8): 696702. pejin, b., vujisić, lj., sabovljević, m., tešević, v., vajs, v. 2012c: fatty acid chemistry of atrichum undulatum and hypnum andoi. hemijska industrija, 66 (2): 207-209. petković, b., tatić, b., ilijin–jug, m. 1988: dve nove zajednice srpske ramondije (ramonda serbica panč.) u gornjem toku sliva reke ibra. glasnik instituta za botaniku i botaničke bašte univerziteta u beogradu, 22: 107–116. petković, b., tatić, b., marin, d. p., dimić, j. 1990: prilog poznavanju zajednica sa edraianthus jugoslavicus lakušić sa mokre gore (jugozapadna srbija). bilten društva ekologa bosne i hercegovine, b5: 131–135. petković, b., tatić, b., marin, p., ilijin-jug, m. 1991: musco-seslerio-edraianthetum jugoslavici, ass. nov. nova zajednica klisura i kanjona tutinskog područja. drugi simpozijum o flori jugoistočne srbije i mogućnostima njenog racionalnog korišćenja, univerzitet u nišu, tehnološki fakultet u leskovcu, leskovac. zbornik radova 1(7-8/89-90): 133–138. petković, b., tatić, b., marin, p., veljić, m. 2000: vegetacija spomenika prirode „đavolja varoš”. 6. simpozijum o flori jugoistočne srbije i susednih područja, sokobanja, zbornik radova: 323–347. pichler, a. 1931: prilog poznavanju mahova tresetara jugoslavije. acta botanica croatica, 6: 47–55. pichler, a. 1939: die lebermossflora auf morschen baumstämmen, vermoderndem holz und auf faulenden baumstümpfen der wälder jugoslaviens. glasnik skopskog naučnog društva, 20: 141–153. pichler, a. 1940: hepatike na kori drveća i grmlja naših šuma. šumski list, 7: 334–354. pócs, t., sabovljević, m., puche, f., segarra moragues, j. g., gimeno, c., kürschner, h. 2004: crossidium laxefilamentosum frey & kürschner (bryopsida: pottiaceae), new to europe and to north africa. journal of bryology, 26: 113–124. podpera, j. 1922: ad bryophytorum haemi peninsulae cognitionen additamentum. acta botanica bohemica, 1: 1–18. popović, m. 1966: prilog poznavanju mahovina u rezervatima i zaštićenim područjima u srbiji. zaštita prirode, 33: 219–228. radulović, n.s., filipović, s.i., zlatković, d.b., đorđević, m.r., stojanović, n.m., randjelović, p.j., mitić, k.v., jevtpvioć-stoimenov, t.m., ranđelović, v.n. 2016: immunomodulatory pinguisane-type sesquiterpenes from the liverwort porella cordaeana (porellaceae): the “new old” furanopinguisanol and its oxidation product exert mutually different effects on rat splenocytes. rsc advances, 6(48), 41847-41860 rajčić, m., nerić, v., vujičić, m., sabovljević, a., sabovljević, m. 2015: the effect of shortand long-termed salt stress on the atrichum undulatum (hedw.) p. beauv. sixth balkan botanical congress, 14-18 september 2015, rijeka, croatia. book of abstracts: 102. rajevski, l. 1951: borove šume u predelima od mokre gore do reke uvac. srpska akademija nauka, zbornik radova 11, institut za ekologiju i biogeografiju, 2: 183–192. ranđelović, v. 1994: geobotanička studija vlasinske 95 biologica nyssana ● 10 (2) december 2019: 87-98 veljić ● bryology in serbia: from floristics to chemosystematics tresave. biološki fakultet, univerziteta u beogradu. ranđelović, v., zlatković, b. 2010: flora i vegetacija vlasinske visoravni. prirodnomatematički fakultet, univerzitet u nišu. rowntree, j. k., pressel, s., ramsay, m.m., sabovljević, a., sabovljević, m. 2011: in vitro conservation of european bryophytes. in vitro cell dev biol – plant, 47: 55–64. rudski, i. 1949a: ekskurzija na žljeb: mokru planinu. prirodnjački muzej srpske zemlje, 23. beograd. rudski, i. 1949b: tipovi lišćarskih šuma jugoistočnog dela šumadije. prirodnjački muzej srpske zemlje, 25: 1–67. sabovljević, a., soković, m., sabovljević, m., grubišić, d. 2006: antimicrobial activity of bryum argenteum. fitoterapia, 77 (2): 144-145. sabovljević, a., sabovljević, m., jocković, n. 2009: in vitro culture and secondary metabolite isolation in bryophytes. in: mohan js, saxena pk, editors. protocols for in vitro cultures and secondary metabolite analysis of aromatic and medicinal plants. methods in molecular biology. new york, ny: humana press, 117–128. sabovljević, m. 1998: the rare bryophytes of šara mountain, yugoslavia, planta europa proceedings of the second european conference on the conservation of wild plants, 2: 159–161. sabovljević, m. 1999: anastrophyllum minutum (schreb.) schust., new to serbia (fr yugoslavia) and its distribution in the balkans. phytologia balcanica, 5(2-3): 93–96. sabovljević, m. 2000: checklist of hepatics of the federal republic of yugoslavia. lindbergia 25: 37– 42. sabovljević, m. 2003a: bryophyte flora of south banat (vojvodina, yugoslavia). cryptogamie, bryologie, 24 (3): 241–252. sabovljević, m. 2003b: données sur la présence et la chorologie des taxons du genre schistidium (grimmiaceae) dans la république fédérale de yougoslavie (serbie et monténégro). bocconea, 16 (2): 991–999. sabovljević, m. 2006: contribution to the bryophyte flora of the djerdap national park (e serbia). phytologia balcanica, 12 (1): 51–54. sabovljević, m. 2015: flora briofita srbije i. tresetnice (sphagnophyta). srpska akademija nauka i umetnosti, 110 p. sabovljević, m., cvetić, t. 2001: rhodobryum ontariense (kindb.) kindb. new to yugoslavia and some notes on the genus rhodobryum (schimp.) limpr. in the federal republic of yug oslavia. ekologija, 36 (2): 145–153. sabovljević, m., cvetić, t. 2003: bryophyte flora of avala mt. (c. serbia, yugoslavia). lindbergia, 28: 90–96. sabovljevic, m., cvetic, t., stevanovic, v. 2004: bryophyte red list of serbia and montenegro. biodiversity and conservation, 13 (9): 1781–1789. sabovljević, m., grdović, s. 2009: bryophyte diversity within urban areas: case study of the city of belgrade (serbia). international journal of botany, 5 (1): 85–92. sabovljević, m., marka, j. 2009: the biological evidence of climate changes: a case study of liverwort lunularia cruciata (l.) dum. ex lindb. in serbia. botanica serbica, 33(2): 185–187. sabovljević, m., sérgio, c. 2002: contribution to the bryoflora of serbia: a bryophyte collection from 1996. portugaliae acta bioogica, 20: 65–74. sabovljević, m., stevanović, v. 1999: moss conspectus of federal republic of yugoslavia. flora mediterranea, 9: 65–95. sabovljević, m., stevanović, v. 2000: sphagnum denticulatum brid., novedad para la brioflora de serbia (yugoslavia). botanica complutensis, 24: 61–63. sabovljević, m., stevanović, v. 2006: contribution to knowledge of the bryophyte flora of bačka (vojvodina, serbia). archives of biological sciences, 58 (2): 135–138. sabovljević, m., papp, b., sabovljević, a., vujičić, m., szurdoki, e., segarra-moragues, j. g. 2012a: in vitro micropropagation of rare and endangered moss enthostodon hungaricus (funariaceae). bioscience journal, 28: 632–640. sabovljević, m. s., segarra-moragues, j. g., puche, f., vujičić, m., cogoni, a., sabovljević, a. 2016: eco-physiological and biotechnological approach to conservation of the world-wide rare and endangered aquatic liverwort riella helicophylla (bory et mont.) mont. acta botanica croatica, 75 (2): 194-198. sabovljević, m. s., nikolić, n., vujičić, m., sinžarsekulić, j., pantović, j., papp, b., sabovljević, a. 2018: ecology, distribution, propagation in vitro, ex situ conservation and native population strenghtening of rare and threatened halophyte moss entosthodon hungaricus in serbia. wulfenia, 25: 117-130. 96 biologica nyssana ● 10 (2) december 2019: 87-98 veljić ● bryology in serbia: from floristics to chemosystematics sabovljević, m., vujičić, m., pantović, j., sabovljević, a. 2014: bryophyte conservation biology: in vitro approach to the ex situ conservation of bryophytes from europe. plant biosystems, 148 (4), 857-868. sabovljević, m., vujičić, m., šinžar-sekulić, j., segarra-moragues, j. g., bapp, b., skorić, m., dragačević, l., sabovljević, a. 2012b: reviving, in vitro differentiation, development and micropropagation of the rare and endangered moss bruchia vogesiaca (bruchiaceae). hortscience, 47 (9): 1347–1350. sabovljević, m. s., vujičić, m., wang, x., garraffo, m., bewley, c. a., sabovljević, a. 2017: production of the macrocyclic bis-bibenzyls in axenically farmed and wild liverwort marchantia polymorpha l. subsp. ruderalis bischl. et boisselier. plant biosystems, 151 (3): 414-418. schiffner, v. 1897: musci bornmüllerian, ein beitrag zum kryptogramenflora des orients. osterr bot zeitschr, 125 p. simić, m. 1892: nekoliko srpskih mahovina, nastavnik, list profesorskoga društva, iii knjiga, 447–450. simić, m. 1897: nekoliko kriptogamskih biljaka u okolini vranjskoj. izveštaj vranjske gimnazije za školsku 1896/97. godinu, 3–8. simić, m. 1898: kriptogamne biljke u okolini vranjskoj. izveštaj vranjske gimnazije za školsku 1897/98. godinu, 13–16. simić, m. 1900: prilog flori mahovina u srbiji, spomenik srpske akademije nauka, 35: 5–46. slavnić, ž. 1956: vodena i barska vegetacija vojvodine. zbornik matice srpske, serija prirodnih nauka, 10: 5–72. soška, t. 1949: pregled mahovina i lišajeva u okolini beograda. glasnik 10. prirodnjackog muzeja srpske zemlje, 1–2: 93–112. stevanović, v., pavić, s., stevanović, b. 1995: diverzitet flore mahovina (bryophyta) jugoslavije sa pregledom vrsta od međunarodnog značaja. – in: stevanović, v., vasić, v.(eds): biodiverzitet jugoslavije sa pregledom vrsta od međunarodnog značaja. biološki fakultet i ecolibri, beograd. tešić, ž., gigov, a., bogdanović, m., milić, č. 1979: tresave srbije. zbornik radova geografskog instituta jovan cvijić, 31: 19–64. tyagi, a.k., bukvički, d., gottardi, d., veljić, m., guеrzoni, m.e., vannini, l., malik, a., marin, p. d. 2013: antimicrobial potential and chemical characterization of serbian liverwort (porella arboris-vitae): sem and tem observations. evidence-based complementary and alternative medicine, article id 382927, veljić, m. 2013: flora mahovina planine kopaonik, srbija. zaštita prirode, 53 (1-2): 73-91. veljić, m., bukvički, d., marin, p. d. 2016a: sphagnum fimbriatum, a species new for the flora of serbia. botanica serbica, 40 (1): 101–103. veljić, m., ljubić, b., marin, p. d., marin, m. 2008: bryophyte flora on the northern slopes of zlatar mountain (southwest serbia). archives of biological sciences, 60 (1): 145–150. veljić, m., marin, p. d. 1997: new moss taxa for the flora of serbia. flora mediterranea, 7: 133–138. veljić, m., marin, p. d., boža, p., petković, b. 1996: flora mahovina vrela grze i crnog timoka (srbija). glasnik instituta za botaniku i botaničke bašte univerziteta u beogradu, 30: 97–106. veljić, m., marin, p. d., boža, p., petković, b. 2001a: bryoflora of some well-springs of the dinaric alps and carpathian karst in serbia. bocconea, 13: 343–351. veljić, m., marin, p. d., petković, b., ljubić, b. 2001b: new species for the bryophyte flora of yugoslavia. cryptogamie, bryologie, 22 (4): 275– 277. veljić, m., marin, p. d., petković, b., ljubić, b. 2004: new records for the bryophyte flora of serbia. archives of biological sciences, 56 (1–2): 11–12. veljić, m., marin, p., lakušić, d., ljubić, b. 2006: bryophyte flora of the uvac river george (southwest serbia). archives of biological sciences, 58 (3): 187–194. veljić, m., tarbuk, m., marin, p. d., ćirić, a., soković m., marin, m. 2008: antimicrobial activity of methanol extracts of some genuine mosses from serbia, pharmaceutical biology, 46 (12), 871-875. veljić, m., ćirić, a., soković, m., janaćković, p., marin, p. d. 2010: antibacterial and antifungal activity of the liverwort (ptilidium pulcherrimum) metanol extract. archives of biological sciences, 62 (2): 381-395. veljić, m., đurić, a., soković, m., ćirić, a., glamočlija, j., marin p. d. 2009: antimicrobial activity of methanol extracts of fontinalis antipyretica, hypnum cupressiforme and ctenidium molluscum. archives of biological sciences, 61 (2): 225-229. veljić, m., rajčević, n., bukvički, d. 2016b: a 97 biologica nyssana ● 10 (2) december 2019: 87-98 veljić ● bryology in serbia: from floristics to chemosystematics revision of the moss collection of the university of belgrade herbarium (beou) from the ostrozub mountain in serbia. biologica nyssana, 7 (1): 1117. vujičić, m. 2016: biohemijski i ekofiziološki odgovori odabranih vrsta mahovina na kontrolisani stres izazvan solima u akseničnim uslovima. phd thesis. univerzitet u beogradu, biološki fakultet. vujičić, m., sabovljević, a., milošević, s., segarra-moragues, j. g., sabovljević, m. 2016: effects of abscisic acid (aba) on development of selected bryophyte species. plant biosystems, 150: 1023-1029. vujičić, m., sabovljević, a., šinžar-sekulić, j., škorić, m., sabovljević, m. 2012: in vitro development of the rare and endangered moss molendoa hornschuchiana (hook.) lindb. ex limpr. (pottiaceae, bryophyta). hortscience, 47: 84–87. wettstein, r. 1890: ueber das vorkommen von trochobryum carniolicum in südserbien. oesterreichische botanische zeitschrift, 40: 170–171. 98 biologica nyssana ● 10 (2) december 2019: 87-98 veljić ● bryology in serbia: from floristics to chemosystematics jovanović et al., 2019, biologica nyssana 10(2) 10 (2) december 2019: 113-123 doi: 10.5281/zenodo.3600187 morphological variability of endemic sempervivum ciliosum subsp. ciliosum (crassulaceae) from the balkan peninsula original article maja jovanović university of niš, faculty of sciences and mathematics, department of biology and ecology, niš, serbia maja.jovanovic1@pmf.edu.rs (corresponding author) dmitar lakušić university of belgrade, faculty of biology, institute of botany and botanical garden “jevremovac”, takovska 43, 11000 belgrade, serbia dlakusic@bio.bg.ac.rs chavdar gussev institute of biodiversity and ecosystem research, department of plant and fungal diversity and resources, bulgarian academy of science, sofia, bulgaria chgussev@gmail.com predrag lazarević university of belgrade, faculty of biology, institute of botany and botanical garden “jevremovac”, takovska 43, 11000 belgrade, serbia predrag.lazarevic@bio.bg.ac.rs bojan zlatković university of niš, faculty of sciences and mathematics, department of biology and ecology, niš, serbia bojanzlat@yahoo.com received: november 12, 2019 revised: december 23, 2019 accepted: december 24, 2019 abstract: sempervivum ciliosum belongs to the group of yellow-flowered houseleek species (s. ciliosum complex) endemic for the territory of the balkan peninsula. concerning the traditional taxonomy of genus sempervivum, there are several taxa included in the above-mentioned group (s. ciliosum, s. jakucsii, s. klepa, s. octopodes and s. galicicum). however, due to highly expressed phenotypic variability, it isn’t possible to certainly establish whether all taxa from that group, including s. ciliosum as a type species, are morphologically clearly defined. the aim of this study was to analyze the level of variability of morphological traits within the populations of s. ciliosum, taken in a narrow sense. measurements of 36 quantitative traits of the vegetative and the flowering region of s. ciliosum subsp. ciliosum, including populations from bulgaria, greece and north macedonia, were processed in statistica 8.0. results of descriptive statistics, correlation analysis and univariate variance analysis (anova) indicate that most of the analyzed traits express moderate to high variability within all examined populations. key words: morphological traits, phenotypic plasticity, variability, balkans apstract: morfološka varijabilnost endemične vrste sempervivum ciliosum subsp. ciliosum (carassulaceae) sa balkanskog poluostrva sempervivum ciliosum pripada grupi žutocvetnih vrsta čuvarkuća (s. ciliosum complex) endemičnih za teritoriju balkanskog poluostrva. u odnosu na tradicionalno shvatanje taksonomije roda sempervivum, nekoliko taksona je uključeno u gore pomenutu grupu (s. ciliosum, s. jakucsii, s. klepa, s. octopodes i s. galicicum). međutim, zbog veoma izražene fenotipske varijabilnosti, nije moguće sa sigurnošću utvrditi da li su svi taksoni ove grupe, uključujući s. ciliosum kao tipsku vrstu, morfološki jasno definisani. cilj ove studije bila je analiza stepena varijabilnosti morfoloških karaktera u okviru populacija s. ciliosum s.s. merenja 36 kvantitativnih karaktera vegetativnog i cvetnog regiona s. ciliosum subsp. ciliosum, kod jedinki populacija iz bugarske, grčke i severne makedonije, obrađena su u programu statistica 8.0. rezultati deskriptivne statistike, korelacione analize i univarijantne analize varijanse (anova) ukazuju da većina analiziranih karaktera ispoljava umerenu do visoku varijabilnost u okviru analiziranih populacija. ključne reči: morfološki karakteri, fenotipska plastičnost, varijabilnost, balkansko poluostrvo introduction genus sempervivum l. belongs to a relatively large family crassulaceae j.st.-hil. the family includes 1300-1500, mostly leaf and stem succulent species divided into 33-35 genera (berger, 1930; thiede & eggli, 2007; mort et al., 2010). although some of the species of this family are extensively studied, highly expressed phenotypic variability as well as the ability of hybridization obscures the already vague picture of their morphological variability, taxonomy, as well as the number of taxa within the family. sempervivum l. is represented by perennial, herbaceous, rosulate, hardy leaf and partially stem succulent plants. according to an earlier, more widely accepted understanding, the genus sempervivum encompasses 40 to 60 species (‘t hart et al., 2005; thiede & eggli, 2007). however, the latest studies indicate that there are 46 species within the genus (karaer et al., 2011; klein & kadereit, 2015; klein © 2019 jovanović et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 113 13th symposium on the flora of southeastern serbia and neighboring regions & kadereit, 2016) as well as 16 heterotypic infraspecific taxa and 16 described natural hybrids sensu ‘t hart et al. (2003). blurred picture of the total number of species within this genus is a consequence of the difficult identification of the representatives as well as the fact that currently there is an extremely high number of the names pertaining to species rank (karaer et al., 2011). the natural habitat of sempervivum species includes rocky and dry terrains of the high mountains of europe and asia (‘t hart et al., 2003). the only outlier of this genus, located outside the main area in europe and asia, is s. atlanticum (hook.f.) ball, endemic of northern africa (karaer et al., 2011). the taxonomic problems within this genus primarily arise from pronounced phenotypic plasticity, reflected through the manifestation of morphological similarity, which is often neglected or overemphasized, when describing species. sempervivum ciliosum taken in a broader sense represents a group of taxa, described from the territory of the central and southern parts of the balkan peninsula. although intriguing taxonomical complex, s. ciliosum is still an under-researched group that includes s. ciliosum craib as a type species, s. jakucsii pénzes, s. klepa micevski, s. octopodes turrill and s. galicicum (a. c. sm.) micevski. sempervivum ciliosum s.s. is a perennial, leafy succulent plant with closed or semi-open rosettes, 2-4 cm in diameter. rosette leaves are oblong-oblanceolate to oblong-lanceolate, acute, characterized by the presence of long trichomes, both on the abaxial and adaxial surface as well as on the margin of leaves which can often be interwoven with each other and with trichomes of adjoining leaves. the flowering stem is erect, 4-10 cm long, cauline leaves are slightly wider than those in the rosette. inflorescence is compact, composed of 9to 14-merous flowers with acute, reddish-brown sepals, lemon yellow to yellow petals (8-12 × 1.5-2 mm), pale green carpels and pale yellow stamens. within s. ciliosum two subspecies have been described (‘t hart, 2002; ‘t hart et al., 2003): sempervivum ciliosum subsp. ciliosum characterized by lemon yellow, 10-12 × 1.5 mm, petals, and s. ciliosum subsp. octopodes recognizable by pale yellow at base purple or lilac petals and purplish filaments. according to ‘t hart et al. (2003) the first subspecies also includes s. ciliosum var. galicicum a. c. sm., described from mt. galičica in north macedonia. howewer, taxonomical position of this taxon is still not completely resolved since micevski (1998) considers it as a separate taxon (s. galicicum) at the species rank. due to highly expressed phenotypic plasticity and scarce literature data available of the morphological features, it is not possible to certainly establish whether all described species of this group are, so-called, valid or well-defined taxa. therefore, the possibility of having more or fewer taxa, than the number already reported in the literature is present. furthermore, taxonomic relations between taxa, nomenclature, and status of species within the complex are extremely vague. studies of the morphological variability of vegetative traits and traits from flowering region, despite the need for more detailed studies, have not been conducted for the taxa within s. ciliosum s.s. in accordance with current taxonomic concept (‘t hart et al., 2003), the species s. ciliosum here was considered in the narrow sense, as s. ciliosum subsp. ciliosum (incl. s. ciliosum var. galicicum a. c. smith). aim of this study was to examine the degree of variability of selected quantitative morphological traits from both previously mentioned regions of individuals from s. ciliosum subsp. ciliosum populations originated from different parts of the balkan peninsula. therefore, the purpose of this study in theoretical terms was to determine the level of morphological variability of s. ciliosum and accordingly to clarify the taxonomic relationships both in the s. ciliosum complex and throughout the genus sempervivum. the practical purpose was to provide additional information for a valid description of the taxa analyzed and to improve the keys for identifying species, and subspecies of this genus. material and methods plant material morphological traits from the vegetative and the flowering region were analyzed on 45 individuals from three populations collected from the territory of central and southern balkan peninsula. from each population, individuals were deposited in the herbarium of the department of biology and ecology, faculty of sciences and mathematics, university of niš (hmn) and herbarium of the university of belgrade (beou). detailed information of habitat characteristics, legators and voucher numbers, as well as localities from where populations were collected, are given in tab. 1 and fig. 1. during the field research, s. ciliosum was collected at a much smaller number of localities than expected because many of the reported distribution data for this species refer to other, morphologically similar species from the genera jovibarba and sempervivum. finally, this species spontaneously hybridizes with other species of the genus sempervivum, resulting in the large presence of hybrid individuals in most of the studied populations, also excluded from the study. in order to eliminate the influence of ecological factors on the variability of morphological traits of selected populations in their natural environment, all individuals, prior to morphometric analysis, were subjected to a period of growing, for a minimum 114 biologica nyssana ● 10 (2) december 2019: 113-123 jovanović et al. ● morphological variability of endemic sempervivum ciliosum subsp. ciliosum (crassulaceae) from the balkan peninsula riod. measurements of morphological traits from the inflorescence and flower (tab. 3) were performed in several ways. quantitative traits such as the number of flowers in the main inflorescence (nf), as well as number of sepals, petals, stamens and carpels (ns, np, sn, nc), as meristic characters, are counted within all examined individuals during the flowering period. the angle of branching of the main inflorescence (abi) was determined using a protractor. measurements of length and width of the main inflorescence (li, wi), length of the pedicel (lp) and diameter of flower (df) are conducted by using a digital caliper. length and width of parts of different segments of the flower are measured on scanned pictures of appropriate permanent slides, using digimizer image analysis software (medcalc software©, belgium). from all examined individuals, to prepare permanent slides, one flower was selected from a different region of the same inflorescence (central, middle and outer). each permanent slide was prepared after floral structures of each flower were carefully dissected and placed on slides. after preparation, slides were scanned on a resolution of 600 dpi on hp scanjet 200. as background, during the process of scanning, blue paper was used in order to provide high-contrast with yellowish floral parts. obtained values of measured floral parts for a flower, from each region of the inflorescence, were statistically processed to obtain mean values of analyzed traits. this way of analyzing floral parts allowed us to reduce differences in dimensions of floral parts between flowers from a different region of an inflorescence. it should be also emphasized that, except for height of the plant (ph), diameter of the stem (ds), length of internodes in the lower and upper part of the stem (lil, liu), the number of flowers in the main inflorescence (nf) and angle of branching of the main inflorescence (abi), measurements of all selected quantitative traits were performed in triplicate. of one year, in a greenhouse. collected individuals from each population were planted in pots of the same diameter, on floradur® substrate (floragard, vertriebs gmbh für gartenbau, germany). after a growing period, 15 individuals from each population were morphometrically processed. morphometric procedures a total of 36 quantitative morphological traits were analyzed for this study i.e., 18 morphological traits each from the vegetative and the flowering region. all morphological traits of the vegetative part (tab. 2), except the height of the plant (ph), which was determined using the ruler, were measured using high precision digital caliper (mahr federal 4107107 16u, esslingen, germany) during the flowering pebiologica nyssana ● 10 (2) december 2019: 113-123 115 jovanović et al. ● morphological variability of endemic sempervivum ciliosum subsp. ciliosum (crassulaceae) from the balkan peninsula table 1. localities, habitat characteristics, legators name and voucher numbers of individuals from analyzed populations of sempervivum ciliosum subsp. ciliosum. locality altitude (m) habitat subtratum legator voucher no. bulgaria, mt. rila, rila 580 cliffs silicate zlatković, b., gussev, ch. hmn 13899 greece, mt. orvilos 1737 subalpine rocky grasslands (daphnofestucetea) limestone lakušić, d., kuzmanović, n., janković, i. beou 46354 north macedonia, mt. galičica, trpejca 1570 rocks limestone zlatković, b., lazarević, p. hmn 13991 fig. 1. locations of analyzed populations of sempervivum ciliosum subsp. ciliosum. statistical analysis for the purposes of this study, variability of selected quantitative morphological traits was analyzed using basic statistical analysis (descriptive statistics), correlation analysis and univariate analysis of variance (anova). all values of measured traits were statistically processed using statistica 8.0 (statsoft, inc., tulsa, usa). 116 results descriptive statistics based on obtained results of descriptive statistics, quantitative morphological traits from the vegetative region manifest a low to moderate degree of variability (tab. 2, fig. 2). morphological traits which stand out by a higher degree of variability, based on highest values of standard deviation, which is the table 2. results of descriptive statistics and anova of morphological traits from the vegetative region of analyzed individuals of sempervivum ciliosum subsp ciliosum. character abbreviation, name and unit n mean min. max. std. dev. cv % f p ph height of the plant (cm) 45 23.97 16.50 36.30 4.99 21 6.33 0.0040** dr diameter of the rosette (cm) 45 3.33 2.15 4.68 0.56 17 4.08 0.0240* llr length of the lower part of the rosettes leaf (mm) 45 21.62 9.05 40.99 7.17 33 0.0001 *** lur length of the upper part of the rosettes leaf (mm) 45 3.88 2.41 5.49 0.71 18 4.67 0.0137 * wlr width of leaves from the middle part of the rosette (mm) 45 6.48 4.68 9.17 1.06 16 0.15 0.8575 n.s. tlr thickness of leaves from the middle part of the rosette (mm) 45 2.55 1.68 3.58 0.55 22 0.0002*** ds diameter of the stem (mm) 45 4.09 2.05 6.72 1.15 28 0.0000*** lil length of internodes in the lower part of the stem (mm) 45 12.19 7.89 17.35 2.22 18 1.44 0.2475 n.s. liu length of internodes in the upper part of the stem 45 8.24 4.77 13.68 2.31 28 2.12 0.1327 n.s. lll length of the lower leaf of the stem (mm) 45 27.27 11.14 40.76 6.08 22 7.33 0.0019 ** wll width of the lower leaf of the stem (mm) 45 6.88 4.07 9.05 1.11 16 2.25 0.1181 n.s. tll thickness of the lower leaf of the stem (mm) 45 2.92 1.18 4.05 0.57 19 2.54 0.0912 n.s. lml length of the middle leaf of the stem (mm) 45 25.23 15.37 36.88 4.70 19 3.19 0.0512 n.s. wml width of the middle leaf of the stem (mm) 45 6.51 3.40 8.93 1.26 19 1.58 0.2181 n.s. tml thickness of the middle leaf of the stem (mm) 45 2.51 1.23 3.59 0.52 21 4.55 0.0162 * lul length of the upper leaf of the stem (mm) 45 19.83 12.69 28.77 3.52 18 5.76 0.0061 ** wul width of the upper leaf of the stem (mm) 45 4.66 3.01 6.72 0.92 20 2.94 0.0640 n.s. tul thickness of the upper leaf of the stem (mm) 45 1.61 1.03 2.10 0.27 17 0.16 0.8513 n.s. (n – number of individuals, mean – mean value, min. – minimum value, max. – maximum value, std. dev. – standard deviation, cv% – coefficient of variation, f – fisher’s coefficient, p – level of significance, ***extremely high statistical significance, **high statistical significance, *low statistical significance; n.s.not significant) biologica nyssana ● 10 (2) december 2019: 113-123 jovanović et al. ● morphological variability of endemic sempervivum ciliosum subsp. ciliosum (crassulaceae) from the balkan peninsula 117 consequence of large scale differences of minimum and maximum values from mean values, are: length of the lower part of the rosettes leaf (llr, 21.62 ± 7.17), length of the lower leaf of the stem (lll, 27.27 ± 6.08), height of the plant (ph, 23.97 ± 4.99) and length of the middle leaf of the stem (lml, 25.23 ± 4.70). values of the most variable trait of the vegetative region, length of the lower part of the rosettes leaf (llr), ranges from 9.05 mm, as a minimum, up to 40.99 mm, as a maximum value. less variable trait, the height of the plant (ph), range from 16.50 cm to 36.30 cm. furthermore, lengths of the leaves of the stem are distributed in the range of minimal and maximal values of 11.14-40.76 mm for the lower leaves (lll) and 15.37-36.88 mm for the middle leaves (lml). morphological traits from the vegetative region exhibiting the lowest degree of variability are the following: thickness of the upper leaf of the stem (tul, 1.61 ± 0.27) with a range of minimal and maximal values of 1.03-2.10 mm, thickness of the middle leaf of the stem (tml, 2.51 ± 0.52) with values in range of 1.23-3.59 mm, thickness of the leaves from the middle part of rosette (trl, 2.55 ± 0.55) which is within a range of 1.683.58 mm, diameter of the rosette (dr, 3.33 ± 0.56) characterized by a minimum value of 2.15 cm and maximal value of 4.68 cm, and thickness of the lower leaf of the stem (tll, 2.92 ± 0.57) with values within a range of 1.18-4.05 mm. obtained values of the coefficient of variation indicate that only length of the lower part of the rosettes leaf is highly variable (llr, cv=33%) while remaining of analyzed morphological traits from vegetative region show moderate degree of variability (cv=10-30%). quantitative morphological traits from flowering region, generally, and according to obtained results, are less variable regarding to traits of the vegetative region. although traits of the inflorescence and flower show a low to a high degree of variability, the number of traits with low variability is significantly higher in regards to highly variable ones. as the most variable traits stand out: length of the main inflorescence (li, 65.92 ± 25.66), width of the main inflorescence (wi, 70.38 ± 24.95), number of flowers in the main inflorescence (nf, 37.75 ± 18.37) and angle of branching of the main inflorescence (abi, 46.20 ± 14.94). the most variable trait, length of the main inflorescence (li) has a range of minimal and maximal values from 20.41 mm to 121.49 mm. thus, the width of the main inflorescence (wi) is within the range of 29.43-137.35 mm, number of flowers in the main inflorescence (nf) varies from 7 to even 103 flowers per inflorescence and minimal value of angle of branching of the main inflorescence (abi) is 24.00° while the maximal value is 90.00°. the morphological traits of lowest variability are as follows: width of carpels (cw, 2.08 ± 0.25), width of petals (wp, 2.11 ± 0.31) width of sepals (ws, fig. 2. box and whisker plots of basic statistic parameters of analyzed vegetative morphological traits of sempervivum ciliosum subsp. ciliosum: height of the plant (ph); dr-diameter of the rosette; dsdiameter of the stem; lillength of internodes in the lower part of the stem; liulength of internodes in the upper part of the stem; llrlength of the lower part of the rosettes leaf; lur-length of the upper part of the rosettes leaf; wlrwidth of leaves from the middle part of the rosette; tlrthickness of leaves from the middle part of the rosette; lll length of the lower leaf of the stem; wllwidth of the lower leaf of the stem; tllthickness of the lower leaf of the stem; lmllength of the middle leaf of the stem; wmlwidth of the middle leaf of the stem; tmlthickness of the middle leaf of the stem; lullength of the upper leaf of the stem; wulwidth of the upper leaf of the stem; tulthickness of the upper leaf of the stem (middle point – mean value; box – mean ± sd; whisker – minimum value maximum value). biologica nyssana ● 10 (2) december 2019: 113-123 jovanović et al. ● morphological variability of endemic sempervivum ciliosum subsp. ciliosum (crassulaceae) from the balkan peninsula 1.58 ± 0.33), length of longer filaments of the flower (llf, 5.13 ± 0.48) and length of shorter filaments of the flower (lsf, 4.63 ± 0.49). based on the values of the coefficient of variation most of the analyzed traits from the flowering region are in the extent of moderate variability, while only four of analyzed traits show a high degree of variability: number of flower in the main inflorescence (nf, cv=49%), length of the main inflorescence (li, cv=39%), width of the main inflorescence (wi, cv=35%) and angle of branching of the main inflorescence (abi, cv=32%). these same traits, as already explained, have been characterized as the most variable, based on the values of standard deviation. thus, length of longer filaments of the flower (llf) and length of petals (lp) with the values of coefficient of variation of 9%, number of carpels (nc) with coefficient of correlation of 8%, number of sepals (ns), as well as number of petals (np) and number of stamens (sn), all characterized with the value of 7% of the same coefficient, are presented as the least variable. results for minimum and maximum values, as well table 3. results of descriptive statistics and anova of morphological traits from the inflorescence and flower of analyzed individuals of sempervivum ciliosum subsp. ciliosum. character abbreviation, name and unit n mean min. max. std. dev. cv % f p li length of the main inflorescence (mm) 45 65.92 20.41 121.49 25.66 39 6.01 0.0050 ** wi width of the main inflorescence (mm) 45 70.38 29.43 137.35 24.95 35 2.78 0.0738 * nf number of flowers in the main inflorescence 45 37.75 7.00 103.00 18.37 49 13.15 0.0000 *** abi angle of branching of the main inflorescence ( ̊ ) 45 46.20 24.00 90.00 14.94 32 12.48 0.0001 *** pl length of the pedicel (mm) 45 1.82 0.92 2.83 0.51 28 13.41 0.0000 *** df diameter of the flower (mm) 45 21.53 16.65 27.12 1.85 9 9.26 0.0005 *** ns number of sepals of the flower (mm) 45 11.58 10.00 13.67 0.82 7 16.55 0.0000 *** ls length of sepals of the flower (mm) 45 4.66 3.45 7.37 0.68 15 18.29 0.0000 *** ws width of sepals of the flower (mm) 45 1.58 1.10 3.12 0.33 21 15.86 0.0000 *** np number of petals of the flower 45 11.59 10.00 13.67 0.84 7 17.63 0.0000 *** lp length of petals of the flower (mm) 45 9.87 8.27 12.11 0.91 9 31.91 0.0000 *** wp width of petals of the flower (mm) 45 2.11 1.49 3.10 0.31 15 15.36 0.0000 *** sn number of stamens of the flower 45 23.09 20.33 27.33 1.71 7 14.06 0.0000 *** lsf length of shorter filaments of the flower 45 4.63 3.40 6.14 0.49 11 0.72 0.4936 n.s. llf length of longer filaments of the flower 45 5.13 4.09 6.47 0.48 9 2.42 0.1011 n.s. nc number of carpels of the flower 45 11.47 10.00 13.67 0.86 8 20.66 0.0000 *** lc length of carpels of the flower (mm) 45 5.70 4.12 6.93 0.72 13 46.39 0.0000 *** cw width of carpels of the flower (mm) 45 2.08 1.53 2.67 0.25 12 15.66 0.0000 *** (n – number of individuals, mean – mean value, min. – minimum value, max. – maximum value, std. dev. – standard deviation, cv% – coefficient of variation, f – fisher’s coefficient, p – level of significance, ***extremely high statistical significance, **high statistical significance, *low statistical significance) 118 biologica nyssana ● 10 (2) december 2019: 113-123 jovanović et al. ● morphological variability of endemic sempervivum ciliosum subsp. ciliosum (crassulaceae) from the balkan peninsula as mean values, standard deviation and coefficient of variation, of all analyzed morphological traits of flowering region, are given in tab. 3 and fig. 3. correlative variability results from correlation analysis of analyzed morphometric traits have shown that all three degrees of linear correlation were represented, both as negative and positive form of correlations. majority of analyzed traits, 54.60%, show a weak positive linear correlation with values of correlative coefficient (r) between 0.00 and 0.50. a weak negative correlation (-0.50 < r < 0.00) is represented in 32.06% pairs of morphological traits. moderate, positive (0.50 < r < 0.80) or negative (-0.50 < r < -0.80), correlation is less represented, i.e. in 10.63% as positive correlation form, respectively, 0.79% as negative correlation form. thus, strong linear connectivity was established only as a positive form of correlation (0.80 < r < 1.00), in three pairs of traits from vegetative and nine pairs of traits from the flowering region (1.90% in total). the pairs of traits with highest degree of correlation (r > 0.90) are: length of shorter filaments of the flower/length of longer filaments of the flower (lsf/llf), number of sepals/ number of petals (ns/np), length of sepals/width of sepals (ls/ws), number of petals/number of carpels (np/nc), number of petals/number of stamens (np/ sn), number of sepals/number of carpels (ns/nc), number of stamens/number of carpels (sn/nc), number of sepals/number of stamens (ns/sn) and thickness of the middle leaf of the stem/thickness of the lower leaf of the stem (tml/tll) (tab. 4). the correlation among pairs of traits from the vegetative region, as well as pairs of traits from the vegetative and the flowering region is mostly represented by weak negative and moderate positive linear correlation, while a correlation between pairs of traits from the flowering region is mostly represented by weak negative correlation. analysis of variance (anova) the significance of mean values differences were tested using analysis of variance (anova) for all selected morphological traits. based on p values (*** p < 0.001), most of the analyzed traits are extremely statistically significant, and as such may be considered as the most significant for general morphological variability of analyzed populations. as the most variable morphological traits, from the vegetative region, stand out: diameter of the stem (ds) with p=0.0000, length of the lower part of the rosettes leaf (llr) with p=0.0001 and thickness of leaves from the middle part of the rosette (tlr) with p=0.0002 (tab. 2). statistically significant traits at extremely high level, from the flowering region, are: diameter of the flower (df, p=0.0005), the angle of branching of the main inflorescence (abi, p=0.0001), as well as all morphological traits of flower parts, all characterized by p=0.0000, except for the length of shorter filaments of the flower (lsf) and length of longer filaments of the flower (llf), which proves to be not significant (tab. 2). among previously mentioned morphological traits, according to the highest obtained values of fisher’s coefficient (f), the fig. 3. box and whisker plots of basic statistic parameters of analyzed morphological traits from flowering region of sempervivum ciliosum subsp. ciliosum: lilength of the main inflorescence; wiwidth of the main inflorescence; nfnumber of flowers in the main inflorescence; abiangle of branching of the main inflorescence; pllength of the pedicel; df-diameter of the flower; nsnumber of sepals; lslength of sepals; wswidth of sepals; npnumber of petals; lplength of petals; wpwidth of petals; snnumber of stamens; lsflength of shorter filaments; llflength of longer filaments; ncnumber of carpels; lclength of carpels; cwwidth of carpels (middle point – mean value; box – mean ± sd; whisker – minimum value maximum value). 119 biologica nyssana ● 10 (2) december 2019: 113-123 jovanović et al. ● morphological variability of endemic sempervivum ciliosum subsp. ciliosum (crassulaceae) from the balkan peninsula following traits can be considered as the most significant: length of carpels (lc, f=46.39), length of petals (lp, f=31.91) number of carpels (nc, f=20.66), length of sepals (ls, f=18.29), and number of petals (np, f=17.63). this morphological traits may be considered as those that best indicate morphological differences between analyzed populations of s. ciliosum. furthermore, traits statistically not significant for delamination of analyzed populations from vegetative region (tab. 2), based on high p values (and extremely low f values), are as follows: width of leaves from the middle part of the rosette (wlr, p=0.8575), width of the middle leaf of the stem (wml, p=0.2181), length of internodes in the lower part of the stem (lil, p=0.2475), length of internodes in the upper part of the stem (liu, p=0.1327), width of the lower leaf of the stem (wll, p=0.1181), thickness of the lower leaf of the stem (tll, p=0.0912), width of the upper leaf of the stem (wul, p=0.0640) and length of the middle leaf of the stem (lml, p=0.0512). above-mentioned set of morphological traits should not be considered as relevant for describing main morphological differences between the analyzed populations of s. ciliosum subsp. ciliosum. discussion morphological variability of any taxa from s. ciliosum complex, including s. ciliosum s.s has not been extensively researched. relatively limited amounts of scientific data, which indicate the degree of variability of morphological traits of previously mentioned taxon, especially those from the flowering region, are available in the current floristic literature (hagemann, 1986; parnell & favarger, 1993; ‘t hart, 2002; ‘t hart et al., 2003). from the other side, understanding of degree and pattern of the variability of morphological traits is the first step leading to an understanding of taxonomical relation between all related yellowflowered taxa from balkan peninsula belonging to s. ciliosum group. obtained results in this study indicate that morphological traits from the vegetative region are characterized by a greater degree of variability than those from the flowering region. a high degree of variability, pronounced within the vegetative morphological traits, especially the leaf traits, may be, to some extent, justified by the possibility of these plants to store water in leaves. but, regardless of the growing t ab le 4 . va lu es o f co rr el at io n co effi ci en t (r ) fo r pa irs o f m or ph ol og ic al t ra its w ith s tro ng p os iti ve c or re la tio n (b ol d va lu es ) w ith in a na ly ze d po pu la tio ns o f s em pe rv iv um c ili os um s ub sp . c ili os um . ll l w ll tl l lm l tm l lu l n s ls w s n p lp s n ls f ll f n c lc ll l 1. 00 0. 81 0. 37 0. 76 0. 28 0. 65 -0 .1 7 -0 .0 1 0. 00 -0 .1 9 0. 39 -0 .1 2 -0 .2 6 -0 .1 5 -0 .3 0 0. 55 w ll 0. 81 1. 00 0. 63 0. 74 0. 56 0. 62 -0 .1 9 0. 14 0. 21 -0 .1 8 0. 27 -0 .1 5 -0 .1 7 -0 .1 6 -0 .2 5 0. 41 tl l 0. 37 0. 63 1. 00 0. 66 0. 91 0. 57 -0 .2 0 0. 15 0. 17 -0 .1 7 -0 .2 1 -0 .1 8 -0 .1 2 -0 .2 1 -0 .1 5 -0 .1 3 lm l 0. 76 0. 74 0. 66 1. 00 0. 67 0. 85 -0 .3 8 -0 .0 5 -0 .0 4 -0 .3 8 0. 07 -0 .3 1 -0 .1 3 -0 .1 3 -0 .4 0 0. 22 tm l 0. 28 0. 56 0. 91 0. 67 1. 00 0. 61 -0 .3 0 0. 01 0. 04 -0 .2 5 -0 .2 8 -0 .2 6 -0 .1 7 -0 .2 6 -0 .1 9 -0 .2 2 lu l 0. 65 0. 62 0. 57 0. 85 0. 61 1. 00 -0 .4 0 -0 .1 7 -0 .1 8 -0 .4 0 0. 04 -0 .3 5 -0 .1 6 -0 .1 5 -0 .4 4 0. 23 n s -0 .1 7 -0 .1 9 -0 .2 0 -0 .3 8 -0 .3 0 -0 .4 0 1. 00 0. 49 0. 39 0. 96 0. 23 0. 92 0. 09 0. 10 0. 94 -0 .1 7 ls -0 .0 1 0. 14 0. 15 -0 .0 5 0. 01 -0 .1 7 0. 49 1. 00 0. 95 0. 48 0. 28 0. 50 0. 12 0. 07 0. 51 0. 01 w s 0. 00 0. 21 0. 17 -0 .0 4 0. 04 -0 .1 8 0. 39 0. 95 1. 00 0. 38 0. 27 0. 43 0. 14 0. 07 0. 42 0. 08 n p -0 .1 9 -0 .1 8 -0 .1 7 -0 .3 8 -0 .2 5 -0 .4 0 0. 96 0. 48 0. 38 1. 00 0. 20 0. 93 0. 08 0. 09 0. 93 -0 .1 9 lp 0. 39 0. 27 -0 .2 1 0. 07 -0 .2 8 0. 04 0. 23 0. 28 0. 27 0. 20 1. 00 0. 22 0. 44 0. 57 0. 08 0. 84 s n -0 .1 2 -0 .1 5 -0 .1 8 -0 .3 1 -0 .2 6 -0 .3 5 0. 92 0. 50 0. 43 0. 93 0. 22 1. 00 0. 08 0. 08 0. 90 -0 .1 8 ls f -0 .2 6 -0 .1 7 -0 .1 2 -0 .1 3 -0 .1 7 -0 .1 6 0. 09 0. 12 0. 14 0. 08 0. 44 0. 08 1. 00 0. 96 0. 07 0. 33 ll f -0 .1 5 -0 .1 6 -0 .2 1 -0 .1 3 -0 .2 6 -0 .1 5 0. 10 0. 07 0. 07 0. 09 0. 57 0. 08 0. 96 1. 00 0. 05 0. 48 n c -0 .3 0 -0 .2 5 -0 .1 5 -0 .4 0 -0 .1 9 -0 .4 4 0. 94 0. 51 0. 42 0. 93 0. 08 0. 90 0. 07 0. 05 1. 00 -0 .3 4 lc 0. 55 0. 41 -0 .1 3 0. 22 -0 .2 2 0. 23 -0 .1 7 0. 01 0. 08 -0 .1 9 0. 84 -0 .1 8 0. 33 0. 48 -0 .3 4 1. 00 120 biologica nyssana ● 10 (2) december 2019: 113-123 jovanović et al. ● morphological variability of endemic sempervivum ciliosum subsp. ciliosum (crassulaceae) from the balkan peninsula procedure that was conducted, morphological traits of the leaves pronounced high degree of variability, which indicates that even under the same condition leaves of populations of s. ciliosum subsp. ciliosum are very variable. furthermore, in this study majority of analyzed traits of flowering region manifest low level of variability with exception of length of the main inflorescence (li), width of the main inflorescence (wi) and number of flowers in the main inflorescence (nf) as well as the angle of branching of the main inflorescence (abi) with higher pronounced degree of variability. low extent of degree of morphological variability within individuals of the analyzed populations is characteristic for flower parts, such as width and length flower parts. it is interesting to emphasize that the width of the sepals (ws) is one of the least variable morphological trait, although very often sepals may exhibit a feature of succulence. one of the reasons for the explained difference in variability between morphological traits of vegetative and the flowering region is probably that morphology of vegetative parts is more ecologically determined, vegetative parts are more prone to store water, which, ultimately, affects on the variability of their morphology. values of the morphological traits of s. ciliosum s.s. represented in the literature (hagemann, 1986; parnell & favarger, 1993; ‘t hart, 2002; ‘t hart et al., 2003) are compared with the corresponding morphological traits of s ciliosum subsp. ciliosum obtained in this study (tab. 5). in comparison with data given in the literature, the obtained values of vegetative traits in this study are significantly higher, whit the exception of values diameter of the rosette (dr) which differs in narrower scale. the differences are most noticeable in the traits height of the plant (ph) and length of rosette leaves (expressed as a sum of the length of the lower part of the rosettes leaf (llr) and length of the upper part of the rosettes leaf (lur)). those variations may be partially explained due to stable growth conditions in which analyzed individuals developed during the period of growing, i.e. amount of water that the plants received during this period were the same. but it is important to note that this is certainly not the only, and most important, cause of such differences. regarding the morphological traits from the flowering region, more traits are available for comparison. table 5. a comparative review of values of morphological traits of s. ciliosum subsp. ciliosum from this study and corresponding traits of s. ciliosum s.s. represented in literature (hagemann, 1986; parnell & favarger, 1993; 't hart, 2002, 't hart et al., 2003). character name and abbreviation this study hagemann, 1986 parnell & favarger, 1993 't hart, 2002 't hart et al., 2003 height of the plant (ph) 23.97-36.30 cm 12 cm 4-10 cm – 13 cm diameter of the rosette (dr) 2.15-4.68 cm 2-3.5 cm 2-3(-5) cm 2-5 cm 2-5 cm length of rosette leaves 11.46-46.48 mm* 15-20 mm 10 mm 7-10(-25) mm 7-10(-25) mm width of leaves from the middle part of the rosette (wrl) 4.68-9.17 mm 3-4(-6) mm 4 mm 3-4(-6) mm 3-4(-6) mm diameter of the flower (df) 16.65-27.12 mm 16-25 mm – – 16-25 mm length of the sepals (ls) 3.45-7.37 mm 3-4 mm – 5-7 mm 5-7 mm length of petals (lp) 8.27-12.11 mm 8-10 mm 10-12(-15) mm 8-12 mm – width of the petals (wp) 1.49-3.10 mm 1.5-2 mm – 1.5-2 mm – length of shorter filaments (lsf) length of longer filaments (llf) 4.63-6.14 mm 5.13-6.93 mm – – ⁓ 6 mm – length of carpels (lc) 4.12-6.93 mm 6-7 mm – 6-7 mm – *expressed as a sum of the length of the lower part of the rosettes leaf (llr) and length of the upper part of the rosettes leaf (lur). 121 biologica nyssana ● 10 (2) december 2019: 113-123 jovanović et al. ● morphological variability of endemic sempervivum ciliosum subsp. ciliosum (crassulaceae) from the balkan peninsula generally, measured values of traits of flower parts are slightly higher, except for the length of carpels (lc) which proves to have lower values, than those presented in the literature (tab. 5). values of the diameter of the flower (df), given in this study, do not deviate significantly, in a morphological sense, from those given in scientific sources. measured values of length of petals (lp), as well as width of the petals (wp), are within the range, or could vary in narrower scale regarding the maximum value, of values given in scientific sources (hagemann, 1986; parnell & favarger, 1993; ‘t hart, 2002). values of the length of filaments are difficult to compare to those stated in literature reports, because in this study variability of filaments was observed through two traits: length of shorter filaments (lsf) and length of longer filaments (llf), while in literature this segregation is nonexistent i.e. all filaments were considered as equals. regardless of this, maximal values of length of the shorter filaments (lsf) and length of the longer filaments (llf) are slightly higher than those suggested by ‘t hart (2002). it is interesting to note that, regarding morphological variability between analyzed populations, values of morphological traits from the flowering region are generally lower in individuals from mt. galičica while values of traits from the vegetative region are higher regarding values of traits of individuals from mt. rila and mt. orvilos. this morphological variation may be due to the fact that the population from mt. galičica belongs to s. ciliosum var. galicicum. as already explained micevski (1998) considers previously mentioned taxon as s. galicicum but to confirm this statement more detailed studies are needed. the number of morphological traits analyzed in this study is much greater than those presented in the referent scientific sources. as explained in the previous chapter, in the current literature, for the purposes of distinguishing s. ciliosum from related species, a greater number of characters of the flowering region are in use. those traits are recognized as more stable i.e. traits in which morphological variability is less expressed, which was also confirmed by this study. obtained differences in values of analyzed morphological traits in this study, from those mentioned in the available literature, may arise from the following reasons: the impact of growing conditions, especially on traits from vegetative region, enhanced phenotypic plasticity, insufficiency of more data of variability as well as that many of data present in literature are outdated and unresolved taxonomical position of taxa within s. ciliosum complex. therefore, further research is necessary. correlative analysis proved similarity in the variation in specified pairs of morphological traits. among analyzed traits, all degrees, weak, moderate and strong, as well as forms, negative and positive, of correlation are expressed. a weak positive correlation is represented by the largest number of pairs, especially between pairs of traits from flowering region, followed by traits from vegetative part. this degree of linear correlation indicates that changes in morphology, i.e. increasing or decreasing, in a dimension of one morphological trait does not greatly affect the dimensions of correlated morphological trait, therefore those traits can be potentially used in examination purposes of morphological differentiation among analyzed populations. strongest linear connection, according to results of this study is established in pairs of traits from the flowering region, such as numbers, lengths and widths of particular parts of a flower. this connection in morphological variation between traits of floral parts, may characterize them as ineligible for purposes of examining the extent of morphological variability between populations of s. ciliosum. but bearing in mind that the numbers of flower segments, sepals, petals, stamens, and carpels, usually changes simultaneously, values of those traits should not be considered as less important in determining degree of variability between analyzed populations, regardless to their high correlation. from vegetative region, high correlations are established between morphological traits of stem leaves, which, to some degree, can be expected. as the length of leaves increases, width and thickness of the leaves also increase, since leaves accumulate larger quantities of water. to the best of our knowledge, analysis of variance (anova), same as correlative analysis, was not conducted within researches of morphological variability of s. ciliosum. according to anova results, the majority of morphological traits from the flowering region are highly statistically significant. the most important, for morphological differentiation, are traits: length of carpels (lc), length of petals (lp), number of carpels (nc), length of sepals (ls) and number of petals (np). it is interesting to point out that parnell & favarger (1990) stated that within population of s. ciliosum s.s., great variation, especially in filaments size occurs. as demonstrated by this study as well, length of filaments, based on the differences in their length among individuals, is characterized as statistically not significant for delamination of analyzed populations. from a vegetative region, traits which highly reflect morphological differences among analyzed population are diameter of the stem (ds), length of the lower part of rosette leaf (llr) and thickness of the leaves from the middle part of rosette (tlr). previous claims again point to the significance of morphological traits from the flowering region, as more reliable to use in purposes of establishing the morphological differences be122 biologica nyssana ● 10 (2) december 2019: 113-123 jovanović et al. ● morphological variability of endemic sempervivum ciliosum subsp. ciliosum (crassulaceae) from the balkan peninsula tween populations of s. ciliosum subsp. ciliosum. previously explained results of this study indicate the existence of difference in morphology within populations of typical subspecies of s. ciliosum from the balkan peninsula, which ultimately can be used to explain pronounced morphological variability of this and related species within s. ciliosum complex. in order to confirm this, the additional analysis (e.g. more detailed morphological, anatomical and other studies) should be conducted in the future. acknowledgements: this study was funded by the ministry of education, science and technological development of the republic of serbia, grant no. 173030. references berger, a. 1930: crassulaceae. in: engler, a., prantl, k. (eds.), die natürlichen pflanzenfamilien. 2nd edition, 18 a: 352–483. leipzig. hagemann, i. 1986: sempervivum l. in: strid, a. (ed.), mountain flora of greece, 1: 338. cambridge university press, cambridge. karaer, f., celep, f., eggli, u. 2011: a taxonomic revision of the sempervivum davisii complex (crassulaceae). nordic journal of botany, 29: 49– 53. klein, j., kadereit, j. 2015: phylogeny, biogeography, and evolution of edaphic association in the european orophytes sempervivum and jovibarba (crassulaceae). international journal of plant sciences, 176 (1): 44–71. klein, j., kadereit, j. 2016: allopatric hybrids as evidence for past range dynamics in sempervivum (crassulaceae), a western eurasian high mountain oreophyte. alpine botany. swiss botanical society. micevski, k. 1998: flora na republika makedonija, tom i, sv. 4. makedonska akademija na naukite i umetnostite. skopje. 1043 p. mort, m., o’leary, t., carrilo-reyes, p., nowell, t., archibald, j., randle, c. 2010: phylogeny and evolution of crassulaceae: past, present, and future. schumannia, 6: 69–86. parnell, j., favarger, c. 1990: notes on sempervivum l. and jovibarba opiz. in: chater, a. o. (ed.), flora europaea notulae systematicae, ser. 2, no. 3. botanical journal of the linnean society, 103: 216–220. parnell, j., favarger, c. 1993: sempervivum l., jovibarba opiz. in: tutin, t. g., burges, n. a., chater, a. o., edmondson, j. r., heywood, v. h., moore, d. m., valentine, d. h., walters, s.m., webb, d.a. (eds.), flora europaea-psilotaceae to platanaceae, 1: 426. cambridge university press, cambridge. ‘t hart, h. 2002: sempervivum l. in: strid, a., tan, k. (eds.), flora hellenica, 2: 309. ruggell (d): a.r.g. gantner verlag k.g. ‘t hart, h., bleij, b., zonneveld, b. 2003: sempervivum. in: eggli, u. (ed.), illustrated handbook of succulent plants, vol 5: 338. springer, berlin. thiede, j., eggli, u. 2007: crassulaceae. in: kubitzki, k. (ed.), families and genera of vascular plants. vol. 9. flowering plants eudicots, 83-118. hamburg: springer. 123 biologica nyssana ● 10 (2) december 2019: 113-123 jovanović et al. ● morphological variability of endemic sempervivum ciliosum subsp. ciliosum (crassulaceae) from the balkan peninsula matejić et al. 2021, biologica nyssana 12(2) 12 (2) december 2021: 87-111 doi: 10.5281/zenodo.5759846 frequency of herbal medicinal products use in southeastern serbia original article matejić jelena university of niš, faculty of medicine, department of pharmacy, bulevar dr zorana đinđića 81, 18000 niš, serbia jekamatejic@gmail.com (corresponding author) pavlović dragana university of niš, faculty of medicine, department of pharmacy, bulevar dr zorana đinđića 81, 18000 niš, serbia stefanović nikola university of niš, faculty of medicine, department of pharmacy, bulevar dr zorana đinđića 81, 18000 niš, serbia stojanović milica university of niš, faculty of medicine, department of pharmacy, bulevar dr zorana đinđića 81, 18000 niš, serbia žarković lazar university of belgrade faculty of biology, institute of botany and botanical garden “jevremovac”, studentski trg 16, 11000 belgrade, serbia marin petar university of belgrade faculty of biology, institute of botany and botanical garden “jevremovac”, studentski trg 16, 11000 belgrade, serbia džamić ana university of belgrade faculty of biology, institute of botany and botanical garden “jevremovac”, studentski trg 16, 11000 belgrade, serbia received: july 18, 2021 revised: october 03, 2021 accepted: november 01, 2021 abstract: the trend of herbal medicine use continues even today, despite the growing number of different synthetic, conventional drugs and the huge progress of the pharmaceutical industry. in recent decades, there has been an increasing use of different herbal medicinal products (hmp) in the prevention of diseases, treatment of chronic and recurrent conditions and diseases, and for maintaining good health. the aim of our paper was to determine the use of hmp in the area of southeastern serbia. the survey method was applied through a pre-compiled questionnaire in the zaječar, sokobanja and aleksinac cities. the results of the research showed that there were 70 different hmp, which were most often used preventively (32.00%), while the rest were used in hypertension (23.50%), prostate hyperplasia (8.50%), diabetes (6.53%), depression (4.60%) and glaucoma (3.90%). the composition of the used products included 84 different herbal species, the most common of which were citrus x aurantium l. (5.90%) and cucurbita moschata duchesne (3.60%). when it comes to prevention and/or treatment of diseases, statistical analysis showed that the respondents living in the aleksinac and surrounding used more plant species compared to people from sokobanja and zaječar, while there was no difference between the respondents in the number of hmp used. respondents who did not have prescribed synthetic drugs, used hmp for the purpose of prevention in a higher percentage. it can be concluded that the respondents mostly use hmp as preventive means, but they also use them as an accompanying therapy. key words: herbal medicinal products, treatment, frequency, prevention, southeastern serbia apstrakt: učestalost korišćenja lekovitih biljnih produkata u jugoistočnoj srbiji trend korišćenja biljnih lekova nastavlja se i danas, bez obzira na postojanje sve većeg broja različitih sintetičkih, konvencionalnih lekova i ogroman napredak farmaceutske industrije. poslednjih decenija je konstatovana sve veća primena biljnih lekova u prevenciji bolesti, lečenju hroničnih i rekurentnih stanja i oboljenja, kao i za održavanje zdravlja. cilj našeg rada bio je da se utvrdi upotreba lekovitih biljnih produkata (lbp) na teritoriji jugoistočne srbije. primenjivana je metoda anketiranja putem unapred sastavljenog upitnika u sledećim gradovima: zaječar, sokobanja i aleksinac. rezultati istraživanja pokazali su da je u upotrebi bilo 70 različitih lbp, koji su najčešće korišćeni preventivno (32,00%), dok su ostali upotrebljavani kod hipertenzije (23,50%), hiperplazije prostate (8,50%), šećerne bolesti (6,53%), depresije (4,60%) i glaukoma (3,90%). sastav korišćenih produkata sadržao je 84 različitih biljnih vrsta, a najčeće su bile zastupljene citrus x aurantium l. (5,90%) i cucurbita moschata duchesne (3,60%). statistička analiza je pokazala da su ispitanici koji žive na teritoriji aleksinca koristili više biljnih vrsta kada je reč o prevenciji i/ili lečenju oboljenja u poređenju sa stanovnicima sokobanje i zaječara, dok nije bilo razlike među ispitanicima kada je reč o broju samih lbp. ispitanici koji nisu imali propisane sintetičke lekove u terapiji u većem procentu su koristili lbp u svrhu prevencije. može se zaključiti da ispitanici u većoj meri koriste lbp kao preventivna sredstva, ali ih u velikom broju koriste i kao prateću terapiju. ključne reči: lekoviti biljni produkti, lečenje, učestalost, prevencija, jugoistočna srbija © 2021 matejić et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 87 introduction herbs have been used as the first drugs in the treatment of diseases, and in recent decades their use has been increasingly important in medicine (barnes, 2012; heinrich et al., 2018; zhang, 2018). herbal therapy is used by 75% of the world’s population. this is due to better acceptability of herbal medicines, fewer side effects that can occur during therapy as well as the fact that they are cheaper (sam, 2019). in addition, patients are more likely to opt for different herbal products as they believe that they are completely safe because they are of natural origin, and consequentlly that their concomitant application with conventional medicines is not dangerous at all. for these reasons, patients usually do not report the use of herbal medicine to their doctors and pharmacists (dimitrijević et al., 2014). yet, in some cases, herbal medicinal products could have a harmful effect on health if they are not used in the prescribed manner and under the supervision of a professional or could lead to harmful interactions with conventional drugs (petrović et al., 2012). herbal medicines, traditional herbal medicines and herbal teas are used for maintaining good health, prevention of diseases, treatment of chronic and recurrent conditions, diseases treatment on the recommendation of a doctor or pharmacist, and more often on the basis of the patient’s decision (petrović et al., 2012; luketina-šunjka et al., 2020). the use of medicinal plants in serbia has a long tradition, and our previous work (matejić et al. 2020) showed the exceptional presence of medicinal plant species in eastern and southeastern serbia that are used for a large number of different indications. the aim of this study was to investigate the frequency of herbal medicinal products (hmp): herbal medicines, traditional herbal medicines, herbal supplements, and different other herbal products that could be found in public pharmacy in southeastern serbia and to determine how much and in what way herbs are used in the treatment of patients in southeastern serbia. in this regard, it was necessary to answer the following questions: (1) what are the most commonly used hmp, (2) for what therapeutic purposes are hmp used, (3) which conventional drug therapy is used in combination with hmp, (4) how does age affect the frequency of hmp use, (5) are there differences in the frequency of herbal use among men and women? materials and methods research area the research was conducted in southeastern serbia, more precisely in three municipalities: zaječar, sokobanja and aleksinac. the municipalities of zaječar and sokobanja belong to the zaječar district, while the municipality of aleksinac belongs to the nišava district. the municipality of zaječar covers an area of 1069 km2 and has 53,509 inhabitants in mid-2019 (statistical office of the republic of serbia, 2020). according to the latest research of the public health institute “timok” zaječar (2020), the total number of residents of this municipality who were ill in 2019 was 39,240. residents mostly suffered from respiratory diseases, i.e., 25.1% of the total number of patients. then they suffered from cardiovascular diseases, about 16.0% of them. musculoskeletal system and connective tissue diseases are in the third place with a share in the total morbidity of 11.0%. they were followed by urogenital system disorders (8.2%) and diseases from the group of symptoms, which includes pathological clinical and laboratory findings with 5.9% of the total morbidity. digestive system diseases are in the sixth place, accounting for 5.0% of the total number of patients. after that, there are diseases related to the endocrine system, nutrition and metabolism, with a share in the total morbidity of 4.9%. in the seventh place are injuries, poisonings and the consequences of external factors (4.7%). skin and subcutaneous tissue diseases account for 3.8%, while mental and behavioral disorders include 3.7% of the total number of patients. there are also diseases from other groups that do not individually contribute much to the number of patients and together make up the remaining 11.7%. in 2019, the most common diagnosis was high blood pressure making 12.5% of the total number of diseases. other common diagnoses were: acute pharyngitis and tonsillitis (11.0%); acute bronchitis and bronchiolitis (6.3%); other back diseases (5.7%); other symptoms, signs and pathological clinical and laboratory findings (4.0%); diabetes (3.8%); cystitis (3.2%); rhinitis, sinusitis and laryngitis (2.9%); degenerative joint disease and skin and subcutaneous tissue diseases (2.6%). the remaining 45% are other diseases. in 2019, the department for women’s health service of the municipality of zaječar reported 1,063 patients with various diseases. most of them suffered from diseases of the urogenital system, as many as 75%. they are followed by diseases from the group of tumors with 13.9%, and then infectious and parasitic diseases with 10.9% of the total number of patients. the diagnoses made in the patients were: menstrual disorders (16.5%), cervicitis (16.3%), menopausal diseases (15.2%), salpingitis and oophoritis (9.0%) and female infertility (8.6%). one third of the total number are other diagnoses that were encountered in 2019 in women. 88 biologica nyssana ● 12 (2) december 2021: 87-111 matejić et al. ● frequency of herbal medicinal products use in southeastern serbia 89 the municipality of sokobanja covers an area of 525 km2 with a total population of 14,755 in mid2016 (statistical office of the republic of serbia, 2017). based on the analysis of the public health institute niš (2017), a total of 23,700 patients were registered in the municipality of sokobanja in 2016. most of them suffered from cardiovascular diseases, namely 31.6% of the total morbidity. respiratory diseases accounted about 24.7%. in the third place are genitourinary diseases with 7.5%, and in the fourth place are musculoskeletal and connective tissue diseases with 7.1% of the total number of patients. these are followed by mental and behavioral disorders with a share in the total morbidity of 4.7%. digestive diseases are in the sixth place, accounting for 3.3%, followed by diseases from the group of symptoms, which includes pathological clinical and laboratory findings with 2.9%. they are followed by diseases from the group of factors that affect the health condition and contact with the health service, about 0.9%. other groups of diseases together make up 17.3% of the total number of patients. the most common diagnosis in 2016 was high blood pressure with 22.0% of the total number of diagnoses. it is followed by: acute pharyngitis and tonsillitis (10.0%), other back diseases (5.0%), tracheitis, emphysema and other obstructive lung diseases (4.5%) and cystitis (3.6%). there are various other diagnoses that together make up the remaining 54.5% of the total number of diseases. during 2016, the department for women’s health service of the municipality of sokobanja registered 934 patients. in as many as 80.5% of cases, the patients suffered from genitourinary diseases. in the second place are diseases from the group of tumors with 7.2%, and in the third are diseases from the group of factors that affect health and contact with the health service with 5.2% of the total number of patients. they are followed by skin and subcutaneous tissue diseases, as well as diseases from the group of symptoms, which includes pathological clinical and laboratory findings, which account for 5.1% of the total morbidity. after that, there are infectious and parasitic diseases, with about 0.6%. conditions belonging to the group of pregnancies, births and postpartum period make up 0.5% of the total number of patients. the five most common diagnoses in women were: pelvic inflammatory diseases (19.9%), menstrual disorders (17.8%), breast disease (13.4%), menopausal symptoms and cystitis (9.9%). these five diagnoses make up as much as 71.6% of all registered diseases. according to data from 2015, the municipality of aleksinac has 49,290 inhabitants on an area of 707 km2 (statistical office of the republic of serbia, 2016). based on the latest research by the public health institute niš (2017), there were a total of 57,242 patients in 2015. cardiovascular diseases took the first place, accounting for 24.3% of the total morbidity. next were respiratory diseases, with about 19.0%. in the third place are musculoskeletal and connective tissue diseases with 8.3%, and in the fourth place are genitourinary diseases with 7.0% of the total number of patients. endocrine diseases make up 5.9%, while mental and behavioral disorders make up 4.4%. there are also diseases from other groups that together contribute to the total morbidity with 31.0%. the most common diagnosis was high blood pressure with 19.9% of the total number of all diseases. other diseases are: acute pharyngitis and tonsillitis (6.6%), back diseases (5.7%), diabetes (4.4%), cystitis (3.7%). other types of diagnoses make up the remaining 59.8%. in the department for women’s health service of the municipality of aleksinac, there were 3,464 registered patients during 2015. women mostly suffered from genitourinary diseases, as many as 78.7%. in the second place are the factors that affect the health condition and contact with the health service with a share in the total morbidity of 9.5%. they are followed by diseases from the group of tumors with 7.8%, then conditions belonging to the group of pregnancies, births and postpartum period with 2.2% and diseases from the group of symptoms, which includes pathological clinical and laboratory findings with 1.0%. blood and blood cell diseases as well as endocrine diseases make up 0.3% of the total number of patients. other registered diseases account for 0.4%. frequent diagnoses in patients were: other pelvic inflammatory diseases (25.5%), menstrual disorders (16.2%), other genitourinary diseases, cystitis and menopause (8.4%) and menopausal diseases (8.2%). the remaining third of total diseases are other types of diagnoses. ethnopharmacological survey the survey has been conducted in a period of three months, more precisely from november 15, 2020 to january 15, 2021. we used a pre-compiled questionnaire, with the help of adonis pharmacy (https://www.adonisapoteka.rs). the questionnaires were completed thanks to employed masters of pharmacy, with the consent of the respondents, in accordance with all codes of ethics, in pharmacies located in the territory of southeastern serbia, in the three surveyed municipalities zaječar, sokobanja and aleksinac. a total of 100 surveys were collected: 13 in zaječar, 66 in sokobanja, and 21 in aleksinac. a total of 46 men and 54 women aged 1 to 88 were surveyed. the questionnaire consisted 6 questions biologica nyssana ● 12 (2) december 2021: 87-111 matejić et al. ● frequency of herbal medicinal products use in southeastern serbia 90 (one close and five open), and gives the following data on the respondents: • gender • age • chronic diseases yes or no • chronic diseases they suffer from, if yes • treatment of chronic disease • name of the hmp they use the obtained data were then analyzed and compared with the literature. chronic disease treatment was classified based on groups of drugs as prescribed by the agency for drugs and medical devices of serbia (2021). the hmp was studied in detail, starting from its pharmaceutical form, through the plants that are part of it and contribute to its action. for each plant species, we specified its folk and latin name according to the plant list (2000) (http://www.theplantlist.org), the medicinal part used and the form in which it is contained in the medicine. then, each hmp was associated with an appropriate chronic disease, the therapy of which it could contribute to. in the end, we did a systematization according to plant species (tab.1). statistical analysis the characteristics of the study group were defined as frequency (with or without percentage). kruskal-wallis test and mann-whitney u test (when data normality was not satisfied) were used to compare continuous data regarding the use of plants preparations and plants species between defined groups of informants. the chi-square (χ2) test was used to compare the distribution of plants preparations and plants species use between defined groups of informants. statistical analyses were performed using spss statistical software (version 20.0) at the significance level set at p<0.05. results and discussion quantitative analyses the results of the research showed that there were 70 different herbal medicines in use. the highest percentage was used preventively (32.00%), while the others were used in hypertension (23.50%), prostate hyperplasia (8.50%), diabetes (6.53%), depression (4.60%), glaucoma (3.90%) and angina pectoris and neurasthenia (2.60%). in their regular therapy, patients used drugs from the following groups: beta blockers (12.30%); ace inhibitors, calcium antagonists and prostate hyperplasia therapy (6.60%); oral antidiabetics (6.20%); antihypertensive (5.30%); benzodiazepines (4.90%) and glaucoma therapy (4.10%). the most commonly used drugs were: betamsal (0.4 mg) and concor cor (1.25 mg) (2.90%); cornelin (10 mg) and tamsol (0.4 mg) (2.50%); bromazepam (3 mg), diazepam (5 mg), tensec (5 mg) and xalatan sol (2.10%); cosopt sol, glucophage (1000 mg) and spironolakton (25 mg) (1.60%) and amaryl (2 mg), binevol (5 mg), cornelin (20 mg), diclofenac duo (75 mg), enalapril (20 mg), prilinda (5 mg) and tritace comp (5 mg + 25 mg) (1.20%). the largest number of hmp was in the form of: capsules (25.50%); tablets (23.60%); herbal tea (17.30%); solution (6.40%) and syrup (5.50%). the most commonly used were: urasan forte (7.30%); comprosta forte (6.40%); phlebodia 600 (4.50%); senna tea (3,60%); bilobil intense, peppermint tea, palisept and venodia plus (2.70%), alpenkräuter cream, gingival gel, hipervag intim, lagosa 150, prospan syrup, prospan effervescent tablets, prostamol uno, prostenal perfect, relax, rtanj tea (winter savory) and venalek (1.8%). herbs whose medicinal ingredients are part of herbal medicines include 84 different species. all species originate from 45 different families, of which the largest number of species originate from the following families: rutaceae (6.30%); rosaceae (3.60%); myrtaceae and urticaceae (2.70%). the most common were the following species: citrus x aurantium l. (5.90%); cucurbita moschata duchesne (3.60%); brassica napus l., foeniculum vulgare mill. and valeriana officinalis l. (3.20%); ginkgo biloba l., melissa officinalis l., senna alexandrina mill. and urtica dioica l. (2.70%); hedera helix l., hypericum perforatum l., mentha x piperita l., rosmarinus officinalis l. and origanum vulgare l. (2.30%); betula pubescens ehrh., calendula officinalis l., eucalyptus globulus labill., humulus lupulus l., matricaria chamomilla l., rosa canina l., vaccinium myrtillus l. and zea mays l. (1.80%); achillea millefolium l., aesculus hippocastanum l., equisetum arvense l., glycyrrhiza glabra l., mentha arvensis l., oenothera biennis l., petroselinum crispum (mill.) fuss, silybum marianum (l.) gaertn. and vitis vinifera l. (1.40%). the parts of the plant that were used as the source of ingredients in the largest percentage were as follows: folium (36.20%); flos (12.70%); herba and radix (10.40%); fructus (10.00%); semen (7.70%) and pericarpium (6.30%). herbal ingredients were most often in the form of an extract (49.30%); then in the form of crushed herbal drugs (17.60%); essential oil (15.80%); isolated substances (7.20%) and in the form of powdered herbal drugs (5.40%). statistical analysis a detailed statistical data processing was used to determine the frequency of use of hmp and plants in southeastern serbia. biologica nyssana ● 12 (2) december 2021: 87-111 matejić et al. ● frequency of herbal medicinal products use in southeastern serbia 91 biologica nyssana ● 12 (2) december 2021: 87-111 matejić et al. ● frequency of herbal medicinal products use in southeastern serbia ta bl e 1. f re qu en cy o f h m p u se in s ou th ea st er n s er bi a l at in n am e, s er bi an na m e an d fa m ily p ar t o f a pl an t f or m p ha rm . f or m n am e of h m p a ll m ed ic in es us ed w it h h m p t he ra py gr ou ps p re se nt d is ea se s w he n h m p w as u se d a bi es s ib ir ic a l ed eb ., si bi rs ka je la , p in ac ea e fo liu m es se nt ia l o il cr ea m a lp sk a kr em a / / / a bi es a lb a m ill ., je la , pi na ce ae fo liu m es se nt ia l o il oi nt m en t pa vl e ba lz am / / / a ch ill ea m ill ef ol iu m l ., ha jd uč ka tr av a, a st er ac ea e he rb a ex tr ac t oi nt m en t, liq ui d a sa m c rv en i ko nj sk i ba lz am , fe m is an a ka pi , f em is an b k ap i b ro m az ep am (3 m g) , si da ta (5 0 m g) , v iv ac e (5 m g) , v iv ac e pl us a c e in hi bi to rs , an tid ep re ss an t dr ug , an tih yp er te ns iv e dr ug , be nz od ia ze pi ne de pr es si on , h yp er te ns io n a es cu lu s hi pp oc as ta nu m l ., di vl ji ke st en , sa pi nd ac ea e se m en ex tr ac t, es se nt ia l o il ge ll, o in tm en t, ta bl et a sa m c rv en i ko nj sk i ba lz am , e rb av en re ta rd , k es te n ge l b ro m az ep am (3 m g) , si da ta (5 0 m g) , v iv ac e (5 m g) , v iv ac e pl us a c e in hi bi to rs , an tid ep re ss an t dr ug , an tih yp er te ns iv e dr ug , be nz od ia ze pi ne de pr es si on , h yp er te ns io n a lc he m ill a xa nt ho ch lo ra r ot hm ., vi ra k, r os ac ea e he rb a ex tr ac t liq ui d fe m is an a ka pi / / / a lo e fe ro x m ill ., al oj a, x an th or rh oe ac ea e su cc us oi l, po w de re d he rb al su bs ta nc e cr ea m , t ab le t t en h er bs , v ita lis s po rt s cr ea m / / ch ro ni c ob st ip at io n a lth ae a offi ci na lis l ., be li sl ez , m al va ce ae fo liu m , r ad ix ex tr ac t, fr ag m en te d he rb al su bs ta nc e he rb al te a, pa st ill es , s yr up b eb a c s ir up be lo g sl ez a, č aj k or en a be lo g sl ez a, č aj p ro tiv br on hi tis a, h er bi ko s ir up be lo g sl ez a, pr ot ec t b el i sl ez b ip re z (2 .5 m g) , c or ne lin (1 0 m g) , e na la pr il (1 0 m g) , k lo m et ol (1 0 m g) , m ac ro pe n (4 00 m g) , m ol ic or (2 m g) a c e in hi bi to rs , ca lc iu m an ta go ni st s, an tia ng in al d ru g, an tie m et ic d ru g, be ta b lo ck er , m ac ro lid e an tib io tic an gi na p ec to ri s, hy pe rt en si on biologica nyssana ● 12 (2) december 2021: 87-111 92 matejić et al. ● frequency of herbal medicinal products use in southeastern serbia a nn on a m ur ic at a l ., gr av io la , a nn on ac ea e fo liu m po w de re d he rb al su bs ta nc e ca ps ul e a m az on sk a gr av io la b is op ro lo l (5 m g) , i ru zi d (2 0 m g+ 1 2. 5 m g) an tih yp er te ns iv e dr ug , b et a bl oc ke r hy pe rt en si on a rc to st ap hy lo s uv aur si (l .) sp re ng ., uv a, e ri ca ce ae fo liu m fr ag m en te d he rb al su bs ta nc e he rb al te a č aj li st a uv e, k ir ko lin a l ux č aj z a m rš av lje nj e b ip re z (2 .5 m g) , c or ne lin (1 0 m g) , m ol ic or (2 m g) ca lc iu m an ta go ni st s, an tia ng in al dr ug , b et a bl oc ke r an gi na p ec to ri s, hy pe rt en si on a rt em is ia a bs in th iu m l ., pe le n, a st er ac ea e he rb a tin ct ur e sy ru p a m ar ap ha rm si ru p / / / b et ul a pu be sc en s e hr h. , b re za , b et ul ac ea e fo liu m ex tr ac t, tin ct ur e, fr ag m en te d he rb al su bs ta nc e he rb al te a, po w de r, liq ui d k ap i z a m rš av lje nj e, u ro bi tr at in st an t č aj , u ro pr ot ek t č aj in ko nt an (1 5 m g) , m ar oc en (5 00 m g) , pa nr az ol (2 0 m g) pr ot on p um p in hi bi to r, qu in ol on e an tib io tic , in co nt in en ce tr ea tm en t in co nt in en ce b ra ss ic a na pu s l ., ul ja na re pi ca , b ra ss ic ac ea e flo s ex tr ac t ta bl et c on pr os ta fo rt e a m ar yl (2 m g) , a m ar yl (4 m g) , a m lo di pi n (5 m g) , b en ep ro st (5 m g) , b et am sa l (0 .4 m g) , b in ev ol (5 m g) , b ro m az ep am (3 m g) , c or ne lin (1 0 m g) , c or ne lin (2 0 m g) , c os op t so l, d at us t (0 .5 m g) , d ila co r (0 .2 5 m g) , g lu co ph ag e (1 00 0 m g) , a c e in hi bi to rs , ca lc iu m an ta go ni st s, an tih yp er te ns iv e dr ug , be nz od ia ze pi ne , be ta b lo ck er , in su lin , ca rd io to ni c ag en t, or al an tid ia be tic dr ug , g la uc om a tr ea tm en t, be ni gn p ro st at ic hy pe rp la si a tr ea tm en t, vi ta m in at ri al fi br ill at io n an d at ri al fl ut te r, de pr es si on , gl au co m a, b en ig n pr os ta tic h yp er pl as ia , hy pe rt en si on , d ia be te s ty pe 2 , d ia be te s ty pe 1 l at in n am e, s er bi an na m e an d fa m ily p ar t o f a pl an t f or m p ha rm . f or m n am e of h m p a ll m ed ic in es us ed w it h h m p t he ra py gr ou ps p re se nt d is ea se s w he n h m p w as u se d biologica nyssana ● 12 (2) december 2021: 87-111 matejić et al. ● frequency of herbal medicinal products use in southeastern serbia 93 l at in n am e, s er bi an na m e an d fa m ily p ar t o f a pl an t f or m p ha rm . f or m n am e of h m p a ll m ed ic in es us ed w it h h m p t he ra py gr ou ps p re se nt d is ea se s w he n h m p w as u se d in su m an b as al so lo st ar (1 00 ij/ m l) , m on op ri l pl us (2 0 m g + 12 .5 m g) , p ri lin da (5 m g) , p ri no rm (1 00 m g) , t am so l (0 .4 m g) , t el m ic or (4 0 m g) , t en se c (5 m g) , t ri ta ce co m p (5 m g + 25 m g) , v ig an to l s ol , x al at an s ol c al en du la o ffi ci na lis l ., ne ve n, a st er ac ea e flo s ex tr ac t, es se nt ia l o il liq ui d, v ag in al ta bl et fe m is an a ka pi , f em is an b k ap i, h ip er va g in tim / / / c am el lia s in en si s (l .) k un tz e, z el en i č aj , t he ac ea e fo liu m fr ag m en te d he rb al su bs ta nc e he rb al te a k ir ko lin a l ux č aj z a m rš av lje nj e / / / c ar um c ar vi l ., ki m , a pi ac ea e fr uc tu s ex tr ac t po w de r h ip p ča j z a do jil je / / / c en ta ur iu m e ry th ra ea r af n, k ič ic a, g en tia na ce ae he rb a tin ct ur e sy ru p a m ar ap ha rm si ru p / / / c en te lla a si at ic a (l .) u rb ., ce nt el a, a pi ac ea e he rb a ex tr ac t ta bl et e rb av en re ta rd / / / c he lid on iu m m aj us l ., liš ei vi ca , p ap av er ac ea e he rb a fr ag m en te d he rb al su bs ta nc e he rb al te a č aj h er be liš ei vi ce / / / c itr us li m on (l .) o sb ec k, li m un , pe ri ca rp iu m es se nt ia l o il ca ps ul e g el om yt ro l fo rt e b ru fe n (4 00 m g) no ns te ro id al si nu si tis biologica nyssana ● 12 (2) december 2021: 87-111 94 matejić et al. ● frequency of herbal medicinal products use in southeastern serbia l at in n am e, s er bi an na m e an d fa m ily p ar t o f a pl an t f or m p ha rm . f or m n am e of h m p a ll m ed ic in es us ed w it h h m p t he ra py gr ou ps p re se nt d is ea se s w he n h m p w as u se d r ut ac ea e an tiin fla m m at ory d ru g c itr us x a ur an tiu m l ., go rk a na ra nd ža , r ut ac ea e pe ri ca rp iu m is ol at ed a ct iv e co m po ne nt s, es se nt ia l o il ca ps ul e, ta bl et d et ra le x, fl ux iv , g el om yt ro l fo rt e, ph le bo di a (6 00 m g) , v en al ek , v en od ia p lu s a m lo di pi n (1 0 m g) , a zo pt so l, b et am sa l (0 .4 m g) , b en ep ro st (5 m g) , b is op ro lo l (2 .5 m g) , b ro m az ep am (3 m g) , b ru fe n (4 00 m g) , c ar di op ir in (1 00 m g) , c on co r co r ( 1. 25 m g) , c or as p (1 00 m g) , c or ne lin (2 0 m g) , d ia ze pa m (5 m g) , e na la pr il (1 0 m g) , e na la pr il (2 0 m g) , f ar in (5 m g) , f le ka ni d (1 00 m g) , h em op re s (5 0 m g + 5 m g) , h yp ol ip (2 0 m g) , l as ix (4 0 m g) , m on iz ol (2 0 m g) , n ev ot en s (5 m g) , pr es ol ol (5 0 m g) , sp ir on ol ak to n (2 5 m g) , t en se c (5 m g) , t ri m et ac or (3 5 m g) , a c e in hi bi to rs , ca lc iu m an ta go ni st s, an tia ng in al d ru g, an tia rr hy th m ic dr ug , be nz od ia ze pi ne , be ta b lo ck er , di ur et ic , hy po lip id em ic dr ug , a nt ip la te le t ag en t, no ns te ro id al an tiin fla m m at or y dr ug , o ra l an tic oa gu la nt , gl au co m a tr ea tm en t, be ni gn p ro st at ic hy pe rp la si a tr ea tm en t an gi na p ec to ri s, de pr es si on , g la uc om a, hy pe rl ip id ae m ia , b en ig n pr os ta tic h yp er pl as ia , hy pe rt en si on , ne ur as th en ia , s in us iti s, ca rd ia c in su ffi ci en cy , ve in th ro m bo si s biologica nyssana ● 12 (2) december 2021: 87-111 matejić et al. ● frequency of herbal medicinal products use in southeastern serbia 95 l at in n am e, s er bi an na m e an d fa m ily p ar t o f a pl an t f or m p ha rm . f or m n am e of h m p a ll m ed ic in es us ed w it h h m p t he ra py gr ou ps p re se nt d is ea se s w he n h m p w as u se d v az ot al (1 0 m g) , v iv ac e (2 .5 m g) , x al at an s ol c uc ur bi ta m os ch at a d uc he sn e, m us ka tn a tik va , c uc ur bi ta ce ae se m en ex tr ac t ca ps ul e u ra sa n fo rt e a m ar yl (2 m g) , c ar di op ir in (7 5 m g) , c on co r co r ( 1. 25 m g) , c or ne lin (1 0 m g) , c os op t s ol , d ak ta no l k re m , d ia ze pa m (5 m g) , m ol si do m in (4 m g) , m on op ri l (1 0 m g) , n eb ile t ( 5 m g) , n itr og lic er in (0 .5 m g) , pa ra va no (2 0 m g) , p la vi x (7 5 m g) , si no de rm n , st ug er on fo rt e, t am so l ( 0. 4 m g) , t ef or (8 50 m g) , t ri ta ce c om p (2 .5 m g + 12 .5 m g) , v az ot al (5 m g) , x al at an so l, z of ec ar d (3 0 m g) a c e in hi bi to rs , ca lc iu m an ta go ni st s, an tia ng in al d ru g, an tih yp er te ns iv e dr ug , an tim yc ot ic dr ug , be nz od ia ze pi ne , be ta b lo ck er , de rm at ic a ge nt , hy po lip id em ic dr ug , a nt ip la te le t ag en t, or al an tid ia be tic dr ug , g la uc om a tr ea tm en t, be ni gn p ro st at ic hy pe rp la si a tr ea tm en t, ve rt ig o tr ea tm en t an gi na p ec to ri s, de pr es si on , g la uc om a, hy pe rl ip id ae m ia , b en ig n pr os ta tic h yp er pl as ia , hy pe rt en si on , c on ta ct de rm at iti s, n eu ra st he ni a, pi ty ri as is , c ar di ac in su ffi ci en cy , d ia be te s ty pe 2 c ur cu m a lo ng a l ., ku rk um a, z in gi be ra ce ae ra di x ex tr ac t ca ps ul e k ur ku m a fo rt e d ic lo fe na c du o (7 5 m g) no ns te ro id al an tiin fla m m at or y dr ug os te op or os is r ut ac ea e an tiin fla m m at ory d ru g c itr us x a ur an tiu m l ., go rk a na ra nd ža , r ut ac ea e pe ri ca rp iu m is ol at ed a ct iv e co m po ne nt s, es se nt ia l o il ca ps ul e, ta bl et d et ra le x, fl ux iv , g el om yt ro l fo rt e, ph le bo di a (6 00 m g) , v en al ek , v en od ia p lu s a m lo di pi n (1 0 m g) , a zo pt so l, b et am sa l (0 .4 m g) , b en ep ro st (5 m g) , b is op ro lo l (2 .5 m g) , b ro m az ep am (3 m g) , b ru fe n (4 00 m g) , c ar di op ir in (1 00 m g) , c on co r co r ( 1. 25 m g) , c or as p (1 00 m g) , c or ne lin (2 0 m g) , d ia ze pa m (5 m g) , e na la pr il (1 0 m g) , e na la pr il (2 0 m g) , f ar in (5 m g) , f le ka ni d (1 00 m g) , h em op re s (5 0 m g + 5 m g) , h yp ol ip (2 0 m g) , l as ix (4 0 m g) , m on iz ol (2 0 m g) , n ev ot en s (5 m g) , pr es ol ol (5 0 m g) , sp ir on ol ak to n (2 5 m g) , t en se c (5 m g) , t ri m et ac or (3 5 m g) , a c e in hi bi to rs , ca lc iu m an ta go ni st s, an tia ng in al d ru g, an tia rr hy th m ic dr ug , be nz od ia ze pi ne , be ta b lo ck er , di ur et ic , hy po lip id em ic dr ug , a nt ip la te le t ag en t, no ns te ro id al an tiin fla m m at or y dr ug , o ra l an tic oa gu la nt , gl au co m a tr ea tm en t, be ni gn p ro st at ic hy pe rp la si a tr ea tm en t an gi na p ec to ri s, de pr es si on , g la uc om a, hy pe rl ip id ae m ia , b en ig n pr os ta tic h yp er pl as ia , hy pe rt en si on , ne ur as th en ia , s in us iti s, ca rd ia c in su ffi ci en cy , ve in th ro m bo si s biologica nyssana ● 12 (2) december 2021: 87-111 96 matejić et al. ● frequency of herbal medicinal products use in southeastern serbia l at in n am e, s er bi an na m e an d fa m ily p ar t o f a pl an t f or m p ha rm . f or m n am e of h m p a ll m ed ic in es us ed w it h h m p t he ra py gr ou ps p re se nt d is ea se s w he n h m p w as u se d e ch in ac ea p ur pu re a (l .) m oe nc h, e hi na ce a, a st er ac ea e he rb a ex tr ac t eff er ve sc en t ta bl et b io gr ip in / / / e qu is et um a rv en se l ., ra st av ić , e qu is et ac ea e he rb a fr ag m en te d he rb al su bs ta nc e he rb al te a č aj p ro tiv br on hi tis a, u ro pr ot ek t č aj b ip re z (2 .5 m g) , c or ne lin (1 0 m g) , m ol ic or (2 m g) ca lc iu m an ta go ni st s, an tia ng in al d ru g, be ta b lo ck er an gi na p ec to ri s, hy pe rt en si on e uc al yp tu s gl ob ul us l ab ill ., eu ka lip tu s, m yr ta ce ae fo liu m es se nt ia l o il ca ps ul e, c re am a lp sk a kr em a, g el om yt ro l fo rt e, v ita lis sp or ts c re am b ru fe n (4 00 m g) no ns te ro id al an tiin fla m m at or y dr ug si nu si tis f oe ni cu lu m v ul ga re m ill ., m or ač , a pi ac ea e fr uc tu s ex tr ac t, es se nt ia l o il, po w de re d he rb al su bs ta nc e , t in ct ur e, fr ag m en te d he rb al su bs ta nc e he rb al te a, oi nt m en t, po w de r, liq ui d, ta bl et a sa m c rv en i ko nj sk i ba lz am , č aj p ro tiv br on hi tis a, h ip p ča j za d oj ilj e, k ap i z a m rš av lje nj e, pl an tla x ča j za g rč ev e, t en h er bs , u ro bi tr at in st an t č aj b ip re z (2 .5 m g) , b ro m az ep am (3 m g) , c or ne lin (1 0 m g) , i nk on ta n (1 5 m g) , m ar oc en (5 00 m g) , m ol ic or (2 m g) , pa nr az ol (2 0 m g) , si da ta (5 0 m g) , v iv ac e (5 m g) , v iv ac e pl us a c e in hi bi to rs , ca lc iu m an ta go ni st s, an tia ng in al d ru g, an tid ep re ss an t dr ug , an tih yp er te ns iv e dr ug , be nz od ia ze pi ne , be ta b lo ck er , pr ot on p um p in hi bi to r, qu in ol on e an tib io tic , in co nt in en ce tr ea tm en t an gi na p ec to ri s, de pr es si on , hy pe rt en si on , ch ro ni c ob st ip at io n, in co nt in en ce f ra ng ul a pu rs hi an a c oo pe r, am er ič ka kr uš in a, r ha m na ce ae co rt ex po w de re d he rb al su bs ta nc e ta bl et t en h er bs / / ch ro ni c ob st ip at io n f ra ng ul a al nu s m ill ., kr uš in a, r ha m na ce ae co rt ex tin ct ur e, fr ag m en te d he rb al su bs ta nc e he rb al te a, li qu id k ap i z a m rš av lje nj e, k ir ko lin a l ux č aj z a m rš av lje nj e / / / biologica nyssana ● 12 (2) december 2021: 87-111 matejić et al. ● frequency of herbal medicinal products use in southeastern serbia 97 l at in n am e, s er bi an na m e an d fa m ily p ar t o f a pl an t f or m p ha rm . f or m n am e of h m p a ll m ed ic in es us ed w it h h m p t he ra py gr ou ps p re se nt d is ea se s w he n h m p w as u se d g en tia na lu te a l ., lin cu ra , g en tia na ce ae ra di x po w de re d he rb al su bs ta nc e , tin ct ur e sy ru p, ta bl et a m ar ap ha rm si ru p, t en he rb s / / ch ro ni c ob st ip at io n g er an iu m r ob er tia nu m l ., zd ra va c, g er an ia ce ae he rb a ex tr ac t liq ui d fe m is an a ka pi / / / g in kg o bi lo ba l ., gi nk o, g in kg oa ce ae fo liu m ex tr ac t ca ps ul e, ta bl et b ilo bi l in te ns e, g in ko m ax , g yn kg ob il, t an ak an a m lo di pi n (1 0 m g) , b ar io s (5 m g) , b ev ip le x, b ip re z (2 .5 m g) , c on co r c or (1 .2 5 m g) , d ila co r ( 0. 25 m g) , e na la pr il (2 0 m g) , i nd ap re s (1 .5 m g) , m ol si do m in (4 m g) , p ol iv it b , p re so lo l ( 10 0 m g) , s ko pr yl (1 0 m g) , s pi ro no la kt on (2 5 m g) , t am so l ( 0. 4 m g) , t ri ta ce c om p (5 m g + 25 m g) , v es tib o (1 6 m g) a c e in hi bi to rs , ca lc iu m an ta go ni st s, an tia ng in al d ru g, an tih yp er te ns iv e dr ug , b et a bl oc ke r, di ur et ic , ca rd io to ni c ag en t, be ni gn pr os ta tic hy pe rp la si a tr ea tm en t, ve rt ig o tr ea tm en t, vi ta m in be ni gn p ro st at ic hy pe rp la si a, hy pe rt en si on , v oc al c or d an d la ry nx p ar al ys is , ca rd ia c in su ffi ci en cy , ve rt ig o an d tin ni tu s g ly ci ne m ax (l .) m er r., s oj a, f ab ac ea e se m en is ol at ed a ct iv e co m po ne nt s ca ps ul e e ss en tia le fo rt e n (3 00 m g) fo lk is (5 m g) , h em ok vi n pl us , l yr ic a (2 5 m g) , pr on is on (2 0 m g) , sa la zo py ri n (5 00 m g) , t am so l (0 .4 m g) an tia ne m ic dr ug fo r m eg al ob la st ic an em ia , an tie pi le pt ic dr ug , an tih yp er te ns iv e dr ug , de pr es si on , b en ig n pr os ta tic h yp er pl as ia , hy pe rt en si on , u lc er at iv e co lit is biologica nyssana ● 12 (2) december 2021: 87-111 matejić et al. ● frequency of herbal medicinal products use in southeastern serbia l at in n am e, s er bi an na m e an d fa m ily p ar t o f a pl an t f or m p ha rm . f or m n am e of h m p a ll m ed ic in es us ed w it h h m p t he ra py gr ou ps p re se nt d is ea se s w he n h m p w as u se d gl uc oc or tic oi d, su lp ho na m id e, be ni gn p ro st at ic hy pe rp la si a tr ea tm en t g ly cy rr hi za g la br a l ., sl ad ić , f ab ac ea e ra di x po w de re d he rb al su bs ta nc e , fr ag m en te d he rb al su bs ta nc e he rb al te a, ta bl et č aj p ro tiv br on hi tis a, č aj s la tk og ko re na , t en he rb s b ip re z (2 .5 m g) , c or ne lin (1 0 m g) , m ol ic or (2 m g) ca lc iu m an ta go ni st s, an tia ng in al d ru g, be ta b lo ck er an gi na p ec to ri s, hy pe rt en si on , c hr on ic ob st ip at io n, li ve r st ea to si s h ed er a he lix l ., br šl ja n, a ra lia ce ae fo liu m ex tr ac t sy ru p, p ow de r, eff er ve sc en t ta bl et in tu si n pu lv is , pr os pa n si ru p, pr os pa n šu m eć e ta bl et e / / / h um ul us lu pu lu s l ., hm el j, c an na ba ce ae flo s ex tr ac t oi nt m en t, liq ui d, ta bl et a sa m c rv en i ko nj sk i ba lz am , k ap i za u m ir en je , r el ax b ro m az ep am (3 m g) , n ov om ix 30 f le xp en (1 00 ij/ m l) , si da ta (5 0 m g) , v iv ac e (5 m g) , v iv ac e pl us a c e in hi bi to rs , an tid ep re ss an t dr ug , an tih yp er te ns iv e dr ug , be nz od ia ze pi ne , in su lin ce lia c di se as e, de pr es si on , hy pe rt en si on , d ia be te s ty pe 1 h yp er ic um p er fo ra tu m l ., ka nt ar io n, h yp er ic ac ea e flo s ex tr ac t, es se nt ia l o il liq ui d, o il ex tr ac t, va gi na l ta bl et h ip er va g in tim , k ap i z a um ir en je , u lje od k an ta ri on a za s po lja šn ju up ot re bu , v ag in al et e sa ka nt ar io no m b ar io s (5 m g) , c on co r c or (1 .2 5 m g) , c or ne lin (1 0 m g) ca lc iu m an ta go ni st s, b et a bl oc ke r hy pe rt en si on la va nd ul a an gu st ifo lia m ill ., la va nd a, l am ia ce ae flo s es se nt ia l o il ge ll k es te n ge l / / / 98 biologica nyssana ● 12 (2) december 2021: 87-111 matejić et al. ● frequency of herbal medicinal products use in southeastern serbia l at in n am e, s er bi an na m e an d fa m ily p ar t o f a pl an t f or m p ha rm . f or m n am e of h m p a ll m ed ic in es us ed w it h h m p t he ra py gr ou ps p re se nt d is ea se s w he n h m p w as u se d m al pi gh ia e m ar gi na ta d c ., ac er ol a, m al pi gh ia ce ae fr uc tu s ex tr ac t eff er ve sc en t ta bl et b io gr ip in / / / m ar ru bi um v ul ga re l ., oč aj ni ca , l am ia ce ae he rb a po w de re d he rb al su bs ta nc e ta bl et t en h er bs / / ch ro ni c ob st ip at io n m at ri ca ri a ch am om ill a l ., ka m ili ca , a st er ac ea e flo s ex tr ac t ge ll, o in tm en t, sy ru p a sa m c rv en i ko nj sk i ba lz am , g in gi va l g el , h er bi ko s ir up be lo g sl ez a a m ar yl (2 m g) , a sp ir in p ro te ct (1 00 m g) , b ro m az ep am (3 m g) , g lu co ph ag e (1 00 0 m g) , s id at a (5 0 m g) , v iv ac e (5 m g) , v iv ac e pl us a c e in hi bi to rs , an tid ep re ss an t dr ug , an tih yp er te ns iv e dr ug , be nz od ia ze pi ne , an tip la te le t ag en t, or al an tid ia be tic d ru g de pr es si on , hy pe rt en si on , d ia be te s ty pe 2 m el is sa o ffi ci na lis l ., m at ič nj ak , l am ia ce ae fo liu m ex tr ac t oi nt m en t, po w de r, liq ui d, ta bl et a sa m c rv en i ko nj sk i ba lz am , fe m is an b ka pi , h ip p ča j z a do jil je , k ap i z a um ir en je , r el ax b ro m az ep am (3 m g) , n ov om ix 30 f le xp en (1 00 ij/ m l) , s id at a (5 0 m g) , v iv ac e (5 m g) , v iv ac e pl us a c e in hi bi to rs , an tid ep re ss an t dr ug , an tih yp er te ns iv e dr ug , be nz od ia ze pi ne , in su lin ce lia c di se as e, de pr es si on , hy pe rt en si on , d ia be te s ty pe 1 m en th a ar ve ns is l ., po ljs ka n an a, l am ia ce ae fo liu m es se nt ia l o il cr ea m a lp sk a kr em a, v ita lis s po rt s cr ea m / / / m en th a x pi pe ri ta l ., pi to m a na na , l am ia ce ae fo liu m es se nt ia l o il, fr ag m en te d he rb al su bs ta nc e he rb al te a, g el l č aj li st a na ne , g in gi va l g el a m ar yl (2 m g) , a sp ir in p ro te ct (1 00 m g) , g lu co ph ag e (1 00 0 m g) an tip la te le t ag en t, or al an tid ia be tic d ru g hy pe rt en si on , d ia be te s ty pe 2 99 biologica nyssana ● 12 (2) december 2021: 87-111 matejić et al. ● frequency of herbal medicinal products use in southeastern serbia l at in n am e, s er bi an na m e an d fa m ily p ar t o f a pl an t f or m p ha rm . f or m n am e of h m p a ll m ed ic in es us ed w it h h m p t he ra py gr ou ps p re se nt d is ea se s w he n h m p w as u se d m yr tu s co m m un is l ., m ir ta , m yr ta ce ae fo liu m es se nt ia l o il ca ps ul e, oi nt m en t g el om yt ro l fo rt e, p av le ba lz am b ru fe n (4 00 m g) no ns te ro id al an tiin fla m m at or y dr ug si nu si tis o ci m um b as ili cu m l ., bo si lja k, l am ia ce ae fo liu m es se nt ia l o il liq ui d si nu so l m aj or an s pr ej za n os a sp ir in p ro te ct (1 00 m g) , b is op ro lo l (5 m g) , b ro m az ep am (3 m g) , d ic lo fe na c du o (7 5 m g) , h em om yc in (5 00 m g) be nz od ia ze pi ne , be ta b lo ck er , an tip la te le t ag en t, m ac ro lid e an tib io tic , no ns te ro id al an tiin fla m m at or y dr ug hy pe rt en si on o en ot he ra b ie nn is l ., no ću ra k, o na gr ac ea e se m en es se nt ia l o il, oi l ca ps ul e, v ag in al ta bl et h ip er va g in tim , s ol ga r ul je n oć ur ka / / / o ri ga nu m v ul ga re l ., vr an ilo va tr av a, l am ia ce ae fo liu m is ol at ed a ct iv e co m po ne nt s, fr ag m en te d he rb al su bs ta nc e he rb al te a, ca ps ul e pa lis ep t, u ro pr ot ek t č aj a ct aw el l (1 00 m g) , b in ev ol (5 m g) , b ro m az ep am (3 m g) , c or ne lin (2 0 m g) , e lic ea (1 0 m g) , i rb en id a pl us (1 50 m g + 12 .5 m g) , k ar vi le ks (1 2. 5 m g) , l ex ili um (3 m g) , l un at a (5 m g) , t ef or (5 00 m g) , t re si ba fl ex t ou ch (2 00 ij/ m l) , t ri ta ce co m p (2 .5 m g + 12 .5 m g) ca lc iu m an ta go ni st s, an tid ep re ss an t dr ug , an tih yp er te ns iv e dr ug , an tip sy ch ot ic dr ug , be nz od ia ze pi ne , be ta b lo ck er , hy pn ot ic d ru g, in su lin , o ra l an tid ia be tic d ru g de pr es si on , hy pe rt en si on , d ia be te s ty pe 1 100 biologica nyssana ● 12 (2) december 2021: 87-111 matejić et al. ● frequency of herbal medicinal products use in southeastern serbia l at in n am e, s er bi an na m e an d fa m ily p ar t o f a pl an t f or m p ha rm . f or m n am e of h m p a ll m ed ic in es us ed w it h h m p t he ra py gr ou ps p re se nt d is ea se s w he n h m p w as u se d o ri ga nu m n m aj or an a l ., m aj or an , l am ia ce ae fo liu m es se nt ia l o il liq ui d si nu so l m aj or an s pr ej za n os a sp ir in p ro te ct (1 00 m g) , b is op ro lo l (5 m g) , b ro m az ep am (3 m g) , d ic lo fe na c du o (7 5 m g) , h em om yc in (5 00 m g) be nz od ia ze pi ne , be ta b lo ck er , an tip la te le t ag en t, m ac ro lid e an tib io tic , no ns te ro id al an tiin fla m m at or y dr ug hy pe rt en si on o rt ho si ph on a ri st at us (b lu m e) m iq ., ja va č aj , l am ia ce ae fo liu m ex tr ac t po w de r u ro bi tr at in st an t č aj in ko nt an (1 5 m g) , m ar oc en (5 00 m g) , pa nr az ol (2 0 m g) pr ot on p um p in hi bi to r, qu in ol on e an tib io tic , in co nt in en ce tr ea tm en t in co nt in en ce p as si flo ra e du lis si m s, p as ifl or a, pa ss ifl or ac ea e he rb a ex tr ac t ta bl et a lo ra u ltr a, sm ir el a no ć c oa m le ss a (4 m g + 5 m g + 1. 25 m g) , e gl on yl (5 0 m g) an tih yp er te ns iv e dr ug , an tip sy ch ot ic dr ug ga st ro -i nt es tin al ne ur os es , h yp er te ns io n p et ro se lin um c ri sp um (m ill .) fu ss , p er šu n, a pi ac ea e fo liu m ex tr ac t, fr ag m en te d he rb al su bs ta nc e he rb al te a, li qu id fe m is an a k ap i, u ro pr ot ek t č aj / / / p im pi ne lla a ni su m l ., an is , a pi ac ea e fr uc tu s ex tr ac t po w de r h ip p ča j z a do jil je / / / p in us s yl ve st ri s l ., sr eb rn i b or , p in ac ea e fo liu m et ar sk o ul je oi nt m en t pa vl e ba lz am / / / p la nt ag o la nc eo la ta l ., bo kv ic a, pl an ta gi na ce ae fo liu m ex tr ac t, fr ag m en te d he rb al su bs ta nc e he rb al te a, li qu id č aj p ro tiv br on hi tis a, pr op ol is k ap i sa b ok vi co m b ip re z (2 .5 m g) , c or ne lin (1 0 m g) , m ol ic or (2 m g) an tia ng in al dr ug , c al ci um an ta go ni st s, be ta b lo ck er an gi na p ec to ri s, hy pe rt en si on p ot en til la e re ct a (l .) r ae us ch ., sr če nj ak , r os ac ea e rh iz om a tin ct ur e ge ll g in gi va l g el a m ar yl (2 m g) , a sp ir in p ro te ct (1 00 m g) , an tip la te le t ag en t, hy pe rt en si on , d ia be te s ty pe 2 101 biologica nyssana ● 12 (2) december 2021: 87-111 matejić et al. ● frequency of herbal medicinal products use in southeastern serbia l at in n am e, s er bi an na m e an d fa m ily p ar t o f a pl an t f or m p ha rm . f or m n am e of h m p a ll m ed ic in es us ed w it h h m p t he ra py gr ou ps p re se nt d is ea se s w he n h m p w as u se d g lu co ph ag e (1 00 0 m g) or al a nt id ia be tic dr ug p ri m ul a ve ri s l ., ja go rč ev in a, pr im ul ac ea e fo liu m , r ad ix ex tr ac t, fr ag m en te d he rb al su bs ta nc e he rb al te a, s yr up b ro nh o sa n ja go rč ev in a, tim ija n, di vi zm a, č aj p ro tiv br on hi tis a b ip re z (2 .5 m g) , c or ne lin (1 0 m g) , m ol ic or (2 m g) ca lc iu m an ta go ni st s, an tia ng in al dr ug , b et a bl oc ke r an gi na p ec to ri s, hy pe rt en si on r ha m nu s al pi na l ., že st ik a, r ha m na ce ae co rt ex po w de re d he rb al su bs ta nc e ta bl et t en h er bs / / ch ro ni c ob st ip at io n r he um p al m at um l ., ra ba rb ar a, po ly go na ce ae ra di x po w de re d he rb al su bs ta nc e ta bl et t en h er bs / / ch ro ni c ob st ip at io n r ib es n ig ru m l ., cr na ri bi zl a, g ro ss ul ar ia ce ae fr uc tu s ex tr ac t pa st ill es is la c as si s b is op ro lo l (2 .5 m g) be ta b lo ck er hy pe rt en si on r ic in us c om m un is l ., ri ci nu s, e up ho rb ia ce ae se m en ex tr ac t oi l e xt ra ct r ic in us ov o ul je / / / r os a ca ni na l ., ši pu ra k, r os ac ea e fr uc tu s ex tr ac t, fr ag m en te d he rb al su bs ta nc e am po ul e, h er ba l te a, li qu id , s yr up c h a lp ha pl us , h er bi ko si ru p be lo g sl ez a, k ap i z a m rš av lje nj e, k ir ko lin a l ux č aj z a m rš av lje nj e d ic lo fe na c du o (7 5 m g) no ns te ro id al an tiin fla m m at or y dr ug ar th ri tis r os m ar in us o ffi ci na lis l ., ru zm ar in , l am ia ce ae fo liu m ex tr ac t, es se nt ia l o il ge ll, c re am , liq ui d a lp sk a kr em a, k ap i za u m ir en je , k es te n ge l, v ita lis s po rt s cr ea m / / / 102 biologica nyssana ● 12 (2) december 2021: 87-111 matejić et al. ● frequency of herbal medicinal products use in southeastern serbia l at in n am e, s er bi an na m e an d fa m ily p ar t o f a pl an t f or m p ha rm . f or m n am e of h m p a ll m ed ic in es us ed w it h h m p t he ra py gr ou ps p re se nt d is ea se s w he n h m p w as u se d r ub us id ae us l ., m al in a, r os ac ea e fo liu m ex tr ac t liq ui d fe m is an a ka pi / / / r us cu s ac ul ea tu s l ., ve pr in a, a sp ar ag ac ea e rh iz om a ex tr ac t ta bl et e rb av en re ta rd / / / sa lix a lb a l ., be la vr ba , s al ic ac ea e co rt ex ex tr ac t eff er ve sc en t ta bl et b io gr ip in / / / sa tu re ja m on ta na l ., rt an js ki č aj , l am ia ce ae he rb a fr ag m en te d he rb al su bs ta nc e he rb al te a r ta nj sk i č aj c on co r c or (1 .2 5 m g) be ta b lo ck er hy pe rt en si on se nn a al ex an dr in a m ill ., se na , f ab ac ea e fo liu m po w de re d he rb al su bs ta nc e , fr ag m en te d he rb al su bs ta nc e he rb al te a, ta bl et č aj li st a se ne , k ir ko lin a l ux č aj z a m rš av lje nj e, t en h er bs a m lo ga l (1 0 m g) , a nt ia gr ex (7 5 m g) , c ar di op ir in (7 5 m g) , c on co r (5 m g) , d ia ze pa m (5 m g ), g lu fo rm in (1 00 0 m g) , h lo rp ro m az in (2 5 m g) , m ic ar di s (8 0 m g) , p ri lin da (5 m g) , t en se c (5 m g) , t re nt al (4 00 m g) , v as ili p (2 0 m g) a c e in hi bi to rs , ca lc iu m an ta go ni st s, an tih yp er te ns iv e dr ug , an tip sy ch ot ic dr ug , be nz od ia ze pi ne , be ta b lo ck er , hy po lip id em ic dr ug , an tip la te le t ag en t, or al an tid ia be tic dr ug , c ir cu la to ry di so rd er s tr ea tm en t de pr es si on , hy pe rl ip id ae m ia , hy pe rt en si on , c hr on ic ob st ip at io n, d ia be te s ty pe 2 se re no a re pe ns (w . b ar ta m ) s m al l, te st er as ta p al m a, a re ca ce ae fr uc tu s ex tr ac t ca ps ul e pr os ta m ol un o, p ro st en al pe rf ec t a m ar yl (3 m g) , a nd ol (1 00 m g) , b er lit hi on 60 0 (6 00 m g) , b er od ua l n (0 .0 5 m g + 0. 02 1 m g) , c ar di pi ne (5 m g) , a c e in hi bi to rs , ca lc iu m an ta go ni st s, an tia ng in al dr ug , an tih yp er te ns iv e dr ug , an gi na p ec to ri s, at ri al fi br ill at io n an d at ri al fl ut te r, be ni gn pr os ta tic h yp er pl as ia , hy pe rt en si on , c hr on ic ob st ru ct iv e pu lm on ar y di se as e, n eu ra st he ni a, di ab et es ty pe 2 103 biologica nyssana ● 12 (2) december 2021: 87-111 matejić et al. ● frequency of herbal medicinal products use in southeastern serbia l at in n am e, s er bi an na m e an d fa m ily p ar t o f a pl an t f or m p ha rm . f or m n am e of h m p a ll m ed ic in es us ed w it h h m p t he ra py gr ou ps p re se nt d is ea se s w he n h m p w as u se d c on co r c or (1 .2 5 m g) , c or ne lin (1 0 m g) , d ia ze pa m (5 m g) , d ur ofi lin (1 25 m g) , g lio ks an (8 0 m g) , g lu fo rm in (1 00 0 m g) , g lu co ph ag e (1 00 0 m g) , l ex ili um (3 m g) , m on os an (2 0 m g) , sk op ry l p lu s, sp ir on ol ak to n (2 5 m g) , t am so l (0 .4 m g) , t ef or (5 00 m g) , t en se c (5 m g) , t ri ta ce (5 m g) be nz od ia ze pi ne , be ta b lo ck er , br on ch os pa sm ol yt ic a ge nt , di ur et ic , a nt ipl at el et a ge nt , or al a nt id ia be tic dr ug , d ia be tic po ly ne ur op ath y tr ea tm en t, be ni gn p ro sta tic h yp er pl as ia tr ea tm en t si ly bu m m ar ia nu m (l .) g ae rt n. , s ik av ic a, a st er ac ea e fr uc tu s ex tr ac t ca ps ul e, ta bl et l ag os a 15 0, si ly m ar in fo lk is (5 m g) , h em ok vi n pl us , l yr ic a (2 5 m g) , pr on is on (2 0 m g) , sa la zo py ri n (5 00 m g) , t am so l (0 .4 m g) an tia ne m ic dr ug fo r m eg al ob la st ic an em ia , an tie pi le pt ic dr ug , an tih yp er te ns iv e dr ug , gl uc oc or tic oi d, su lp ho na m id e, be ni gn p ro st at ic hy pe rp la si a tr ea tm en t de pr es si on , b en ig n pr os ta tic h yp er pl as ia , hy pe rt en si on , l iv er st ea to si s, u lc er at iv e co lit is so lid ag o vi rg aau re a l ., zl at ni ca , a st er ac ea e he rb a ex tr ac t po w de r u ro bi tr at in st an t č aj in ko nt an (1 5 m g) , pr ot on p um p in hi bi to r, in co nt in en ce 104 biologica nyssana ● 12 (2) december 2021: 87-111 matejić et al. ● frequency of herbal medicinal products use in southeastern serbia l at in n am e, s er bi an na m e an d fa m ily p ar t o f a pl an t f or m p ha rm . f or m n am e of h m p a ll m ed ic in es us ed w it h h m p t he ra py gr ou ps p re se nt d is ea se s w he n h m p w as u se d m ar oc en (5 00 m g) , pa nr az ol (2 0 m g) qu in ol on e an tib io tic , in co nt in en ce tr ea tm en t st yp hn ol ob iu m ja po ni cu m (l .) sc ho tt, ja pa ns ki b ag re m , fa ba ce ae flo s is ol at ed a ct iv e co m po ne nt s ta bl et e rb av en re ta rd , f lu xi v c on co r c or (1 .2 5 m g) , fl ek an id (1 00 m g) an tia rr hy th m ic dr ug , b et a bl oc ke r ca rd ia c in su ffi ci en cy sy m ph yt um offi ci na le l ., ga ve z, b or ag in ac ea e ra di x ex tr ac t ge ll b ei nw el l g av ez g el / / / ta ra xa cu m ca m py lo de s g .e .h ag lu nd , m as la ča k, c om po si ta e ra di x po w de re d he rb al su bs ta nc e ta bl et t en h er bs / / ch ro ni c ob st ip at io n th ym us s er py llu m l ., m aj ki na d uš ic a, l am ia ce ae he rb a fr ag m en te d he rb al su bs ta nc e he rb al te a č aj p ro tiv br on hi tis a b ip re z (2 .5 m g) , c or ne lin (1 0 m g) , m ol ic or (2 m g) ca lc iu m an ta go ni st s, an tia ng in al d ru g, be ta b lo ck er an gi na p ec to ri s, hy pe rt en si on th ym us v ul ga ri s l ., tim ija n, l am ia ce ae he rb a ex tr ac t sy ru p b ro nh o sa n ja go rč ev in a, tim ija n, di vi zm a / / / tu ss ila go fa rf ar a l ., po db el , a st er ac ea e fo liu m fr ag m en te d he rb al su bs ta nc e he rb al te a č aj p ro tiv br on hi tis a b ip re z (2 .5 m g) , c or ne lin (1 0 m g) , m ol ic or (2 m g) ca lc iu m an ta go ni st s, an tia ng in al d ru g, be ta b lo ck er an gi na p ec to ri s, hy pe rt en si on u rt ic a di oi ca l ., ko pr iv a, u rt ic ac ea e fo liu m , h er ba , ra di x ex tr ac t ca ps ul e, p ow de r, ta bl et h ip p ča j za d oj ilj e, pr os te na l pe rf ec t, v en od ia p lu s a m ar yl (3 m g) , a nd ol (1 00 m g) , a zo pt so l, b er od ua l n (0 .0 5 m g + 0. 02 1 m g) , a c e in hi bi to rs , ca lc iu m a nt ag oni st s, a nt ia ng in al dr ug , b en zo di az ep in e, b et a bl oc ke r, an gi na p ec to ri s, de pr es si on , gl au co m a, b en ig n pr os ta tic h yp er pl as ia , hy pe rt en si on , c hr on ic ob st ru ct iv e pu lm on ar y di se as e, 105 biologica nyssana ● 12 (2) december 2021: 87-111 matejić et al. ● frequency of herbal medicinal products use in southeastern serbia l at in n am e, s er bi an na m e an d fa m ily p ar t o f a pl an t f or m p ha rm . f or m n am e of h m p a ll m ed ic in es us ed w it h h m p t he ra py gr ou ps p re se nt d is ea se s w he n h m p w as u se d b is op ro lo l (2 .5 m g) , b ro m az ep am (3 m g) , c or as p (1 00 m g) , c or ne lin (1 0 m g) , c or ne lin (2 0 m g) , d ia ze pa m (5 m g) , e na la pr il (2 0m g) , fa ri n (5 m g) , g lu co ph ag e (1 00 0 m g) , m on os an (2 0 m g) , p re so lo l (5 0 m g) , t am so l (0 .4 m g) ,t en se c (5 m g) , tr im et ac or (3 5 m g) , t ri ta ce (5 m g) , v iv ac e (2 .5 m g) , x al at an so l br on ch os pa sm oly tic a ge nt , a ntip la te le t a ge nt , or al a nt id ia be tic dr ug , o ra l a nt ico ag ul an t, gl au co m a tr ea tm en t, be ni gn p ro sta tic h yp er pl as ia tr ea tm en t, ve in th ro m bo si s ne ur as th en ia , d ia be te s ty pe 2 , v ei n th ro m bo si s v ac ci ni um m yr til lu s l ., bo ro vn ic a, e ri ca ce ae fo liu m ex tr ac t ta bl et e rb av en re ta rd , v en od ia p lu s a zo pt s ol , b is op ro lo l (2 .5 m g) , b ro m az ep am (3 m g) , c or as p (1 00 m g) , c or ne lin (2 0 m g) , d ia ze pa m (5 m g) , e na la pr il (2 0m g) , fa ri n (5 m g) , pr es ol ol (5 0 m g) , t ri m et ac or (3 5 m g) , v iv ac e (2 .5 m g) , x al at an s ol a c e in hi bi to rs , ca lc iu m an ta go ni st s, an tia ng in al d ru g, be nz od ia ze pi ne , be ta b lo ck er , an tip la te le t ag en t, or al an tic oa gu la nt , gl au co m a tr ea tm en t an gi na p ec to ri s, de pr es si on , g la uc om a, hy pe rt en si on , ne ur as th en ia , v ei n th ro m bo si s 106 biologica nyssana ● 12 (2) december 2021: 87-111 matejić et al. ● frequency of herbal medicinal products use in southeastern serbia l at in n am e, s er bi an na m e an d fa m ily p ar t o f a pl an t f or m p ha rm . f or m n am e of h m p a ll m ed ic in es us ed w it h h m p t he ra py gr ou ps p re se nt d is ea se s w he n h m p w as u se d v ac ci ni um m ac ro ca rpo n a ito n, a m er ič ka bo ro vn ic a, e ri ca ce ae fr uc tu s fr ag m en te d he rb al su bs ta nc e he rb al te a č aj p lo da br us ni ce / / / v al er ia na o ffi ci na lis l ., va le ri ja na , c ap ri fo lia ce ae ra di x ex tr ac t oi nt m en t, liq ui d, ta bl et a sa m c rv en i ko nj sk i ba lz am , fe m is an b ka pi , k ap i za u m ir en je , r el ax , sm ir el a no ć, v al er ija na ka pi b ro m az ep am (3 m g) , c oa m le ss a (4 m g + 5m g + 1. 25 m g) , n ov om ix 3 0 fl ex pe n (1 00 ij/ m l) , s id at a (5 0 m g) , v iv ac e (5 m g) , v iv ac e pl us a c e in hi bi to rs , an tid ep re ss an t dr ug , an tih yp er te ns iv e dr ug , be nz od ia ze pi ne , in su lin ce lia c di se as e, de pr es si on , hy pe rt en si on , d ia be te s ty pe 1 v er ba sc um p hl om oi de s l ., di vi zm a, sc ro ph ul ar ia ce ae flo s ex tr ac t sy ru p b ro nh o sa n ja go rč ev in a, tim ija , di vi zm a / / / v is cu m a lb um l ., im el a, s an ta la ce ae he rb a ex tr ac t liq ui d fe m is an b ka pi / / / v iti s vi ni fe ra l ., gr ož đe , v ita ce ae fo liu m , s em en ex tr ac t oi nt m en t, ta bl et a sa m c rv en i ko nj sk i ba lz am , e rb av en re ta rd , s m ir el a no ć b ro m az ep am (3 m g) , c oa m le ss a (4 m g + 5 m g + 1. 25 m g) , si da ta (5 0 m g) , v iv ac e (5 m g) , v iv ac e pl us a c e in hi bi to rs , an tid ep re ss an t dr ug , an tih yp er te ns iv e dr ug , be nz od ia ze pi ne de pr es si on , h yp er te ns io n ze a m ay s l ., ku ku ru z, po ac ea e st ig m a fr ag m en te d he rb al su bs ta nc e he rb al te a č aj k uk ur uz ne sv ile , k ir ko lin a l ux č aj z a m rš av lje nj e, u ro pr ot ek t č aj c on co r ( 5 m g) , d ia ze pa m (5 m g) , g lu fo rm in (1 00 0 m g) , h lo rp ro m az in (2 5 m g) , p ri lin da (5 m g) , t en se c (5 m g) , t re nt al (4 00 m g) a c e in hi bi to rs , an tip sy ch ot ic dr ug , be nz od ia ze pi ne , be ta b lo ck er , or al a nt id ia be tic dr ug , c ir cu la to ry di so rd er s tr ea tm en t de pr es si on , hy pe rt en si on , d ia be te s ty pe 2 107 the results showed that the respondents living in the municipality of aleksinac used more plant species for prevention and/or treatment of diseases compared to people from sokobanja and zaječar, while there was no difference between the respondents when it comes to the number of hmp (tab. 2). tab. 3 shows that women used more herbal species when it comes to prevention and/or treatment of the disease compared to men, while there was no difference between the subjects when it comes to the number of hmp. 108 biologica nyssana ● 12 (2) december 2021: 87-111 matejić et al. ● frequency of herbal medicinal products use in southeastern serbia location number of hmp per patient number of ps per patient aleksinac 1.05±0.22 3.33±2.48 sokobanja 1.12±0.33 1.95±1.87 zaječar 1.08±0.28 1.77±1.09 kruskal-wallis test chi-square=1.037; p=0.596 chi-square=9.749; p=0.008 mann-whitney u test post hoc / aleksinac vs. sokobanja, p=0.002 aleksinac vs. zaječar, p=0.038 sokobanja vs. zaječar, p=0.816 table 2. number of hmp and species per respondent in relation to location ps-plant species table 3. number of hmp and species per respondent in relation to location gender number of hmp per patient number of ps per patient male 1.97±0.25 1.67±1.73 female 1.13±0.34 2.69±2.12 mann-whitney u test z=-1.065; p=0.287 z=-3.210; p=0.001 age number of hmp per patient number of ps per patient ≤35 1.05±0.22 2.33±1.56 36-50 1.14±0.36 2.61±2.38 51-64 1.09±0.29 2.27±2.29 ≥ 65 1.10±0.31 1.72±1.62 kruskal-wallis test chi-square=1.223; p=0.747 chi-square=3.504; p=0.320 mann-whitney u test post hoc / / table 4. number of hmp and plant species per respondent in relation to age the analysis showed (tab. 4, tab. 5) there were no differences between the number of used hmp and plant species per subject in relation to age, and that they did not differ in the number of used hmp and plant species per subject in relation to the presence of chronic diseases. also, in our study it was concluded (tab. 6) that the subjects did not differ in the number of used hmp and herbal species per subject in relation to the number of conventional medicines per patient. respondents who did not have finished pharmaceutical products in therapy used hmp for the purpose of prevention in a higher percentage (tab. 7). table 5. number of hmp/species per respondent in relation to chronic diseases presence of chronic diseases number of hmp per patient number of ps per patient yes 1.12±0.33 2.04±2.06 no 1.08±0.28 2.41±1.95 mann-whitney u test z=-0.597; p=0.550 z=-1.141; p=0.254 in tab. 8, it can be seen that most finished pharmaceutical products and hmp were used for the same indications. as expected, in the group of patients who used hmp for an indication different from the one for which the respondents used conventional medicines, 63.5% of them did not use medicines at all (tab. 9). comparative analysis the area of southeastern serbia is known for its rich tradition of using herbal preparations and herbal medicines. the work by matejić et al. (2020), shows that in the villages of the timok and svrljig region, people often opt for the use of plants from nature. this is the first measure of prevention, and even treatment, while they later decide to visit the doctor. according to the results of this ethnobotanical research conducted from 2015 to 2017, the local population used a total of 195 species in traditional treatments, specifically 31 species were used by the population of the timok region, 77 both by the population of the timok and svrljig regions, and 87 by the population of the srvrljig region. 109 biologica nyssana ● 12 (2) december 2021: 87-111 matejić et al. ● frequency of herbal medicinal products use in southeastern serbia table 6. selection of indications for the use of hmp in relation to the number of medicines indication for the use of hp number of hmp per patient number of ps per patient 0 medicines 1.12±0.32 2.58±2.24 1-3 medicines 1.10±0.31 2.10±3.78 ≥ 4 medicines 1.07±0.26 1.64±1.42 kruskal-wallis test chi-square=0.387; p=0.824 chi-square=3.981; p=0.137 mann-whitney u test post hoc / / table 7. the purpose of the use of hmp in relation to the number of finished pharmaceutical products purpose of using hp 0 medicines 1-3 medicines ≥ 4 medicines prevention (n=79) 50 (63.29%) 11 (13.92%) 18 (22.78%) treatment (n=21) 2 (9.52%) 9 (42.86%) 10 (47.62%) χ2 test χ2=19.821; p<0.001 table 8. the most common indications for the use of conventional medicines and indications for the use of hmp indication n (%) indication n (%) cardiovascular diseases 37% cardiovascular diseases 18% benign prostatic hyperplasia 13% benign prostatic hyperplasia 19% mental disorders 12% mental disorders 11% diabetes 10% ocular disorders 6% other 24% patients without comorbidity 49 table 9. indications for the use of hmp in relation to the number of medicines indication for the use of hp 0 medicines 1-3 medicines ≥ 4 medicines same as in the case of finished pharmaceutical products (n=20) 1 (5%) 9 (45%) 10 (50%) different in relation to the indication of finished pharmaceutical products (n=80) 51 (63.75%) 11 (13.75%) 18 (22.50%) χ2 test χ2=19.821; p<0.001 the timok region has a total of 108 species that have been used in the traditional treatment of the population. people most often used the following: hypericum spp., matricaria chamomilla, mentha x piperita, urtica dioica, juglans regia and achillea millefolium. all plant species that were used in the timok region can be classified into a total of 47 families. among them, most species are members of the families lamiaceae, rosaceae, asteraceae, apiaceae and brassicaceae. depending on the type of plant, different parts of it were used to make herbal preparations, and in as many as 94.6% of cases the aerial parts, flower, fruit, leaf and root were used. the population most often used herbs to prepare tea, in the form of compresses, as a tincture, fresh and cooked. most of the plants were used internally (for internal use), 79.5% of the total number of plants, while a smaller number of plants were used externally (for external use), that is 20.5%. plants from the timok region were used in the treatment of the following systems: digestive, endocrine, metabolic and nutritional, integumentary, respiratory, circulatory and urinary, with 83.7% of all treated systems (matejić et al. 2020). the svrljig region is characterized by a total of 164 different plant species that the population used for traditional medical purposes. in the svrljig region, the most commonly used were: satureja montana, sambucus nigra, polygonum aviculare, marrubium vulgare and teucrium chamaedrys. all species are classified in 64 families, and the largest percentage of them belongs to the lamiaceae, rosaceae, asteraceae, polygonaceae and amaryllidaceae. the most used parts of plants were: aerial parts, leaf, flower, fruit and root. in 90.8% of cases, the plants were used in the form of tea, compresses, tinctures, for ritual purposes and fresh. in the largest percentage, the population used plants internally (for internal use), as much as 76.0%, while externally (for external use) 20.8% of the total number of plant species were used. however, 3.2% of species were used both externally and internally. the population used the plants in the treatment of various systems: digestive, endocrine, metabolic and nutritional, circulatory, urinary, integumentary, musculoskeletal and general and nonspecific, which represents 78.7% of all treated systems. the research by zlatković et al. (2014), conducted from 2011 to 2012, shows that 45 different species were in use on mount rtanj. the most commonly used was hypericum perforatum. the largest percentage of the used plants belonged to the families lamiaceae, rosaceae and asteraceae. they were used primarily in the treatment of the immune, respiratory and digestive systems. the work by jarić et al. (2015) showed that in the region of suva planina the population used 128 different plant species in the period from 2012 to 2014. most of them used the following species: achillea millefolium, allium cepa, allium sativum, arctostaphyllos uva-ursi, gentiana lutea, hypericum perforatum, juglans regia, matricaria chamomilla, mentha x piperita, plantago lanceolata, plantago major, salvia officinalis, sempervivum tectorum, tilia cordata and thymus serpyllum. all used species are classified according to families, and the most common families were: asteraceae, rosaceae, brassicaceae and alliaceae. these herbs were used to treat mainly the respiratory system, disorders of the genitourinary and the digestive system. the research by janaćković et al. (2019), realized during 2016, indicates that 37 wild species were used among the population of negotin valley, most often the following: matricaria chamomilla, urtica dioica, hypericum perforatum, salvia officinalis, plantago major, achillea millefolilium, calendula officinalis and taraxacum campylodes. the largest percentage belonged to the families lamiaceae, asteraceae and rosaceae, and they were primarily used to treat the immune, digestive and respiratory systems, as well as skin diseases. all these ethnopharmacological researches are related to the plants used in traditional medicine, while our study gives a review of the plant species and hmp which are ingredients of pharmacological products and are distributed in public pharmacies. the number of people who use these pharmacological products increase even in rural areas. conclusions obtained results reflect a high frequency of the use of hmp in southeastern serbia. residents of this area are more likely to opt for the use of various natural products than for conventional medicines. they use hmp to treat many diseases, and often in their prevention. the most commonly used preparation was urasan forte. the composition of this preparation includes nutmeg (cucurbita moschata duchesne), one of the most common plant species. it is used in prostate hyperplasia, which together with other diseases of the urinary system often occurs in the inhabitants of this area. the most common species was bitter orange (citrus x aurantium l.), an integral part of many hmp used in the treatment of hypertension, hyperlipidemia, venous thrombosis and sinusitis. most of the inhabitants suffered from hypertension, as confirmed by the relevant research conducted in this area. the results showed that the respondents from aleksinac used more species for prevention and/or treatment of diseases compared biologica nyssana ● 12 (2) december 2021: 87-111 110 matejić et al. ● frequency of herbal medicinal products use in southeastern serbia to people from sokobanja and zaječar, while there was no difference between the respondents when it comes to the number of hmp. respondents who did not have conventional medicines in therapy used hmp for prevention in a higher percentage. the local population prefers simple consumption of preparations in the form of capsules and tablets, but the traditional use of herbal teas is also very common. acknowledgements. the authors thank the ministry of education, science and technological development of the republic of serbia for financial support (grant no: 451-03-68/2020-14/ 200178, 451-03-9/2021-14/200113). we owe a special thanks to the adonis pharmacy (https:// www.adonisapoteka.rs), which made this research possible. references agency for drugs and medical devices of serbia 2021. national drug registry (belgrade). barnes, j. 2012: adverse drug reactions and pharmacovigilance of herbal medicines. in: talbot, j., aronson, j.k. (eds.), stephens’ detection and evaluation of adverse drug reacrions: principles and practice, willeyblackwell, chichester. dimitrijević, m., catić-đorđević, a., stefanović, n., pavlović, d., veličković-radovanović, r. 2014: self-medication in primary healthcare in the teritiry of the city of niš. acta medica medianae, 53(3): 19-24. heinrich, m., barnes, j., prietogracia, j., gibbons, s., williamson, e. 2018: fundamentals of pharmacognosy and phytotherapy. 3rd edition. elsevier. janaćković, p., gavrilović, m., savić, j., marin, p.d., dajić stevanović, z. 2019: traditional knowledge on plant use from negotin krajina (eastern serbia): an ethnobotanical study. indian journal of traditional knowledge, 18(1): 25–33. jarić, s., mačukanović-jocić, m., đurđević, l., mitrović, m., kostić, o., karadžić, b., pavlović, p. 2015: an ethnobotanical survey of traditionally used plants on suva planina mountain (southeastern serbia). journal of ethnopharmacology, 175: 93–108. luketina-šunjka, m., rančić, n., mihailović, n., radević, s., dagović, s., jakovljević, m. 2020: sealth self-evaluation of complementary and alternative medicine users in serbia: cross-sectional national study. acta medica medianae, 59(4): 3442. matejić, j., stefanović, n., ivković, m., živanović, n., marin, d.p., džamić, m.a. 2020: traditional uses of autochthonous medicinal and ritual plants and other remedies for health in eastern and southeastern serbia. journal of ethnopharmacology, 261: 113186. petrović, s., kukić-marković, j., pavlovićdrobac, m. 2012: biljni lekoviti proizvodi: uslovi za bezbednu primenu. archive of pharmacy, 62(2): 119-35. public health institute “timok” zaječar, 2020: analysis of the health status of the population of zaječar district in the period from 2015 to 2019 (zaječar). http://www.zavodzajecar.rs/images/pdf/ analizaokrugzajecar2015-2019.pdf public health institute niš, 2017: analysis of the health status of the population of the municipality of sokobanja in the period from 2012 to 2016 (niš). http://www.izjz-nis.org.rs/ d o w n l o a d / i n f o r m a t i k a / 2 0 1 7 % 2 0 a n a l i z a % 2 0 zdravstvenog%20stanja%20stanovnistva%20 opstine%20sokobanja%20%202012-2016.pdf public health institute niš, 2017: analysis of the health status of the population of the municipality of aleksinac for 2015 (niš). sam, s. 2019: importance and effectiveness of herbal medicines. journal of pharmacognosy and phytochemistry, 8(2): 354-357. statistical office of the republic of serbia, 2016. municipalities and regions in the republic of serbia 2016 (belgrade). https://pod2.stat.gov.rs/ objavljenepublikacije/g2016/pdf/g20162020.pdf statistical office of the republic of serbia, 2017. municipalities and regions in the republic of serbia 2017 (belgrade). https://publikacije.stat.gov.rs/ g2017/pdf/g201713044.pdf statistical office of the republic of serbia, 2020. municipalities and regions in the republic of serbia 2020 (belgrade). https://publikacije.stat.gov.rs/ g2020/pdf/g202013047.pdf zhang, l. 2018: pharmacovigilance of herbal and traditional medicines. in: bate, a. (ed.), evidencebased pharmacovigilance clinical and quantitative aspects, springer nature, humana press. zlatković, b.k., bogosavljević, s.s., radivojević, a.r., pavlović, m.a., 2014: traditional use of the native medicinal plant resource of mt. rtanj (eastern serbia): ethnobotanical evaluation and comparison. journal of ethnopharmacology, 151: 704–713. 111 biologica nyssana ● 12 (2) december 2021: 87-111 matejić et al. ● frequency of herbal medicinal products use in southeastern serbia anticancer compounds from medicinal plants biologica nyssana 5 (1)  september 2014: 11-15 šarac, z. et al.  application of canonical discriminant analysis… 11 original article received: 28 june 2014 revised: 8 july 2014 accepted: 10 september 2014 application of canonical discriminant analysis in differentiation of natural populations of pinus nigra in serbia based on terpene composition zorica šarac 1* , srdjan bojović 2 , biljana nikolić 3 , bojan k. zlatković 1 , and petar d. marin 4 1 university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia 2 university of belgrade, institute for biological research “siniša stanković”, boulevard despota stefana 142, 11060 belgrade, serbia 3 university of belgrade, institute of forestry, kneza višeslava 3, 11000 belgrade, serbia 4 university of belgrade, faculty of biology, institute of botany and botanical garden “jevremovac“, studentski trg 16, 11000 belgrade, serbia * e-mail: saraczorica@gmail.com abstract: šarac, z., bojović, s., nikolić, b., zlatković, b., marin, p.: application of canonical discriminant analysis in differentiation of natural populations of pinus nigra in serbia based on terpene composition. biologica nyssana, 5 (1), september 2014: 11-15. the canonical discriminant analysis (cda) was performed in order to check the hypothesis of chemical separation infraspecific taxa of pinus nigra j.f. arnold (ssp. nigra, var. gocensis, ssp. pallasiana, and var. banatica) in serbia based on variability of the needle terpenes. the cda, which maximizes variations between a priori groups, showed division of seven native p. nigra populations into three groups, which belong to three taxonomically recognized taxa (ssp. nigra, ssp. pallasiana, and var. banatica). the most important characters in discrimination were (e)-caryophyllene, α-humulene, terpinolene, germacrene d, αpinene, and myrcene. the individuals of pallasiana group were poorer in (e)-caryophyllene and terpinolene and richer in α-humulene contrary to the individuals of nigra group. the individuals of banatica group had the highest content of α-pinene and myrcene. the obtained results were compared with recently published data on chemodiversity of p. nigra infraspecific taxa on the territory of serbia. key words: canonical, chemotaxonomy, discriminant analysis, needle terpenes, pinus nigra. apstract: šarac, z., bojović, s., nikolić, b., zlatković, b., marin, p.: primena diskriminantne kanonijske analize u diferencijaciji autohtonih populacija pinus nigra u srbiji na osnovu sastava terpena. biologica nyssana, 5 (1), september 2014: 11-15. diskriminatna kanonijska analiza (cda) izvedena je u cilju provere hipoteze o fitohemijskom razdvajanju infraspecijskih taksona pinus nigra j.f. arnold (ssp. nigra, var. gocensis, ssp. pallasiana i var. banatica) u srbiji na osnovu varijabilnosti terpena u četinama. cda, koja naglašava varijabilnost između a priori definisanih grupa, pokazala je rezdvajanje sedam autohtonih p. nigra populacija u tri grupe (ssp. nigra, ssp. 5 (1) • september 2014: 11-15 biologica nyssana 5 (1)  september 2014: 11-15 šarac, z. et al.  application of canonical discriminant analysis… 12 pallasiana i var. banatica). najvažniji karakteri u diskriminaciji bili su (e)-kariofilen, α-humulen, terpinolen, germakren d, α-pinen i mircen. individue grupe pallasiana siromašnije su u sadržaju (e)-kariofilena i terpinolena, a bogatije u sadržaju α-humulena, suprotno od individua grupe nigra. individue grupe banatica imaju veći sadržaj α-pinena i mircena. dobijeni rezultati upoređeni su sa nedavno objavljenim podacima o hemodiverzitetu p. nigra infraspecijskih taksona na teritoriji srbije. ključne reči: kanonijska, hemotaksonomija, diskriminantna analiza, terpeni u ;etinarima, pinus nigra. introduction pinus nigra j.f. arnold (pinaceae) is a species of the tertiary origin belonging to the group of taxa typical for mediterranean and submediterranean coniferous forests (b o g u n i c et al., 2007). it is one of the widespread, morphologically as well as taxonomically very variable pine species in europe (g a u s s e n et al. 1993), with highly fragmented distribution range that extends from north africa through the northern mediterranean and eastwards to the black sea. in the flora of serbia, j o v a n o v i ć (1992) recognized two subspecies of black pine that can be geographically differentiated, each further divided into several varieties. generally, in the western, southwestern and central parts of serbia, p. nigra ssp. nigra is widespread, within which a distinct variety is recognized (var. gocensis đ o r đ e v i ć ), while in eastern serbia p. nigra ssp. pallasiana (d. don) holmboe has an extremely narrow distribution range. namely, crimean black pine (p. nigra ssp. pallasiana) was only recorded in southeastern serbia (r a j e v s k i , 1950) on the mountain crnook (jarešnik). apart from this, in eastern and northeastern serbia three additional populations of black pine are known, which j o v a n o v i ć (1992) describes as var. banatica georg. et ion. within ssp. pallasiana. the chemodiversity aspect of black pine on the territory of serbia has been addressed concerning n-alkane (b o j o v i ć et al., 2012) and terpene (š a r a c et al., 2013) variability. in the second study, 58 essential-oil components were identified in the needles of 195 trees from seven populations. unlike principal component analyses (pca), which showed an overlap of all populations, cluster analyses (ca) separated analysed samples into three basic groups: the first group consisted of populations from western (banjska stena, omar, zmajevački potok), southwestern (priboj) and central serbia (goč) (considered as ssp. nigra group), the second of population from southeastern serbia (jarešnik) (ssp. pallasiana group), and the third of population from northeastern serbia (lazareva reka), which had the most distinct oil composition (ssp. banatica group) (š a r a c e t a l ., 2013). bearing this in mind, the aim of the present study was to check the hypothesis of chemical separation of infraspecific taxa of p. nigra from serbia by performing a canonical discriminant analysis (cda). this method maximizes variations between a priori defined groups and thus characterize the degree of divergence among analyzed populations (j a m e s & m c c u l l o c h , 1990). also, cda was performed in order to determine the relative importance of terpenes as discriminators between a priori groups. material and methods plant material the samples from seven typical populations of p. nigra infraspecific taxa i banjska stena, ii omar, iii zmajevački potok (ssp. nigra), iv priboj-crni vrh, v goč-gvozdac (var. gocensis), vi jarešnik (ssp. pallasiana), and vii lazareva reka (var. banatica), growing wild in serbia were analyzed. details about sampling, locations and ecological conditions of the selected populations, numbering of populations, the extraction of the essential oils, the gc-fid and gc/ms analyses, and the identification of terpenes have been reported previously (š a r a c e t a l ., 2013). statistical analysis the cda was carried out in order to check the hypothesis that the analyzed sample was composed of discrete groups, which are chemically differentiated one from other. from the total data set of 75 original compounds (for details s e e š a r a c e t a l . , 2013), 14 compounds were selected for cda, viz., α-thujene, α-pinene, camphene, β-pinene, myrcene, limonene, (e)-β-ocimene, terpinolene, (e)caryophyllene, α-humulene, γ-muurolene, germacrene d, δ-cadinene, and germacrene d-4-ol. the 61 compounds which were found in traces (content <0.5%, according to r u d l o f f e t a l . , 1975; l i e u t i e r e t a l ., 1991) were excluded from further analysis. the cda was computed on the selected data set (195 samples x 14 variables) with a priori defined groups. statistical analyses were performed using the software statistica 5.1 (s t a t s o f t , 1997). biologica nyssana 5 (1)  september 2014: 11-15 šarac, z. et al.  application of canonical discriminant analysis… 13 results and discussion the cda based on individuals from seven native p. nigra populations showed that the first two discriminant functions participated in 79.6% of the total discrimination, of which the first function with 64.7% (tab. 1). the first discriminant function is approximately equal determined by the content of terpinolene, α-thujene, (e)-caryophyllene, α-pinene, and germacrene d. the second function is mostly defined by the content of germacrene d, α-pinene, and β-pinene (tab. 1). table 1. standardized coefficients for the first two canonical axes (ca) of variation in terpene variables from the discriminant functional analysis of 7 a priori groups. variables ca1 ca2 α-thujene -1.087 0.404 α-pinene 1.017 -1.823 camphene -0.408 -0.482 β-pinene 0.829 -1.618 myrcene 0.683 0.643 limonene -0.086 -0.408 (e)-β-ocimene 0.498 -0.494 terpinolene 1.260 -0.758 (e)-caryophyllene 1.053 -0.140 α-humulene -0.880 -0.840 γ-muurolene 0.016 -0.051 germacrene d 1.010 -2.305 δ-cadinene 0.729 -0.230 germacrene d-4-ol 0.432 -0.959 eigenvalue 1.493 0.344 % explained variation 64.7 79.6 the first discriminant function mainly separated population vi while the second function separated population vii from the other populations (tab. 2, fig. 1). the cda with 7 populations was correctly classified only 53% of individuals on average (classification matrix values, data from the program). however, its results are sufficient to assume the existence of three groups of individuals: (1) populations i, ii, iii, iv, and v which can be designated as ssp. nigra, (2) population vi as ssp. pallasiana, and (3) population vii as ssp. banatica, in accordance with previous results of terpene analysis (š a r a c e t a l . , 2013). however, departing from the first group of individuals (ssp. nigra), population v was partially overlapped with the second group (ssp. pallasina) by axis 1. differentiation suggestion obtained from seven populations was the inducement for performing the cda with three a priori defined groups. table 2. means of canonical variables. population ca1 ca2 i 1.414 -0.241 ii 0.481 -0.247 iii 0.468 -0.149 iv 0.881 -0.106 v -0.654 0.041 vi -2.366 -0.273 vii -0.053 1.988 fig. 1. canonical discriminat analysis (cda) based on the content of 14 terpenes isolated from 195 pinus nigra samples of seven populations (i-vii). the cda based on individuals from three groups (taxa) showed that the first discriminant function explained 73.3% and the second 26.7% of the total discrimination (tab. 3). the first function was mostly determined by the content of (e)caryophyllene, α-humulene and terpinolene (tab 3). the second function was mainly defined by the content of germacrene d, α-pinene, and myrcene. the first discriminant function separated nigra and pallasiana group, while the second function banatica group from the other groups (fig 2). the individuals of pallasiana group had lower content of (e)-caryophyllene and terpinolene and higher content of α-humulene (considering that the majority of individuals was on the right side of the figure). the individuals of nigra group had higher content of (e)-caryophyllene and terpinolene, and lower content of α-humulene (the majority of individuals was on the left side of the figure). the individuals of banactica group had higher content of α-pinene and myrcene. the cda with 3 a priori defined gropus was correctly classified 88% of individuals on average (classification matrix values, data from the program). biologica nyssana 5 (1)  september 2014: 11-15 šarac, z. et al.  application of canonical discriminant analysis… 14 table 3. standardized coefficients for the first two canonical axes (ca) of variation in terpene variables from the discriminant functional analysis of 3 a priori groups. variables ca1 ca2 α-thujene 0,741 -0,436 α-pinene -0.548 1,682 camphene 0,496 0,411 β-pinene -0,464 1,523 myrcene -0,739 -0,621 limonene 0,329 0,281 (e)-β-ocimene -0,348 0,464 terpinolene -0,781 0,785 (e)-caryophyllene -1,069 0,294 α-humulene 0,942 0,670 γ-muurolene 0,177 -0,102 germacrene d -0,477 2,169 δ-cadinene -0,719 0,345 germacrene d-4-ol -0,128 0,832 eigenvalue 0,922 0,336 % explained variation 0,733 1,00 fig. 2. canonical discriminat analysis (cda) based on the content of 14 terpenes isolated from 195 pinus nigra samples of three groups (ssp. nigra, ssp. pallasiana and var. banatica). in accordance with previously published results of ca (š a r a c e t a l ., 2013), cda confirmed that based on the terpene variability in native populations of black pine in serbia, three discrete groups can be distinguished. the first group (nigra) consisted of populations described as var. nigra (populations i-iii) and var. gocensis (populations iv and v) within ssp. nigra according to existing morho-anatomical and phytocoenological studies (j o v a n o v i ć , 1992). also, our very recent (š a r a c et al. (2013) and present study, showed that populations determined as var. gocensis exhibited a moderate tendency of separation from the first group (nigra) (population iv in ca and population v in cda). based on n-alkanes variability, the populations assigned as var. gocensis (iv and v) showed even grater tendency of splitting (bojović et al., 2013). population v has been classified into the first group (nigra), characterized as chemotype 1 by b o j o v i ć et al. (2012), while population iv belonged to the second group (pallasiana), assigned as chemotype 2. it was assumed that populations considered as var. gocensis could be transitional form between subspecies nigra in the west and subspecies pallasiana in the southest of serbia. taxonomic position of var. gocensis within p. nigra complex is still not completely resolved. this variety was primarily described by đ o r đ e v i ć (1931) from the slopes of the mountain goč in central serbia. later, v i d a k o v i ć (1955), determined the wider distribution of this variety, and based on the leaf anatomy raised it to the subspecies level (ssp. gocensis vid.). comparing to the typical var. nigra it is different in some tree traits (bark of mature trees had prominent transverse and longitudinal furrows, resembling the bark of pinus heldreichii h. christ), as well as in needles structure. however, according to results of our analyses there is no such clear phytochemical distinction between var. gocensis and typical var. nigra. the second group is composed mainly of trees of population vi from southeastern serbia belonging to ssp. pallasiana. the presence of crimean pine in serbia was originally reported by a d a m o v i ć (1909), but without precise data on its distribution. r a j e v s k i (1950) was the first who established the position of this population within the territory of serbia. the single enclava of crimean pine is situated at the locality jarešnik in the surroundings of bosilegrad growing on the crystalline slates. the population in serbia represents the northernmost disjunction of the crimean black pine area spreading from the southern parts of balkan peninsula. based on , nalkane and terpene composition, ssp. pallasiana is clearly distinguished as separate group (taxon and chemotype) from ssp. nigra according to this and previous studies (bojović et al., 2012; šarac et al., 2013). finally, the third group consisted of trees from population lazareva reka, determined as var. banatica within ssp. pallasiana (j o v a n o v i ć , 1992). this taxon is previously described in romania (domogled valea cernei) and according to the literature data the black pine populations from northwestern romania have a very controversial taxonomic position (b o ş c a i u & b o ş c a i u , 1999). in the second edition of flora europea, g a u s s e n et al. (1993) treated these populations as an independent species, i.e., p. banatica. analysis biologica nyssana 5 (1)  september 2014: 11-15 šarac, z. et al.  application of canonical discriminant analysis… 15 of the chemodiversity of the black pine populations from serbia, based on both n-alkane and terpene composition, supports the view that var. banatica should be considered as distinct subspecies or even species (b o j o v i ć et al., 2012; š a r a c et al., 2013). conclusion the canonical discriminant analysis (cda) of recently reported terpene composition of p. nigra in serbia (š a r a c et al., 2013), also showed division of populations into three discrete groups. the cda confirmed the attitude that terpenes are good taxonomic markers at infraspecific level in the pinales. nevertheless, all these assumptions should be checked by further extending studies to the entire balkan peninsula and the mediterranean region, and especially including the classical locality (locus classicus) of p. nigra var. banatica from romania (domogled valea cernei). also, detail molecular analysis is needed to clarify relationships witin this extremely variable and complex taxon. references adamović, l. 1909: die vegetationsverhältnisse der balkanländer, leipzig, 258-262. bogunic, f., muratovic, e., ballian, d., siljakyakovlev, s., brown, s.c. 2007: genome size stability of five subspecies of pinus nigra arnold s.l. environmental and experimental botany, 59: 354-360. bojović, s., šarac, z., nikolić, b., tešević, v., todosijević, m., veljić, m., marin, p. d. 2012: composition of n-alkanes in natural populations of pinus nigra from serbia – chemotaxonomic implications. chemistry & biodiversity, 9: 27612774. boşcaiu, n., boşcaiu, m. 1999: on the presence of pinus nigra subsp. pallasiana in romania. wissenschaftliche mitteilungen aus dem niederösterreichischen landesmuseum, 12: 2124. đorđević, p. 1931: pinus nigra arn. var. gočensis, n. var. izdanje ministarstva šuma i rudnika, beograd. gaussen, h., heywood, v.h., charter, a.o. 1993. pinus l.. in: tutin, t.g., heywood, v.h., burges, n.a., valentine, d.h., walters, s.m., webb, b.a. (eds.), flora europea 1: 40-44, cambridge university press, cambridge. james, f.c., mcculloch, c.e. 1990: multivariate analysis in ecology and systematics: panacea or pandoras box?. annuual reviews of ecology and systematics, 21: 129-166. jovanović, b. 1992. p. nigra arn. in: sarić, m. (ed.), flora srbije 1: 200-202, srpska akademija nauka i umetnosti, beograd. lieutier, f., berryman, a.a., millstein, j.a. 1991: preliminary study of the monoterpene response of three pines to ophiostoma clavigerum (ascomycetes: ophiostomatales) and two chemical elicitors. annals of forest science, 48: 377-388. rajevski, l. 1950: nalazište pinus nigra arn. var. pallasiana (lamb.) u okolini bosilegrada. zbornik radova instituta za ekologiju i biogeografiju, srpska akademija nauka i umetnosti, beograd. statsoft 1997. statistica for windows, version 5.1. statsoft inc., tulsa. šarac, z., bojović, s., nikolić, b., tešević, v., đorđević, i., marin, p. d. 2013: chemotaxonomic significance of the terpene composition in natural populations of pinus nigra j. f. arnold from serbia. chemistry & biodiversity, 10: 1507-1520. vidaković, m. 1955: značenje anatomske građe iglica kod svojta crnog bora u jugoslaviji. šumarski list, zagreb, 79: 244-253. von rudloff, e. 1975: volatile leaf oil analysis in chemosystematic studies of north america conifers. biochemical systematics and ecology, 2: 131-167. biologica nyssana 5 (1)  september 2014: 11-15 šarac, z. et al.  application of canonical discriminant analysis… 16 balo et al. 2021, biologica nyssana 12(1) 12 (1) september 2021: 71-78 doi: 10.5281/zenodo.5523045 bats assemblages in awasian water forest reserve, tandag city, surigao del sur, philippines original article allyka eve g. balo college of teacher education, surigao del sur state university-main campus, rosario, tandag city, surigao del sur, philippines allykaevebalo@gmail.com mark lee o. sabandal college of teacher education, surigao del sur state university-main campus, rosario, tandag city, surigao del sur, philippines malesab017@gmail.com johnny b. maglasang college of teacher education, surigao del sur state university-main campus, rosario, tandag city, surigao del sur, philippines johnnymaglasang12345@gmail.com lovely jean b. cosmiano college of teacher education, surigao del sur state university-main campus, rosario, tandag city, surigao del sur, philippines cosmianolovelyjean14@gmail.com arturo g. gracia jr. college of teacher education, surigao del sur state university-main campus, rosario, tandag city, surigao del sur, and department of natural sciences and mathematics, college of arts and sciences, surigao del sur state university-main campus, rosario, tandag city, surigao del sur, philippines artzgracia@gmail.com (corresponding author) received: mart 19, 2020 revised: october 11, 2020 accepted: june 23, 2021 abstract: bats are an integral part of the ecosystem that plays a vital role in its stability. however, their existence is being threatened by uncontrollable anthropogenic activities, and its basic ecological information, especially in eastern mindanao, is limited. thus, the study was conducted to evaluate the assemblage of bats in one of the underexplored sites of eastern mindanao biodiversity corridor. mist-netting for a total of 9 net nights was carried out to document the species. a total of 10 species was observed. among these species, cynopterus brachyotis had the highest relative abundance comprising 53% of the population. species diversity was relatively higher in the secondary mixed-dipterocarp forest with a shannon-weiner diversity index (h’) of 1.41 compared to the primary dipterocarp forest (h’=1.19). the species ptenochirus jagori and rhinolophus subrufus were the only bats observed with aggregated population distribution pattern. among the recorded species, six (60%) were assessed as endemic comprising 5 philippine and 1 mindanao endemic, while eonycteris robusta and rhinolophus subrufus were the near-threatened species recorded. based on the results, awasian water forest reserve houses an array of bat species with a high percentage of endemicity. key words: assessment, diversity, endemism, volant mammals apstract: zajednica slepih miševa u rezervatu awasian water forest, tandag, surigao del sur, filipini slepi miševi su integralni deo ekosistema koji igraju vitalnu ulogu u njegovoj stabilnosti. ipak, njihovo postojanje je ugroženo nekontrolisanim antropogenim aktivnostima i osnovne ekološke infomacije o njima su ograničene, posebno u istočnom minadau. zbog toga, sprovedeno je istraživanje kako bi se procenio raspored slepih miševa na jednoj od neistraženih lokacija koridora biodiverziteta u istočnom minadau. kako bi se dokumentovale vrste, izvršeno je postavljanje mreže tokom ukupno 9 noći. utvrđeno je ukupno 10 vrsta. među njima, cynopterus brachyotis imao je najveću relativnu učestalost sačinjavajući 53% populacije. diverzitet vrsta bio je relativno veći u sekundarnoj mešovitoj dipterokarpnoj šumi sa shannon-weiner indeksom diverziteta (h) od 1,41 u poređenju sa primarnom dikterokarpnom šumom (h=1,19). vrste ptenochirus jagori i rhinolophus subrufus su jedini slepi miševi čije su populacije bile grupisanog rasporeda distribucije. među zabeleženim vrstama, šest (60%) procenjene su kao endemične od koji 5 pripadaju filipinskim a 1 mindanao endemitima, dok vrste eonycteris robusta i rhinolophus subrufus predstavljaju skoro ugrožene vrste. na osnovu rezultata, rezervat awasian water forest je stanište za spektar vrsta slepih miševa sa visokim procentom endemizma. ključne reči: procena, diverzitet, endemizam, leteći sisari © 2021 balo et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 71 introduction the clade chiroptera includes two extant clades, megachiroptera (old world fruit bats) and microchiroptera (echolocating bats). bats are considered as an ecological indicator because they control pests, pollinate fruit trees, and regenerate forests (kasso and balakrishnan, 2013). in the philippines, bats are deemed as one of the most diverse mammals with at least 80 total extant species comprised of 26 megachiropteran and 54 microchiropteran species (sedlock et al., 2020). of these, approximately 40% are reported to be endemic. however, uncontrolled anthropogenic activities, habitat destruction, and disturbances are among the threats that threaten these taxa (quibod et al., 2019). the philippines is previously known for its dense forest in the early 20th century, with a reported forest cover of 95% of the total landmass. however, the continuous deforestation resulted in massive forest denudation, which is estimated to be around 80 to 90% (butler, 2014). bats are highly forest-dependent animals, and changes in their habitat could decrease their population and lead to eventual extinction (mickleburgh et al., 2002). this report is further supported by murphy and romanuk (2014) that mammalian species diversity, abundance, and along its mean biomass tend to decrease with increasing human disturbance and continuous habitat loss. for these reasons, the philippines has been identified as an important area for bat diversity that prioritizes conservation efforts globally (tanalgo and hughes, 2018). awasian water forest reserve is one of the watersheds of mt. hilong-hilong, a biodiversity corridor that lies between the provinces of agusan and surigao in the north eastern mindanao. although reported to exhibit rich flora and fauna, these claims are mainly based on scientific expeditions done on the western side of the mountain ecosystem (agusan); and only a few to none scientific data can be retrieved to represent the eastern side (surigao). the availability of ecological data is considered vital since it serve as a basis for setting conservation priorities and guidelines (goodman and benstead, 2005; mallari, 2009). thus, this study was conducted to address the scarcity of scientific information on bat community in the underexplored area of mt. hilong-hilong; and provide pragmatic findings that can be utilized for a better understanding of bat assemblage in the area. materials and methods place and duration of the study the study was conducted in the awasian water forest reserve located at barangay awasian, tandag city surigao del sur philippines (fig. 1). the site is geographically located at 9°04’28.9”n and 126°08’34.7”e. the site is one of the watersheds of mt. hilong-hilong, situated in the south-eastern part of the mountain ecosystem. the study was conducted from october 1 to 9, 2017, covering nine days of sampling. the site’s topography is generally plain, rolling, and gently sloping. the climatic condition in the area falls under the philippines’ type ii climate condition, which is characterized by rainfall distributed throughout the year, with a negligible short dry season (pagasa, 2011). establishment of sampling stations and habitat assessment two 1-km transect lines were established in the study site with an aerial distance from each other of 200 meters. the first transect was considered as station 1 that cut-across the dipterocarp forest. it was situated at an elevation that extends from 50 to 150 meters above sea level (masl). the dominant plant species were mixtures of shorea spp., ficus spp., and cultivated fruit trees like lansium domesticum, durio zibithenus, and artocarpus odoratissimus. the soil-litter was thin and dry. the area’s distance to the nearest water body (stream) was around 5 to 100 meters. during the sampling period, the temperature ranges from 24 to 30 °c, with a general weather condition of calm-night with a clear sky. the second transect was designated as station 2, established along the secondary mixed-dipterocarp forest. it was situated at an elevation of 100 to 250 m a.s.l. with a slightly-closed to closed canopy cover. the station’s dominant plant species were shorea spp. of the family dipterocarpaceae and ficus spp. of family moraceae. soil-litter was dry but thicker as compared with station 1. the area’s distance to the nearest stream was around 700 m, and 10 to 100 m from the small spring and stagnant canals. the tem72 biologica nyssana ● 12 (1) september 2021: 71-78 balo et al. ● bats assemblages in awasian water forest reserve, tandag city, surigao del sur, philippines fig. 1. location map of mt. hilong-hilong and spot map of the study site. (birdlife international, 2020) 73 perature during the sampling period ranged from 20 to 24 °c, with a general weather condition of gloomy with intermittent rain. data collection and identification along these transects, 21 mist-nets with a dimension of 4.5 meters long and 12 meters wide with a mesh size of 33 mm were established to capture the bats. checking of nets was performed from 6:00 pm to 5:00 am with 30 minutes to 1-hour interval to monitor if there were entangled bats and avoid possible mortality. captured species were placed into a cloth bag to prevent further stress on the organism and were brought to the processing area for taxonomic examination. the morphological metrics, which include ear (e), forelimb (fl), hindfoot (hf), tail (t), total length (tl), total body length (tbl), and teeth formation, were noted. these morphological metrics served as the bases for the identification of bat species. the dichotomous key for philippine bats by ingle and heaney (1992) was used for species identification and classification. after taking all the taxonomic information, bats were fed with a sucrose solution and were released back to their captured habitat. however, upon the release, markings for each individual were considered to avoid count duplication if recaptured. on the other hand, dead bat individuals were prepared as voucher specimens and were preserved directly in a 10% formalin solution. data analysis the shannon-weiner diversity index (h’) was used as the primary index to represent bats’ diversity. along with this, the species diversity equitability (hmax) or the highest possible diversity index that can be obtained based on the given data set was also determined to assess how close the h’ index to the theoretical value. moreover, the simpson diversity index (1-d) and species evenness (j’) were calculated to assess the level of species dominance and how evenly distributed the community’s population. with an interpretation that if the obtained 1-d and j’ values are closer to the value of one, means more diverse, and if it is closer to zero, indicates the opposite. the species rarefaction curve was generated to represent the estimated species richness of bats in the sampled area and assess the bat sampling adequacy. for determining the species distribution, the statistical analysis – chi-square was used to evaluate if the bat species’ populations are either random or aggregated. the level of significance used was 0.05 percent. the distribution probability values that are greater than the level of significance was given a random remark. in contrast, those with below the significance value are considered to have an aggregated distribution pattern. all these analyses were done using the biodiversity professional software 2.0 by macleece (1997). all ecological and conservation assessments were based on the international union conservation for nature (iucn) redlist of 2020. results and discussion species richness and diversity a total of 196 individuals of bats belonging to three families and nine genera representing ten species were recorded. the species richness is comparatively lower than the records in loboc watershed, mt. irid, mt. kanlaon, mt. malindang, mt. matutum, mt. palali, polilo islands, and sikatuna protected landscape. the discrepancy of the number of species observed is attributed to the sample size. the aforementioned studies had a larger sample area that crossed a broader elevational gradient and various vegetation types. on the other note, the result is observed to have a higher richness as compared to the records in bagumbayan, bega watershed, lowland forest of upland cavite, mt. apo, mt. kalatungan, mt. kitanglad, mt. natib, and cagayan de oro river. the findings even equate with the number of species observed in the expansion site of mt. hamiguitan in davao oriental and clarin river in misamis occidental even though these studies also covered a more extensive sampling area with at least four sampling stations (tab. 1). with this, it could entail that awasian water forest reserve houses more bats with respect to the ratio and proportion between the species observed and the sampled area. the findings could also imply that the area has better ecological support for bat assemblage since it can hold various species that even surpasses some of those major mountain ecosystems in the philippines like mt. apo and mt. kalatungan. a condition that conforms with the findings of mohagan et al. (2015) that bat richness tends to be higher in a forest ecosystem with more evident ecological support. not to mention that probable new species could be added to the list if the sampling effort was extended as the species rarefaction depicts (fig. 2). however, the chance is narrowing down as the data saturation is gradually flattening. as for diversity measures, the secondary mixeddipterocarp forest showed to have a consistently better result in all the calculated diversity analysis compared to the dipterocarp forest (fig. 3). this result is attributed to a more complex plant structure that results in broader food options for bats. it was noted that at the course of the study, the explicitly observed fruit trees in the secondary mixed-dipterocarp forest biologica nyssana ● 12 (1) september 2021: 71-78 balo et al. ● bats assemblages in awasian water forest reserve, tandag city, surigao del sur, philippines 74 like l. domesticum, d. zibithenus, and a. odoratissimus were fruiting, thus, possibly attracting more species and population of bats in the said habitat. not to mention that the dominant bat-guild observed was fruit bats; hence, the likelihood for the species to congregate in the area is plausible. the observation aligns with the report of hodgkison and balding (2004) and shafie et al. (2011) that bat diversity in a particular area is highly influenced by food availability. meanwhile, the overall all bat diversity in the area was revealed to be moderate. among the species, cynopterus brachyotis and ptenochirus jagori were the well-documented bats, wherein the two species comprised 53 and 25 percent of the population, respectively (fig. 4). the result conforms with various studies conducted in the philippine forests (tab. 1). according to tan et al. (1998), c. brachyotis is widely distributed in the southeast asian region and is common in the philippine forest. the species also has a wide range of habitat preferences, extending from primary forest to disturbed habitats. while the species p. jagori is a common philippine endemic bat observed in the lowland forests (sedlock et al., 2008; iucn, 2020). table 1. summary table of some of the bat studies conducted in the philippines site species richness endemism (%) dominant species observed references bagumbayan, sultan kudarat 8 38 cynopterus brachyotis tanalgo (2017) bega watershed, agusan del sur 8 50 cynopterus brachyotis monteclaro and nuñeza (2015) cagayan de oro river, misamis oriental 8 25 cynopterus brachyotis lobite et al. (2013) clarin river, misamis occidental 10 40 ptenochirus jagori del socorro et al. (2018) loboc watershed, bohol 15 40 cynopterus brachyotis joe et al. (2012) lowland forest, upland cavite 6 67 * lagat and causaren (2018) mt. apo, north cotabato 8 38 cynopterus brachyotis achondro et al. (2014) mt. hamiguitan, davao oriental 10 40 cynopterus brachyotis amoroso et al. (2019) mt. irid, southern sierra madre 16 19 cynopterus brachyotis balete et al. (2013) mt. kalatungan, bukidnon 6 50 haplonycteris fischeri mohagan et al. (2018) mt. kanlaon, negros occidental 23 35 cynopterus brachyotis deligero et al. (2015) mt. kitanglad, bukidnon 4 75 alionycteris paucidentata mohagan et al. (2015) mt. malindang, misamis occidental 19 47 cynopterus brachyotis nuñeza et al. (2006) mt. matutum, saranggani 15 47 cynopterus brachyotis nuñeza et al. (2015) mt. natib, bataan province 9 33 cynopterus brachyotis rickart et al. (2013) mt. palali, caraballo mountains 12 42 otopteropus cartilagonodus alviola et al. (2011) polilo islands, calabarzon 25 24 * alviola (2010) sikatuna protected landscape, bohol 11 44 cynopterus brachyotis van vegchel (2003) fig. 2. species rarefaction plot in awasian water forest reserve biologica nyssana ● 12 (1) september 2021: 71-78 balo et al. ● bats assemblages in awasian water forest reserve, tandag city, surigao del sur, philippines species distribution pattern across habitats, endemicity, and conservation status out of the ten bat populations, the population of p. jagori (fruit bat) and r. subrufus (insectivorous bat) were noted as aggregated (p<0.05). on the other hand, the rest of the bat populations were noted as randomly distributed (p>0.05) (tab. 2). the results could suggest two major points: first, for the case of p. jagori and r. subrufus, necessary ecological support like food source is only found in a particular area or habitat and hence, the reason why the populations of the two bats tend to cluster. secondly, as for those bat populations with random distribution, the ecological factors that support the population are sporadically distributed in the area; hence, its population is also randomly distributed. this observation conforms with the report of de jong and ahlen (1991) and kush et al. (2004) that food source and supply are essential indicators for bat’s regional distribution, especially for insectivorous bats. among the ten documented species, five bats are assessed as philippine endemic, and one is noted as mindanao endemic. the assessment leads to com75 table 2. distribution pattern of bat’s population across habitats taxon variance mean chi-sq df. probability remarks pteropodidae cynopterus brachyotis 24.5 52.5 0.4667 1 0.501796 random eonycteris robusta** 4.5 1.5 3 1 0.079335 random haplonycteris fischeri** 0.5 2.5 0.2 1 0.659200 random harpyionycteris whiteheadi** 0.5 1.5 0.3333 1 0.571066 random macroglossus minimus 12.5 7.5 1.6667 1 0.193468 random ptenochirus jagori** 420.5 24.5 17.1633 1 0.000066 aggregated ptenochirus minor* 18 5 3.6 1 0.054660 random rhinolophidae rhinolophus subrufus** 12.5 2.5 5 1 0.023993 aggregated vespertilionidae miniopterus schreibersi 0.5 0.5 1 1 0.318676 random pipistrellus sp. 0.5 0.5 1 1 0.318676 random note: names with a double asterisk (**) are philippine endemic bat species. while names with a single asterisk (*) are mindanao endemic fig. 3. comparative bar graph of shannon-weiner diversity index (h’), maximum diversity index value (hmax), species diversity equitability (h’/hmax), and simpson diversity index (1-d) biologica nyssana ● 12 (1) september 2021: 71-78 balo et al. ● bats assemblages in awasian water forest reserve, tandag city, surigao del sur, philippines prehensive documentation of 60% (6/10) total bat endemicity in the area. this total endemism is observed to be relatively higher than the bat endemism reported in various philippine forests (tab. 1), with an endemism percentage difference that ranges from 10 to 41 percent. thus, suggesting that the area is an ideal abode to various endemic species. meanwhile, among the bats, two species (e. robusta and r. subrufus) are evaluated to be under the near-threatened category based on the iucn 2017 and 2020 assessments. under this criterion, the species are considered to have a decreasing population and are subject to a threatened status if pressures on its existence will be unaddressed. according to iucn (2020), the primary threat that affects these species’ populations is habitat loss. an evident threat in the buffer zones of the watershed. conclusions awasian water forest reserve harbors various species of bats with high species endemicity. the presence of bat species with random distribution patterns is advantageous to the area, for it contributes to a wider dispersion of ecological services like pollination and seed dispersal. near-threatened species’ presence suggests further conservation efforts considering that threats, especially from anthropogenic pressures, are evident. acknowledgment. the authors would like to express its heartfelt gratitude to the department of environment and natural resources (denr), tandag water district (twd), and local government unit of tandag for the permits and logistical support. to mr. gerry bernadas, aldrin dua, jose buaya, jesus verano, and all the local guides for the extended support during the study’s conduct. references achondo, m.j.m.m., casim, l.f., tanalgo, k.c., agduma, a.r., lloyd, b., bretaña, p., mancao, l.s., salem, j.g.s., bello, v.p. 2014: occurrence and abundance of fruit bats in selected conservation areas of north cotabato, philippines. asian journal of conservation biology, 3(1): 3-7. alviola, p.a. 2000: the distribution and ecology of bats in the polillo islands, philippines. wildlife of polillo island, philippines. university of oxford-university of the philippines at los banos polillo, 99. alviola, p.a., duya, m.r.m., duya, m.v., heaney, l.r., rickart, e.a. 2011: mammalian diversity patterns on mount palali, caraballo mountains, luzon. fieldiana life and earth sciences, 2011(2): 61-74. amoroso, v., mohagan, a., coritico, f., laraga, s., lagunday, n., domingo, k. l., colong, r.d., ponce, r. 2019: status of mammals in the expansion sites of the mt. hamiguitan range 76 fig. 4. comparative abundance of bat species in awasian water forest reserve. species name with single asterisk before the scientific name are fruit bats. the rest are insectivorous bats biologica nyssana ● 12 (1) september 2021: 71-78 balo et al. ● bats assemblages in awasian water forest reserve, tandag city, surigao del sur, philippines wildlife sanctuary, mindanao, philippines. journal of environmental science and management, 22(2): 6-12. balete, d.s., heaney, l.r., rickart, e.a. 2013: the mammals of mt. irid, southern sierra madre, luzon island, philippines. nat. mus. phil.: j. nat. hist, 1: 17-31. butler, r. 2014: mongabay rainforet profiles: philippines. https://rainforests.mongabay. com/20philippines.htm de jong, j., ahlén, i. 1991: factors affecting the distribution pattern of bats in uppland, central sweden. ecography, 14(2): 92-96. del socorro, m.m l., enguito, m.r.c., mapiot, e.f. 2018: species diversity of bats in clarin river, misamis occidental, philippines. journal of multidisciplinary research, 7(1): 209-223. deligero, j.a., warguez, d.a., doble, k.j.s., paguntalan, l.m.j., jakosalem, p g.c. 2015: forest bat diversity, abundance and habitat selection in mt. kanlaon natural park, negros island. philippine biodiversity symposium, 1-23. goodman, s.m., benstead, j.p. 2005: updated estimates of biotic diversity and endemism for madagascar. oryx, 39(1): 73-77. hodgkison, r., balding, s.t. 2004: temporal variation in the relative abundance of fruit bats (megachiroptera: pteropodidae) in relation to the availability of food in a lowland malaysian rain forest. biotropica, 36(4):522–533. ingle, n.r., heaney, l.r. 1992: a key to the bats of the philippine islands. fieldiana, zoology, 69: 1-44. iucn. 2017: the iucn red list of threatened species. http://www.iucnredlist.org/. iucn. 2020: the iucn red list of threatened species. http://www.iucnredlist.org/. jose, r.p., labonite, m.a., bullecer, r.c., ancog, a.b., butron, n g., bullecer, r.p. 2012: indigenous knowledge and taxonomy of bats in loboc watershed forest reserve, bohol, philippine. in biology education for social and sustainable development (pp. 127-144). kasso, m., balakrishnan, m. 2013: ecological and economic importance of bats (order chiroptera). isrn biodiversity, 2013. kusch, j., weber, c., idelberger, s., koob, t. 2004: foraging habitat preferences of bats in relation to food supply and spatial vegetation structures in a western european low mountain range forest. folia zoologica, praha, 53(2): 113-128. lagat, r.d., causaren, r.m. 2019: initial terrestrial vertebrate diversity assessment in upland cavite, philippines. philippine journal of systematic biology, 12(2): 70-91. lobite, n.j.s. japos, g.v., lubos, l.c. 2013: a preliminary assessment of the chiropteran fauna of the oro river, cagayan de oro city, philippines. asian journal of biodiversity, 4(1): 119-134. mallari, n.a.d. 2009: maximising the value of ecological and socioeconomic data in support of conservation planning for key understorey bird species in palawan, philippines (doctoral dissertation, phd thesis, manchester, uk: manchester metropolitan university). mcaleece, n., gage, j.d.g., lambshead, p.j.d., paterson, g.l.j. 1997: biodiversity professional statistics analysis software. jointly developed by the scottish association for marine science and the natural history museum london. mickleburgh, s.p., hutson, a.m., racey, p.a. 2002: a review of the global conservation status of bats. oryx, 36(1): 18-34. mohagan, a.b., toledo-bruno, a.g., bonghanoy, a.m., flavia, c., balite, c.f.b. 2018: bat diversity and local conservation initiatives in the montane forest of mt. kalatungan in pangantucan, bukidnon, philippines. journal of biodiversity and envrionmental sciences, 13(5): 57-70, mohagan, a.b., nuneza, o.m., escarlos jr, j.a., gracia jr, a.g., selpa, e.c.t., baguhin, l.j. b., coritico, f.p., amoroso, v.b. 2015: diversity and endemism of terrestrial mammals in four long term ecological research sites in mindanao, philippines. asia life sciences, 24(1): 219-233. monteclaro, r.a.o., nuñeza, o.m. 2015: species diversity of fruit bats in bega watershed, prosperidad, agusan del sur, philippines. journal of biodiversity and envrionmental sciences, 6(4): 12437. murphy, g.e., romanuk, t.n. 2014: a meta‐ analysis of declines in local species richness from human disturbances. ecology and evolution, 4(1), 91-103. nuñeza, o.m., ates, f.b., alicante, a.a., calizoenguito, m.r., toledo-bruno, a.g., labajo, y.i., dejarme, s.m. 2006: vertebrate faunal diversity and relevant interrelationships of critical resources in mt. malindang1. the mt. malingdang experience, 37. nuneza, o.m., non, m.l.p., makiputin, r.c., 77 biologica nyssana ● 12 (1) september 2021: 71-78 balo et al. ● bats assemblages in awasian water forest reserve, tandag city, surigao del sur, philippines oconer, e.p. 2015: species diversity of bats in mt. matutum protected landscape, philippines. journal of biodiversity and environmental sciences, 6(6): 377-390. philippine atmospheric geophysical and astronomic services administration (pagasa)department of science and technology. 2011. climate change in the philippines. funded under the mdgf-1656 strengthening the philippines institutional capacity toadapt to climate change. quibod, m.n.r.m., alviola, p.a., de guia, a p.o., cuevas, v.c., lit jr, i.l., pasion, b.o. 2019: diversity and threats to cave-dwelling bats in a small island in the southern philippines. journal of asiapacific biodiversity, 12(4): 481-487. rickart, e.a., heaney, l.r., balete, d.s., alviola, p.a., duya, m.r.m., duya, m.v., sedlock, j.l. 2013: the mammals of mt. natib, bataan province, luzon, philippines. national museum of the philippines journal of natural history, 1: 31-44. shafie, n.j., sah, s.a. m., latip, n.s.a., azman, n.m., khairuddin, n.l. 2011: diversity pattern of bats at two contrasting habitat types along kerian river, perak, malaysia. tropical life sciences research, 22(2): 13. sedlock, j.l., weyandt, s.e., cororan, l., damerow, m., hwa, s.h., pauli, b. 2008: bat diversity in tropical forest and agro-pastoral habitats within a protected area in the philippines. acta chiropterologica, 10(2): 349-358. sedlock, j.l., heaney, l.r., balete, d.s., ruedi, m. 2020: philippine bats of the genus kerivoula (chiroptera: vespertilionidae): overview and assessment of variation in k. pellucida and k. whiteheadi. zootaxa, 4755(3): 454-490. tan k.h, zubaid a, kunz t.h. 1998: food habits of cynopterus brachyotis (muller) (chiroptera: pteropodidae) in peninsular malaysia. journal of tropical ecology 14(03): 299-307. tanalgo, k.c., hughes, a.c. 2018: bats of the philippine islands—a review of research directions and relevance to national-level priorities and targets. mammalian biology, 91(1): 46-56. tanalgo, k.c., casim, l.f., tabora, j.a.g. 2017: a preliminary study on bats in a smallscale mining site in southcentral mindanao, philippines. ecological questions, 25: 85-93. van vegchel, f.j.p. 2002. a survey of the bats of rajah sikatuna protected landscape, bohol island, philippines and local attitudes towards them. sylvatrop, the technical journal of philippine ecosystems and natural resources, 13(1-2): 1-14. 78 biologica nyssana ● 12 (1) september 2021: 71-78 balo et al. ● bats assemblages in awasian water forest reserve, tandag city, surigao del sur, philippines jušković et al. 2021, biologica nyssana 12(1) 12 (1) september 2021: 11-21 doi: 10.5281/zenodo.5522956 a comparative anatomical study on two closely related astragalus l. taxa (fabaceae) from the central part of the balkan peninsula original article marina jušković department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, niš, serbia marinaju@pmf.ni.ac.rs (corresponding author) dragana jenačković gocić department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, niš, serbia draganaj@pmf.ni.ac.rs danijela nikolić department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, niš, serbia danid@pmf.ni.ac.rs bojan zlatković department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, niš, serbia bojanzlat@yahoo.com vladimir ranđelović department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, niš, serbia vladar@pmf.ni.ac.rs received: april 29, 2021 revised: june 29, 2021 accepted: september 01, 2021 abstract: the present study involves species a. monspessulanus l. and a. spruneri boiss. section incani dc. from the area of central balkans. due to high polymorphism, this section is taxonomically the most problematic group and delimitations of its species are not completely clear. the main aims of the study were to determine the micro-morphological and anatomical variability of populations of these species based on quantitative characters of leaflet and leaf petiole, and also to show the potential degree of differentiation within species. additional goal was to determine anatomic parameters that may be used in taxonomy of studied species and their encompassing section. the results indicate high variability of analyzed characters and anatomical differentiation of populations of each species. results of statistical analyses indicate that characters which refer to petiole anatomy have higher taxonomic value than characters which refer to leaflet anatomy. anatomical features described in this paper mostly agree with characters found in previous studies of other astragalus species, and they have potential taxonomic significance. key words: astragalus monspessulanus, a. spruneri, anatomical characters, taxonomy apstract: uporedna anatomska istraživanja dva veoma srodna astragalus l. taksona sa centralnog dela balkanskog poluostrva u ovom radu su analizirane vrste a. monspessulanus l. i a. spruneri boiss., sekcije incani dc. sa područja centralnog balkana. sekcija incani dc. predstavlja taksonomski složenu sekciju, sa velikim brojem endemičnih vrsta, u okviru koje, zbog preklapanja morfoloških karaktera i velike fenotipske plastičnosti, odnosi između vrsta još uvek nisu u potpunosti razjašnjeni. glavni cilj rada bio je utvrđivanje mikro-morfološke i anatomske varijabilnosti populacija ovih vrsta na osnovu kvantitativnih karakteristika lamine i lisne drške, kao i prikaz potencijalnog stepena diferencijacije unutar vrsta. cilj je bio i otkriti anatomske karaktere koji se mogu koristiti u taksonomiji proučavanih vrsta, kao i sekcije kojoj one pripadaju. rezultati ukazuju na visoku varijabilnost analiziranih karaktera i anatomsku diferencijaciju populacija svake vrste. rezultati statističkih analiza takođe pokazuju da karakteristike koji se odnose na anatomiju lisne drške imaju veću taksonomsku vrednost od karakteristika koji se odnose na anatomiju lamine. anatomske karakteristike opisane u ovom radu uglavnom se slažu sa karakteristikama opisanim u prethodnim istraživanjima drugih vrsta roda astragalus i imaju potencijalni taksonomski značaj. ključne reči: astragalus monspessulanus, a. spruneri, anatomska diferencijacija, taxonomy introduction the genus astragalus l. is one of the largest genera of vascular plants, with 2500-3000 species (podlech, 1986; maassoumi, 1998; ranjbar & karamian, 2002). this genus is widespread, mostly in the northern hemisphere and in south america, with the diversity centers in arid and semiarid mountainous areas. the place of the greatest diversity and evolutionary differentiation is in southwestern asia (maassoumi & ranjbar, 1998). the region of southern europe, including balkan peninsula, contains a particularly large number of astragalus species (60). this is one of the taxonomically most interesting polymorphic genera of the balkan flora. there are 17 species in © 2021 jušković et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 11 flora of serbia according to diklić (1972). additional floristic research also provided evidence of species a. exscapus l. (boža, 1979), so with addition of the recently recorded species astragalus wilmottianus the genus astragalus is presently represented in serbia with 19 species (ranđelović et al., 2002). many of which are narrow-range endemics. in spite of numerous studies, genus astragalus is still characterized by insufficiently resolved taxonomic issues. the goal of the previous studies was to evaluate interspecies relationships within the genus astragalus, including both taxonomy and phylogeny and using morphological, phytogeographical, molecular systematics, palynological and karyological characters (podlech, 1986; sharawy et al., 2003; osaloo, 2003; taeb et al., 2007; mourad & sharawy, 2010; meher et al., 2012; el-sahhar et al., 2013; amini et al., 2018). this genus shows high morphological variability and complexity. certain characters, including characteristics of trichomes and leaves, were used in subgeneric classification of genus astragalus, introducing subgenera (podlech, 1982; zarre, 2000; zarre, 2003; meher et al., 2012). there is high significance of the overall shape of hairs and detailed micromorphological studies on hairs. zarre (2003) has shown that several characters of hairs may be used for phylogeny reconstruction in astragalus. the anatomy of vegetative organs of astragalus species has not been studied extensively. on the other hand, the anatomical parameters have been shown to greatly contribute to solving of significant taxonomic issues within various taxonomic groups (zarre et al., 2010; jušković et al., 2016, 2017; raca et al., 2017; stojanović et al., 2019). in the present paper, micro-morphological and anatomical variability of populations a. monspessulanus l. and a. spruneri boiss. were analyzed according to quantitative characters of leaflet and leaf petiole. intraspecies variability plays a key role in both long-term and shortterm responses of species toward variations in environmental factors. species a. monspessulanus and a. spruneri belongs to the section incani dc. this is one of the most species-rich sections of astragalus, with 140 species. due to extensive overlaps in morphological characters and high phenotype plasticity, section incani dc. represents a taxonomically interesting group. the selection of studied anatomical characters was based on previous research on astragalus taxa by haddad & barnett, 1989; zarre, 2003; pirani et al., 2006. the analyzed characters were sampled in three populations each of a. monspessulanus and a. spruneri from the central part of the balkan peninsula. the goal was to determine anatomic characters that may be used in determining taxonomy of the studied species as well as the section to which they belong. materials and methods the specimens of a. monspessulanus l. and a. spruneri boiss. were collected during the flowering season, from six native populations from the balkan peninsula. voucher specimens were deposited in the herbarium moesiacum niš, department of biology and ecology, faculty of sciences and mathematics, university of niš, serbia (hmn) (tab. 1). the collected plant material was either placed in the herbarium or fixed in 50% ethanol. the anatomical analysis of leaflet and leaf petiole was performed on permanent and temporary slides prepared by the standard histological method for light microscopy (ruzin, 1999). manual 12 biologica nyssana ● 12 (1) september 2021: 11-21 jušković et al. ● a comparative anatomical study on two closely related astragalus l. taxa from the central part of the balkan peninsula table 1. species analysed, with voucher numbers, and collection sites and dates taxon collection site collection date voucher number astragalus monspessulanus serbia: village petačinci 11-may 2012 hmn-7311 astragalus monspessulanus serbia: gorge of the river jerma, village vlasi 11-may 2012 hmn-7312 astragalus monspessulanus montenegro: morača river canyon, bioče 20-may 2012 hmn-7306 astragalus monspessulanus bulgaria: kyustendil, village staro selo 21-april 2012 hmn-7314 astragalus spruneri macedonia: matka canyon, monastery „matka“ 15-april 2012 hmn-7313 astragalus spruneri macedonia: mariovo, village zović 14-april 2012 hmn-7309 13 microtome (gligorijević & pejčinović, 1983) was utilized in order to make cross-sections. the cross sections of the petiole were taken in the middle, between the stem an lamina, following the observation by howard (1979). epidermal peels, for surface structure and stomata analyses, were prepared by using jefferson’s solution (10% nitric acid and 10% chrome-trioxide, 1:1), stained in safranin and alcian blue, and after the dehydration, mounted in canada balsam. the morphoanatomic measurements were performed on the microscope leica dm 2500-leica dfc490-leica qwin standard (leica microsystem, germany). in the present study, statistical analyses were carried out for 31 quantitative characters related to the leaflet and leaves petiole anatomy. statistical significance of differences between the species in regard to the analyzed characters was assessed by t-test for independent groups. the degree of variability and morpho-anatomical differentiation on level of population and species were established using principal component analysis (pca), canonical discriminant analysis (cda) and agglomerative hierarchical classification (single linkage method). these analyses were performed on the three data sets. one of them was included all characters analysed (leaflet and petiole anatomy), while the other two data sets included only the characters related to leaflet anatomy or petiole anatomy, respectively. all statistical analyses were done in statistical package statistica 8.0 (statsoft, 2007). results and discussion leaflet anatomy of a. monspessulanus the leaflets from all populations in species a. monspessulanus have shown similar anatomical patterns. internal leaflet tissues are organized in biologica nyssana ● 12 (1) september 2021: 11-21 jušković et al. ● a comparative anatomical study on two closely related astragalus l. taxa from the central part of the balkan peninsula fig. 1. cross sections of the leaflet midrib (left) and lamina (right) in investigated astragalus species. a – a. monspessulanus (vlasi), b – a. monspessulanus (petačinci), c – a. monspessulanus (bioče), d – a. spruneri (monastery „matka“), e – a. spruneri (staro selo), f – a. spruneri (zović). scale bars = 50 µm. abbreviations: ade – adaxial epidermis, abe – abaxial epidermis, ph – phloem, pp – palisade parenchyma, sp – spongy parenchyma, st – stomata, vb – vascular bundle, vs – vascular sheath, xy – xylem. 14 an isolateral structure (fig. 1). the leaflets are densely hairy, with bifurcate hairs with two equal or unequal arms, usually opposed in the same plane, tip pointed; arms with verrucose surface are observable particularly on the major vein surface (fig. 2). the presence of bifurcate hairs is a common character in the astragalus. number of hairs at the abaxial side of the leaflet is 0-12 and number on the adaxial side is 0-11. two epidermal layers develop on adaxial and abaxial surfaces of the leaflet. the cells of adaxial epidermis are visibly larger compared to the cells of abaxial epidermis. the outer walls are cutinized. cuticle is thin on both sides of leaf: 1.3-5.6 μm (fig. 1). the epidermal cells of the leaflet are polygonal or irregular in shape. the outline of the anticlinal walls has been described as straight, sinuous or sinuate (fig. 2). the stomata apparatus in astragalus species is present at both surfaces of leaflet, so it is called an amphistomatic leaf. the stomata are tiny, at the same level or slightly above the level of the other epidermal cells. stomata frequency varies, ranging from 57 to 181 per mm2 at the abaxial side and 0 to 161 per mm2 at the adaxial side. the greatest number of stomata (86-181 per mm2 at the abaxial and 0-161 per mm2 at the adaxial side) was recorded in plants from the village vlasi, while the smallest (57-161 per mm2 at the abaxial side and 60 – 128 per mm2 at the adaxial side) was observed in plants from bioče in montenegro (tab. 2). the dominant stomatal type in astragalus is anisocytic (fig. 2). the average recorded thickness of leaflet ranged from 255 μm (population at bioče and village vlasi) to 276 μm (petačinci population), with minimum recorded values of 178 μm and maximum recorded values of 329 μm (tab. 2). internal structure of leaflet does not differ significantly among the studied populations and species. the palisade parenchyma is 2-3 layered with cells arranged parallel to each other, where one layer is better developed. the spongy tissue is composed of 2-3 layers of chlorenchymatous cells with small intercellular spaces. there is a layer of atypical palisade tissue at the abaxial side in the biologica nyssana ● 12 (1) september 2021: 11-21 jušković et al. ● a comparative anatomical study on two closely related astragalus l. taxa from the central part of the balkan peninsula fig. 2. comparative overview of adaxial (left) and abaxial (right) epidermis (a-c; e-g) and trichomes on abaxial epidermis (d, h) in investigated astragalus species. a, d – a. monspessulanus (vlasi), b – a. monspessulanus (petačinci), c – a. monspessulanus bioče, e, h a. spruneri (monastery „matka“), f a. spruneri (staro selo), g – a. spruneri (zović). scale bars = 50 µm. 15 biologica nyssana ● 12 (1) september 2021: 11-21 jušković et al. ● a comparative anatomical study on two closely related astragalus l. taxa from the central part of the balkan peninsula characteristics species t-test p value a. monsspesulanus a. sprunerii mean±sd mean ±sd leaf thickness (µm) 249.026±33.263 264.208±38.386 0.005 palisade tissue thickness (µm) 105.563±20.982 113.989±21.562 0.009 spongy tissue thickness (µm) 72.518±12.567 84.512±17.181 0.000 largest thickness of the leaf blade (µm) 328.854±31.797 325.899±41.043 0.590 height of adaxial epidermal cells (µm) 38.647±7.729 35.984±5.643 0.009 height of abaxial epidermal cells (µm) 35.894±6.347 34.354±4.939 0.071 cuticle thickness on the leaf adaxial epidermis (µm) 2.649±0.703 2.489±0.306 0.049 cuticle thickness on the leaf abaxial epidermis (µm) 2.475±0.7097 2.458±0.294 0.826 petiole surface area (μm²) 829225.244±184868.229 756978.071±229110.570 0.021 petiole central cylinder surface area (μm²) 484291.184±139401.760 404279.434±141626.392 0.000 petiole epidermis thickness (µm) 39.304±4.727 35.997±3.944 0.000 petiole cortex thickness (µm) 122.697±18.476 131.099±17.211 0.002 surface xylem area of the central vasculare bundle petiole (μm²) 37483.420±9592.423 21245.137±8586.570 0.000 surface phloem area of the central vasculare bundle petiole (μm²) 26875.076±7015.183 15728.974±6315.581 0.000 surface sclerenchym area of the central vasculare bundle of the petiole (μm²) 33323.145±9757.989 15173.440±5963.925 0.000 surface xylem area of the left vasculare bundle petiole (μm²) 9268.319±3833.309 6285.631±2333.143 0.000 surface phloem area of the left vasculare bundle petiole (μm²) 10338.462±4893.622 5889.551±2500.453 0.000 surface sclerenchym area of the left vasculare bundle of the petiole (μm²) 12358.751±4661.728 5589.495±2753.462 0.000 surface xylem area of the right vasculare bundle petiole (μm²) 8630.900±3309.513 6230.853±2189.006 0.000 surface phloem area of the right vasculare bundle petiole (μm²) 9294.905±2778.404 5772.199±2063.511 0.000 surface sclerenchym area of the right vasculare bundle of the petiole (μm²) 11416.189±4396.266 5320.995±2563.347 0.000 number of adaxial stomata (/mm2) 118.184±37.340 117.966±31.301 0.966 number of abaxial stomata (/mm2) 105.274±27.850 106.452±21.090 0.749 number of adaxial hair (/mm2) 3.354±2.619 5.110±2.585 0.000 number of abaxial hair (/mm2) 3.302±2.447 5.257±2.640 0.000 length of adaxial stomata (µm) 27.026±3.352 26.184±3.529 0.103 width of adaxial stomata (µm) 25.161±3.088 24.589±2.391 0.166 surface area of adaxial stomata (µm2) 538.831±122.565 510.127±113.029 0.104 length of abaxial stomata (µm) 27.614±3.633 25.787±3.199 0.000 width of abaxial stomata (µm) 25.418±3.265 24.250±2.293 0.006 surface area of abaxial stomata (µm2) 557.974±134.615 494.227±100.144 0.000 table 2. comparison of morphometric characteristics (mean ± sd and range) for a. monsspesulanus and a. spruneri mesophyll. individual cells or a group of cells match palisade parenchyma in shape and arrangement. intercellular spaces are present between cells in this part of mesophyll. the thickness of palisade tissue is 68-158 μm while thickness of spongy tissue is 48-118 μm. differences related to dimensions of palisade and spongy tissues correspond to differences in the total thickness of leaflets. mesophyll cells become rounded at median ribs (fig. 1). vascular bundles are abundant and surrounded by large cells of parenchyma. mechanical tissue is poorly represented. leaflet anatomy of a. spruneri the internal tissue in leaflets of species a. spruneri is organized in the same way as in the leaflets of a. monspessulanus (fig. 1). densely packed t-shaped hairs with equal, tip-pointed arms are present at both adaxial and abaxial sides of the leaflet, and they are particularly densely distributed at the main vein (fig. 2). differences in indumentum in comparison to species a. monspessulanus include density and distribution of trichomes. number of hairs varies in range of 1.7-12.4 at the abaxial side and 1.44-11.02 at the adaxial side. epidermis has single layers both on abaxial and adaxial sides, with thin cell walls and cuticle. cuticle on both sides is less developed and thinner (2-3.2 μm) than in populations of species a. monspessulanus. the outer epidermal cells are larger than the inner ones (fig. 1). the epidermal cells of leaflets are polygonal or irregular in shape. the outline of the anticlinal walls was described as straight, sinuous or sinuate (fig. 2). stomata are present both on abaxial and adaxial side (amphistomatic leaves). stomata frequency varies: 57-243 per mm2 at the abaxial side and 60161 per mm2 at the adaxial side. the greatest number of stomata (86-181 per mm2 at the abaxial side and 90-161 per mm2 at the adaxial side) was recorded in plants from the matka canyon, while the lowest number, 57-161 per mm2 at the abaxial side and 60 – 132 per mm2 at the adaxial side) was observed in plants from mariovo – zović (tab. 2). stomata are small and similar in size on both sides of epidermis. they are of anisocytic type. the mean thickness of the leaflets was 237 at kyustendil old village, 240 μm at mariovo-zović and 276 μm at matka canyon, with minimum at 187 μm and maximum at 336 μm (tab. 2). palisade tissue is usually composed of 1-2 layers of welldeveloped cells, and the spongy tissue has 2-3 cell layers. the thickness of the palisade tissue was 71159 μm and thickness of spongy tissue was 46-110 μm. lateral vascular bundles are present alongside the entire leaf. they are situated in the mesophyll, between spongy and palisade tissue (fig. 1). comparation between a. monspessulanus and a. spruneri according to the results of t-test (tab. 2), there is a significant statistical difference between a. monspessulanus and a. spruneri (p<0.05) in terms of the following leaf anatomy characters: leaf thickness, palisade tissue thickness, spongy tissue thickness, height of adaxial epidermal cells, thickness of cuticle at the adaxial side of the leaf, number of trichomes on abaxial and adaxial sides, length, width and surface area of stomata at the abaxial side of the leaf (tab. 2). although the results of t-test show statistically significant differences in 10 out of 18 characters related to leaf anatomy, results of pca and cda do not show clear separation of individuals of these two species on ordination diagrams (fig. 4, fig. 5). separation of individuals along the first pca ordination axis (surface area of stomata at the abaxial side of the leaf) and second pca ordination axis (leaf thickness) is statistically significantly contributed to only by a single character (stated in brackets). results of cda and classification analysis (fig. 6) indicate presence of clear differentiation in population of a. monspessulanus sampled in the canyon of morača valley in comparison to all other studied populations. the greatest recorded degree of similarity in leaflet anatomy was present between populations of a. spruneri sampled in north macedonia (matka canyon) and bulgaria (kyustendil) and between populations of species a. monspessulanus sampled in serbia. the anatomical results of this study are generally closely matching the referenced results for other species of genus astragalus (boughalleb et al., 2014). leaflets of species a. monspessulanus and a. spruneri are characterized by certain xeromorphic characteristics – relatively tiny and soft leaves with hairs and tiny stomata present both on adaxial and abaxial sides of the leaf, well-developed palisade tissue both on adaxial and abaxial sides of the leaf, and a high number of vascular bundles. the results of statistical analyses are generally indicating low utilitarian value of characters related to leaf anatomy for taxonomy purposes. according to literature sources, micromorphology of hairs is an important taxonomic character for genus astragalus and used in their systematics (taeb et al., 2007; zarre, 2000, 2003; zarre & podlech, 1996, 2001a,b,c; pirani et al., 2006). ghahremani-nejad (2004) examined value of trichome characteristics for separation of bifurcating-hair genus astragalus at the sectional level. the results point to a small range of hair character variation within the section incani dc. according to that author, in order to illuminate 16 biologica nyssana ● 12 (1) september 2021: 11-21 jušković et al. ● a comparative anatomical study on two closely related astragalus l. taxa from the central part of the balkan peninsula intersectional relationships, bifurcate hair characters should be studied more closely. petiole anatomy of a. monspessulanus in cross section, the outline of the petiole in species a. monspessulanus varies among populations from suborbicular to elliptic (fig. 3). the epidermis of all populations consists of a single layer of subcircular to subrectangular cells. cortex is situated below the epidermis, in form of a continuous ring composed of 3–5 layers of collenchymatous cells and parenchymatous cells. all populations have three prominent, primary vascular bundles, one dorsal median bundle and two ventral lateral bundles. the dorsal median bundle (dmb) is larger than the ventral lateral bundles. two to six smaller, secondary bundles accompany the three primary bundles. the vascular bundles are collateral and arranged in a circle, separated from one another by parenchymatic tissue. each vascular bundle is surrounded by a thick sclerenchymatous sheath composed of very thickwalled extraxylary fibers. the pith in all species is composed of large parenchyma cells (fig. 3). petiole anatomy of a. spruneri the outline of cross section of the petiole in species a. spruneri has a semi-triangular outline in which their abaxial sides were convex and their adaxial sides either flat or only slightly convex (fig. 3). just as in species a. monspessulanus, 2/3 of the petiole surface is composed of the central cylinder and 1/3 of the primary cortex. single–layered epidermis is covered with denser indumentum than the petiole epidermis of a. monspessulanus petiole. the cortex is composed of 2-3 layers of collenchyma and several layers of parenchyma cells. the central cylinder includes several vascular bundles, the largest main bundle in the center and two lateral, somewhat smaller vascular bundles. the number and size of vascular bundles varied between individuals of the same population as well as among populations: 5-11 bundles were recorded in different individuals. the vascular bundles are surrounded by well-developed sclerenchyma tissue. the central region is composed 17 biologica nyssana ● 12 (1) september 2021: 11-21 jušković et al. ● a comparative anatomical study on two closely related astragalus l. taxa from the central part of the balkan peninsula fig. 3. cross sections of the petiole in investigated astragalus species. a – a. monspessulanus (vlasi), b – a. monspessulanus (petačinci), c – a. monspessulanus (bioče), d – a. spruneri (monastery „matka“), e – a. spruneri (staro selo), f – a. spruneri (zović), g – dorsal median vascular bundle in a. monspessulanus (bioče), h, i – details of the spine petiole anatomy in a. spruneri (monastery „matka“). scale bars = 200 µm (a-f); 50 µm (g-i). abbreviations: co – cortex, dva – dorsal ventral axis, dmb dorsal median vascular bundle, e – epidermis, ph – phloem, pi – pith, sb – subsidiary vasculare bundle, sbs – sclerenchymatous bundle sheath, st – stomata, t – trichoma, va – ventral axis, vlb – ventral lateral vascular bundle, xy – xylem. biologica nyssana ● 12 (1) september 2021: 11-21 18 jušković et al. ● a comparative anatomical study on two closely related astragalus l. taxa from the central part of the balkan peninsula fig. 4. results of the principal component analysis (pca) and canonical discriminant analysis (cda) for populations of investigated astragalus species based on: all characters of leaflet and petiole anatomy, characters of leaflet anatomy and characters of petiole anatomy. fig. 5. results of the principal component analysis (pca) for populations of investigated astragalus species based on: all characters of leaflet and petiole anatomy, characters of leaflet anatomy and characters of petiole anatomy. of large, thin-walled parenchyma cells, just as in species a. monspessulanus (fig. 3). comparation between a. monspessulanus and a. spruneri according to the results of t-test, the studied species show statistically significant differences regarding the petiole anatomy (tab. 2). according to pca results, individuals of a. monspessulanus are almost completely differentiated from individuals of species a. spruneri along the first ordination axis (fig. 4, fig. 5). only very few individuals of a. monspessulanus sampled in the canyon of river morača show a more significant degree of similarity with individuals of species a. spruneri. on the other hand, a few individuals of a. spruneri sampled in bulgaria (kyustendil) show a significant degree of similarity with individuals of species a. monspessulanus (serbia: village vlasi). the list of characters making the highest contribution to differentiation along the first ordination axis of pca diagram includes all the analyzed characters except for thickness of petiole epidermis and thickness of primary cortex of petiole. cda diagram shows almost complete differentiation of species (fig. 5). individuals of species a. monspessulanus are concentrated in the positive part of cda diagram, while individuals of species a. spruneri are positioned in the negative part of the cda diagram. the results of agglomerative hierarchical classification support the results of other statistical analyses, i.e. existence of clear interspecies differentiation regarding the leaf stalk anatomy (fig. 6). although the statistical analysis of the dataset including all studied characters of leaflets and petioles has also supported interspecies differentiation, analysis of dataset including only the characters of petioles has provided more illustrative results. according to metcalfe & chalk (1950), the petiole has considerable taxonomic importance, as it is not heavily influenced by environmental changes. the general description of petiole cross-section is in accordance with previous data for other astragalus species (howard, 1979; haddad & barnett, 1989; pirani et al. 2006). research on european species of astragalus has shown that anatomic characteristics of the petiole have a limited taxonomic value, while characters of type of tissue in parenchyma of central region and amount of collenchyma tissue around the vascular bundles have high taxonomic importance for recognizing the two main species groups (haddad & barnett, 1989). species a. monspessulanus and a. spruneri belong to a group of taxa characterized by a relatively small number of thick-walled parenchyma cells in the central part of the petiole and a relatively high fig. 6. cluster analysis (single linkage method) for populations of investigated astragalus species based on: all characters of leaflet and petiole anatomy, characters of leaflet anatomy and characters of petiole anatomy. 19 biologica nyssana ● 12 (1) september 2021: 11-21 jušković et al. ● a comparative anatomical study on two closely related astragalus l. taxa from the central part of the balkan peninsula biologica nyssana ● 12 (1) september 2021: 11-21 20 jušković et al. ● a comparative anatomical study on two closely related astragalus l. taxa from the central part of the balkan peninsula amount of collenchyma tissue around the vascular bundles. conclusions our results show which anatomical and micromorphological characteristics can be used for taxonomic differentiation of species from the astragalus sect. incani. anatomical features described here largely agree with previous characters found in other astragalus species. anatomical and micromorphological characters described here have potential taxonomic and significance. results of statistical analyses indicate that characters which refer to petiole anatomy have higher taxonomic value than characters which refer to leaflet anatomy. acknowledgements. this study was supported by the project of ministry of science and technological development of republic of serbia (contract no. 451-039/2021-14/200124). references amini, e., kazempour‐osaloo, s., maassoumi a. a., zare‐maivan, h.. 2018: phylogeny, biogeography and divergence times of astragalus section incani dc. (fabaceae) inferred from nrdna its and plastid rpl32-trnl(uag) sequences. nordic journal of botany, 2019: e02059. boughalleb, f., raoudha abdellaoui, r., benbrahim, n., mohammed neffati, m. 2014: anatomical adaptations of astragalus gombiformis pomel. under drought stress. central european journal of biology, 9(12): 1215-1225. boža, p. 1979: prilog flori srbije. biosistematika, 5 (1). diklić, n. 1972: rod astragalus l. in: josifović, m. (ed.): flora sr srbije 4: 274-301. srpska akademija nauka i umetnosti. beograd. el-sahhar, k.f., emara, kh. s., ali, w.a. 2013: comparative systematic studies of astragalus in flora of arab republic of egypt and syrian arab republic: plant morphology, sem of lamina surface and sds-page of proteins. research journal of agriculture and biological sciences, 9(6): 271-286. ghahremani-nejad, f. 2004: value of trichome characteristics for the separation of bifurcating hairy astragalus l. (fabaceae) at the sectional level. turkish journal of botany, 28: 241-245. gligorijević, s., pejčinović, d. 1983: contribution to the methodology of anatomical sections and preparation. acta biologiae et medicinae experimentalis, 8: 43-45. haddad, r.s., barnett, j.r. 1989: variation in petiole anatomy of the european spiny species of astragalus l. (leguminosae-papilionoideaegalegeae). botanical jurnal of the linnean society, 101: 241–247. howard, r.a. 1979: the petiole. in metcalfe, c.r., chalk, l. (eds), anatomy of the dicotiledones, (2) i: 88-96, oxford: clarendon press. jušković, m., vasiljević, p., savić, a., jenačković, d., stevanović, b. 2016: morpho-anatomical differentiation of the populations of daphne cneorum l. (thymelaeaceae) from serbia. biologica nyssana, 7(1): 1-9. jušković, m., vasiljević, p., savić, a., jenačković, d., stevanović, b. 2017: comparative morphoanatomical analysis of the leaves and stems of daphne (thymelaeaceae) species. biologia, 72(7): 709—721. maassoumi, a.a. 1988: astragalus in the old world, check list. tehran: research institute of forests and rangelands. maassoumi, a.a., ranjbar, m. 1998: revision of the genus astragalus l. sect. leucocercis bunges (papilionaceae) from iran. iranian journal of botany, 7: 239-248. meher, r. sh. a., maassoumi, a.a., saidi, a., osaloo, sh.k., nohooji, m.g. 2012: morphological cladistic analysis of some bifurcate hairy sections of astragalus (fabaceae) in iran. turkish journal of botany, 36: 434-442. mehrnia, m., zarre, s., sokhan-sanj, a. 2005: intraand inter-specific relationships within the astragalus microcephalus complex (fabaceae) using rapd. biochemical. systematics and ecology, 33: 149–158. metcalfe, c.r., chalk, l. 1950: anatomy of dicotiledons, vol. ii. clarendon press, oxford, uk. mourad, m.m., sharawy, s.m. 2010: the interspecific relationships of astragalus species in egypt assessed by the morphoanatomical characters of the pod. feddes repertorium, 121(1-2): 38-58. osaloo, s.k., maassoumi, a.a., murakami, n. 2003: molecular systematics of the genus astragalus l. (fabaceae): phylogenetic analyses of nuclear ribosomal dna internal transcribed spacers and chloroplast gene ndhf sequences. plant systematics and evolution, 242: 1-32. pirani, a., zarre, sh., tillich, h. j., podlech, d., niknam, v. 2006: spine anatomy and its systematic biologica nyssana ● 12 (1) september 2021: 11-21 jušković et al. ● a comparative anatomical study on two closely related astragalus l. taxa from the central part of the balkan peninsula application in astragalus sect. rhacophorus s. l. (fabaceae) in iran. flora, 201(3): 240–247. podlech, d. 1982: neue aspekte zur evolution und gliederung der gattung astragalus l. mitteilungen der botanischen staatssammlung münchen, 18: 359-378. podlech, d. 1986: taxonomic and phytogeographical problems in astragalus of the old worldland south west asia. proceeding of the royal society of edinburgh, 89b: 37-43. podlech, d. 2008: the genus astragalus l. (fabaceae) in europe with exclusion of the former soviet union. feddes repertorium, 119(5–6): 310– 387. raca, i., ljubisavljević, i., jušković, m., ranđelović, n., ranđelović, v. 2017: comparative anatomical study of the taxa from series verni mathew (crocus l.) in serbia. biologica nyssana, 8(1): 15-22. ranđelović, v., zlatković, b., jušković, m. 2002: astragalus wilmottianus stoj. nova vrsta u flori srbije. proceeding of 7th symposium on flora of southeastern serbia and neighbouring regions, niš, 1-4. ranjbar, m., karamian, r. 2002: astragalus sect. astragalus (fabaceae) in iran, complementary notes with a key to the species. nordic journal of botany, 22: 177-181. ruzin s.e. 1999. plant microtechnique and microscopy. oxford university press, new york, 322 pp. sharawy, s.m., mourad, m.m., al-nowaihi, a.s. 2003: the assessment of the morphoanatomical characters of the spermoderm in the delimitation of some astragalus taxa growing in egypt. bulletin of pharmaceutical sciences, 32(2-d): 325–346. statsoft 2007. statistica for windows, version 8.0. statsoft inc., tulsa. stojanović, j., raca, i., jelena jevtić, j., jušković, m., ranđelović, v. 2019: comparative morphoanatomical analysis of gagea pratensis (pers.) dumort. (liliaceae) from serbia and montenegro. biologica nyssana, 10(2): 125-133. taeb, f., zarre, s., podlech, d., tillich, j.h., kazempour, osaloo, s., maassoumi, a.a. 2007: a contribution to the phylogeny of annual species of astragalus (fabaceae) in the old world using hair micromorphology and other morphological characters. feddes repertorium, 118(5-6): 206-227. zarre, s. 2000. systematic revision of astragalus sect. adiaspastus, sect. macrophyllium and sect. pterophorus. englera, 18: 1–219. zarre, s. 2003: hair micromorphology and its phylogenetic application in thorny species of astragalus (fabaceae). botanical jurnal of the linnean society, 143: 323-330. zarre, s., podlech, d. 1996: taxonomic revision of astragalus l. sect. hymenostegis bunge (leguminosae). sendtnera, 3: 255−312. zarre, s., podlech, d. 2001a: a short contribution to genus astragalus l. (fabaceae) in turkey. pakistan journal of botany, 33(2): 153−155. zarre, s., podlech, d. 2001b: taxonomic revision of astragalus sect. acanthophace (fabaceae). sendtnera, 7: 233−255. zarre, s., podlech, d. 2001c: astragalus sect. semnanenses (fabaceae): a new monotypic section from iran. nordic journal of botany, 21(5): 485−491. 21 anticancer compounds from medicinal plants biologica nyssana 3 (2)  december 2012: 105-107 georgiev, d.  new species of stygobiotic belgrandiella… 105 original article new species of stygobiotic belgrandiella from north bulgaria (gastropoda: hydrobiidae) dilian georgiev department of ecology and environmental conservation, university of plovdiv, tzar assen str. 24, bg-4000 plovdiv, bulgaria * e-mail: diliangeorgiev@abv.bg abstract: georgiev, d.: new species of stygobiotic belgrandiella from north bulgaria (gastropoda: hydrobiidae). biologica nyssana, 3 (2), december 2012: 105-107. a new stygobiotic species of belgrandiella from a cave in north bulgaria was described. nine shells were collected by the author on 09.03.2013 at the entrance of the water cave (vodnata) near village of musina, north bulgaria, n43 13 14.9 e25 25 39.6, 198 m alt. the systematic position of the genus belgrandiella was also discussed. key words: aquatic, bulgaria, endemic, subterranean, snail introduction the genus belgrandiella wagner, 1927 (gastropoda: risooidea) consists of minute snails inhabiting the springs and the subterranean waters of south-west and central europe to the caucasus (k a b a t , h e r s l e r , 1993; a r c o n a d a , r a m o s , 2003). this is one of the most diverse genera from the risooidea superfamily in bulgaria having 11 species known till now (g e o r g i e v , 2011a, g e o r g i e v , 2013). there are two ecological groups of species from the genus: stygobiotic (belgrandiella hessei wagner, 1927, belgrandiella pusilla angelov, 1959, belgrandiella bulgarica angelov, 1972, belgrandiella bureschi angelov, 1976, belgrandiella stanimirae georgiev, 2011, belgrandiella maarensis georgiev, 2013) and spring-living species (belgrandiella angelovi pintér, 1968, belgrandiella zagoraensis glöer & georgiev, 2009, belgrandiella dobrostanica glöer & georgiev, 2009, belgrandiella bachkovoensis glöer & georgiev, 2009, belgrandiella pandurskii georgiev, 2011, the last one also found in cave waters) (w a g n e r , 1927; a n g e l o v , 1959, 1972, 1976; p i n t é r , 1968; h u b e n o v , 2005, 2007; g l ö e r , g e o r g i e v , 2009; g e o r g i e v , 2011b, c, d). in this paper i describe a new stygobiotic species of belgrandiella from a cave in north bulgaria showing that the subterranean waters of the country are still poorly known considering their fauna and many new species could be found in further research. material and methods the shells were collected by sieving the cave river deposits by 1x1 and 2x2 mm width of the mesh sieves. the material from the smaller meshed sieve was then brought to the laboratory and dried. after that it was again put into water and the floating shells were collected by a strainer and small brush. the measurements were carried out by means of ceti stereo microscope and an eye-piece micrometer, and photographs were made with camera system with a digital adapter. the material is stored in the national museum of natural history, sofia, bulgaria. abbreviations used: h shell height, w shell width, ah aperture height, aw aperture width, lh last whorl height. 3 (2) • december 2012: 105-107 biologica nyssana 3 (2)  december 2010: 105-107 georgiev, d.  new species of stygobiotic belgrandiella… 106 table 1. shell measurements of belgrandiella hubenovi n. sp. (for abbreviations see “materials and methods”) n h w ah aw lh w/h ah/h aw/ah lh/h 1 1.49 0.76 0.54 0.54 1.06 0.51 0.37 1.00 0.71 2 1.49 0.89 0.56 0.56 1.02 0.60 0.38 1.00 0.69 3 1.67 0.89 0.63 0.59 1.12 0.53 0.38 0.95 0.67 4 1.73 0.99 0.71 0.63 1.19 0.57 0.41 0.88 0.69 5 1.55 0.89 0.56 0.59 1.12 0.57 0.36 1.06 0.72 6 1.62 0.86 0.63 0.59 1.12 0.53 0.39 0.95 0.69 7 1.49 0.87 0.56 0.59 1.09 0.59 0.38 1.06 0.73 8 1.68 0.87 0.63 0.59 1.19 0.52 0.37 0.95 0.71 average 1.59 0.88 0.60 0.59 1.11 0.55 0.38 0.98 0.70 fig. 1. front and side view of the holotype of belgrandiella hubenovi n. sp. results and discussion genus belgrandiella wagner, 1927 type species: belgrandia kusceri wagner, 1914 belgrandiella hubenovi n. sp. material examined: 9 shells, 09.03.2013, the water cave (vodnata) near village of musina, north bulgaria, n43 13 14.9 e25 25 39.6, 198 m alt., d. georgiev leg. holotype: h = 1.49 mm, w = 0.76 mm, ah = 0.54 mm, aw = 0.54 mm, lh = 1.06 paratypes: 3 shells, national museum of natural history, sofia, bulgaria, 5 shells coll. d. georgiev. locus typicus: water cave near village of musina, north bulgaria, n43° 13' 14.9'' e25° 25' 39.6'', 198 m alt. differential diagnosis: by its bent periostome the new species differs from all known bulgarian species from the genus belgrandiella with an exception of b. hessei – another stygobiont known from temnata dupka cave near lakatnik town, about 167 km to the west. from this species b. hubenovi n. sp. differs by its smaller size (h max = 1.73 mm versus 2.3 mm in b. hessei), and not so thick and developed aperture lip. description: shell: the shell is ovate-conic to ovate-cylindrical, white, whorls are 3-4, regularly growing with, shining surface, with deep suture, fine growth lines sometimes forming small, irregular smooth ribs. the umbilicus is slit-like, the aperture is egg-shaped with sharp periostome which is bent to the outside from the lateral view. the operculum and the soft body are not known. h = 1.49-1.73 mm, w = 0.76-0.99 mm, ah = 0.54-0.71 mm, aw = 0.54-0.63 mm, lh = 1.02-1.19 mm, w/h = 0.510.60, ah/h = 0.36-0.41, aw/ah = 0.88-1.06, lh/h = 0.67-0.73. etymology: named after assoc. prof. dr zdravko hubenov (national museum of natural history, sofia, bulgaria) who contributed so much in the studies of the bulgarian aquatic malacofauna and paid me attention of some springs as potential localities of freshwater snails. distribution: known only from the type locality, possibly local endemic species. habitat and ecology: a stygobiotic species found only as empty shells in the river deposits at the entrance of vodnata cave near village of musina. this cave can be explored only by boat and experienced divers for searching living snails on the bottom substrate of the inner stream. the systematic position of the genus belgrandiella wagner, 1927 is unclear. it is referred by various authors to different family groups as hydrobiidae, orientalinidae or belgrandiellidae (radoman, 1983; kabat & hersler, 1993; angelov, 2000; arconada & ramos, 2003). the genus consists of minute risooid snails with different shell shapes, mainly ovoid-conical but also conical or ovoid and different penis morphology (simple long, biologica nyssana 3 (2)  december 2012: 105-107 georgiev, d.  new species of stygobiotic belgrandiella… 107 simple short, conical or triangular, or same shape but with one small lobe on the left side). it can be supposed (as for other extremely subterranean genera, for example bythiospeum bourguignat, 1882) that belgrandiella is a complex of more still undefined genera. however it is a very hard task and sometimes is impossible for the scientists to reach the habitats of some of the stygobiotic species. references angelov, a. 1959. neue gastropoden aus den unterirdischen gewässern bulgariens. archiv für molluskenkunde, 88(1/3): 51-54. angelov, a. 1972. neue hydrobiidae aus höhlengewässern bulgariens. archiv für molluskenkunde, 102(1/3): 107-112. angelov, a. 1976. ein neuer vertreter der gattung belgrandiella a. wagner, 1927 (gastropoda, hydrobiidae) von grundwassern bulgariens. acta zoologica bulgarica, 4: 78-80. angelov, a. 2000. mollusca (gastropoda et bivalvia) aquae dulcis, catalogus faunae bulgaicae. – pensoft & backhuys publ., sofia, leiden, 54 p. arconada, b., ramos, m. 2003. the ibero-balearic region: one of the areas of highest hydrobiidae (gastropoda, prosobranchia, risooidea) diversity in europe. graellsia, 59 (2-3): 91-104. georgiev, d. 2011a. check list of the bulgarian minor freshwater snails (gastropoda: risooidea) with some ecological and zoogeographical notes. zoonotes, 24: 1-4. georgiev, d. 2011b. a new species of belgrandiella (wagner 1927) (mollusca: gastropoda) from caves in northern bulgaria. acta zoologica bulgarica, 63 (1): 7-10. georgiev, d. 2011c. new species of snails (mollusca: gastropoda: risooidea) from cave waters of bulgaria. buletin shkenkor, ser. shkenkat natyrore, 61: 83-96. georgiev, d. 2011d. new localities of four bulgarian hydrobiidae species (gastropoda: risooidea). zoonotes, 24: 1-4. georgiev, d. 2013. catalogue of the stygobiotic and troglophilous freshwater snails (gastropoda: rissooidea: hydrobiidae) of bulgaria with descriptions of five new species. ruthenica, 23 (1): 59-67. glöer, p., georgiev, d. 2009. new rissooidea from bulgaria (gastropoda: rissooidea). – mollusca, 27 (2): 123-136. hubenov, z. 2005. malacofaunistic diversity of bulgaria. in: current state of bulgarian biodiversity – problems and perspectives, petrova a. (ed.) bulgarian bioplatform, sofia, 199-246. (in bulgarian). hubenov, z. 2007. fauna and zoogeography of marine, freshwater, and terrestrial mollusks (mollusca) in bulgaria. in: fet, v., a. popov (eds.). biogeography and ecology of bulgaria. springer, dodrecht, 141-198. kabat, a., hersler, r. 1993. the prosobranch family hydrobiidae (gastropoda: rissooidea): review of classification and supraspecific taxa. smithsonian contributions to zoology, 547: 194. pintér, l. 1968. eine neue wasserschnecke aus bulgarien. archiv für molluskenkunde, 98 (1/2): 61-63. radoman, p. 1983. hydrobioidea a superfamily of prosobranchia (gastropoda). i. systematics. monographs 547, serbian academy of sciences and arts, (department of science), 256 pp. wagner, a. 1927. studien zur molluskenfauna der balkanhalbinsel mit besonderer berücksichtigung bulgariens und thraziens, nebst monographischer bearbeitung einzelner gruppen. annales zoologici muzei polonici hist. nat., 6 (4): 263-399. microsoft word bn020206 marko lazic biologica nyssana 2 (2) december 2011: 00-00 nahirnić a. supplements of butterfly fauna… 131 short communication ! accessory femoral pores in podarcis muralis from southern serbia marko m. lazić1, jelka crnobrnja-isailović1, 2 1university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia 2institute for biological research „siniša stanković“, university of belgrade, serbia * e-mail: markol@pmf.ni.ac.rs abstract: lazić, m.l., crnobrnja-isailović, j.: accessory femoral pores in podarcis muralis from southern serbia, biologica nyssana, 2 (2), december 2011: 131-133. abnormalities in scalation are common in lizards. bilateral asymmetries are frequent, as are supernumerary scales. factors possibly responsible for such developmental anomalies are inbreeding and environmental stress. here, we report two cases of accessory femoral pores in a podarcis muralis from southern serbia. key words: abnormal scalation, podarcis muralis, femoral pores, developmental instability, environmental stress developmental stability is the ability of individuals to undergo stable development of their phenotype under a range of environmental conditions (m ø l l e r , 1997). the origin of the disturbances is assumed to be genetic, environmental, or the product of a genotypeenvironment interaction (m a r k o w , 1995). deviations from developmental stability arise when organism fails to buffer such disturbances. developmental anomalies and asymmetries in bilateral traits are common in lizards (b r a ñ a & j i , 2000; c m o b m j a -i s a i l o v i c et al., 2005). there is generally negative correlation between developmental instability and fitness components such as growth, fecundity and longevity and these relations can be either direct and indirect (m ø l l e r , 1997). here, we report two cases of supernumerary femoral pores in common wall lizard (podarcis muralis) from southern serbia. podarcis muralis is small lacertid lizard species, distributed in central and southern europe, but its range extends to northwestern asia minor (g a s c et al., 1997; a r n o l d & o v e n d e n , 2002). it is one of the most widespread lizard species in the central balkans (r a d o v a n o v i ć , 1951), and, consequently, susceptible to various kinds of negative anthropogenic impacts, as pesticide use (g u i l l a u m e , 1997). in spring 2011, during collection of adult p. muralis in southern serbia, adult female (63.0 mm svl) and adult male (52.3 mm svl) with supernumerary femoral pores were sampled. female was sampled in the niš fortress (n 43° 19.477’ e 021° 53.864’) in the center of the city of niš. the habitat consisted of tall stone walls up to 10 meters with open grass. accessory rows of femoral pores were noted in both hind limbs (figure 1).the accessory pores (left n =14, right n =8) were smaller and located parallel. on left hind limb they were located along the entire length of normal series and on the right they were in a central position and posterior to the normal series (left n = 20, right n = 20). this was the only case of supernumerary femoral pores in 155 individuals examined from this population (appr. 0.64% of entire sample). male was 2 (2) • december 2011: 131-133 biologica nyssana 2 (2) december 2011: 131-133 lazić m.l., crnobrnja-isailović j. accessory femoral pores in podarcis… 132 collected in sićevo gorge, 15 km from the city of niš (n 43° 20.307’ e 022° 05.000’). habitat consisted of stone walls up to 4 meters high, large and small piles of stones and knocked down trees. accessory rows of femoral pores (n=4) were noted on left hind limb (figure 2) but above the normal series, in central position and anterior of the normal series (left n=16, right n=17). this was the only one with this type of anomaly in 126 examined individuals (appr. 0.79% of entire sample). after photographing and measuring, animals were released to the site of capture. there are few records of accessory femoral pores in lizards. in portugal, supernumerary femoral pores were found in adult female podarcis bocagei (k a l i o n t z o p o u l o u & c a r r e t e r o , 2006). as in this study, frequency of occurrence was quite low, one individual from 37 sampled (2.70%). moreover, they didn’t find any such case in 380 lizards of the same species sampled from seven localities. accessory femoral pores were recorded in the collared lizard, crotaphytus collaris (w a l k e r , 1980) with 63.7% of males and 36.3% of females displaying the anomaly, which was attributed to inbreeding since the population was small and isolated. these anomalies were also recorded among iguanians (t a n n e r & a v e r y , 1964). figure 1. female specimen of podarcis muralis from niš fortress, southern serbia. the white arrows on each side delimit the additional row of femoral pores figure 2. male specimen of podarcis muralis from sićevo gorge, southern serbia. the white arrows on each side delimit the additional row of femoral pores biologica nyssana 2 (2) december 2011: 131-133 lazić m.l., crnobrnja-isailović j. accessory femoral pores in podarcis… 133 two populations of common wall lizard from serbia analysed here are not completely isolated from the nearby ones. both human settlements and natural habitats (rocks, gorges) are more or less connected by some sort of corridors (railways, stone piles, edges of magistral roads, traditional houses) that provide opportunities for migration. therefore, abnormal scalation found in studied individuals is more likely to be caused by environmental stress from environmental polution (as pesticide use), than by inbreeding. however, it is important to mention that another two population samples, collected from additional two localities (one urban and one rural), did not contain individuals with accessory femoral pores. our preliminary analises showed that environmental stress impact occur in another urban locality, at least (l a z i ć et al., submitted), but this type of anomaly was not detected. acknowledgements. this work was supported by grant no. 173025 of ministry of education and science of republic of serbia. references arnold, e.n., ovenden, d.w. 2002: a field guide to the reptiles and amphibians of britain and europe. harper collins publishers ltd., london, 288 pp. braña, f., ji, x. 2000: influence of incubation temperature on morphology, locomotor performance, and early growth of hatchling wall lizards (podarcis muralis). the journal of experimental zoology, 286 (4): 422-433. crnobrnja-isailović j., aleksić i., bejaković, d. 2005: fluctuating asymmetry in podarcis muralis populations from southern montenegro: detection of environmental stress in insular populations. amphibiareptilia, 26 (2): 149-158. gasc, j-p., cabela, a., crnobrnja-isailović, j., dolmen, d., grossenbacher, k., haffner, p., lescure, j., martens, h., martinez-rica, j.p., maurin, h., oliveira, m.l., sofianidou, t.s., veith, m. and zuiderwijk, a. (eds), 1997: atlas of amphibians and reptiles in europe. societas europaea herpetologica and museum national d' histoire naturelle (iegb/spn), paris. guillaume, cl.p. 1997: podarcis muralis (laurenti, 1768). in: casc, j.p., cabela, a., crnobrnjaisailovic, j., dolmen, d., grossenbacher, k., haffner, p., lescure, j., martens, h., martinezrica, j.p., maurin, h., oliveira, m.e., sofianidou, t.s., veith, m., zuiderwijk, a. (eds), atlas of amphibians and reptiles in europe, societas europaea herpetologica and museum national d’ histoire naturelle (iegp/spn), paris, p. 286-287. kaliontzopoulou, a., carretero, m.a. 2006: podarcis bocagei (bocage's wali lizard) abnormal scalation. herpetological review, 37(4): 470-471. markow, a.t. 1995: evolutionary ecology and developmental instability. annu. rev. entomol, 40: 105-120 møller, p.a. 1997: developmental stability and fitness: a review. the american naturalist, 149 (5): 916-932. radovanović, m. 1951: vodozemci i gmizavci naše zemlje. beograd (srpsko biološko društvo). tanner, w.w., avery, f.d. 1964: a new sauromalus obesus from the upper colorado basin of utah. herpetologica, 20 (1): 38-42. walker, j.m. 1980: accessory femoral pores in a colony of the collared lizard, crotaphytus collaris in texas. journal of herpetology, 14 (4): 417-418. microsoft word bn-oa-0201-04 stancic et al biologica nyssana 2 (1) september 2011: 35-38 stančev, i. et al. yield and morphological characteristics of some types… 35 original article ! yield and morphological characteristics of some types of cms hybrids of tobacco prilep ivica stančić*, saša petrović, jelica živić college of agriculture and food technology, prokuplje * e-mail: istancic@medianis.net abstract: stančić, i., petrović, s., živić, j.: yield and morphological characteristics of some types of cms hybrids of tobacco prilep. biologica nyssana, 2 (1), september 2011: 35-38. this paper presents characteristics of the four newly created hybrid varieties of tobacco prilep. the survey was conducted in period from year 2005. to 2006, and was performed at the experimental field of ad selekcija in aleksinac. the aim was to perform a comparative analysis of the newly created cms hybrid varieties of tobacco type prilep through testing the yield and morphological characteristics. relating morphological traits, all tested hybrids showed the characteristic of the oriental tobacco type prilep. the hybrid p-3 had a slightly greater plant height, whereas the hybrid p-2 achieved the greatest number of leaves per plant and the hybrid p-4 some greater size of the largest leaf as well as the length of internodes. the hybrid p-3 exhibited a much greater yield of dry leaves than hybrids p-2 and p-4. the same hybrid achieved significantly better features in proportional representation of high classes compared to the hybrid p-2. key words: tobacco, prilep, hybrid, dry leaf yield, morphological characteristics introduction ! production area of tobacco (nicotiana tabacum l.) has decreased in the recent period in serbia. according to the report of the serbian chamber of commerce, the production was only 320.000 kilograms in 2009. production of oriental tobacco is mainly conducted in the regions of south morava and kosovo and metohija. one of the reasons for production decrease is the present assortment which does not satisfy either the yield or the quality (s t a n č i ć et al., 1998). for this reasons, breeders are trying to create hybrids based on cytoplasmic male sterility (cms). in this way, the illegal seed production will be prevented, and conditions for achieving varietal uniformity and good yield and quality will be also created in the entire production area. knowledge in gene pool of tobacco could assist in selection of materials for breeding or genetic investigations (m o o n et al., 2009). on the basis of the needs for oriental tobacco that is present 8-10% in the manufacture of cigarettes, the goal of the work was to perform a comparative analysis of the newly created cms hybrid varieties of tobacco type prilep through testing the yield and morphological characteristics. materials and methods the survey comprised four hybrids based on cms of type prilep: p-1, p-2, p-3 and p-4. the process of plant breeding was carried out through a series of crossbreeding, where sterile analogues were conducted at first. the subsequent procedure comprised the research of combining potentials of the achieved material in order to find the best parental genotypes of f1 hybrids. the survey was conducted from year 2005. to 2006., 2 (1) • september 2011: 35-38 biologica nyssana 2 (1) september 2011: 35-38 stančev, i. et al. yield and morphological characteristics of some types… 36 table 1. morphological characteristics of tested tobacco hybrids hybrids plant height including flower (cm) number of leaves per plant internodes length (cm) length of leaf (cm) width of leaf (cm) ratio leaf length / width p-1 49 36 1,6 13,1 7,9 1,66 p-2 50 39 1,8 13,2 8,1 1,63 p-3 55 38 2,2 13,7 8,1 1,69 p-4 49 35 2,5 15,2 8,9 1,71 table 2. yield of leaves (kg/ha) and percentage of high classes (i and ii) hybrids yield of leaves (kg/ha) rank % representation (i and class ii) rank p-1 1410 2 26,56 2 p-2 1356 4 22,31 4 p-3 1494 1 28,76 1 p-4 1361 3 23,38 3 lsd 0,05 0,01 93,72 130,23 6,12 8,32 cv 5,62 3,21 at the experimental field of ad selekcija in aleksinac. the experiment was set up according to a completely randomized block design with five replications. during the experiments the optimal agricultural practices for oriental tobacco cultivation of were applied. yield of dry leaves (kg/ha) and calculation of the proportional representation of high classes (i and ii) (j o v a n o v i ć , d., 2001) were obtained after harvesting, drying and grading. investigation of the morphological characteristics was carried out during the vegetation period, and ten plants from each trial were used as a representative sample. the measurement was done on the insertion of the middle leaf and it included the height of the plants with a flower, the number of leaves per plant, the length of internodes in the middle of the stem, the size of leaves and the ratio of leaf length and width. results and discussion morphological traits of the tested hybrids were obtained during the vegetation period (tab. 2). the hybrid p-3 achieved the highest height (55 cm), while hybrids p-1 and p-4 expressed some lower values. regarding the number of leaves per plant, the hybrid p-2 had the greatest value (39), whereas the hybrid p-4 the lowest (35). investigation of the genetic variability of plant height and number of leaves per plant in some varieties of oriental tobacco and their f1 hybrids is a significant precondition for high and stable yield (k o r u b i n -a l e k s o s k a , a., 2010). the highest (2.5 cm) and lowest (1.6) length of internodes were found for hybrids p-4 and p1respectively. the greatest leaf size was found in the hybrid p-4 with an average length of 15.2 cm and an average width of 8.9 cm. the hybrid p-1 had the smallest leaf with an average length of 13.1 cm and an average width of 7.9 cm. m i c e s k a et al. 2003 obtained higher values for the examined morphological characteristics of tobacco type prilep. regarding the ratio of leaf length and width, the hybrid p-4 showed the greatest value (1.71), while the lowest value (1.66) was found for the hybrid p1. bearing in mind the correlation between leaf size and its chemical composition, relation of length and width of the leaf is an important parameter for the quality of oriental tobacco. the parameters of morphological characteristics of tested hybrids were typical for the oriental tobacco type prilep. the leaf yield is a trait that is influenced by many interrelated factors. genetic properties of tobacco hybrids and agro-ecological conditions of production are the decisive factors in formation of organic and mineral complex of substances, affecting the corresponding leaf yield. the hybrid p3 achieved the greatest dry leaf yield (1494 kg / ha), while the hybrids p-2 and p-4 exhibited lower values (tab.1). statistically significant differences were found among the hybrids. the hybrid p-3 achieved significantly greater yield comparing to hybrids p-2 and p-4. no significant differences were obtained among other tested hybrids. tobacco breeders focus their program on those quantitative traits that are highly associated with leaf yield (d r a z i c et al., 2010). the quality of tobacco was expressed through the percentage of high classes of the leaves (i and ii, tab. 1). the hybrid p-3 exhibited the highest percentage of high class representation (28.76%), biologica nyssana 2 (1) september 2011: 35-38 stančev, i. et al. yield and morphological characteristics of some types… 37 while the hybrid p-2 had the lowest participation of high classes in dry leaf (22.31%). the hybrid p-3 showed some better characteristics compared to the hybrid p-2 in term of the proportional distribution of high classes. there was no statistically significant difference among other tested hybrids. the obtained yield values and the percentage of high classes of the leaves are in accordance with the results obtained by m i c e s k a & d i m i t r i e s k i (2000). conclusion the obtained mean values and variability parameters show that the tested hybrids exhibited a high variability in yield and morphological traits. the genotypes usually varied in leaf yield, the percentage of high classes, plant height and internodes length. the hybrid p-3 achieved the highest yield of dry leaves. it also had the best proportional distribution of high classes, whereas morphological characteristics of this hybrid were typical for the oriental tobacco type prilep. genetic potential and morphological characteristics of the hybrid p-3 could be included in breeding program of oriental tobacco. references dražić, s., prodanović, s., živanović, t. 2010: identification of donor lines containing favorable alleles for quantitative traits in burley tobacco (n. tabacum l.). genetika, vol 42, no. 2, 287 297. jovanović, d. 2001: priručnik za proizvodnju duvana.digp “prosveta”, niš korubin-aleksoska, a., nikova, v., aleksoski, j. 2010: regression analysis of the interitance of leaf size in f1 and f2 propgenies in various tobacco genotypes. biotechnology & biotechnological equipment. eq. volume 24, number 2. p. 401-406. miceska, g., dimitreski, m. 2000: some characteristics tobacco variety p 10-3/2 (in vitro), transplanted in field conditions. tutun/tobacco science and profession.vol. 50, no 4-6, p. 61-69. miceska, g., dimitreski, m., sspasenoski, m. 2003: the effect of common mosaic virus (tmv) on morphological characteristic of plants from tobcco type prilep. tutun/tobacco science and profession. vol. 53, no 9-10, p. 268-277. moon, h. s., j. m. nifong, j. s. nicholson, a. heineman, k. lion, r. van der hoeven, a. j. hayes & r. s.lewis 2009: microsatellite-based analysis of tobacco (nicotiana tabacum l.). crop sci, 49,2149-2159. stančić, i., popović, r., pešić, v. 1998: analiza fizičkih i hemijskih osobina lista kod nekih cms hibrida orijentalnih duvana. tutun/tobacco. vol. 48 no. 1-6. p.58-64. biologica nyssana 2 (1) june 2011: x-x čavlović d. et al. allochthonous woody taxa in zasavica ecosystem 38 ćirković et al. 2023, biologica nyssana 14(1) 14 (1) june 2023: 31-38 doi: 10.5281/zenodo.8027114 results of the first analysis of tadpoles’ diet and determination of microplastics presence of rana, bufo and bufotes species from different localities in serbia original article glorija ćirković department of biology and ecology, faculty of science, university of kragujevac, radoja domanovića 12, kragujevac, serbia glorija.cirkovic@pmf.kg.ac.rs (corresponding author) aleksandra rakonjac department of biology and ecology, faculty of science, university of kragujevac, radoja domanovića 12, kragujevac, serbia rastko ajtić department of biology and ecology, faculty of science, university of kragujevac, radoja domanovića 12, kragujevac, serbia received: october 14, 2022 revised: april 19, 2023 accepted: may 02, 2023 abstract: nutrition is one of the main processes in living beings because it provides energy. amphibians are an essential link in food chains, but they have not been sufficiently studied. it is believed that most anuran larvae and tadpoles are omnivorous, but the trophic status of many tadpoles has not been precisely determined. microplastics (mps) are one of the major threats to aquatic ecosystems and can be ingested by various groups of aquatic organisms, such as plankton, shellfish, fish, and amphibians, but current knowledge of mps occurrence in these aquatic organisms is still scarce. in this research, the intestinal contents of tadpoles of four anuran species were analyzed to assess nutrition and the presence of microplastics. the results show that the largest percentage of tadpoles’ diet consists of diatoms from the genera achnanthidium and navicula, and detritus. the remains of some invertebrates were also present in the material. microplastic analysis proved the presence of mps in all investigated samples. the data obtained in this research represent the first concrete results due to which it is necessary to continue extensive research at several levels. key words: tadpoles, nutrition, microplastics, rana, bufo, bufotes apstrakt: rezultati prve analize ishrane punoglavaca i utvrđivanje prisustva mikroplastike kod vrsta iz rodova rana, bufo i bufotes sa različitih lokaliteta u srbiji ishrana je jedan od ključnih procesa živih bića jer predstavlja glavni izvor energije. vodozemci su važna karika u lancima ishrane, ali nisu dovoljno proučavani. veruje se da je većina larvi bezrepih vodozemaca, odnosno punoglavaca, omnivorna, ali trofički status mnogih punoglavaca nije precizno utvrđen. mikroplastika (mp) je jedan od glavnih zagađivača u vodenim ekosistemima pa je različite grupe vodenih organizama, kao što su plankton, školjke, ribe i vodozemci mogu uneti u organizam, ali trenutno znanje o prisustvu mp u ovim vodenim organizmima je još uvek nedovoljno. u ovom istraživanju analiziran je sadržaj creva punoglavaca četiri vrste bezrepih vodozemaca kako bi se procenila ishrana i prisustvo mikroplastike. rezultati pokazuju da najveći procenat ishrane punoglavaca čine silikatne alge iz rodova achnanthidium i navicula, kao i detritus. takođe, u sadržaju creva bili su prisutni i ostaci nekih beskičmenjaka. analize mikroplastike dokazale su da su čestice mp prisutne u svim ispitivanim uzorcima. podaci dobijeni u ovom istraživanju predstavljaju prve konkretne rezultate zbog kojih je neophodno nastaviti dodatna istraživanja na više nivoa ključne reči: punoglavci, ishrana, mikroplastika, rana, bufo, bufotes introduction knowing a species’ trophic status and spectrum of nutrition is of particular importance, given that food is the primary link between the species and its habitat. nutrition is an ongoing process and therefore can be crucial to the ecology of a particular species. also, feeding is the most important activity of living beings, primarily because of obtaining the energy needed to perform life activities. amphibians © 2023 ćirković et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 31 are vertebrates that can be at the top of food chains, although they are more often found in the middle of the food chains and form the link between lower taxa, such as insects, and higher ones, such as birds and mammals (cogălniceanu et al., 2000). they are a very important link in food chains because, due to their life history, they connect aquatic and terrestrial ecosystems. adults are, in most cases, carnivores. they feed on most invertebrates, and on some vertebrates. in contrast to adults, larvae of tailed and anuran amphibians show much greater variability in diet types. some feed on plankton, some are microphagous, herbivorous, or detritivorous, and there are also predatory larvae (mcdiarmid & altig, 2010). even though they are very important, they have been insufficiently investigated and the trophic status of many species of amphibians, both larvae and adults, has not been sufficiently studied. in the literature related to the balkan peninsula, there is very little information about the diet of representatives of the order anura (popović et al., 1992; simić et al., 1992; bjelić et al., 1996; paunović, 2000; paunović et al., 2010; paunović, 2011; crnobrnja-isailović et al., 2012; čađenović, 2014), and the diet and morphology of the larval anuran, i.e. tadpoles, have been particularly poorly researched (sidorovska et al., 2002; ilić, 2020). most aquatic tadpoles are primary consumers that metamorphose into terrestrial adults, thereby transferring energy from aquatic habitats to food chains in terrestrial ecosystems (trakimas et al., 2011). because of their different habitats and the variety of life forms and feeding structures, different species of anuran amphibian larvae use several food sources including periphyton and detritus (ranvestel et al., 2004). many tadpoles are benthic in their habitats, spending most of their time at the bottom of ponds, and relatively little in free water, so the composition of the benthos in the aquatic ecosystems they inhabit is important for their nutrition. tadpoles from the bufonidae and ranidae families are, among others, with these characteristics. however, some species from these families sometimes, except for benthic organisms, feed on organisms from the surface of the water (bacterial film) (rana dalmatina), scraping periphyton, corpses, and eggs of the same or other species or plankton (rana temporaria, bufo bufo) (wells, 2007). species r. dalmatina, r. temporaria and b. bufo in serbia can be syntopic. they enter aquatic habitats only during a short reproductive period in early spring, where tadpoles develop after hatching from eggs. the species r. dalmatina, b. bufo and bufotes viridis inhabit the entire territory of serbia, while the species r. temporaria is found on 10-50% of the entire territory of serbia (vukov et al., 2013). the increased use of plastic products in human activities has caused concern for the environment. when plastic reaches (directly or indirectly) aquatic ecosystems, it undergoes degradation, after which there is a change in particle size and density. however, most plastic products are very resistant to biochemical degradation and therefore persist for a long time in the environment. over time, they break down into smaller and smaller fractions called microplastics (from 1 to 5 μm in size) (pastorino et al., 2022). there are two types of microplastics: primary and secondary. the primary represents plastic particles that are produced and that enter the composition of various products (for example cosmetics), and the secondary is created by the decomposition of macroplastics or during the use and disposal of plastic products such as plastic packaging (pastorino et al., 2022). microplastics (mps) are one of the major threats to aquatic ecosystems and can be ingested by various groups of aquatic organisms, such as plankton, shellfish, fish, and amphibians, but current knowledge of mps occurrence and impact on these aquatic organisms is still scarce (hu et al., 2018; kolenda et al., 2020). also, despite the key role of amphibians in the food webs and the potential to transfer contaminants through trophic levels and from freshwater to terrestrial ecosystems, the data on their capacity to accumulate mps are very scarce compared to other vertebrates (pastorino et al., 2022). there are only a few published field studies about mps accumulation by tadpoles (hu et al., 2018; karaoğlu & gül, 2020; kolenda et al., 2020). the main goal of this research is to obtain results on the nutrition of tadpoles from different habitats in serbia and thus supplement knowledge about the nutrition of anuran larvae and contribute to determining their trophic status. another goal of this study was to determine whether tadpoles digest microplastics which can serve as a good initial step for further analyses of whether the mps affect the development and metabolism of tadpoles. materials and methods sampling we used for the analysis 130 tadpoles of rana dalmatina, r. temporaria, bufo bufo and bufotes viridis from 11 localities in serbia (tab. 1; fig. 1). the tadpoles were collected using standard nets for amphibians and then preserved in alcohol. the collection was obtained during spring (april, may) 2021. and 2022. the 70 tadpoles were used to investigate tadpoles’ diet, and 60 tadpoles 32 biologica nyssana ● 14 (1) june 2023: 31-38 ćirković et al. ● results of the first analysis of tadpoles’ diet and determination of microplastics presence of rana, bufo and bufotes species from different localities in serbia biologica nyssana ● 14 (1) june 2023: 31-38 ćirković et al. ● results of the first analysis of tadpoles’ diet and determination of microplastics presence of rana, bufo and bufotes species from different localities in serbia 33 were analyzed to estimate the presence of microplastics (tab. 1). the selected four species for this study were the most adequate for analysis because they were the most abundant in investigated localities. for future research, we plan to include other species and more localities. the tadpoles were from different developmental stages (most of them were 25 and 27), according to gosner’s simplified table for staging anuran embryos and larvae (gosner, 1960). determination of species tadpoles can be identified using the details of the overall body proportions, but the position of the spiraculum and the arrangement of tooth rows and other mouthpart details allow a confident identification to be made. for the identification of the tadpole samples, we used an identification key (ajtić & ćirković, in prep.) based on the specific arrangement of tooth rows and other mouthpart details. nutrition analysis to assess nutrition, intestinal contents were analyzed. a whole intestine was removed from each tadpole and contents were placed in ethanol (96%) and homogenized. depending on the abundance of particles, either the entire sample or a subsample was observed under the microscope at table 1. localities and a number of tadpoles used for intestinal content analysis, and developmental stages according to gosner (1960) species localities number of specimens for nutrition analysis number of specimens for microplastic analysis developmental stages rana temporaria golijska river 10 20 34, 38 trešnjica river (povlen) 5 10 25 srednji povlen 5 / 29 rana dalmatina medvednik 5 / 24,25 cer 5 5 25,38 golija 5 / 36 bufo bufo tometino polje 5 / 37 cer 5 5 27 zlatibor 5 10 26 besna kobila 5 / 25 bufotes viridis starac 10 10 28 zasavica 5 / 34 fig. 1. localities 34 biologica nyssana ● 14 (1) june 2023: 31-38 800x magnification. the observation was made by transects, photographed and, if possible, identified. algae were identified to the genera level using standard identification keys (krammer & langebertalot, 1986, 1988, 1991; john et al., 2011; jüttner et al., 2015; wehr et al., 2015). microplastics analysis the presence of microplastics was analyzed using the method described by hu et al. (2018) with some modifications. the specific number of tadpoles from each investigated species was separated in glass petri dishes. then, they were washed three times with distilled water and digested using h2o2 (30%) for no more than 72 h. the digested solutions were filtered through 47 mm diameter polycarbonate filters with a pore size of 5 µm. all filters were incubated at 60 °c for 4 h in a dryer and stored in dry glass petri dishes until further observation. mps were then searched for by means of a stereomicroscope. to avoid contamination, a cotton laboratory coat was worn during procedures, and before analysis, all liquids (pure water and h2o2) were filtered using filter paper (5 µm porosity), and all the equipment used during analysis was rinsed in distilled water. the procedural blanks were performed to assess whether the materials, solutions, equipment, or workspace were contaminated with microplastics. results and discussion the intestinal content analysis confirmed that the most significant percent of tadpoles’ nutrition were diatoms, i.e., algae from class bacillariophyceae, and the most present were specimens from the genera achnanthidium and navicula, and in a smaller amount, specimens from the genera surirella, pinnularia, and nitzschia (tab. 2). this may be a consequence of the period in which the tadpoles were feeding, as not all algae develop at the same time. their development depends on the season, the physical properties of substrata, and different environmental factors (murdock & dodds, 2007). the presence of algae from cl. xanthophyceae, especially genus tribonema was also noted. in some intestinal content, the representatives from the genus phacus (cl. euglenophyceae, phylum euglenozoa; specimens from srednji povlen) (tab. 2) were found as well. detritus was also found in intestinal content, as well as the remains of invertebrates that could not be determined. our results showing that the diet of tadpoles is not limited to algae is consistent with other studies (altig et al., 2007), where it was reported that tadpoles consume a range of different particles in their diet, including benthic or planktonic algae, vegetation, bacteria, fungi, zooplankton, and animal flesh, as well as inorganic particles, such as sand. it is assumed that the diverse diet of most tadpoles actually reflects an indiscriminate diet. the type and dynamics of tadpole nutrition are related to the structure and morphology of the oral apparatus, but also to the availability and diversity of nutritional resources in the habitats where metamorphosis takes place. a smaller number of studies (peterson & boulton, 1999) have focused on tadpoles that feed on algae by scraping from the substrate, but herbivorous tadpoles have been shown to alter the structure and/or biomass of algae in permanent lentic aquatic ecosystems. some researchers claim that the contents of tadpoles’ guts represent an inexhaustible sample of algae (huang et al., 2003; skelly & golon, 2003), which is confirmed by our results. it is believed that tadpoles, many of which are primary consumers, greatly influence on the structure and functioning of ecosystems by changing the composition of algae, i.e. the composition of primary producers, as well as on the dynamics of organic matter in many aquatic habitats. previous research has shown that primary production, nutrient cycling, plant residue decomposition, and invertebrate populations change when tadpoles are removed from habitats or reduced in numbers (whiles et al., 2006). the substrate in aquatic ecosystems is a significant source of food for tadpoles. the tadpoles use organic matter from detritus in their nutrition, benthic algae from phyla bacillariophyta and chlorophyta, and remains of all other algae, invertebrates, and vertebrates. this refers to tadpoles of many species in the families bufonidae, discoglossidae, hylidae, leptodactylidae, megophryidae, myobatrachidae, pelobatidae, ranidae, and rhacophoridae (wells, 2007). the results of the skelly & golon (2003) study confirm that tadpoles are affected by variations in the benthic particles as a food source. the same authors showed that tadpoles are nevertheless selective when it comes to nutrition and that the variety and availability of nutrient sources in and between aquatic habitats significantly impact their development. in serbia, there are no studies of microplastics’ influence on anurans, especially not on their larvae. there is some information about the presence of mps in some fish species (nikolić et al., 2022) which suggests that there are particles of mps in our aquatic ecosystems and that’s why we could assume that it can also be found in tadpoles because they are extremely sensitive to changes in the environment. after examination of the remains of tadpoles used for microplastic analyses, it was confirmed that ćirković et al. ● results of the first analysis of tadpoles’ diet and determination of microplastics presence of rana, bufo and bufotes species from different localities in serbia ta bl e 2. a lg ae c om po si tio n in ta dp ol es ’ i nt es tin e b uf o bu fo b uf ot es v ir id is r an a da lm at in a r an a te m po ra ri a c er t om et in o po lje b es na ko bi la z la tib or st ar ac z as av ic a m ed ve dn ik c er g ol ija g ol ijs ka r iv er t re šn jic a r iv er (p ov le n) sr ed nj i po vl en c l. b ac ill ar io ph yc ea e a ch na nt hi di um ++ + ++ + ++ + ++ ++ ++ + ++ + ++ + ++ + ++ + ++ + n av ic ul a ++ + ++ + + ++ + ++ + ++ + ++ + ++ + ++ + ++ + m el os ir a + + ++ d en tic ul a ++ + ++ su ri re lla + + + + + + ++ n itz sc hi a ++ + ++ + + + + + + p in nu la ri a ++ + + + + + + + ++ + + ++ + g om ph on em a ++ ++ + + + + + u ln ar ia + + + + + + f ra gi la ri a ++ + + + h an na ea + ++ + + c l. c hl or op hy ce ae m ic ro sp or a ++ + c l. x an th op hy ce ae t ri bo ne m a + ++ + + ++ + ++ + + + + + ++ c l. e ug le no ph yc ea e p ha cu s + + ++ + biologica nyssana ● 14 (1) june 2023: 31-38 ćirković et al. ● results of the first analysis of tadpoles’ diet and determination of microplastics presence of rana, bufo and bufotes species from different localities in serbia ++ + pr es en t i n ea ch s am pl e; + + pr es en t i n ev er y sa m pl e, b ut in a s m al le r p er ce nt ag e; + p re se nt in le ss th an 5 0% o f s am pl es ; n ot p re se nt 35 36 biologica nyssana ● 14 (1) june 2023: 31-38 ćirković et al. ● results of the first analysis of tadpoles’ diet and determination of microplastics presence of rana, bufo and bufotes species from different localities in serbia that mps particles are present in tadpoles (karaoğlu & gül, 2020). pastorino et al. (2022) showed that mps can be found in the digestive tract of adult specimens of rana temporaria. the presence of microplastics in tadpole samples from different localities in serbia represents the first concrete results, so it is necessary to continue the elements of microplastics were present in all investigated species. the mps in most cases were fibrils in different colors (tab. 3, fig. 2). this finding provides novel insights into the accumulation of mps in tadpoles from different localities in serbia. as early life stages of amphibians, tadpoles show extensive feeding activity that exposes them to mps table 3. results of microplastic analysis species localities number of specimens presence of microplastics characteristics of microplastics (shape, color) bufo bufo cer 5 + black particles bufotes viridis starac 10 + red filaments rana dalmatina cer 5 + black filament rana temporaria golijska river 20 + blue and red filaments trešnjica (povlen) 10 + black filaments fig. 2. microplastic filaments in tadpole samples + present elements present in the aquatic environment (hu et al., 2018). our results match with the results of da costa araújo et al. (2019) which have shown that mps can be ingested by physalaemus cuvieri (fam. leptodactylidae) tadpoles. mps can accumulate in their organs and cause morphological, cytotoxic, and mutagenic changes that may affect their health and development. kolenda et al. (2020) study showed that tadpoles of pond-breeding anurans from central europe are exposed to mps pollution. the same authors also suggest that tadpoles can be a significant vector of mps transfer from aquatic to terrestrial ecosystems. investigation of mps in tadpoles of species pelophylax ridibundus and rana macrocnemis (fam. ranidae) from turkey confirmed extensive research at several levels. for future analysis, we plan to include other species and more localities. tadpoles are extremely sensitive to changes in the environment and can give scientists valuable insight into how an ecosystem is functioning, and because they have an important role in food chains, many other animals are affected by them. the investigation of mps in amphibians is of critical importance due to their endangerment and decline, and the accumulation of mps in amphibians can be a pathway for transfering these important pollutants between freshwater and terrestrial ecosystems (pastorino et al., 2022). 37 biologica nyssana ● 14 (1) june 2023: 31-38 acknowledgements. this work was funded by the ministry of science, technological development and innovation of the republic of serbia. agreement no. 451-03-68/2022-14/ 200122. references altig, r., whiles, m.r., & taylor, c.l. (2007). what do tadpoles really eat? assessing the trophic status of an understudied and imperiled group of consumers in freshwater habitats. freshwater biology, 52. bjelić, o., horvat, a., kostić, d., popović, e., & simić, s. (1996). food analysis and helmintes of rana temporaria l., 1758 (amhibia. anura) at šar planina mountain in serbia. 1st congress of biologists of macedonia, former yugoslav. republic of macedonia, ohrid, 18-21 september 1996. book of proceedings, p.140. cogălniceanu, d., palmer, w. m., & ciubuc, c. (2000). feeding in anuran communities on islands in the danube floodplain. amphibia-reptilia, 22, 1-19. crnobrnja-isailović, j., ćurčić, s., stojadinović, d., tomašević-kolarov, n., aleksić, i., & tomanović, ž. (2012). diet composition and food preferences in adult common toads (bufo bufo) (amphibia: anura: bufonidae). journal of herpetology, 46(4). čađenović, n. (2014). promene trofičkih karakteristika obične krastače bufo bufo, mertens and müller, 1928 (bufonidae: anura) tokom postmetamorfoze. [changes in trophic characteristics of common toad bufo bufo, mertens and müller, 1928 (bufonidae: anura) during postmetamorphosis] (doctoral dissertation, university of novi sad) https://nardus.mpn.gov.rs/handle/123456789/9136 da costa araújo, a.p., de melo, n.f.s., de oliveira junior, a.g., rodrigues, f.p., fernandes, t., de andrade vieira, j.e., rocha, t.l., & malafaia, g. (2019). how much are microplastics harmful to the health of amphibians? a study with pristine polyethylene microplastics and physalaemus cuvieri. journal of hazardous materials, 382(121066) gosner, k.l. (1960). a simplified table for staging anuran embryos and larvae with notes on identification. herpetologica, 16, 183-190. https:// www.jstor.org/stable/3890061 hu, l., chernick, m., hinton, d.e., & shi, h. (2018). microplastics in small waterbodies and tadpoles from yangtze river delta, china. environmental science & technology, 52, 8885– 8893. huang, c., liang, m., & kam, y. (2003). the fatty acid composition of oophagous tadpoles (chirixalus eiffingeri) fed conspecific or chicken egg yolk. comparative biochemistry and physiology, 135, 329-336. ilić, m. (2020). morfološka diferencijacija larvenih stadijuma odabranih vrsta žaba rodova rana i bufo na području srbije. [morphological differentiation of larval stages of selected anuran rana and bufo species in serbia] (doctoral dissertation, university of belgrade). john, d.m., whitton, b.a., & brook, a.j. (2011). freshwater algal flora of the british isles. cambrige university press, cambrige. 878 p. jüntter, i., williams, d.m., levkov, z., falasco, e., battegazzore, m., cantonati, m., van de vijver, b., angele, c., & ector, l. (2015). reinvestigation of the type material for odontidium hyemale (roth) kützing and related species, with description of four new species in the genus odontidium (fragilariaceae, bacillariophyta). phytotaxa, 234(1), 1-36. karaoğlu, k. & gül, s. (2020). characterization of microplastic pollution in tadpoles living in small water-bodies from rize, the northeast of turkey. chemosphere, 255. krammer, k. & lange-bertalot, h. (1986). bacillariophyceae 1, teil: naviculaceae. in: ettl, h., gerloff, j., heying, h., mollenhauer, d. (eds.) süβwasserflora von mitteleuropa. [freshwater flora of central europe] (p. 596). gustav fisher verlag, jena. krammer, k. & lange-bertalot, h. (1988). bacillariophyceae 2, teil: bacilariaceae, epithemiaceae, surelliaceae. in: ettl, h., gerloff, j., heying, h., mollenhauer, d. (eds.) süβwasserflora von mitteleuropa. [freshwater flora of central europe] (p. 596). gustav fisher verlag, stutgart and jena. krammer, k. & lange-bertalot, h. (1991). bacillariophyceae 4, teil: achnanthaceae. kritische ergänzungen zu navicula (lineolatae) und gomphonema. in: ettl, h., gartner, g., gerloff, j., heying, h., mollenhauer, d. (eds.) süβwasserflora von mitteleuropa. [freshwater flora of central europe] (p. 437). gustav fisher verlag, stutgart and new york. kolenda, k., kuśmierek, n., & pstrowska, k. (2020). microplastic ingestion by tadpoles of pondbreeding amphibians—first results from central europe (sw poland). environmental science and pollution research, 27, 33380–33384. mcdiarmid, r.w. & altig, r. (2010). morphology 37 ćirković et al. ● results of the first analysis of tadpoles’ diet and determination of microplastics presence of rana, bufo and bufotes species from different localities in serbia 38 of amphibian larvae. in: dodd j.c.k. (ed.) amphibian ecology and conservation – a handbook of techniques. oxford university press, oxford. murdock, j.n. & dodds, w.k. (2007). linking benthic algal biomass to stream substratum topography. journal of phycology, 43, 449–460. nikolić, m.d., milošković, a.m., jakovljević, m.m., radenković, m.d., veličković, t.z., đuretanović, s.r., kojadinović, n.m., nikolić, m.m., & simić, v.m. (2022). the first observation of the presence of microplastics in wild common bleak (alburnus alburnus l.) and standardization of extraction protocols. kragujevac journal of science, 44, 267-282. pastorino, p., prearo, m., di blasio, a., barcelò, d., anselmi, s., colussi, s., alberti, s., tedde, g., dondo, a., ottino, m., pizzul, e., & renzi, m. (2022). microplastics occurrence in the european common frog (rana temporaria) from cottian alps (northwest italy). diversity, 14(66). paunović, а. (2000). ishrana anura (amphibia) petrovaradinskog rita. [diet of anura (amphibia) of petrovaradin rit] (master thesis, university of novi sad). paunović, a., bjelić-čabrilo, o., & simić, m. (2010). the diet of water frogs (pelophylax esculentus “complex”) from the petrovaradinski rit marsh (serbia). archive of biological sciences, 62(3), 797-804. paunović, a. (2011). ishrana češnjarki (pelobates spp.) u uslovima sintopije i alotopije. [the diet of syntopic and allotopic spadefoot toads (pelobates spp.).] (doctoral dissertation, university of novi sad). peterson, c.g. & boulton, a.j. (1999). stream permanence influences microalgal food availability to grazing tadpoles in arid-zone springs. oecologia, 118(3), 340-352. popović, e., simić, s., & tallósi, b. (1992). food analysis of some rana species in the habitat of carska bara (yu). tiscia, 26, 1-3. biologica nyssana ● 14 (1) june 2023: 31-38 ranvestel, a.w., lips, k.r., pringle, c.m., whiles, r.m., & bixby, r.j. (2004). neotropical tadpoles influence stream benthos: evidence for the ecological consequences of decline in amphibian populations. freshwater biology, 49, 274-285. sidorovska, v., ljubisavljevic, k., džukić, g., & kalezić, m.l. (2002) tadpole morphology of two spadefoot toads (pelobates fuscus and p. syriacus) (amphibia, anura, pelobatidae). spixiana, 25, 183191. simić, s., tallósi, b., & popović, e. (1992). seasonal changes in feeding of rana ridibunda palas (amphibia: anura) in backwater tisza. tiscia, 26, 5-7. skelly, k.d. & golon, j. (2003). assimilation of natural benthic substrates by two species of tadpoles. herpetologica, 59(1), 37-42. trakimas, g., jardine, d.t., barisevičiūtė, r., garbaras, a., skipitytė, r., & remeikis, v. (2011). ontogenetic dietary shifts in european common frog (rana temporaria) revealed by stable isotopes. hydrobiologia, 675(1), 87-95. vukov, t., kalezić, m.l., tomović, lj., krizmanić, i., jović, d., labus, n., & džukić, g. (2013). amphibians in serbia: distribution and diversity patterns. bulletin of the natural history museum, 6, 90-112. wehr, j.d., sheath, r.g., & kociolek, j.p. (2015). freshwater algae of north america, ecology and classification (1066 p.). academic press, san diego. wells, k.d. (2007). the ecology and behaviour of amphibians. (1141 p.) the university of chicago press, chicago. whiles, m.r., lips, k.r., pringle, c.m., kilham, s.s., bixby, r.j., brenes, r., connelly, s., colongaud, j.c., hunte-brown, m., huryn, a.d., montgomery, c., & peterson, s. (2006). the effects of amphibian population declines on the structure and function of neotropical stream ecosystems. frontiers in ecology and the environment, 4(1), 2734. ćirković et al. ● results of the first analysis of tadpoles’ diet and determination of microplastics presence of rana, bufo and bufotes species from different localities in serbia microsoft word 0404_cavar_et_al biologica nyssana 1 (1-2) december 2010: 99-103 čavar, s. et al. comparison of essential oil composition of stachys … 31 original article ! comparison of essential oil composition of stachys menthifolia vis. from two natural habitats in croatia sanja ćavar1,2*, milka maksimović1, marija edita šolić3 1university of sarajevo, faculty of science, department of chemistry, zmaja od bosne 33-35, 71000 sarajevo, bosnia and herzegovina 2university of ljubljana, faculty of chemistry and chemical technology, aškerčeva 5, 1000 ljubljana, slovenia 3institute „mountain and sea“, franjevački put 1, 21300 makarska, croatia e-mail: sanja.cavar@pmf.unsa.ba abstract: ćavar, s., maksimović, m., šolić, m. e.: comparison of essential oil composition of stachys menthifolia vis. from two natural habitats in croatia. biologica nyssana, 1 (1-2), december 2010: 99-103. stachys menthifolia vis. is an endemic species from the balkan peninsula. aerial parts of the plant were collected from its natural habitat near dubrovnik. hydrodistilled volatile oil obtained from the plant material of s. menthifolia was subjected to gas chromatographic analysis coupled to mass spectrometry. more than 70 compounds were identified, representing 94.5% of the total oil. the major constituents of the oil were diterpenoid abietatriene (11.7%), and sesquiterpene hydrocarbons α-bisabolene (8.4%), and β-caryophyllene (7.4%). presented results are comparable to our previous findings on essential oil composition of the same species from biokovo mountain, with small differences in quantitative and qualitative constitution of the oil. although plants belonging to the stachys genus show significant variability in their chemical compositions depending on the location and stage of plant development, this work indicates that chemical polymorphism of endemic s. menthifolia does not manifest in the region of croatian mediterranean area. key words: abietatriene, essential oil, gc-ms, stachys menthifolia vis. introduction ! the genus stachys is one of the largest representative genera of the lamiaceae family, and includes about 300 species in the subtropical and tropical regions of both hemispheres (m a b b e r l e y , 1997). like most of species belonging to the family lamiaceae, stachys species produce essential oils, but the composition of volatile compounds is known only in a small number of species (m a l y , 1985; c a k i r et al., 1997; m a r i o t t i et al., 1997; p é l i s s i e r at al., 1999; c h a l c h a t et al., 2001; k u k i ć et al., 2006; p a l i ć et al., 2006; r a d u l o v i c et al., 2007). available literature data indicate the existence of a chemical polymorphism of essential oil composition among stachys taxa. stachys menthifolia vis. is an endemic species of the west balkan region. š i l i ć & š o l i ć (2001) discovered its natural habitat in the region of biokovo mountain in croatia. the first location in croatia was near dubrovnik, discovered by roberto visiani, who was the first who described this species. continuing our phytochemical research on s. menthifolia, this paper presents the essential oil composition of the plant collected from its natural habitat near dubrovnik. 10th sfses • 17-20 june 2010, vlasina lake1 (1-2) • december 2010: 99-103 biologica nyssana 1 (1-2) december 2010: 99-103 ćavar, s. et al. comparison of essential oil composition of stachys … 32 material and methods plant material was collected in may 2008 from the natural habitat near dubrovnik. voucher specimens have been deposited at the department of chemistry, faculty of science, university of sarajevo, bosnia and herzegovina. all applied reagents were of the highest purity available and purchased from the sigmaaldrich chemical company. essential oil from the air-dried parts of s. menthifolia was isolated by hydrodistillation according to european pharmacopoeia for 2 h. the oil was extracted with dichloromethane and dried over anhydrous sodium sulphate and stored at 4°c in the dark until the analysis. gas chromatography-mass spectrometry analysis was carried out on a hewlett-packard 6890 series ii gas chromatograph fitted with a fused silica hp-5 (5% phenyl methyl siloxane) capillary column (30 m × 0.252 mm, 0.25 μm film thickness), coupled to a hp 6890 series ii mass selective detector (msd). column temperature was programmed from 60°c to 240°c at 3°c min-1, and helium was used as carrier gas. other operating conditions were as follows: inlet pressure 9.43 psi, injector temperature 250°c, detector temperature 280°c, split ratio 1:25, injection volume 1 μl. ionization of the sample components was performed in the ei mode, (70 ev), with scan range 20-555 amu, and scan time 1.60 s. the linear retention indices, ri, for all compounds were determined by injection of the hexane solution containing the homologous series of c8-c26 n-alkanes (v a n d e n d o o l & k r a t z , 1963). the identification of the essential oil constituents was accomplished by the visual interpretation, comparing their retention indices and mass spectra with literature data (a d a m s , 2007) by computer library search (hp chemstation computer library nbs75k.l, nist/epa/nih mass spectral library 2.0 and mass finder 4 computer software and terpenoids library), and in the laboratory own database. semi-quantitative analysis was carried out directly from peak areas in the gc profile. results and discussion the yield of s. menthifolia hydrodistilled oil was 0.09% based on the dry weight of the plant material. exactly seventy-six compounds were identified in the essential oil, representing 94.5% of the total oil. the components identified in investigated sample of s. menthifolia, their retention indices, and percentage composition is summarized in table 1. the major constituents of the oil were diterpenoid abietatriene (11.7%), and sesquiterpene hydrocarbons α-bisabolene (8.4%), and βcaryophyllene (7.4%). in general, sesquiterpenoid compounds predominate in the oil with 59.1%, where 35.1% are sesquiterpene hydrocarbons. presented results can be compared to essential oil composition of s. menthifolia from biokovo mountain (ć a v a r et al., 2010), with some differences in quantitative and qualitative constitution of the oil (figure 1). oxygenated sesquiterpenes were the most abundant volatiles in s. menthifolia from biokovo (table 1), followed by diterpene hydrocarbons. this sample was characterized as the oil of abietatriene– 8−α−acetoxyelemol chemotype. significant differences between these samples were in the content of aromatic compounds, particularly 4’methoxyacetophenone, which was not found in the population from dubrovnik. moreover, the content of sesquiterpene hydrocarbons was much higher in the sample from dubrovnik. the observed differences might be explained by the fact that this plant material was harvested one month earlier than the sample from biokovo. figure 1. gc chromatograms of essential oil of s. menthifolia from dubrovnik (1) and biokovo (2) literature survey on studies related to the essential oils obtained from the plants belonging to stachys genus from the balkan peninsula, showed significant variability in their chemical composition depending on location and stages of plant development. essential oil obtained from s. milanii petrović (p a l i ć et al., 2006) had borneol and terpinen-4-ol as the most abundant compounds, while the major constituents of essential oil from bosnian s. alpina l. spp. dinarica murb. were βbiologica nyssana 1 (1-2) december 2010: 99-103 čavar, s. et al. comparison of essential oil composition of stachys … 33 caryophyllene and germacrene d (k u k i ć et al., 2006). dehydroabietane is the predominant compound in the essential oil of s. plumosa griseb. (p e t r o v i ć et al., 2006). sesquiterpenoids were the main class of the constituents in croatian stachys species as follows: (e)-nerolidol in s. alpina; germacrene d in s. officinalis, s. recta subsp. subcrenata, s. salviifolia, and s. sylvatica, caryophyllene oxide, along with 1-octen-3-ol, in s. palustris, while s. recta subsp. recta contained βionone as the dominant volatile constituent (b i l u š i ć v u n d a c et al., 2006). table 1. comparison of essential oil composition of s. menthifolia from croatia # ri compound dubrovnik ra† (%) biokovo* ra (%) 1. 801 hexanal 0.3 tr‡-0.2 2. 847 (2e)-hexenal 0.6 tr-0.2 3. 861 n-hexanol 0.3 4. 874 2-methyl butyl acetate 0.2 5. 930 α-pinene 0.3 tr-1.0 6. 975 1-octen-3-ol 0.9 tr-0.5 7. 1026 limonene 1.0 0.1-3.4 8. 1041 benzene acetaldehyde 0.1 tr 9. 1056 γ-terpinene 0.2 tr 10. 1099 linalool 3.6 0.7-1.7 11. 1103 nonanal 0.2 tr 12. 1107 2-methyl butyl isovalerate 0.5 13. 1111 1-octen-3-yl acetate 1.9 14. 1122 3-octanol acetate 0.4 15. 1189 α-terpineol 0.3 tr-0.3 16. 1190 methyl salycilate tr tr 17. 1255 linalool acetate 0.3 18. 1286 trans-linalool oxide acetate (pyranoid) 0.2 19. 1373 α-copaene 0.5 tr-0.3 20. 1381 β-bourbonene 0.3 tr 21. 1416 β-caryophyllene 7.4 0.5-2.8 22. 1422 β-maaliene 0.2 23. 1434 β-guaiene 0.1 tr 24. 1450 α-humulene 3.4 tr-0.2 25. 1456 (e)-β-farnesene 1.4 26. 1478 γ-curcumene 0.3 27. 1481 ar-curcumene 0.3 tr-0.2 28. 1492 α-muurolene 0.6 tr-0.2 29. 1499 (e,e)-α-farnesene 1.3 30. 1503 α-zingiberene 3.8 31. 1508 α-bisabolene 8.4 tr-0.1 32. 1512 γ-cadinene 1.7 tr-0.4 33. 1522 δ-cadinene 3.3 34. 1530 trans-cadina-1,4-diene 0.3 35. 1535 α-cadinene 0.9 tr-0.2 36. 1547 elemol 1.4 2.2-3.6 37. 1564 2-epi-(e)-β-caryophyllene 0.3 38. 1574 spathulenol 0.7 0.7-1.7 39. 1579 caryophyllene oxide tr 2.4-5.2 40. 1581 clovenol 1.1 41. 1588 globulol 1.9 1.5-2.4 42. 1600 guaiol tr tr-0.3 43. 1606 β-oplopenone 0.3 tr 44. 1627 trans-isolongifolanone 0.9 1.2-1.6 45. 1629 γ-eudesmol 0.9 1.1-1.9 biologica nyssana 1 (1-2) december 2010: 99-103 ćavar, s. et al. comparison of essential oil composition of stachys … 34 r a d u l o v i c et al., (2007) suggested that high degree of variation in the main volatiles of the species of stachys genus, especially of germacrene d and its congeners, was correlated with possible rearrangements under hydrodistillation conditions. according to the presence and quantity of dominant compounds, the essential oil of investigated populations of s. menthifolia significantly differs from the previously published data concerning this species (s k a l t s a et al., 2003). greek s. menthifolia essential oil had abietatriene (13.7%), kaurene (9.0%) and 13-epimanoyl oxide (7.5%) as the most abundant compounds. the observed differences in qualitative and quantitative composition of the essential oils between these two geographically isolated populations of s. menthifolia, confirm the influence of the environmental conditions on the volatiles # ri compound dubrovnik ra† (%) biokovo* ra (%) 46. 1633 caryophylla-4(15),8(13)-dien-5-α-ol 0.4 tr-0.4 47. 1640 epi-α-murrolol 1.7 1.8-2.6 48. 1647 β-eudesmol 2.3 2.4-5.0 49. 1650 α-eudesmol 1.5 1.4-3.3 50. 1652 α-cadinol 1.5 2.0-3.1 51. 1655 allo-aromadendrene epoxide 1.3 52. 1669 valeranone 4.5 53. 1676 n-tetradecanol 0.5 0.5-0.6 54. 1682 α-bisabolol 0.4 55. 1695 isolongifolol 2.7 56. 1761 benzyl benzoate tr 57. 1788 8-α-acetoxyelemol 2.1 6.9-21.3 58. 1807 cryptomeridiol 0.3 0.5-6.7 59. 1844 6,10,14-trimethylpentadecan-2-one 0.2 60. 1903 isopimara-9(11),15-diene 0.6 0-5-1.1 61. 1954 pimaradiene 0.5 0.2-0.8 62. 1963 n-hexadecanoic acid 0.7 63. 1983 manool oxide 1.9 0.7-2.3 64. 2002 (e)-labda-7,12,14-triene 0.3 0.5-2.7 65. 2006 13-epi-dolabradien tr 0.3-0.6 66. 2050 abietatriene 11.7 3.5-21.1 67. 2073 abietadiene 0.6 tr-0.8 68. 2141 abienol 0.4 0.7-0.9 69. 2206 2-keto manool oxide 0.7 0.7-1.2 70. 2226 7-α-hydroxy manool 0.4 0.2-0.3 71. 2258 larixol 0.5 0.4-1.1 72. 2288 dehydroabietal 1.9 1.5 73. 2298 n-tricosane tr tr 74. 2499 n-pentacosane 0.3 tr-0.2 75. 2599 n-hexacosane tr tr-0.2 76. 2698 n-heptacosane 0.6 aliphatic compounds 7.4 0.5-1.4 aromatic compounds 2.2 4.5-17.0 monoterpene hydrocarbons 1.5 0.1-4.6 oxygenated monoterpenes 4.4 1.7-2.9 sesquiterpene hydrocarbons 35.1 1.4-6.4 oxygenated sesquiterpenes 24.0 48.4-58.9 diterpene hydrocarbons 13.1 3.5-25.2 oxygenated diterpenes 6.8 1.5-5.8 total identified 94.5 86.8-90.8 *data taken from ć a v a r et al., (2010); †ra-relative area; ‡tr-trace (<0.1%). biologica nyssana 1 (1-2) december 2010: 99-103 čavar, s. et al. comparison of essential oil composition of stachys … 35 found in plants. it is essential to notify that these prominent variations were not retrieved in the populations from croatia. conclusion in conclusion, gc-ms analysis on the essential oil of endemic s. menthifolia populations from croatian mediterranean area indicated similarities in qualitative, but small differences in quantitative composition of their essential oils. although plants belonging to stachys genus show significant variability in their chemical compositions depending on the location and stage of plant development, this work indicates that chemical polymorphism of endemic s. menthifolia does not significantly manifest in the region of croatian mediterranean area. references adams, r. p. 2007: identification of essential oil components by gas chromatography/mass spectrometry, 4th ed. allured publ., carol stream, il. bilušić vundac,v., pfeifhofer, h. w., brantner, a. h., males, z., plazibat, m. 2006: essential oil of seven stachys taxa from croatia. biochemical systematics and ecology, 34, 875-881. cakir, a.,  duru, m. e., harmandar, m., izumi, s., hirata, t. 1997: the volatile constituents of stachys recta l. and stachys balansae l. from turkey. flavour and fragrance journal, 12, 215-218. ćavar, s, maksimović, m., vidic, d., šolić, m. e. 2010: chemical composition of the essential oil of stachys menthifolia vis. pharmaceutical biology, 48, 170-176. chalchat, j. c. petrović, s. d., maksimović z. a., gorunović, m. s.  2001: essential oil of stachys officinalis l. (trevis), lamiaceae, from montenegro. journal of essential oil research, 13, 286-287. kukić, j. petrović, s., pavlović, m., couladis, m., tzakou, o., niketić, m. 2006: composition of the stachys alpina l. ssp. dinarica murb. flavour and fragrance journal, 21, 539-542. mabberley, d. j. 1997: the plant-book, 2nd ed. cambridge university press, cambridge, new york, melbourne. maly, e. 1985: paper chromatography of the essential oils occurring in the genus stachys. journal of chromatography, 333, 288-289. mariotti, j. p., costa, j., bianchini, a., bernardini, a.f., casanova, j.  1997: composition and variability of the essential oil of stachys glutinosa l. from corsica (france). flavour and fragrance journal, 12, 205-209. palić, r. m., lazarević, j. s., stojanović, g. s., ranđelović, v. n.  2006: chemical composition and antimicrobial activity of the essential oil of stachys milanii petrovic. journal of essential oil research, 18, 290-292. pélissier, y., marion, s., rapior, s. 1999: volatile constituents of stachys corsica (pers.) (lamiaceae). journal of essential oil research, 11, 63-64. petrovic, s., ristic, m., milenkovic, m., kukic, j., antic-stankovic, j., niketic, m. 2006: composition and antimicrobial activity of essential oil of stachys plumosa griseb. flavour and fragrance journal, 21, 250-252. radulović, n., lazarević, j., ristić, n., palić, r. 2007: chemotaxonomic significance of the volatiles in the genus stachys (lamiaceae): essential oil composition of four balkan stachys species. biochemical systematics and ecology, 35, 196-208. šilić, č., šolić, m. e. 2002: the taxonomy, chorology and ecology of stachys menthifolia vis. (lamiaceae) in the north-west part of its distribution area. acta botanica croatica, 61, 5156. skaltsa, h. d. demetzos, c., lazari, d., soković, m. 2003: essential oil analysis and antimicrobial activity of eight stachys species from greece. phytochemistry, 64, 743-752. van den dool, h., kratz, p. d. 1963: a generalization of the retention index system including linear temperature programmed gasliquid partition chromatography. journal of chromatography, 11, 463-471. microsoft word bn-oa-0201-06 jaksic biologica nyssana 2 (1) september 2011: 45-50 jakšić p. butterfly species (lepidoptera: hesperioidea and papilionoidea)... 45 original article ! butterfly species (lepidoptera: hesperioidea and papilionoidea) new to the serbian fauna predrag jakšić* university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia * e-mail: jaksic@pmf.ni.ac.rs abstract: jakšić, p.: butterfly species (lepidoptera: hesperioidea and papilionoidea) new to the serbian fauna. biologica nyssana, 2 (1), september 2011: 45-50. a species pyrgus trebevicensis (warren, 1926), melitaea telona (fruhstorfer, 1908) and coenonympha orientalis (rebel, 1913) are here recorded in serbia for the first time. the key morpho-anatomical parameters for identification were done. original pictures of c. orientalis in situ and photos of genital apparatuses are given. original map of distribution examined species in serbia was made. key words: butterflies, serbia, faunistics introduction ! the first fauna list of serbian butterfly species was provided by g r a d o j e v i ć (1931), where 150 species are mentioned. for this crucial work the knowledge about the serbian butterfly fauna has been significantly increased due to activity of national and foreign lepidopterists. until now, the existence of 193 good species in serbia has been verified (j a k š i ć & đ u r i ć , 2008). on the basis of morphometric study on the male genitalia, statistic analysis of populations and the dna sequences analysis, a new contemporary approach gives us the possibility to solve old taxonomic problems, mostly among the sibling species. the „sibling species“ concept denotes morphologically similar or identical natural populations that have been reproductively isolated. soon after the formation of this concept, h e y d e m a n n (1943) published a list of 40 sibling species. recently, descimon and mallet (2009) have published a long paper about sibling species among butterflies. sibling species are easy to distinct by the differences in their habits, early stages and ecophysiology. but, their fine variations in morphology and anatomy, howewer, are also essential for their differentiation. as a rule, these differences are most dificult to discover. all the three newly established species shown here belong to a grup of sibling species. materials and methods the adults material has been collected on the field using entomological net. after preparing, we determined the specimens by the wing-patterns and in all cases the identification has been also carried out by an examination of the male genitalia. the preparations were carried out following the well known standard procedure: maceration by boiling in potash, dissecting and cleaning, clearing in xylolum and mounting in canada balsam. the photos of genital parameters were taken using the "leica dm 1000" microscope with the "camera leica dec 290"; photos of in situ specimen were taken using “olympus” sp-510uz (7,1 megapixel and 10x optical zoom). all the material (specimens and 2 (1) • september 2011: 45-50 biologica nyssana 2 (1) september 2011: 45-50 jakšić p. butterfly species (lepidoptera: hesperioidea and papilionoidea)... 46 genitalia slides) are deposited in the author's collection. figure 1. male genitalia (valva) of: p. armoricanus, serbia, šar-planina mt., brezovica, 1400 m, octob., 3rd, 1975., jakšić p. leg., prep. no. srb-5148 (a); p. alveus, serbia, kopaonik mt., žljeb, 1770 m, july, 21st, 2005., jakšić p. leg., prep. no. srb-2278 (b); p. trebevicensis, serbia, tara planina mt., kaluđerske bare, 1020 m, july, 8-9th, 1985., jakšić p. leg., prep. no. srb-638 (c). results pyrgus trebevicensis (warren, 1926) in the new list, the butterfly species of europe (s w a a y et al., 2010) this species does not exist as a distinct species separated from pyrgus alveus. as a good species within the national faunas it has been listed by the following authors: austria (r e n n i e r , 1991; g r o s & e m b a c h e r , 1998; h a b e l e r , 1999); czech republic (b e n e š et al. 2001); deutschalnd (r e n n i e r , 1991; w a g n e r , 2002); bosnia and herzegovina (s c h a w e r d a , 1918; r e n n i e r , 1991; l e l o , 2007). s h a w e r d a (1918) described this species as p. reverdini: „etwas grösser, oberseits heller braun, nicht so dunkel wie die nennform, normal stark weiss gefleckt. unterseits sind die weissen flecke grösser, das braun lichter, breitere flügelspannung meist 29 mm. 5 ♂, 2 ♀, trebevic, vucijabara. von mir im juli 1907 und 1912 erbeutet.“ w a r r e n (1926) proposed to replace the name trebevicensis into reverdini schawerda, 1918, because reverdini is a primary junior homonym of p. reverdini oberhur. he citated s c h a w e r d a (1918) that reverdini differs from the type in being slightly larger, with the ground-colour of the upperside lighter brown and not so black as in typical alveus, and with the white markings strongly pronounced. the underside has all the white markings broader and the groundcolour of the hind-wings paler yellow. all the wings are said to be a litle broader than in the type.“ new data for serbia: material examined: serbia, tara planina mt., kaluđerske bare, 1020 m (x = 430 54' 33"; y = 190 32' 55") 1m, 8-9. vii 1985., jakšić p. leg., prep. no. srb-638; serbia, užice, 670 m (x = 430 50' 44"; y = 190 49' 25") 1m, 4. vi 1982., toševski i. leg., prep. no. srb-100; serbia, divčibare, 950 m (x = 440 06' 09"; y = 190 59' 20") 1m, 27. vi 2006., đurić m., leg., prep no. srb-2323; serbia, fruška gora mt., 130 m (x = 450 12' 47"; y = 190 50' 02") 1 m, vii 1929., rogulja m. leg., prep no. srb-96; serbia, golija mt., 1800 m (x = 430 20' 20"; y = 200 16' 38") 1 m, 22. vii 1937., vagner o. leg., prep. no. srb-60; serbia, golija mt., 1800 m (x = 430 20' 20"; y = 200 16' 38") 1 m, 22. vii 1937., vagner o. leg., prep. no. srb-87; serbia, stol mt., 900 m (x = 440 10' 24"; 220 07' 20") 1 m, 28. vii 1974., zečević m. leg., prep. no. srb-82 (fig. 4: ●) melitaea telona (fruhstorfer, 1908) in the new list the butterfly species of europe (s w a a y et al., 2010) this species exists as a distinct species separated from m. phoebe. as a good species within the national faunas it has been listed by the following authors: hungary (t ó t h & va r g a , 2010); macedonia (ve r o v n i k et al., 2010). tóth and v a r g a (2010) pointed their basic biologica nyssana 2 (1) september 2011: 45-50 jakšić p. butterfly species (lepidoptera: hesperioidea and papilionoidea)... 47 characteristics in male genitalia: „in males the depth of the central notch of the saccus proved to be the most important difference. except two characters all show significant differences, but if these differences are considered separately we can found major overlaping. in general we observed that in m. telona we can see a more notched saccus, and more symetric shape of processus posterior (in same side) because the inner process in the processus posterior is shorter than m. phoebe. these two characters are essentially the same as the distinctive characters in the original description of m. „phoebe“ kovacsi.“ according to p e c s e n y e et al. (2007) – cited by t ó t h & va r g a (2010) enzyme electrophoretic study of hungarian populations has also shown obvious difference between m. phoebe and m. telona without any mark of hybridisation. new data for serbia: material examined: serbia, stara planina mt., janja, 520 m (x = 430 24' 47"; 220 31' 04") 1 m, 8. vii 2010., jakšić p. leg., prep. no. srb-2484; serbia, stara planina mt., janja, 520 m (x = 430 24' 47"; 220 31' 04") 1 m, 8. vii 2010., jakšić p. leg., prep. no. srb-2485 (fig. 4: ▲) coenonympha orientalis (rebel, 1913) in the new list, the butterfly species of europe (s w a a y et al., 2010) this species exists as a distinct species separated from c. gardetta. as a good species within the national faunas it has been listed by c o u t s i s & g h a v a l á s (2005) for the territory of greece. b o i l l a t (1990) gives the first detailed presentation of arguments suppotring c. orientalis as a „bona species“. according to c o u t s i s & g h a v a l a s (2005): „... c. orientalis is being raised to specific level apparently on the basis of what is believed to be unique external characters, that do not relate to any of the other taxa under consideration, of sympatry with c. leander (though no actual syntopism is involved here), and of the supposed absence of intermediates between the two.“ in our specimens of c. gardetta the underside of fore-wing apex is grey and there is no ocelli; in c. orientalis the underside of fore-wing apex is brown-orange, with one or two ocelli. analysing the available material it can be concluded that c. gardetta is not present whereas c. orientalis is present in this part of the balkan peninsula. figure 2. melitaea telona (fruhstorfer, 1908): saccus and valva, srbija, stara planina mt., janja, 520 m, july, 8th, 2010., jakšić p. leg., prep. no. srb2484; new data for serbia: material examined: -serbia, tutin, smolućka reka, crkvine, 950 m (x = 430 02' 44"; y = 200 21' 40") 1 m, 23. vi 2006., jakšić p. leg; serbia, kopaonik mt., žljeb, 1750 – 1770 m (x = 430 18' 46"; y = 200 50' 00") 2 mm, 21. vii 2005., jakšić p. leg.; serbia, kopaonik mt., bele stene, 1760 m (x = 430 18' 30"; y = 200 49' 53") 1m, 23. vii 2010., jakšić p. leg. (fig. 4: ■). biologica nyssana 2 (1) september 2011: 45-50 jakšić p. butterfly species (lepidoptera: hesperioidea and papilionoidea)... 48 figure 3. c. orientalis (rebel, 1913), specimen from serbia, kopaonik mt., bele stene, 1760 m, july, 23rd, 2010., jakšić p. leg. discussion and conclusion the combination of morpho-anatomical, physiological-biochemical characteristics, ecological characteristics, as well as taxa biogeography present a good basis for solving the boundary taxonomic cases. even today, we are still impressed by the sensibility of the classical entomologists who were insightful enough to describe new taxa a century ago. out of three sibling species analysed here, two were raised to the status of species. in the near future combinig the morphological and dna analysis, the taxon pyrgus trebevicensis could be classified as a „bona species“ after this review 195 butterfly species have been noted in the territory of serbia: 22 species of hesperioidea and 171 species of papilionoidea (j a k š i ć , 2008) + two new species. this excludes c. gardetta, but includes the presence of c. orientalis instead. related to the butterfly fauna of europe (482 species, s w a a y et al., 2010), the species present in serbia make up 40%. references dražić beneš, j., konvička, m., & fric, z., 2001. faunistic records from the czech republic – 137 lepidoptera: hesperiidae, pyrgus trebevicensis warren, 1926. klapalekiana, 37: 152. boillat, h. 1990. coenonympha (superspecies gardetta) orientalis rebel mise au point taxinomique (lepidoptera nymphalidae satyrinae). alexanor 16(7): 395–412. figire 4. utm distribution map of examined species pyrgus trebevicensis (warren, 1926) ●; melitaea telona (fruhstorfer, 1908) ▲ and coenonympha orientalis (rebel, 1913) ■. coutsis, g.j. and ghavalás, n., 2005. a recently discovered new locality for coenonympha leander in greece, and notes about the taxonomic position of the species-group taxon coenonympha orientalis (lepidoptera: nymphalidae, satyrinae). phegea, 33(4): 121128. davenport, d. 1941. the butterflies of the satyrid genus coenonympha. bulletin of the museum of comparative zoology, harvard college 87: 215349. descimon, h. and mallet, j., 2009. bad species. in: ecology of butterflies in europe, eds. j. settele, t. shreeve, m. konvička and h. van dyck. cambridge university press. pp.: 219-249. gradojević, m., 1930-31. leptirovi srbije – diurna. (les papillons de serbie i. diurna). glasnik jugoslovenskog entomoloskog društva, v-vi (12): 133-158, beograd. gros, p. und embacher, g., 1998. pyrgus warrenensis (verity, 1928) und p. trebevicensis (warren, 1926), zwei für die fauna salzburgs biologica nyssana 2 (1) september 2011: 45-50 jakšić p. butterfly species (lepidoptera: hesperioidea and papilionoidea)... 49 neue dickkopffalterarten (lepidoptera: hesperiidae, pyrginae). z. arb. gem. őst. ent., 50: 3-16. habeler, h., 1999. lepidopterologische nachrichten aus der steiermark, 17 (lepidoptera). joannea zool., 1: 13-19. heydemann, f., 1943. die bedeutung der sogenannten dualspecies (zwillingsarten) für unsere kenntnis der artund rassenbildung bei lepidopteren. stettiner entomol. ztg., 104: 116142. jakšić, p., 2008. butterflies in the environment of serbia. in: jakšić, p. (ed.): prime butterfly areas in serbia. habiprot, beograd. pp.: 9-17. jakšić, p. i đurić, m., 2008. srpski nazivi dnevnih leptira (lepidoptera: hesperioidea i papilionoidea). (serbian names for butterflies). proceeding of the 9th symposium of flora of southeastern serbia and neighbouring regions, niš. pp.: 231-237. jong de, r., 1972. systematics and geographic history of the genus pyrgus in the palaearctic region (lepidoptera, hesperiidae). tijdschrift voot entomologie, 115(1): 1-121+pl. 1-6. lelo, s., 2007. contribution to knowledge of the fauna of butterflies in bosnia and herzegovina. acta entomologica serbica, 12(2): 73-92. pamperis, l., 2009. the butterflies of greece. koan editions, athens. pp. 1-766. renner, f., 1991. neue untersuchungsergebnisse aus der pyrgus alveus hübner gruppe in der palaearktis unter besonderer berücksichtigung von süddeutschland (lepidoptera: hesperidae). neue entomologische nachrichten aus dem entomologischen museum dr. ulf eitschberger, marktleuthen.28: 4-157. russell, p. et al., 2007. further investigations into melitaea telona fruhstorfer, 1908 (ogygia fruhstorfer, 1908; = emipunica verity, 1919)(lepidoptera: nymphalidae), with observation on biology and distribution. entomologist's gazette, 58(3): 137-166. schawerda, k., 1918. elfter nachtrag zur lepidopterenfauna bosniens und der herzegowina. verh. zool.-botan-ges. wien, 68: 19-36. swaay van, c. et al., 2010. european red list of butterflies. butterfly canservation europe, iucn species programme and iucn regional office for pan-europe, luxembourg pp.: 1-47. tóth, j.p. and varga, z., 2010. morphometric study on the genitalia of sibling species melitaea phoebe and m. telona (lepidoptera: nymphalidae). acta zoologica academiae scientiarum hungaricae, 56(3): 273-282. verovnik, r., micevski, b., đurić, m., jakšić, p., keymeulen, a., swaay van, c., veling, k., 2010. contribution to the knowledge of the butterfly fauna of the republic of macedonia (lepidoptera: papilionoidea & hesperioidea). acta entomologica slovenica, 18(1): 31-46. wagner, w., 2002. zur őkologie von pyrgus trebevicensis (warren, 1926) und pyrgus alveus (hübner, [(1803])(lepidoptera: hesperiidae) auf der schwäbischen alb (baden-württemberg). entomologische zeitschrift stuttgart., 112(5): 145-156. warren, b.c.s., 1926. monograph of the tribe hesperiidi (european species) with revised classification of the subfamily hesperiinae (palaearctic species) based on the genital armature of the males. transactions of the entomological society of london, 74: 1-170. biologica nyssana 2 (1) september 2011: 45-50 jakšić p. butterfly species (lepidoptera: hesperioidea and papilionoidea)... 50 živković et al. 2022, biologica nyssana 13(2) 13 (2) december 2022: 141-151 doi: 10.5281/zenodo.7437274 study of eutypa lata isolates originating from serbia original article sanja živković faculty of agriculture, university of niš, kruševac, serbia zivkovic.sanja@ni.ac.rs (corresponding author) tanja vasić faculty of agriculture, university of niš, kruševac, serbia darko jevremović fruit research institute, 32102 čačak, serbia mitra debasis icar-indian institute of horticultural research, karnataka 560 089, india received: june 08, 2022 revised: october 25, 2022 accepted: november 04, 2022 abstract: the potential influence of different types of light on the growth and sporulation of eutypa lata isolates was assessed, and detection at the molecular level was also performed. by applying in vitro observations, the influence of different types of light was monitored, (24 h exposure to uv light; alternating 12 h uv and 12 h darkness; alternating 12 h artificial fluorescent light and 12 h darkness), on the radial growth and sporulation of the anamorphic stage of three isolates of eutypa lata fungus (el117, el153 and el199) by comparison with two reference isolates of eutypa lata, bx1.10 and 8f, obtained from the inra, france. after the molecular detection using of the specific primer pair lata 1/ lata 2.2, all the studied isolates were found to belong to the species eutypa lata (syn. eutypa armeniacae), anamorph libertella blepharis. it was also found that the most suitable type of light for radial growth and sporulation of the studied isolates is exposure to 24 h uv light for 30 days. key words: isolates, molecular detection, breeding traits, light types, eutypa lata apstrakt: izučavanje izolata eutypa lata poreklom iz srbije u ovom radu je proučavan jedan od uzročnika odumiranja čokota vinove loze gljiva eutypa lata. praćen je uticaj različitih tipova svetlosti na porast i sporulaciju izolata eutipa lata, a izvršena je i detekcija na molekularnom nivou. naime, u in vitro uslovima praćen je uticaj različitih tipova svetlosti (24 h izlaganje uticaju uv svetla (uv); alternativno, 12 h uv i 12 h tama (uv-t); alternativno, 12 h veštačko fluorescentno svetlo i 12 h tama (st)) na radijalni porast i sporulaciju anamorfnog stadijuma tri izolata gljive eutypa lata (el117, el153 and el199) upoređivanjem sa dva referentna izolata eutypa lata bx1.10 i 8f dobijenih sa institute national de la recherche agronomique, inra, france. takođe je urađena detekcija izolata na molekularnom nivou primenom specifičnog para prajmera lata 1/lata 2.2. nakon sprovedene molekularne detekcije, utvrđeno je da svi proučavani izolati pripadaju vrsti eutypa lata (syn. eutypa armeniacae), anamorf libertella blepharis. takođe je ustanovljeno da je najpogodniji tip svetlosti za radijalni porast i sporulaciju, proučavanih izolata izlaganje, 24 h uv svetlu u trajanju od 30 dana. ključne reči: izolati, molekularna detekcija, odgajivačke odlike, tipovi svetlosti, eutypa lata introduction eutypa dieback, esca complex and botryosphaeria dieback (sosnowski & mccarthi, 2017) cause the greatest economic damage to vines as the cause of grapevine death (gtd), affecting plant growth, reducing yield and quality of grapes and premature decay of grapevines (gramaje et al., 2018). grapevine dieback disease is caused by multiple fungal pathogens that act individually or in combination and can infect a grapevine plant at all stages of growth (fontaine et al., 2016). it must be noted that these fungi cause similar and even the same symptoms on the leaves and shoots, as well as on the trunk of the vine. this complicates the separation of symptoms and the precise identification of disease causes in vineyards (gubler et al., 2005; gramaje et al., 2018). the dieback of the grapevine occurs in almost all countries of the world where the grapevine is grown commercially. one of the causes of grapevine dieback (gtd), which will be discussed in this text, is the fungus eutypa lata. this fungus is a vascular pathogen that infects the vine through fresh pruning wounds. the disease is spread by ascospores (carter, 1994; rolshausen et al., 2015). © 2022 živković et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 141 the fungus e. lata is very difficult to identify because this fungus does not form a teleomorphic stage (ascospores) in regions where annual rainfall is less than 330 mm (ju et al., 1991), as well as on artificial nutrient media (carter, 1994; munkvold, 2001; živković, 2019). adam (1938) caused an infection on the apricot tree, for the first time, by using a suspension of conidia of this fungus. glawe et al. (1982) determined this fungus based on host symptoms, pathogenicity tests on apricot and identification of anamorphs in culture (loc cit. ju et al., 1991). fungus eutypa lata can be easily grown on conventional laboratory media, but not all isolates sporulate (ju et al., 1991; mckemy et al., 1993; munkvold, 2001). for these reasons, a number of authors have studied the anamorphic stage of the fungus e. lata (rolshusen et al., 2006; trouillas & gubler, 2010). lecomte et al. (2000) were the first to design specific primers for the determination of e. lata species. the lata 1/lata 2.2 primer pair was particularly successful in separating e. lata isolates from isolates of over fifty different species of grapevine microorganisms. the aim of this study was to determine the affiliation of three studied isolates of eutypa lata (syn. eutypa armeniacae), anamorph libertella blepharis, with the help of molecular detection, using a specific pair of primers lata 1/lata 2.2 for the species eutypa lata. also for this purpose, sporulation and radial increase of the anamorphic stage of three isolates (el117, el153 and el199) on the nutrient medium of pda under the influence of different types of light were monitored. materials and methods samples and fungal isolation examination of vineyards in several sites of grape growing in our country in the period from 2004 to 2021, revealed symptoms of dieback of grapevine plants. symptoms on the leaves of diseased grapevine plants are manifested in the form of small, chlorotic spots, distributed along the edge of the leaves, while the central part of the leaf blade has a wrinkled appearance. the edges of the leaves are worn and bent downwards, and in severe infections, the surface of the leaves is mostly covered with necrotic spots. the shoots are light green in color, have a shortened appearance and the so-called zigzag growing of internodes. in the later stages, the disease is manifested by the appearance of a large number of very short shoots that grow from the same place on the branch of the vine, very close to each other. the first signs of the disease first develop around larger pruning wounds. by removing the dead bark, necrotic parts can be seen that extend along the stem and can be several tens of centimeters long. necrosis first affects the surface, and then penetrates into the interior, affecting the central part of the vine. dead tissue is dry, light or dark brown in color. on the crosssection of the diseased tree, more or less pronounced necrosis is manifested, which is in the initial phase in the shape of the letter „v”. for isolation, 1 cm fragments of grapevine stem and cordons were used, from the junction between the healthy and diseased parts. to isolate the pathogen, diseased parts of grapevine plants were surface-sterilized with 5% sodium hypochlorite for 2 min, followed by three washes with sterile distilled water. surface sterilized tissue was transferred to sterile filter paper and placed on potato dextrose agar (pda) containing streptomycin, and incubated at 24 °c. after incubation for 3 to 5 days, colonies of fungi developed around the fragments, which were sieved on potato dextrose agar (pda) medium and served to obtain monosporial isolates. based on the color and appearance of the fungal mycelium on potato dextrose agar (pda) medium, as well as the anamorphic stage, isolates were determined to the genus level. individual conidia were selected and transferred directly to the pda plate according to the procedures described by dhingra & sinclair (1995), and stored on pda in tubes at 4 °c. in order to check the pathogenicity of the obtained monosporial isolates of the fungus, artificial inoculations of the vine were performed. the pathogenicity test of the isolates was performed by inoculation of non-rooted grapevine cuttings (peros & berger, 1994). the cabernet sauvignon variety was used for the inoculation of cuttings in the experiment. breeding traits (radial growth and sporulation under the influence of three types of light) of 3 isolates of eutypa spp. (el117, el153 and el199) isolated from serbia and two control isolates from international collections were studied after obtaining monosporial cultures and confirming the pathogenicity of fungi isolates. influence of light on the development and sporulation of the studied anamorphic phase isolates of eutypa spp. to study the influence of different variants of artificial fluorescent light and darkness on colony growth and sporulation of the studied isolates of eutypa spp. pure cultures of 3 isolates and 2 reference isolates of e. lata grown on pda at 25 °c were used. the seeded petri plates were then exposed to 3 variants of artificial fluorescent light and darkness, as follows: 142 biologica nyssana ● 13 (2) december 2022: 141-151 živković et al. ● study of eutypa lata isolates originating from serbia biologica nyssana ● 13 (2) december 2022: 141-151 živković et al. ● study of eutypa lata isolates originating from serbia 143 1. 24 h exposure to uv light (uv); 2. alternatively, 12 h uv and 12 h darkness (uvt); 3. alternating 12 x artificial fluorescent light and 12 x darkness (s-t). three 40 w bulbs served as a source of fluorescent light, and two uv bulbs, with a wavelength of 366 nm, which were about 50 cm away from the petri plate, served as a source of ultra-violet light. mycelial growth was monitored by measuring the diameter of the colony for 10 days until the first isolate completely covered the petri plate. the first measurement of colony growth was performed after three days, and then, during the next ten days, until the first isolate completely filled the petri plate, the linear growth of colonies was monitored by linear diameter measurement. the average daily colony growth was calculated according to the formula (brasier and weber, 1987): (d2-d1)/(t2-t1)=daily growth level where d1 and d2 are the diameters of the colony after the first and second measurements, and t1 and t2 days elapsed from seeding to the first and second measurements (loc. cit. živković, 2019). determination of sporulation levels, i.e. the number of conidia per ml of suspension, was performed using a hemocytometer by thom with 16 fields, whose size is 1 mm2 and depth is 0.2 mm. the number of conidia was determined in order to determine the differences in the intensity of sporulation of 3 isolates of eutypa spp. studied in this paper. for this purpose, a suspension of spores was prepared by adding 5 ml of distilled water to a petri plate with a culture of the studied isolates of eutypa spp. the resulting conidia suspension together with the mycelial fragments was poured into a clean tube and then squeezed through a polyethylene mesh to remove the mycelial fragments. the counting was performed in 2 x 16 repetitions (32 fields), and from that, the average number of conidia per 1 mm2 was calculated. the number of conidia thus obtained was taken to calculate the density of spores in 1 cm3 (1 ml) of the suspension according to the formula: n=nx5x1000 where: n number of spores in 1 ml of suspension (spore density/1ml), n – the average number of spores per 1 mm2. the density of the spores is obtained by multiplying the average number of spores by the number 5 (because the depth of the pits is 0.2 mm). to obtain the number of spores in 1 mm3, and then multiplying the obtained number by 1000, since 1 cm3 contains 1000 mm3, to obtain a spore density in a volume of 1 cm3 (1 ml). the number of conidia per 1 mm2 of the colony was determined based on the number of conidia/ml and the area of the colony, which was calculated from the proportion of paper weight on which the contours of colonies were traced and a paper weight of 1 dm2 (živković, 2019). the level of sporulation is expressed according to the scale by quesad and lopez (1980), where: +=weak sporulation (<5.000 spores/ml), ++=medium sporulation (5.000-10.000 spores/ml) and +++=abundant sporulation (>10.000 spores/ml) (loc. cit. živković, 2019). photographing the test results was performed 30 days after sowing the substrate. two reference isolates, 8f and bx1.10, were used as controls. statistical analysis statistical analysis was performed in order to determine the relationship between eutypa spp. isolate and two reference isolates 8f and bx1.10. data were analyzed by variance analysis (anova) using computer software (proc glm, sas, system, version 8.1; sas institute, cary, nc). to satisfy the assumptions of the anova, the arcsine transformation of the proportion was used (y=2xarcsine√p). the homogeneity of groups was assessed using duncan’s test with p=0.05. molecular detection detection of three isolates of eutypa spp. from serbia and 2 reference isolates of e. lata was performed using the polymerase chain reaction (pcr) method. pcr was performed using 1 pair of specific primers. visualization of the obtained products was performed by electrophoretic separation in an agarose gel. dna extraction colonies of the tested isolates were grown on pda in the dark, at a temperature of 25 °c for 7 days. dna extraction was performed according to the method described by day & shattock (1997). in the first step, the colonies of the tested isolates were scraped from the surface of the substrate with a sterile spatula and transferred to a 2 ml tube with liquid nitrogen. after evaporation of the liquid nitrogen, 800 μl of 2% ctab buffer was poured into a tube and incubated at 65 °c for 1 h. during incubation, the content of the tube was shaken vigorously every 15 min. after incubation, 800 μl of chloroform was added to each microtube and vortexed, then centrifuged for 10 min at 11000 rpm and 4 °c (eppendorf 5804 r, germany). the resulting supernatant (about 700 μl) was pipetted into a new 1.5 ml tube, about 420 μl of isopropanol was added and centrifuged for 15 min at 11000 rpm and 4 °c. after centrifugation, 144 biologica nyssana ● 13 (2) december 2022: 141-151 živković et al. ● study of eutypa lata isolates originating from serbia the supernatant was carefully drained, and 1 ml of ice-cold 70% ethanol was added to the tubes, which was then carefully drained. the open microtubes were left for 10-15 min at room temperature. after drying, the dna pellet was resuspended in 100 μl te buffer. chain amplification of nucleic acid fragments for the detection of the studied isolates of eutypa spp. the pcr reaction was applied using a specific primer pair lata 1 and lata 2.2 (tab. 1). pcr amplification of the samples was performed in a thermocycler (mastercycler® eppendorf, germany). lata 1/lata 2.2 primers enable fragment amplification in the its1 and its2 regions. this primer pair serves for the specific detection of eutypa lata species by amplifying a fragment of about 385 bp located in the its1 and its2 regions (lecomte et al., 2000) (tab. 1). the 25 μl pcr mix contained: 12.5 μl 2 x master mix, 9 μl sterile distilled h2o, 1 μl 10 μm lata 1 and lata 2.2 primer and 1.5 μl dna. pcr amplification of the samples was performed according to the same program as with primers its1-its4. as a negative control in all pcr reactions, sterile water (pcr grade) was used instead of the target dna sample (pcr mixture with rnase-free water). reference isolates of e. lata from international collections were also used in pcr reactions as positive controls. visualization and analysis of pcr reaction products analysis of pcr products was performed after electrophoretic separation of the obtained products in 1.5% agarose gel and 0.5 x tbe buffer. the agarose gel was prepared by dissolving 3.6 g of agarose (merck, germany) in 240 ml of 0.5 x tbe buffer and heating to boiling point in a microwave oven. after cooling, the gel was poured into the molds of an electrophoresis apparatus (serva, germany). a comb immersed in the appropriate mold trays is immersed in the gel. after the polymerization of the gel, the comb was removed and the gel mold was placed in an electrophoresis apparatus. 0.5 x tbe buffer was added to the apparatus to a level where the gel was completely immersed in the buffer. before pipetting into the wells, 5 μl of the pcr reaction product of each sample was mixed with 1.5 μl of infusion dye (loading dye, mbi fermentas, lithuania). in electrophoresis, a 50 bp ladder marker (sigma aldrich, germany) was used to determine the size of the product by comparison with the expected size of the dna fragments of the marker. electrophoresis was performed at 100 v for 30 min. staining was performed by immersing the agarose gel in 0.5 μg/ml ethidium bromide solution for 15 min. amplified fragments in the gel were observed under uv light using a transilluminator (biometer, uk). results samples and fungal isolation examination of vineyards in several localities where grapevine is grown in our country, in the period from 2004 to 2019, showed symptoms of dying of grapevine vines. symptoms on the leaves of diseased vines are manifested in the form of small, chlorotic spots, distributed along the edge of the leaves, while the central part of the leaf blade has a wrinkled appearance. the edges of the leaves are worn and bent downwards, and in severe infections, the surface of the leaves is mostly covered with necrotic spots. the shoots are light green in color, have a shortened appearance and the so-called zigzag growth of internodes. in the later stages, the disease is manifested by the appearance of a large number of very short shoots that grow from the same place on the branch of the vine, very close to each other. the first signs of the disease first develop around larger pruning wounds. by removing the dead bark from the vine, necrosis is noticed, which develops around older pruning wounds. necrotic parts extend along the stem and can be several tens of centimeters long. necrosis first affects the surface, and then penetrates into the interior, affecting the central part of the grapevine. dead tissue is dry, light or dark brown in color. due to the growth of a healthy part of the wood around the infected one, the cracks appear on the stem surface along the necrotic belt, whereby outside, on the dead part of the stem tissue, smaller or larger longitudinal cracks appear (fig. 1). on the cross-section of the diseased stem, more or less pronounced necrosis is manifested, which is in the initial phase in the shape of the letter „v” on table 1. specific primer pair for detection of eutypa lata targeted sequence primer name sequence 5’-3’ fragment size literal source its-its2 lata 1 gagctaccctgtagcccgctg ~385 bp lecomte et al. (2000)lata 2.2 gacgtcagccgtgacacacc 145 biologica nyssana ● 13 (2) december 2022: 141-151 živković et al. ● study of eutypa lata isolates originating from serbia to three variants of artificial light and darkness, as follows: 1) 24 h exposure to uv light (uv), 2) alternating 12 h uv and 12 h darkness (uv-t); 3) alternating 12 h of artificial fluorescent light and 12 h of darkness (s-t). the aim of this experiment was to examine which isolate has the largest radial increase, daily increase, and degree of sporulation. partially or completely dried limbs and sleeves, the bark peels and falls off over time. on a stem without bark, especially around older sections from pruning, slight surface depressions of dead tissue can be noticed. samples were taken from diseased vines by cutting fragments 10 to 20 cm long. from these fragments, pieces about 1 cm long were cut from the junction of healthy and diseased tissue and seeded on a sterile pda medium. on potato-dextrose (pda) medium, 24 h after sowing, the studied isolates of eutypa spp. form the beginning of white mycelium. the mycelium is hyaline, milky white in color, more or less airy. over time, the mycelium becomes light gray-white in color with a thick, cottony, airy mycelium adhering to the substrate, reminiscent of down feathers. all studied isolates showed pathogenic properties and caused changes in the inoculated tissue by inoculation of unrooted cuttings of cabernet sauvignon grapevine. influence of light on the development and sporulation of the studied isolates of eutypa spp. during these studies, the radial growth of colonies and sporulation of all examined isolates under the influence of 12 h uv and 12 h darkness, under the influence of 12 h light and 12 h darkness and under the influence of 24 h uv (fig. 2) in a period of 30 days were studied. to study the influence of different variants of artificial light and darkness on colony growth and sporulation of the five studied isolates, three isolates eutypa spp., from grapevine, originating from serbia, and two determined control isolates of e. lata, originating from italy and france, were used. the radial growth of colonies and sporulation of five selected isolates was studied on a potato-dextrose medium by exposing seeded petri dishes at 25 °c fig. 1. eutypa spp.: tissue cracking and deformations due to thе growth of healthy tissue radial colony growth the radial growth of colonies of all five studied isolates was statistically significantly influenced by different variants of artificial light and darkness to which the seeded cultures were exposed (tab. 2). thus, during these studies, it was found that the highest increase in colonies of all studied isolates was under the influence of alternating 12 h uv and 12 h darkness, a slightly smaller increase was under the influence of 24 h uv over a period of 30 days, and the weakest increase was under the influence of 12 h light and 12 h darkness (tab. 2). it is also observed that isolates el153, el199 and reference isolate bx1.10, under the influence of 12 h uv and 12 h darkness have a statistically significantly higher increase compared to other tested isolates. while these isolates under the influence of 12 h of light and 12 h of darkness achieve a statistically significantly smaller rise compared to other tested isolates. (tab. 2). average daily growth the average daily colony growth in all five studied isolates of different variants of artificial fluorescent light and darkness was statistically significantly fig. 2. eutypa spp.: appearance of a colony of el199 isolates on a pda medium exposed to 24 h of ultraviolet light (uv) for a period of 30 days 146 biologica nyssana ● 13 (2) december 2022: 141-151 živković et al. ● study of eutypa lata isolates originating from serbia affected. considering this trait, some isolates differed significantly. it was found that all tested isolates (el117, el153 and el199, 8f and bx1.10) achieved a statistically significantly higher daily gain when exposed to 12 light and 12 h of darkness compared to other types of light (tab. 2). after statistical analysis of the data, it can be concluded that isolate el117 has a statistically significantly higher daily yield compared to other tested isolates at all types of light, except isolate 8f, which, under the influence of treatment with light 12 h uv and 12 h darkness, achieved statistically significantly higher growth compared to isolate el117 (tab. 2). sporulation (number of conidia per mm2 of colony) the type of light greatly affects the ability of the studied eutypa spp. isolates to form conidia in greater or lesser numbers. thus, different types of light in all tested isolates influenced the level of sporulation, which can be seen from tab. 3. the number of conidia formed per mm2 of colony was statistically significantly conditioned by the variant of artificial light and darkness in all five studied isolates of eutypa spp. in terms of this trait, some isolates differed significantly. thus, under the influence of 12 h of light and 12 h of darkness after 30 days of exposure, no sporulation of the el153 and el199 isolates occurred, while in isolates el117, as well as two reference isolates 8f and bx1.10, sporulation was abundant (tab. 3). when all five studied isolates were exposed to 24 h uv light for 30 days and a combination of 12 h uv and 12 h darkness, sporulation occurred in all studied isolates of eutypa spp., as shown in tab. 3. under the influence of both types of light, the el117 isolate exhibited statistically significantly higher extent of sporulation compared to other tested isolates, as well as two reference isolates. molecular detection and identification of the studied isolates the molecular method of polymerase chain reaction has been successfully applied for the detection of the studied isolates of eutypa spp. after extraction of the total dna of the tested isolates, pcr reaction was performed with one pair of specific primers (lata 1/lata 2.2) in order to detect the tested isolates to the level of the species. the specific primer pair lata 1/lata 2.2 for the determination of e. lata species enables amplification of the its region of ribosomal dna and 5.8 rrna (its1/its2 region). by comparing the amplified fragments of the tested isolates, with the marker (m) used, the presence of amplicons of the expected size of about 385 bp was found in all isolates, which confirms that all three tested isolates belong to the e. lata species. no amplification occurred with the negative control (fig. 3). discussion during the multi-year period (2004-2021) monitoring in vineyards in 14 localities, but also on individual vines in yards in serbia, it was found that table 2. eutypa spp.: influence of light on colony growth and sporulation of studied isolates of this parasite isolates different light types 24 h uv 30 days 12 h uv+12 h darkness 12 h light + 12 h darkness colony diameter (mm) el117 88.00 a 89.00 ab 88.00 a el153 86.60 ab 90.00 a 85.40 ab el 199 88.40 a 90.00 a 87.40 a 8f 87.80 a 89.00 ab 86.00 ab bx1.10 87.00 a 90.00 a 85.40 ab daily growth (mm)** el117 5.80 a 5.80 ab 7.16 a el153 5.16 a 5.72 ab 6.48 ab el199 4.76 ab 5.70 ab 6.52 ab 8f 5.12 a 6.08 a 6.08 ab bx1.10 5.32 a 4.68 b 6.24 ab *data in columns labeled with the same letters were not statistically significantly different based on the duncan test (p=0.05); **average daily increment calculated by formula (d2-d1 )/(t2-t1 ) 147 biologica nyssana ● 13 (2) december 2022: 141-151 živković et al. ● study of eutypa lata isolates originating from serbia table 3. eutypa spp.: influence of light on colony growth and sporulation of studied isolates of this parasite isolates different light types 24 h uv 30 days 12 h uv+12 h darkness 12 h light + 12 h darkness sporulation after 30 days *** el117 +++ +++ +++ el153 +++ +++ el199 +++ +++ 8f +++ +++ +++ bx1.10 +++ +++ +++ number of conidia after 30 days per mm2 el117 40.94 a 40.75 a 33.75 a el153 12.13 bc 16.19 b 0.00 b el199 15.00 b 15.38 b 0.00 b 8f 33.94 ab 34.44 ab 33.31 a bx1.10 33.31 ab 38.12 ab 32.44 ab *data in columns labeled with the same letters were not statistically significantly different based on the duncan test (p=0.05); ***sporulation: +=weak, ++=medium, and +++=abundant fig. 3. amplified dna fragments of eutypa lata about 385 bp in size obtained using lata 1/lata 2.2 primer pairs. el117, el153, el199 studied isolates; 8f and bx1.10 reference isolates; negative control; m-50 bp dna step ladder the fungus eutypa lata causes symptoms on shoots 25 to 50 cm long, and later as the disease progresses, on the stem and branches of diseased plants. this has been proven by the isolation of pathogens, and then by artificial inoculations on the cuttings of a large number of grapevine varieties. during the mentioned period of monitoring in vineyards in 14 localities in serbia, the presence of symptoms that the species eutypa lata causes both on inflorescences and berries of grapes. according to day and carter (1976), symptoms on shoots can be observed when shoots have 3 or 4 internodes. the symptoms are very difficult to notice and detect later, during the vegetation, because the diseased leaves are sheltered by healthy shoots and leaves. diseased leaves stop growing, are chlorotic and bend towards the back. the internodes are shortened and are often zigzagged, and the clusters are poorly developed and sparse (munkvold et al., 1993, 1994). cancerous wounds are caused by the growth of healthy tissue at the junction of healthy and diseased tissue, they are brown in color and are usually covered with tree bark. the grapevine dieback when the necrosis spreads to the entire stem (péros and berger 1994; sosonowski et al. 2007; 2013; sosnowski, 2016). symptoms on grapevine shoots may indicate the presence of this fungus in the stem or branches of the vine, and as the disease progresses, the symptoms spread to the entire plant. the growth of the fungus in the stem is slow, 10-20 cm per year, which is why after infection, the symptoms of dying on herbaceous organs do not appear in the first 2 to 3 vegetation seasons (munkvold et al., 1993). the first symptoms are noticed only in the third or fourth year after the infection. such a long incubation of the fungus in the stem and branches makes this disease „insidious”, and due to the slow development of the disease, economic losses are manifested in vineyards older than eight years. also, the fungus does not infect biologica nyssana ● 13 (2) december 2022: 141-151 živković et al. ● study of eutypa lata isolates originating from serbia 148 new vines younger than 5 years, and symptoms are rarely seen on a vine younger than 8 years (gubler et al., 2005; sosnowski et al. 2007; 2013; živković et al., 2012 a, b; sosnowski, 2016; živković, 2019). tissue necrosis spreads in a circle over time, covering an increasing part of the cross-section, and at the same time, it spreads along the stem, up and down, and reaches the root neck. drying of the limbs, vines and sleeves, which occurs as a consequence of disease progression, is best observed during the winter (munkvold et al., 1993; sosnowski et al., 2013; živković, 2019). fungus eutypa lata can be easily grown on conventional laboratory media, but not all isolates sporulate. to encourage sporulation, the cultures are exposed to a light regime of 12 x uv light 12 x darkness, 12 x light 12 x darkness or 24 h uv radiation and sporulation occurs after 30 days. mckemy et al. (1993) stimulated sporulation by exposing e. lata colonies to a shift of 12 h of light 12 h of darkness. in this regime, sporulation occurs after 30 days. ju et al. (1991) stimulated the sporulation of e. lata isolates by re-seeding a 2% pda medium with 5 g/l of yeast extract. the re-seeded cultures of e. lata were kept for a period of three weeks at 20 °c and exposed to 12 h of fluorescent light 12 h of darkness. glawe & rogers (1982) and glawe et al. (1982), in order to accelerate sporulation, exposed e. lata isolates on a pda substrate to shifting of 12 h of fluorescent light 12 h of darkness for 30 days. carter (1994) stimulated sporulation of e. lata isolates on a pda medium by exposing it to 24 h of uv light for 30 days or a combination of 12 h of uv light 12 h of darkness, as well as a combination of 12 h of light 12 h of darkness. munkvold (2001) stated that sporulation of conidia of e. lata isolates on pda medium is stimulated by constant exposure to uv rays for 30 days. dye & carter (1976), glawe et al. (1982) and mckemy et al. (1993) described cotton-white colonies of e. lata isolates on a pda medium. over time, the color of the colony turns to cream. under the influence of a shift of 12 h of light 12 h of darkness and at alternating temperatures of 19 °c and 15 °c, sporulation occurs after 30 days, which agrees with the statements of carter (1994) and munkvold (2001). the same authors state that in some cultures a gray pigment is formed after 15 days and the back turns black. three to four weeks after seeding, small black pycnidia appear in the culture under the influence of constant uv light. conidia are excreted from the pycnidia in the form of a cream to an orange gelatinous mass (rolshusen et al., 2006; trouillas & gubler, 2010). the test results show that not all studied light types have a statistically significant effect on the radial growth of the tested isolates, but they do cause a significant statistical difference in their sporulation. thus, different types of light, in all tested isolates, influenced the level of sporulation. isolates el117, 8f, and bx1.10 form a statistically significantly higher number of conidia at 24 h uv light and then under the influence of 12 h uv and 12 h darkness (glawe & rogers, 1982; glawe et al., 1982; munkvold, 2001). identification of the fungus e. lata is difficult due to the large morphological similarities of related species of the genus eutypa (rolshausen et al., 2014). therefore, many authors have developed molecular methods for the detection of eutypa lata species (descenzo et al., 1999; lecomte et al., 2000; rolshausen et al., 2004, 2006, 2014; catal et al., 2007; trouillas & gubler, 2010; urbez-torres et al., 2012). thus descenzo et al. (1999) for the first time used sequences of the its region to study 115 isolates of e. lata, isolated from ten plant hosts, of different geographical origin. these authors found that in all studied e. lata isolates, the 5.8 s rdna sequences were identical, regardless of the host and origin of the isolates. then, lecomte et al. (2000) use dna sequences to identify e. lata species. based on the its region rdna, they synthesized 3 pairs of primers lata1/ lata2-1, lata1/lata2-2 and lata3/lata2-1, as well as 3 pairs of primers based on the fragment of the rapd sequence sca 10a/sca 10b, scb 02a/ scb 02b and scd 18 a/scd 18 b. using the above primer pairs, they tested 60 isolates of e. lata of different geographical origin, isolated from vines. however, the lata1/lata2-2 primer pair was found to be the most specific for the detection of e. lata species. rolshausen et al. (2004) using a universal primer pair its1-its4 and the restriction enzyme alui, by analysis of rflp products in an agarose gel, separated over 30 isolates of e. lata from different hosts and different geographical origins, from other species from the family diatrypaceae. rolshausen et al. (2006) analyzed the sequences of 46 fungal isolates representing 15 different species from the diatrypaceae family, including 25 e. lata isolates collected from different plant hosts and different geographical origins, using its rdna and the protein part of the ß-tubulin gene. catal et al. (2007) based on the its1-its2 region designed a specific pair of el1/el4 primers, the application of which proved successful for the detection of american isolates of e. lata, while in european and australian isolates there was no amplification. trouillas & gubler (2010) successfully determined 35 isolates of e. lata as well as 7 isolates of other fungi, originating from different plant hosts, 149 biologica nyssana ● 13 (2) december 2022: 141-151 živković et al. ● study of eutypa lata isolates originating from serbia using universal primers its1-its4, sequencing of products of about 566 bp in all tested isolates, and sequence analysis likewise, úrbez-torres et al. (2012) used its region, efi-α and β-tubulin for sequence analysis in order to separate the grapevine dieback fungus complex. in this way, as many as 17 different species from the family diatrypaceae have been identified, which contribute to the formation of cancer, including the species e. lata. due to the above, this paper approaches, among other things, the application of molecular detection for the determination of the studied isolates to species levels. molecular methods, as a modern approach to the study of plant pathogens, have a great advantage in the precise identification and characterization of various pathogens. due to its simplicity and reliability, the pcr method has become one of the most commonly used methods in the detection of phytopathogenic fungi. molecular detection of the studied isolates of eutypa spp. it was performed after isolation of total dna. the extracted dna was intact and suitable for further successful amplification in the pcr reaction, enabling successful detection of all isolates used in this work. using a specific primer pair lata 1/lata 2.2 (lecomte et al., 2000), a fragment of about 385 bp in length was successfully amplified in all three studied isolates. these primers were selected based on the recommendation given by lecomte et al. (2000) in a protocol for rapid and efficient detection of this pathogen. in the studied isolates, profiles corresponding to the e. lata species were obtained, which fully corresponds to the identification using the conventional and molecular methods. 8f isolates originating from italy and bx1.10 originating from france, previously identified as e. lata, were also used as reference isolates in these studies. based on the breeding and molecular characteristics of the three isolates of eutypa spp. originating from parts of the grapevine with symptoms of extinction, collected from serbia, and comparing them with two reference isolates of e. lata originating from italy and france, it was determined that grapevine dieback in our country is caused by species of the genus eutypa,eutypa lata (syn. eutypa armeniacae), anamorph libertella blepharis. conclusions based on many years of research, the results were obtained from which the following can be concluded: examination of vineyards in several localities in serbia, in the period from 2004 to 2021, showed the appearance of symptoms of death of grapevine vines. from the breeding traits to the development of colonies of the studied isolates of eutypa lata the type of light had the most significant influence. under the effect of light type 12 h of light and 12 h of darkness, the isolates el153 and el199 did not sporulate, while under the action of 24 h of uv light, and at 12 h of uv and 12 h of darkness these isolates, as well as other tested isolates, achieved abundant sporulation. molecular detection of isolates was performed using a specific primer pair lata 1/lata 2.2, which allows amplification of the its region of ribosomal dna and 5.8 rrna (its1/its2 region). using these primers in all studied isolates, as well as reference isolates from international collections, a fragment of 385 bp was multiplied, which confirmed that it belongs to e. lata species. in the studied isolates, profiles corresponding to e. lata species were obtained, which fully corresponds to the identification using the conventional method. references carter, m.v. 1994: wood and root diseases caused by fungi. eutypa dieback. in: pearson r.c. and goheen a.c. (ed.), compendium of grape diseases. 3rd ed: 32-34, aps press. st. paul, minnesota. catal, m., jordan, s.a., butterworth, s.c., schilder, a.m.c. 2007: detection of eutypa lata and eutypella vitis in grapevine by nested multiplex polymerase chain reaction. phytopathology, 97: 737-747. day, j.p., shattock, r.c. 1997: aggressiveness and other factors relating to displacement of populations of phytophthora infestans in england and wales. european journal of plant pathology, 103: 379–91. descenzo, r.a., engel, s.r., gomez, g., jackson, e.l., munkvod, g.p., weller, j., irelan, n.a. 1999: genetic analysis of eutypa strains from california supports the presence of two pathogenic species. phytophtapogy, 89: 884-893. dhingra, o.d., sinclair, j.b. 1995: basic plant pathology methods. crc press, boca raton, florida. dye, m.h., carter, m.v. 1976: association of eutypa armeniacae and phomopsisviticola with a dieback disease of grapevines in new zealand. australasian plant pathology society newsletter, 5: 6-7. fontaine, f., pinto, c., vallet, j., clément, c., gomes, a.c., spagnolo, a. 2016. the effects of grape vine trunk diseases (gtds) on vine biologica nyssana ● 13 (2) december 2022: 141-151 živković et al. ● study of eutypa lata isolates originating from serbia 150 physiology. european journal of plant pathology, 144: 707–721. glawe, d.a., rogers, j.d. 1982: observations on the anamorphs of six species of eutypa and eutypella. mycotaxon, 14: 334-346. glawe, d.a., skotland, c.b., moller, w.j. 1982: isolation and identification of eutypa armeniacae from diseased grapevines in washington state. mycotaxon, 16: 123-132. gramaje, d., úrbez-torres, j.r., sosnowski, m.r. 2018. managing grapevine trunk diseases with respect to etiology and epidemiology: current strategies and future prospects. plant disease, 102: 12-39. gubler, w.d., rolshausen, p.e., trouillas, j.r., urbez, j.r., voegel, t. 2005: grapevine trunk disease in california. http:www.practicalwinery. com. sad. ju, y.m., glawe, d.a., rogers, j.d. 1991: conidial germination in eutypa armeniacae and selected other species of diatrypaceae: implications for the systematics and biology of diatrypaceous fungi. mycotaxon, xli(1): 311-320. lecomte, p., péros, j.p., blancard, d., bastien, n., délye, c. 2000: pcr assays that identify the grapevine dieback fungus eutypa lata. applied and environmental microbiology: 4475-4480. mckemy, j.m., glawe, d.a., munkvold, g.p. 1993: a hyphomycetous synanamorph of eutypa armeniacae in artificial culture. mycologia, 85(6): 941-944. munkvold, g.p., duthie, j.a., marois, j.j. 1993: spatial patterns of grapevines with eutypa dieback in vineyards with or without perithecia. phytopathology, 83: 1440-1448. munkvold, g.p., duthie, j.a., marois, j.j. 1994: reductions in yield and vegetative growth of grapevines due to eutypa dieback. phytopathology, 84: 186-192. munkvold, g.p. 2001: eutypa dieback of grapevine and apricot. online. plant health progress, doi: 10.1094/php-2001-0219-01-dg. peros, j.p., berger g. 1994: a rapid method to assess the aggressiveness of e. lata isolates and the susceptibility of grapevine cultivars to eutypa dieback. agronomie, 14: 515-523. rolshausen, p.e., trouillas, f., gubler, w.d. 2004: identification of eutypa lata by pcr-rflp. plant disease 88: 925-929. rolshausen, p.e., mahoney, n.e., molyneux, r.j., gubler, w.d. 2006: a reassessment of the species concept in eutypa lata, the causal agemt of eutypa dieback of grapevine. phytopathology, 96: 369-377. rolshausen, p.e., baumgartner, k., travadon, r., pouzoulet, j. 2014: identification of eutypa spp. causing eutypa dieback of grapevine in eastern north america. plant disease, 98: 483-491. rolshausen, p.e., sosnowski, m., trouillas, f.p., gubler, w.d. 2015: diseases caused by fungi and oomycetes. eutypa dieback. in: wilcox w.f., gubler w.d, uyemoto j.k. (ed.) compendium of grape diseases, disorders, and pests, 2nd ed: 5761, aps press, st. paul, minnesota. sosnowski, m.r., lardner, r., wicks, t.j., scott, e.s. 2007: the influence of grapevine cultivar and isolate of eutypa lata on wood and foliar symptoms. plant disease, 91: 924-931. sosnowski, m.r., loschiavo, a. p., wicks, t.j., scott, e.s. 2013: evaluating treatments and spray application for the protection of grapevine pruning wounds from infection by eutypa lata. plant disease, 97: 1599-1604. sosnowski, m.r. 2016: best practices management guide. eutypa dieback. available at: http: www. barossa.com/uploads/214/20160621 eutypa – dieback – best – practice – management.guide.pdf [accessed on 5 june 2017]. sosnowski, m., mccarthy, g. 2017: economic impact of grapevine trunk disease management in sauvignon blanc vineyards of new zealand. wine & viticulture journal, 32: 42–48. trouillas, f.p., gubler, w.d. 2010: host range, biological variation, and phylogenetic diversity of eutypa lata in california. phytopathology, 100(10): 1048-1056. úrbez-torres, j.r., peduto, f., striegler, r.k., urrea-romero, k.e., rupe, j.c., cartwright, r.d., gubler, w.d. 2012: characterization of fungal pathogens associated with grapevine trunk diseases in arkansas and missouri. journal fungal diversity, 52(1): 169-189. živković, s., vasić, t., anđelković, s., jevremović, d., trkulja, v. 2012a: identification and characterization of eutypa lata on grapevine in serbia. plant disease, 96(6): 913. živković, s., vasić, t., trkulja, v., krnjaja, v., marković, j. 2012b: pathogenicity on grapevine and sporulation of eutypa lata isolates originating from serbia. romanian biotechnological letters, 17(3): 7379-7388. živković, s. 2019: karakterizacija eutypa lata, prouzrokovača odumiranja čokota vinove loze u srbiji i osetljivost sorti. phd thesis. univerzitet u beogradu, poljoprivredni fakultet, beograd. biologica nyssana ● 13 (2) december 2022: 141-151 živković et al. ● study of eutypa lata isolates originating from serbia 151 microsoft word 0601_gnjatovic_zikic biologica nyssana 1 (1-2) december 2010: 111-115 gnjatović, i.,žikić, v. cerambycids of southeast serbia… 111 original article ! cerambycids of southeast serbia (coleoptera, cerambycidae) ivan gnjatović, vladimir žikić university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia * e-mail: ivangnjatovic81@gmail.com abstract: gnjatović, i., žikić, v.: cerambycids of southeast serbia (coleoptera, cerambycidae). biologica nyssana, 1 (1-2), december 2010: 111-115. the cerambycids (longhorn beetles) are known as very attractive insects, as well as pests, especially in forestry. our investigation and collecting the specimens on the territory of serbia has started in the middle of the nineteenth century. up today, 242 species were registered in the fauna of serbia (ilić, 2005). the first information about cerambycids in serbia was published by bobić (1871). significant contribution in researching of the family cerambycidae was given by košanin (1904). finally, there were several domestic authors occupied by longhorn beetles: adamović (1950), mikšić (1963), ćurčić et al. (2003) and ilić (2005). in this review forty-nine species reviewed from 34 genera which belong to five subfamilies: prioninae, lepturinae, spondylidinae, cerambycinae and lamiinae. examined material has been collected in the southeastern serbia. the most numerous were the subfamily cerambycinae, comprising 18 species from 15 genera. key words: coleoptera, checklist, southeast serbia introduction ! the cerambycidae comprises about 25,00030,000 species distributed in all zoogeographical regions worldwide. all known species are phytophagous; majority of their larvae are xylophagous (86%); some members feed on herbaceous plants (13%). they could cause serious damages on forest habitats, so they are marked as pests (bílý and mehl, 1989). there are about 2.500 species in the palaearctic fauna of which 625 occur in europe (b í l ý and m e h l , 1989; i l i ć , 2005). pančić was the first investigator who started collecting the specimens of coleoptera in serbia since 1846, but the first information about cerambycids in serbia has been published by b o b i ć (1891). significant contribution in researching of the family cerambycidae was given by k o š a n i n (1904). he has noted 72 species from various localities in serbia. long after that, a d a m o v i ć (1950) complements košanin’s coleopteran collection and reporting 25 new species. m i k š i ć (1963) published 278 species for the fauna of ex yugoslavia. for the territory of serbia he has noticed 141 species, based on following data of s v i r č e v (1938), k u t h y (1896), ž i v o j i n o v i ć (1950) and a d a m o v i ć (1950). in additional research m i k š i ć at al. (1971, 1973, and 1985) presented 283 species from 105 genera. in the latest publication ć u r č i ć et al. (2003), reported 5 subspecies, along with 49 species, belonging to 49 genera as well as two new species in serbia: leiopus femoratus faimaire and pachytodes cerambyciformis (schrank). up to year of 2005, it was described 242 species, 40 subspecies from 123 genera, 15 subgenera, 44 tribes and 6 subfamilies in serbia (i l i ć , 2005). most recent data shows there are 259 species of longhorn beetles in serbia (p i l & s t o j a n o v i ć , 2009). 10th sfses • 17-20 june 2010, vlasina lake1 (1-2) • december 2010: 111-115 biologica nyssana 1 (1-2) december 2010: 111-115 gnjatović, i.,žikić, v. cerambycids of southeast serbia… 112 material and methods greater part of samples comes from the student’s collections of insects. our analisys included the specimens from family cerambycidae, with except of the tribe of dorcadionini. investigated material was collected on 31 localities with different vegetation and habitat in southeastern serbia (details are given in fig. 1). samples were preserved in 70% ethyl-alcohol, and deployed in wooden boxes held at about 22 °c. all specimens are deposited in department of biology and ecology, faculty of sciences, university of niš, serbia. identification of the collected samples is performed by the keys: m i k š i ć and g e o r g i j e v i ć (1971, 1973); bense (1995). this investigation related to all representatives of the family cerambycidae, with the exception of the tribe dorcadionini. results and discussion a total number of 49 species were identified from 34 genera, from 20 tribes, belonging in 5 subfamilies: prioninae (2 species), lepturinae (15 species), spondylidinae (2 species), cerambycinae (18 species), lamiinae (12 species). new records for the fauna of investigated territory are marked by an asterisk (*) (tab. 1). subfamily prioninae tribe megopidini aegosoma scabricornis (scop. 1763), synonym megopis scabricornis – 3♀, serbia: bela palanka: čiflik, 26.06.2006, leg. i. gnjatović; 3♀, serbia: suva mt., 23.05.2007, leg. o. stojnev. prionus coriarus (l. 1758) – 1♂, serbia: niš 15.08.2005, leg. s. lukić; 2♂, serbia: stara mt., 07.07.2006, leg m. jovanović; 1♂, serbia: bela palanka: čiflik, 26.06.2006, leg. i. gnjatović; 1♂, serbia: vladičin han, 18.07.2008, leg. i. gnjatović. subfamily lepturinae tribe anaglyptini anastrangalia sanguinolenta l. 1761 – 1♂, serbia: čestin, 27.05.2007, leg. o.stojnev; 1♂, serbia: suva mt, 16.07.2007, leg. i. gnjatović; 1♂, serbia: vladičin han, 16.07.2008, leg. i. gnjatović. 1♀, serbia: pirot, 15.08.2006, leg. i. gnjatović. anastrangalia dubia (scop. 1763) – 1♀, serbia: vitkovac, 27.05.2008, leg. i. gnjatović. tribe asemini *brachyta balcanica (hampe 1870) – 1♂, serbia: niš, 16.07.2009, leg. i. gnjatović; 1♀, serbia: niš: bojanine vode, 16.07.2008, leg. i. gnjatović; 1♀, serbia: donji dušnik, 09.06.2007, leg. i. gnjatović. tribe lepturini cortodera flavimana (walt 1838) – 1♀, serbia: donji dušnik, 03.06.2006, leg. i. gnjatović; 1♂, serbia: donji dušnik, 01.06.2007, leg. i. gnjatović; 1♂, serbia: bosilegrad, 10.08.2008, leg. o. stojnev. corymbia scutellata (fab. 1781) – 1♀, serbia: niš, 16.07.2009, leg. i. gnjatović. leptura maculata poda, 1761, synonym rutpela maculata (poda 1761) – 1♀, 1♂, serbia: donji dušnik, 06.06.2006, leg. i. gnjatović; 1♂, serbia: novo selo, 19.07.2007, leg. j. mirković; 2♂, serbia: niš, 07.06.2009, leg. m. ristić; 1♀, serbia: vladičin han, 18.07.2008, leg. i. gnjatović; 1♂, serbia: vladičin han, 13.08.2008, leg. i. gnjatović; 1♀, 1♂, serbia: suva mt., 25.06.2007, leg. o.stojnev; 1♂, serbia: niš, 15.04.2009, m. stamenković; 1♂, serbia: babička gora, 05.07.2008, leg. i. gnjatović; 1♂, serbia: vrelo, 03.06.2006, leg. i. gnjatović; 1♂, serbia: stara mt., 11.07.2007, leg. i. gnjatović. leptura quadrifasciata l. 1758, -1♀, serbia: vladičin han, 10.08.2008, leg. i. gnjatović . pachytodes cerambyciformis (sch. 1787) -1♂, serbia: vladičin han, 23.08.2008, leg. i. gnjatović; 1♂, serbia: niš, 15.07.2007, leg. o. stojnev; 1♂, serbia: čestin, 27.06.2007, leg. i. gnjatović. pachytodes erraticus (dalman 1817) -1♂, serbia: vladičin han, 21.07.2008, leg. i. gnjatović. tribe rhagiini rhagium mordax deg. 1785 – 2♂, serbia: suva mt., 12.08.2008, leg. i. gnjatović; 1♀, serbia: vladičin han, 22.08.2008, leg. i. gnjatović. rhagium inquisitor l., 1758, – 1♀, serbia: suva mt., 12.08.2008, leg. i. gnjatović. rhagium bifasciatum fab. 1775 – 2♀, serbia: vladičin han, 22.08.2008, leg. i. gnjatović. rhagium sycophanta (sch. 1781) – 1♀, serbia: vladičin han, 22.08.2008, leg. i. gnjatović. tribe stenopterini stenurella bifasciata (mull. 1776) – 1♀, serbia: niš, 15.07.2008, leg. i. gnjatović. tribe tragosomatini vadonia unipunctata (fab. 1787) – 1♂, serbia: donji dušnik, 05.06.2006, leg. i. gnjatović. subfamily spondylidinae tribe asemini arhopalus ferus (mul. 1839) – 1♀, serbia: niška banja, 08.08.2007, leg. i. gnjatović. arhopalus rusticus (l. 1758) – 1♂, serbia: niška banja, 04.09.2007, leg. i. gnjatović; 1♀, serbia: niš: malča, 09.30.2007, leg. i. gnjatović. biologica nyssana 1 (1-2) december 2010: 111-115 gnjatović, i.,žikić, v. cerambycids of southeast serbia… 113 subfamily cerambycinae tribe callichromini aromia moschata (l. 1758) – 3♀, 4♂, serbia: suva mt., 07.07.2006, leg. m. jovanović; rosalia alpina (l.1758) – 1♂, serbia: leskovac, 17.07.2006, leg. i. gnjatović; 1♂, serbia: zlot, 10.08.2007, leg. d. gligorijević; 4♂, serbia: suva mt. 27.07.2007, leg. i. gnjatović; tribe callidini callidium violaceum (l. 1758) – 2♀, serbia: šišmanovac, 03.07.2007, leg. j. mirković; 1♂, serbia: vlasina lake, 22.06.2007, leg. i. gnjatović. hylotrupes bajulus (l. 1758) – 1♀, serbia: vranje, 15.07.2008, leg. i. gnjatović. phymatodes testaceus (l. 1758) – 1♂, serbia: vlasina lake, 22.06.2007, leg. i. gnjatović; 1♂, serbia: niš, 17.05.2005, leg. v. žikić; 1♀, serbia: izvor, 25.07.2006, leg. i. gnjatović; 2♂, 1♀, serbia: niš: novo selo, 22.08.2007, leg i. gnjatović. pyrrhidium sanguineum (l. 1758) – 1♂, serbia: bujanovac, 12.06.2006, leg. i. gnjatović; 2♂, serbia: niš, 12.08.2008, leg. a. savić. ropalopus clavipes (fab. 1775) – 1♀, serbia: vlasina lake, 07.06.2007, leg. i. gnjatović. tribe clytini chlorophorus figuratus (scop. 1763) – 1♂, serbia: vladičin han, 16.08.2008, leg. i. gnjatović. chlorophorus varius (mull. 1766) – 1♀, serbia: niš, 13.08.2009, leg. i. gnjatović. clytus arietis l. 1768, -1♀, serbia: vladičin han, 04.08.2008, leg. i. gnjatović. isotomus speciosus (schn. 1787) – 1♀, 2♀, serbia: niška banja, 23.09.2007, leg. i. gnjatović; 1♀, serbia: niš, 26.09.2009, leg. v. žikić. plagionotus arcuatus (l. 1758) – 1♀, 1♂, serbia: niš, 11.06.2007, leg. i. gnjatović; 2♂, serbia: novo selo, 22.08.2007, leg. i. gnjatović; 1♂, serbia: niš, 16.08.2008, leg. i. gnjatović; 1♂, serbia: zaječar, 16.08.2009, leg. i. gnjatović; 1♀, serbia: suva mt., 09.07.2007, leg. i. gnjatović; plagionotus floralis (pall. 1776) – 1♀, serbia: niš, 11.06.2007, leg. i. gnjatović; 1♂, serbia: niš, 04.07.2008, leg. i. gnjatović; 1♂, serbia: niš, 05.08.2008, leg. i. gnjatović; 1♂, serbia: niš, 15.04.2009, leg. m. stamenković; 1♀, serbia: kukavica mt., 07.06.2008, leg. i. gnjatović; 1♀, serbia: bujanovac, 04.08.2006, leg. i. gnjatović; 1♂, serbia: donji dušnik, 25.06.2006, leg. i. gnjatović; 1♀, serbia: bujanovac, 13.08.2006, leg. i. gnjatović; 1♂, serbia: petrova reka, 12.06.2006, leg. i. gnjatović. tribe cerambycini cerambyx cerdo (l. 1758) – 1♂, serbia: niš, 06.05.2008, leg. i. gnjatović; 4♀, 4♂, suva mt., 19.05.2008, leg. i. gnjatović. cerambyx scopolii fessly 1775 – 3♀, 2♂, serbia: niš, 06.05.2008, leg. i. gnjatović; 1♂, čestin, 15.06.2008, leg. i. gnjatović; 1♀, 3♂, stara mt., 29.05.2006, leg. i. gnjatović; 1♀, serbia: suva mt.: ličje 23.05.2007, i. gnjatović. tribe purpuriceini purpuricenus kaehleri (l., 1758) – 1♂, serbia: niš, 19.06.2009, leg. m. mladenović. tribe hesperophanini stromatium unicolor (oli., 1795) – 1♀, serbia: niš, 12.08.2007, m. jovanović; 1♂, serbia: niš: jelašnica, 26.08.2007 leg. i. gnjatović; 1♂, serbia: niš: gabrovac, 06.08.2006, leg i. gnjatović; 1♀, serbia: vlasina lake, 03.06.2008, leg. i. gnjatović; 1♂, serbia: niš: jelašnica, 01.10.2007, leg. i. gnjatović; serbia: 1♂, vladičin han 07.08.2008, leg. i. gnjatović; serbia: 1♀, stara mt., 13.07.2007, leg. i. gnjatović; 4♀, 3♂, serbia: niš, 15.07.2009, leg. a. vidojković. *trichoferus fasciculatus (fald.,1837) – 1♀, serbia: ličje, 21.05.2007, leg. i. gnjatović. subfamily lamiinae tribe agapanthini agapanthia cardui (l. 1767) – 1♀, 1♂, serbia: niš, 20.07.2006, leg. i. gnjatović. agapanthia dahli (rich. 1821) – 1♀, 1♂, serbia: niš, 11.07.2009, leg. a. vidojković; 1♂, serbia: niš: čegar, 12.06.2008, leg. i. gnjatović; 1♂, serbia: petrova reka, 12.06.2006, leg. i. gnjatović. agapanthia violaceae (fab. 1775) – 1♂, serbia: niš, 21.08.2008, leg. i. gnjatović; 1♂, serbia: niš, 20.07.2009, leg. i. gnjatović; 1♀, serbia: vlasina lake, 16.06.2007, leg. i. gnjatović; 1♀, serbia: niška banja, 17.07.2008, leg. i. gnjatović; 1♀, serbia: vranje, 10.06.2007, leg. i. gnjatović. agapanthia villosoviridescens (de geer 1775) – 1♀, 1♂, srebia: niška banja, 10.09.2007, leg. i. gnjatović; 1♀, 1♂,, serbia: niš; novo selo, 22.08.2007, leg. i. gnjatović; 2♀, serbia: zlot, 18.08.2007, leg. d. gligorijević; 1♀, serbia: batinac, 10.04.2007, leg. s. pištoljević. tribe mesosini mesosa nebulosa (fab. 1781) – 1♀, serbia: vlasina lake, 16.06.2007, leg. i. gnjatović; 1♀, serbia: niš, 04.07.2009, leg. i. gnjatović. tribe monochamini monochamus galloprovincialis (oli. 1795) – 1♂, serbia: niš, 16.06.2007, leg. i. gnjatović. tribe lamiini morimus funereus muls. 1863 – 1♂, serbia: niš: delijski vis, 13.04.2009, leg. m. stanković; 1♂, serbia: niš: delijski vis, 16.09.2009, leg. m. stanković; 4♀, 1♂, serbia: suva mt., 12.08.2008, leg. i. gnjatović. biologica nyssana 1 (1-2) december 2010: 111-115 gnjatović, i.,žikić, v. cerambycids of southeast serbia… 114 tribe phytoeciini *opsilia molybdaena (dal. 1817) – 1♂, serbia: niš, 06.07.2007, leg. i. gnjatović. phytoecia icterica (schall. 1783) – 1♂, serbia: niška banja, 25.05.2008, leg. i. gnjatović. phytoecia pustulata (sch., 1776) – 1♂, serbia: niš, 06.07.2007, leg. i. gnjatović. phytoecia virgula (char., 1825) – 1♂, serbia: niš, 08.08.2008, leg. i. gnjatović. tribe tetropini tetrops starkii chev. 1859 – 1♂, serbia: bovan lake, 01.05.2009, leg. v. žikić. table 1. the list of new species of investigated teritory. subfamily species locality prioninae brachyta balcanica (hampe, 1870) niš cerambycinae trichoferus fasciculatus (fald.,1837) ličje lamiinae opsilia molybdaena (dal., 1817) niš figure 1. map of serbia (utm 10x10), showing sites were investigated material was collected: en06 bujanovac; en17 vranje; en28 vladičin han, babička gora; en37 kukavica mt.; en53 šišmanovac; en77 leskovac; en79 donji dušnik, ličje; en89 suva mt., bojanine vode; en96 novo selo; en97 niš, gabrovac, delijski vis; en98 niška banja, jelašnica; ep07 čegar, vrelo; ep28 batinac; ep35 bovan lake; ep77 zlot; fn02 bosilegrad; fn20 vlasina lake; fn61 izvor; fn80 bela palanka – čiflik; fn81 petrova reka; fp02 vitkovac; vp30 stara mt.; vp50 zaječar; dp čestin conclusion we presented 49 species of the family cerambycidae from 34 genera. new records for the fauna of southeast serbia are: brachyta balcanica (prioninae), trichoferus fasciculatus (cerambycinae) and opsilia molybdaena (lamiinae). acknowledgment. the 1st author wish to express his thankful to dr p. jakšić (niš, serbia) for his help during research. the 2nd author wish to express his sincere thanks to the ministry of science and environment protection of the republic of serbia (grant no. 143006b). references adamović, ž. 1950: zbirka cerambycidae u prirodnjačkom muzeju srpske zemlje, 1. deo: prionini, cerambycini. glasnik prirodnjačkog muzeja srpske zemlje, beograd. b, (3-4), 343357. bense, u. 1995: longhorn beetles: illustrated key to the cerambycidae and vesperidae of europe. margraf verlag, weikersheim, 512 pp. bílý, s., mehl, o. 1989: longhorn beetles (coleoptera, cerambycidae) of fennoscandia and denmark. e. j. brill/scandinavian science press ltd., 22, fauna entomologica scandinavica, 206 pp. ćurčić, s.b., brajković, m.m., tomović, v.t., mihajlova, b. 2003: contribution to the knowledge of longicorn beetles (cerambycidae, coleoptera) from serbia, montenegro, the repablic of macedonia and greece. arch. biol. sci., belgrade, 55 (1-2), 33-38. biologica nyssana 1 (1-2) december 2010: 111-115 gnjatović, i.,žikić, v. cerambycids of southeast serbia… 115 ilić, n. 2005: strižibube srbije (coleoptera, cerambycidae) – faunistički pregled. autorsko izdanje, beograd, 179 pp. košanin, n. 1904: spisak koleoptera u muzeju srpske zemlje. muzej srpske zemlje, 3, beograd, 26 pp. mikšić, r. 1963: prilog poznavanju faune strižibuba (cerambycidae) jugoslavije. acta biologica zagreb 3, 55-166. mikšić, r., georgijević, e. 1971: cerambycidae jugoslavije, 1. deo. akademija nauka i umjetnosti bosne i hercegovine, sarajevo, 175 pp. mikšić, r., georgijević, e. 1973: cerambycidae jugoslavije, 2. deo. akademija nauka i umjetnosti bosne i hercegovine, sarajevo., 153 pp. mikšić, r., korpič, m. 1985: cerambycidae jugoslavije, 3. deo. akademija nauka i umjetnosti bosne i hercegovine, sarajevo, 145 pp. pil, n., stojanović, d. 2009: theophilea subcilindricollis hladil, 1988 a new longhorn beetle (coleoptera: cerambycidae) for serbian fauna. acta entomologica serbica, 14 (1): 125128. microsoft word 0303_mitrovic_et_al biologica nyssana 1 (1-2) december 2010: 61-64 mitrović, b. et al. chenopodium rubrum l. as a model plant… 61 original article ! chenopodium rubrum l. as a model plant for physiological and biochemical investigations of ontogenesis in vitro aleksandra mitrović*, branka živanović, tanja dučić, jelena bogdanović pristov, ksenija radotić hadži-manić institute for multidisciplinary research, university of belgrade, kneza višeslava 1, 11030 belgrade, serbia * e-mail: mita@imsi.rs abstract: mitrović, a., živanović, b., dučić, t., bogdanović-pristov, j., radotić hadži-manić, k.: chenopodium rubrum l. as a model plant for physiological and biochemical investigations of ontogenesis in vitro. biologica nyssana, 1 (1-2), december 2010: 61-64. chenopodium rubrum l. , is a suitable model plant for studying ontogenesis in vitro as an early flowering species. culture of intact plants in vitro and antioxidative enzymes detection were performed. growth pattern to the end of ontogenesis, flowering and seed development are all determined by the photoperiod seedlings experience during induction and evocation of flowering. different phases of vegetative and reproductive development are characterized by changes in antioxidative enzymes activities. we showed sequential expression of antioxidative enzymes during seed germination. prior to radicule protrusion, cat and sod showed maximal activity, while pod activity appeared later. the highest catalase (cat) activity was measured at the time of flowering while peroxidases (pods) are involved in determination of growth and development in accordance with the environmental clues. the absence of some superoxide dismutase (sod) isoforms could be the indicator of senescence. seed ageing affect changes in antioxidative status of seeds, germination, seedling growth and flowering. key words: catalase, chenopodium rubrum, germination, flowering, peroxidase, superoxide dismutase introduction ! chenopodium rubrum l. belongs to the family chenopodiaceae, genus chenopodium. this is a short-day weedy annual, widely distributed in europe, asia and northern america. ecotypes of this species differ in their photoperiodic characteristics. sel. 184 is a qualitative short-day plant with strictly defined critical night length of 8h (t s u c h i y a & i s h i g u r i , 1981). it is sensitive to photoperiodic stimulus for flowering as early as at cotyledonary stage (s e i d l o v á & o p a t r n á , 1978), when 6 adequate photoperiodic cycles are sufficient for photoperiodic flower induction. c. rubrum plants modify their growth and development in accordance with photoperiod they are exposed to (c o o k , 1975; m i t r o v i ć et al., 2007). as an early flowering species (c u m m i n g , 1967), it is a suitable model plant for studying ontogenesis in vitro. under the adequate photoperiodic conditions, plant flowers in vitro after 15 days (ž i v a n o v i ć et al., 1995), and produces seeds after 10 weeks (m i t r o v i ć et al., 2007). moreover different phases of vegetative and reproductive development are characterized by changes in antioxidative enzymes activities. thus seed germination, seedling growth, flowering, seed maturation and seed ageing are defined by the changes in their antioxidative status (m i t r o v i ć , 2007). it is well known that reactive oxygen species (ros) and their scavenging enzymes participate in protection against pathogens or abiotic stress (h e n d r y & c r a w f o r d , 1994). ros also function as signaling molecules at low 10th sfses • 17-20 june 2010, vlasina lake1 (1-2) • december 2010: 61-64 biologica nyssana 1 (1-2) december 2010: 61-64 mitrović, b. et al. chenopodium rubrum l. as a model plant… 62 concentrations in contrast to high concentrations of ros which can lead to phytotoxicity (f o y e r , 1997). the capacity of ros to serve as signaling molecules highlights the importance of antioxidants to specifically regulate different ros in various cellular compartments. the lack of data obtained from ros measuring in plants is due to technical difficulties associated with quantification of endogenous levels of these very reactive and shortlived species. so, most of the evidences for ros levels has ben provided by studies of antioxidants (d a t et al., 2000). antioxidative enzymes, catalase (cat), superoxide dismutase (sod) and peroxidase (pod) are engaged in the scavenging of ros (v a n l o o n , 1986; b o w l e r et al., 1992; k h a n & p a n d a , 2002) and therefore participate in regulation of plant growth and developmental processes or protection against patogens or abiotic stress. sod plays a crucial role in the antioxidative system by catalysing dismutation of o2·¯ to h2o2 and o2. cat has a high reaction rate, but a low affinity for h2o2, thereby removes the bulk of h2o2. inversely pod has a higher affinity for h2o2, allowing for the scavenging of small amounts of h2o2 in more specific locations (d a t et al., 2000). changes in cat activity are linked to desiccation during seed maturation (b a i l l y et al., 2004), seed germination (b a i l l y et al., 2002; p r o d a n o v i ć et al., 2007; b o g d a n o v i ć et al., 2008) and plant growth and development (b a i l e y & m c h a r g u e , 1943; m a t t e r s & s c a n d a l i o s , 1986; mitrović & bogdanović, 2008). pods are the most investigated enzymes since they have a role in very important physiological processes like seed germination, seedling growth (b e l a n i et al., 2002; d u č i ć et al., 2003/4; p r o d a n o v i ć et al., 2007; b o g d a n o v i ć et al., 2008), root growth (k u k a v i c a et al., 2007), plant growth and development (b a i l e y & m c h a r g u e , 1943; m i t r o v i ć & b o g d a n o v i ć , 2008), and lignin biosynthesis in cell walls (b r u c e & w e s t , 1989). this specific review summarizes some of the data obtained on model plant chenopodium rubrum l. in our laboratory in order to improve the understanding of involvement of antioxidative enzymes activities (and circumstantially ros) in regulation of c. rubrum vegetative and reproductive development. results and disscusion seed germination seed germination starts with imbibition, and ends with radicule protrusion. it is a complex process, associated with many metabolic, cellular and molecular events. accumulation of reactive oxygen species (ros), during seed imbibition, leads to germination (b a i l l y et al., 2004). therefore, antioxidant enzymes have a particular importance for the completion of germination. protein content increased during c. rubrum germination (d u č i ć et al., 2003/4), since proteins are both released from protein storage or synthesized de novo during and after the imbibition phase of germination, as building and regulatory material in emerging seedlings (r o b e r t s , 1972). during c. rubrum seed germination sequential expression of antioxidative enzymes occurred (d u č i ć et al., 2003/4). cat and sod showed the highest activity at the time preceding radicule protrusion, while significant expression of pod occurred after this term. increase in pod activity corresponds to the expression of new pod isoforms (d u č i ć et al., 2003/4). the appearance and increase of pod activity during germination could be specifically linked with final phases of seed germination or early seedling growth (s c h o p f e r et al., 2001; d u č i ć et al., 2003/4; b o g d a n o v i ć et al., 2008). sequential expression of antioxidative enzymes during c.rubrum seed germination points out that the decrease in h2o2 level coincide with final phases of seed germination and early seedling growth. the photoperiodic control of growth and development as already stated, in c. rubrum sel. 184 critical night length and sensitivity to photoperiod are well defined (t s u c h i y a & i s h i g u r i , 1981; s e i d l o v á & o p a t r n á , 1978). altering day length in such a plant, is a valuable source of information about regulation of plant development in accordance with the photoperiod. thus with the increase of day length, plant height is increased, flowering is delayed, seed development occured earlier, and plants produced more seeds (m i t r o v i ć et al., 2007). c. rubrum growth pattern to end of ontogenesis, flowering and seed development, are all determined by the photoperiod the seedlings experience during early phases of reproductive development induction and evocation of flowering (m i t r o v i ć et al., 2007; c o o k , 1975). natural c. rubrum flowering induction works in line with minimizing seed weight and maximizing seed number, favorizing physiological mechanisms that works under suboptimal photoperiods, maximizing probability to survive (c o o k , 1975). in addition to photoperiod temperature also affected seed weight, as previously reported for chenopodium quinoa (b e r t e r o et al., 1999) and c. rubrum (m i t r o v i ć et al., 2007). biologica nyssana 1 (1-2) december 2010: 61-64 mitrović, b. et al. chenopodium rubrum l. as a model plant… 63 antioxidative enzymes activities during ontogenesis in vitro by altering photoperiods in plants with well defined critical night length and sensitivity to photoperiod, it is possible to separate on the time scale different developmental phases (vegetative growth, flowering, seed development and maturation) in plants of the same age. the activities of antioxidative enzymes changes with both, phase of development and photoperiod plants are exposed to (l a l l & n i k o l o v a , 2003; m i t r o v i ć & b o g d a n o v i ć , 2008). c. rubrum flowering in vitro was associated with the highest cat activity. the intensities of 17 pod isoforms differed with day length plants were exposed to. so, it was suggested that pods are involved in determination of c. rubrum growth and development in accordance with seasonal changes of day length. pod isoform pi 4.6 could be associated with stress, induced both, by exposure to continuous light and by senescence (m i t r o v i ć & b o g d a n o v i ć , 2008). according to previously reported data for arabidopsis thaliana it was shown that ⋅o2is involved in the induction and development of senescence (a b a r c a et al., 2001). the absence of some sod isoforms could be the indicator of c. rubrum senescence in vitro which starts during the phase of seed maturation (m i t r o v i ć & b o g d a n o v i ć , 2008). seed aging seed aging is a natural process, starting during, or shortly after harvesting due to irreversible changes leading to loss of viability (v i l l i e r s , 1972). during seed aging chromosome and membrane damage occurs, as well as the damage of the enzyme structure, caused by ros. in the same time ros have the functional significance in seed ageing and germination (s c h o p f e r et al., 2001). seeds sampled during ageing showed an increase in the time needed to start germination possibly due to the need for repair and replacement processes to occur. protein de novo synthesis starts later during imbibition in aged seeds (v i l l i e r s , 1972). protein content, as well as cat and sod activity, was lower in aged c. rubrum seeds (2.5h darkness imbibed), compared to young seeds (m i t r o v i ć et al., 2005). viability declines with seed aging (b e w l e y & b l a c k , 1982) and germination of 3 years old c. rubrum seeds was delayed compared to 3 months old ones (m i t r o v i ć et al., 2005). so, antioxidant enzyme activities may be involved in the evaluation of seed viability in seed ageing. seed age also affect seedling growth and flowering. plants derived from aged seeds shows inhibited growth in vitro (m i t r o v i ć et al., 2005), and delayed flowering (m i t r o v i ć et al., 2005; k a d m a n z a h a v i & p e i p e r , 1987). conclusion this review has summarized some of data obtained on model plant chenopodium rubrum l. grown in vitro in our laboratory during last decade. our results improved knowledge of c. rubrum sensitivity to day length and also confirmed the involvement of antioxidative enzymes in regulation of plant development, from seed germination to seed maturation. acknowledgments. this work was supported by a grant (№. 143043) from the ministry of science and technological development, republic of serbia. references abarca, d., martín, m., sabater, b. 2001: differential leaf stress responses in young and senescent plants. physiol. plantarum, 113: 409-415. bailey, l.f., mchargue, j.s. 1943: enzyme activity in tomato fruits and leaves at different stages of development. american j. of bot., 30: 763-766. bailly, c., bogatek-leszczynska, r, come, d., corbineau, f. 2002: changes in activities of antioxidant enzymes and lipoxygenase during growth of sunflower seedlings from seeds of different vigour. seed science research, 12: 47-55. bailly, c., leymarie, j., lehner, a., rousseau, come, d., corbineau, f. 2004: catalase activity and expression in developing sunflower seeds as related to drying. journal of experimental botany, 55: 475-483. bellani, l.m., guarnier, m., scialabba, a. 2002: differences in the activity and distribution of peroxidases from three different portions of germinating brassica oleracea seeds. physiologia plantarum, 114:102-108. bertero, h.d., king, r.w., hall, a.j. 1999: photoperiod-sensitive development phases in quinoa (chenopodium quinoa willd.). field crops research, 60: 231-243. bewley j.d., black, m. 1982. viability, dormancy, and environmental control. in: physiology and biochemistry of seeds in relation to germination,, 2: 60-199. springer-verlag berlin heidelberg new york. bogdanović, j., radotić, k., mitrović, a. 2008: changes in activities of antioxidant enzymes during chenopodium murale seed germination. biologia plantarum, 52: 396-400. biologica nyssana 1 (1-2) december 2010: 61-64 mitrović, b. et al. chenopodium rubrum l. as a model plant… 64 bowler, c., van montagu, m., inzé, d. 1992: superoxide dismutase and stress tolerance. annual review of plant physiology and plant molecular biology, 43: 83-116. bruce, r.j., west, c.a. 1989: elicitation of lignin biosinthesis and isoperoxidase activity by pectic fragments in suspension culture of castor bean. plant physiology, 91: 889-897. cook, r.e. 1975: the photoindidtive control of seed weight in chenopodium rubrum l. american journal of botany, 62: 427-431. cumming, b.g. 1967. early flowering plants. in: will, f.h.,, wesselss , n.k., cromwell, t. y. (eds.), methods in developmental biology, 277299, new york. dat, j., vandenabeele, s., vranova, e., van montagu, m., inze,  d., van breusegem, f. 2000: dual action of the active oxygen species during plant stress responses. cellular and molecular life sciences, 57: 779-795. dučić, t., lirić-rajlić, i., mitrović, a., radotić, k. 2003/4: expression of antioxidant systems in chenopodium rubrum seed germination. biol. plantarum, 47: 527-533. foyer, c.h., lopez–delgado, h., dat., j.f., scott, i.m. 1997: hydrogen peroxide and glutathione– associated mechanism of acclamatory stress tolerance and signalling. physiol. plantarum, 100: 241-254. hendry, g.a.f., crawford, r.m.m. 1994: oxigen and envirnmental stress in plants – an overview. proceedings of the royal society of edinbourgh, 102b: 1-10. kadman-zahavi, a., peiper, d. 1987: effects of moonlight on flower induction in pharbitis nil, using a single dark period. annals of botany, 60: 621-623. khan, m.h., panda, s.k. 2002: induction of oxidative stress in roots of oryza sativa l. in response to salt stress. biol. plantarum 45: 625-627. kukavica, b., mitrović, a., mojović, m., veljovićjovanović, s. 2007: effect of indole-3-acetic acid on pea root growth, peroxidase profiles and hydroxyl radical formation. arch. biol. sci., 59: 319-326. lall, n., nikolova, r.v. 2003: developmental changes of superoxide dismutase, peroxidase and catalase isoenzyme profiles in leaves of impatiens flanaganiae hemsl. associated with variations in light intensity. south african journal of botany, 68: 518-524. matters,g.l., scandalios, j.g. 1986: effect of elevated temperature on catalase and superoxid dismutase during maize development. differentiation, 30: 190-196. mitrović, a. 2007: physiological and biochemical characteristics of vegetative and reproductive development in vitro of photoperiodic sensitive plant chenopodium rubrum l. phd thesis, faculty of science, univ. of belgrade, serbia. mitrović, a., bogdanović, j. 2008: activities of antioxidative enzymes during chenopodium rubrum l. ontogenesis in vitro. archives of biological sciences, 60: 223-231. mitrović, a., dučić, t., lirić-rajlić, i., radotić, k., živanović, b. 2005: changes in chenopodium rubrum seeds aging. annals of the new york academy of sciences, 1048: 505-508. mitrović, a., giba, z., ćulafić, lj. 2007: the photoperiodic control of growth and development of chenopodium rubrum l. plants in vitro. arch. biol. sci., 59: 203-208. prodanović, o., prodanović, r., bogdanović, j., mitrović, a., milosavić, n., radotić, k. 2007: antioxidative enzymes during germination of two lines of serbian spruce [picea omorika (panč.) purkynĕ]. arch. biol. sci., 59: 209-216. roberts, e.h. 1972. oxidative processes and the control of seed germination. in: heydecker, w. (ed.), seed ecology: 189-218, butterworts, london. schopfer, p., plachy, c., frahry, g. 2001: release of reactive oxygen intermediates (superoxide radicals, hydrogen peroxide, and hydroxyl radicals) and peroxidase in germinating radish seeds controlled by light, gibberellins, and ascorbic acid. plant physiology, 125: 1591-1602. seidlová, f., opatrná, j. 1978: change of growth correlation in the shoot meristem as the cause of dependance of flowering. zeitschrift fuer pflanzenphysiologie, 89: 377-392. tsuchiya, t., ishiguri, y. 1981: role of the quality of light in the photoperiodic flowering responce in four latitudinal ecotypes of chenopodium rubrum l. plant and cell physiology, 22: 525532. van loon, l.c. 1986. the significance of changees in peroxidase in diseased plants. in: greppin, h., penel, c., gaspar, t. (eds.), molecular and physiological aspects of plant peroxidases: 405418. university of geneva, geneva. villers, t. 1972. ageing and the longevity of seeds in field conditions. in: heydecker, w. (ed.), seed ecology: 266-285, proceedings of the nineteenth easter school in agricultural science, university of nottingham, butterworths, london.. živanović, b., ćulafić, lj., filipović, a. 1995: the effects of hormones and saccharides on growth and flowering of green and herbicides-treated chenopodium rubrum l. plants. biol. plantarum, 37: 257-264. microsoft word bn-sc-0201-10 stankovic m biologica nyssana 2 (1) september 2011: 77-81 stanković, m. rare, threatened and relict species in flora… 77 short communication ! rare, threatened and relict species in flora of snr zasavica stanković mihajlo*1 1special nature reserve „zasavica“, s. mitrovica, serbia * e-mail: zasavica@zasavica.org.rs abstract: stanković, m.: rare, threatened and relict species in flora of snr zasavica. biologica nyssana, 2 (1), september 2011: 77-81. in group of biodiversity important plant species there are 23 taxa. 20 taxa are mentioned in „preliminary red list of flora of serbia and montenegro with iucn 2001 conservation statuses“ in following categories: two as critically endangered (aldrovanda vesiculosa l. and hottonia palustris l.), four as endangered (hippuris vulgaris l., lindernia palustris hartm., ranunculus lingua l. and urtica kioviensis rogow.), five as vulnerable (achillea aspleniifolia vent., dryopteris carthusiana (vill.) h. p. fuchs, leucojum aestivum l. subsp. aestivum, stratiotes aloides l. and thelypteris palustris (schott) subsp.palustris, while 9 are with indefinite categories (cr-vu), due to data deficient (dd). special nature reserve „zasavica“ is the only habitat in serbia for aldrovanda vesiculosa l., which was until 2005. considered as extinct from serbia. key words: distribution, european adder, vipera berus, vlasina plateau introduction ! the first data on the flora and vegetation of north mačva (peripannonian serbia), and therefore the site of today's special nature reserve "zasavica" give josif p a n č i ć (1867) and ernest d o m b r o w s k i (1895). then followed a period of over half a century of sporadic research by small groups and individual researchers. in 1995 an initiative to protect river and a decree was adopted on the previous protection (official gazette rs, 51/95). at the suggestion of the institute for nature protection of serbia government of the 1997th the declared special nature reserve "zasavica (official gazette rs, 19/97). this paper aims to show the total value of the diversity of flora srp "zasavica. zasavica area is included in the ipa region of serbia s t e v a n o v i ć & š i n ž a r s e k u l i ć (2009). material and methods based on our field studies conducted on the territory of the special nature reserve “zasavica” and adjacent areas from 1997 to 2010. as well as on herbarium and literature sources, the distribution data of vascular plant taxa with great conservation value and some rare taxa were collected. voucher specimens were deposited in herbarium of institute for nature protection of vojvodina province, herbarium beou, beo, buns and herbarium of special nature reserve “zasavica” in sremska mitrovica. identification and revision of plant material were carried out according to t u t i n et al. (1968-1980; 1996), g a j i ć & k a r a d ž i ć (1991) and j á v o r k a & c s a p o d y (1975). threatened status of plant taxa is represented according to “preliminary red list of flora of serbia and montenegro with iucn 2001 conservation statuses” s t e v a n o v i ć (2002). the floristic elements are given according to the classification of g a j i ć (1980). 2 (1) • september 2011: 77-81 biologica nyssana 2 (1) september 2011: 77-81 stanković, m. rare, threatened and relict species in flora… 78 results and discussion in group of biodiversity important plant species there are 23 taxa. according to „ proclamation & regulation act of strictly protected and protected wild plant, animal and fungi species“ (official gazette rs, 36/09) 11 species are ranked as strictly protected and 46 as protected. also, 20 taxa are mentioned in „preliminary red list of flora of serbia and montenegro with iucn 2001 conservation statuses“ s t e v a n o v i ć (2002) in following categories: two as critically endangered (cr): aldrovanda vesiculosa l., hottonia palustris l.), four as endangered (en): hippuris vulgaris l., lindernia palustris hartm., ranunculus lingua l., urtica kioviensis rogow., five as vulnerable (vu): achillea asplenifolia vent., dryopteris carthusiana (vill.) h. p. fuchs, leucojum aestivum l., stratiotes aloides l. and thelypteris palustris (schott) subsp.palustris, while 9 are with indefinite categories (cr-vu), due to data deficient (dd). special nature reserve „zasavica“ is the only habitat in serbia for aldrovanda vesiculosa l., which was until 2005. considered as extinct from serbia (j a n k o v i ć & s t e v a n o v i ć 1999: 100; s t a n k o v i ć 2007: 35-37). on appendix i of convention on the conservation of european wildlife and natural habitats (strictly protected flora species) as well as on ipa criterion a, threatened species list are 3 species (aldrovanda vesiculosa l., lindernia palustris hartm., salvinia natans (l.) all.). one species (galanthus nivalis l.subsp.nivalis) is protected by cites convention (convention on international trade in endangered species of wild fauna and flora, аppendix 2). according turrill (1929) followt tertiary relicts of balkan peniusula are recorded: stratiotes aloides l., trapa natans agg., butomus umbellatus l., erythronium dens-canis l. subsp.dens-canis, humulus lupulus l., hydrocharis morsus-ranae l., isopyrum thalictroides l., loranthus europaeus jacq., tamus communis l.subsp.communis, viscum album l.subsp.album, aldrovanda vesiculosa l. some species abserved in zasavica reserve inchude tertiary relicts of southern europe wetlands as indicated by pollen analysis of peatbogs d i k l i ć (1984) include nymphaea alba l., nuphar lutea (l.) sibthorp. & sm. subsp.lutaea, salvinia natans (l.)allioni i nymphoides peltata (s.g.gmelin) o. kuntze. or hippuris vulgaris is the only representative of genus hippuris oligotips cirkumholoarctic in europe, which includes two species in america and asia. the sites in serbia are on the southern border area of its kind in europe. v u č k o v i ć & p a n j k o v i ć (1999), while hottonia palustris belongs oligotips relict genus hottonia that includes more h. inflata elliot. b u t o r a c (1999). in the area of the reserve hippuris vulgaris is in the process of withdrawal and hottonia palustris and ranunculus lingua show a tenddency to spread. by 2005. the largest population among the zasavica relic species had stratiotes aloides, which was covered with up to 90% of watercourses, and now appears with emerse belt, so that the steady increase in water level former caps (congestion) rivers have disappeared in many parts of zasavica item. floating reed islands inhabited by urtica kioviensis rogow. and schoenoplectus triqueter (l.) palla for which the peri serbian southern border area b r a n k o v i ć et al. (1996), while achillea asplenifolia vent. pannonian subendemic taxa found on the marshy meadows and are located on the southern border of its area. s o ό (1970). present u. kioviensis has riven range in eastern and central europe with the southern limit of its range in the pannonian plain and the possibility of its occurrence in neoendem wetlands pannonian plain b r a n k o v i ć et al. (1996). the patient was only present in vojvodina part. this type tends to spread within the reserve. the period of glaciation with its interglacial had significantly poorer flora in relation to the warm tertiary. by e n g l e r (1905) after the first glaciation there was a steppe interglacial stage, where in central europe rather than form a steppe tundra vegetation and some of those plants in the period represented postglacial endemic steppe periods. during the upcoming second glaciation in central and southern part of europe remained in part as glacial and partly as interglacial species ceratophyllum demersum l.subsp. demersum riparian forests are the habitat of relict species thelypteris palustris j a n k o v i ć et al. (1997). some species such as utricularia vulgaris, sparganium erectum subsp. erectum, the species myriophyllum spicatum, which were very present in marsh interglacial flora in europe, and still are widespread. four relict species that grow on zasavica are critically endangered species of flora of serbia. conclusion in group of biodiversity important plant species there are 23 taxa. according to „ proclamation & regulation act of strictly protected and protected wild plant, animal and fungi species“, 11 species are ranked as strictly protected and 46 as protected. also, 20 taxa are mentioned in „preliminary red list of flora of biologica nyssana 2 (1) september 2011: 77-81 stanković, m. rare, threatened and relict species in flora… 79 serbia and montenegro with iucn 2001 conservation statuses“ in following categories: two as critically endangered (cr) species, four as endangered (en) species, five as vulnerable (vu) species, while 9 are with indefinite categories (cr-vu), due to data deficient (dd). special nature reserve „zasavica“ is the only habitat in serbia for aldrovanda vesiculosa l., which was until 2005. considered as extinct from serbia. on appendix i of convention on the conservation of european wildlife and natural habitats (strictly protected flora species) as well as on ipa criterion a (a n d e r s o n , 2002, a n d e r s o n et al., 2005), threatened species list are 3 species. one species is protected by cites convention. to understand the importance of international protection zasavica shows and its nominations for the 2005 ipa area as well received as a member of the federation europark 2001 and the proclamation of the ramsar site in 2009. table 1. plant species significant for biodiversity conservation taxa preliminary red list of flora of serbia and montenegro st ri ct ly pr ot ec te d iucn conservation status ip a c ri te ri on b er n c on ve nt io n 1 achillea asplenifolia vent. vu 2 aldrovanda vesiculosa l. cr 3 arum orientale bieb. vu-nt (dd) 4 callitriche palustris l. en-vu (dd) 5 cyperus glomeratus l. nt-lc (dd) 6 dryopteris carthusiana (vill.) h. p. fuchs vu 7 erysimum cheiranthoides l. vu-nt (dd) 8 hesperis sylvestris crantz vu-nt (dd) 9 hippuris vulgaris l. en 10 hottonia palustris l. cr 11 leucojum aestivum l. vu 12 lindernia palustris hartm en 13 lygia passerina (l.) fassano vu-nt (dd) 14 nuphar lutea (l.) sibthorp. & sm. subsp.lutaea 15 nymphaea alba l. 16 ranunculus lingua l. en 17 salvinia natans (l.) all. 18 scirpus triqueter l. cr-vu (dd) 19 stratiotes aloides l. vu 20 thelypteris palustris (schott) subsp. palustris, vu 21 urtica kioviensis rogow. en 22 utricularia australis r. br. en-vu (dd) 23 zannichellia palustris l. vu-lc (dd) biologica nyssana 2 (1) september 2011: 77-81 stanković, m. rare, threatened and relict species in flora… 80 references anderson, s., 2002: identifying important plant areas. london, uk: plantlife international. anderson, s., kušík, t., radford, e. (eds), 2005, important plant areas in central and eastern europe. plantlife international. branković, d., budakov, lj., kovačev, n., mijović d., mikeš, b., pavkov, g., puzović, s., sekulić, n., stojšić, v., habjan-mikeš, v., garovnikov, b., stanković, m., 1996: predlog za zaštitu dobra "zasavica" kao specijalni rezervat prirode. zavod za zaštitu prirode srbije beograd -novi sad. butorac, b., 1999: hottonia palustris l.. in: v. stevanović, ed.: crvena knjiga flore srbije 1, ministarstvo za životnu sredinu r srbije, biološki fakultet univ. u beogradu i zavod za zaštitu prirode r srbije, 296-298. convention on international trade in endangered species of wild fauna and flora, аppendix 2. bonn, 1979. convention on the conservation of european wildlife and natural habitats (strictly protected flora species, appendix 1). bern, 1979. diklić, n., 1984: životne forme biljnih vrsta i biološki aspekti flore sr.srbije. in: sarić, m. (ed.), 1984: vegetacija sr srbije, i. sanu, odeljenje prirodno-matematičkih nauka, beograd, 291-317. dombrovski, e., 1895: osnovi ornitologije sjeverozapadne srbije. glasnik zemaljskog muzeja bih, sarajevo. engler, r.a., 1905: grundzüge der entwicklung der flora europas seit der tertiärzeit. bot. jahrb. b., 36, leipzig gajić, m., 1980: pregled vrsta flore sr srbije sa biljnogeografskim oznakama. glasnik šumarskog fakulteta, serie a “šumarstvo”, 54: 111-141. belgrade. gajić, m., karadžić, d., 1991: flora ravnog srema sa posebnim osvrtom na obedsku baru, šg, sremska mitrovica. janković, b., mišić v., dinić, a., vukićević, e., 1997: vegetacija srbije, ii tom, šumske zajednice 1. sanu, odeljenje prirodnomatematičkih nauka, beograd. janković, m., stevanović, v., 1999: aldrovanda vesiculosa. in: v. stevanović, ed.: crvena knjiga flore srbije 1, ministarstvo za životnu sredinu r srbije, biološki fakultet univ. u beogradu i zavod za zaštitu prirode r srbije, 299-301 jávorka, s., csapody, v., 1975: közép-európa délkeleti részének flórája képekben iconographia florae partis austro-orientalis europae centralis]. akadémiai kiadó. budapest pančić, j., 1869: građa za faunu kneževine srbije. glasnik srpskog učenog društva, beograd. službeni glasnik republike srbija br 51/95: rešenje prethodnog zaštite prirodnog dobra zasavica. službeni glasnik republike srbija br.36/09 zakon o zaštiti prirode, anex i i ii strogo zaštićene i zaštićenih divljih biljaka, životinja i gljiva. službeni glasnik rs,19/97 uredba o zaštiti zasavice kao specijalni rezervat prirode. soό,r., 1964-1970: a magyar flόra ès vegetáció rendszertani-növènyföldrajzi kèzikönyve i-iv, akadèmiai kiadó budapest. stanković m.2007. rezultati istraživanja globalno ugrožene vrste aldrovanda vesiculosa l. (fam.droseraceae) tokom 2006. godine u specijalnom rezervatu prirode zasavica, zbornik naučno-stručnog skupa „zasavica 2007“sa međunarodnim učešćem, sremska mitrovica stevanović v. (ed.) 2002. preliminarna crvena lista flore srbije i crne gore prema kriterijumima iucn-а iz 2001 godine. belgrade. (manuscr.) stevanović v., šinžar-sekulić, 2009.serbia in: conserving importand plant areas: investingin the gren gold of southeast europaea (eds.radfordea and obé b) plant life international,sa lisaury, p.63 turrill w.b.1929, the plant-life of the balkan peninsula,oxford,leipzig tutin t. g., heywood v h., burges n.a.. moore d. m.,valentine d. h,walters s. m., and webb d. a. (eds.), 1968-1980: flora europaea ii-v. cambridge university press. tutin, t.g., burges, n.a., chater, a.o., edmondson, j.r., heywood, v.h.., moore, d. m., valentine, d. h., walters, s. m. and webb, d.a. (eds.) 1993: flora europaea i, 2nd ed. cambridge university press. vučković, m. & panjković, b., 1999: hippuris vulgaris l. in: v. stevanović, ed.: crvena knjiga flore srbije 1, ministarstvo za životnu sredinu r srbije, biološki fakultet univ. u beogradu i zavod za zaštitu prirode r srbije, 299-301. microsoft word 0305_zivanovic_et_al biologica nyssana 1 (1-2) december 2010: 71-75 mitrović, b. et al. chenopodium rubrum l. as a model plant… 31 original article ! chenopodium murale l., a long-day plant as a model for physiological and biochemical research branka živanović1, aleksandra mitrović1*, jelena bogdanović pristov1, ksenija radotić hadži-manić1, ljubinka ćulafić2 1 institute for multidisciplinary research, university of belgrade, kneza višeslava 1, 11030 belgrade, serbia 2 institute of botany and botanical garden "jevremovac", takovska 43, faculty of biology, university of belgrade, 11000 belgrade, serbia * e-mail: mita@imsi.rs abstract: živanović, b., mitrović, a., bogdanović-pristov, j., radotić hadži-manić, k., ćulafić, lj.: chenopodium murale l., a long-day plant as a model for physiological and biochemical research. biologica nyssana, 1 (1-2), december 2010: 71-75. chenopodium murale l. genus chenopodium family chenopodiaceae is a weedy annual widely distributed in serbia. this is a long-day plant and an early flowering species. we used culture of intact plants in vitro and antioxidative enzymes detection in order to examine the effect of gibberellic acid (ga3) on two key processes during ontogenesis – germination and flowering. our results showed a sequential expression of antioxidative enzymes during germination. in dry seeds and during early imbibition catalase (cat) and superoxide dismutase (sod) activities showed no changes, while peroxidase (pod) activity was under the level of delectability. during radicule protrusion cat and sod activity increased. early seedling development correlates with decrease in sod, increase in cat and appearance of pod activity. ga3 delayed and synchronized germination. c. murale photoperiodic sensitivity for flowering shows age-dependant oscillatory changes. glucose and ga3 have cumulative stimulatory effect on its flowering in vitro. the exposure of 2 months old vegetative plants to continuous darkness, in the presence of ga3 in culture media, resulted in flowering. therefore transferring to darkness canceled photoperiodic control in c.murale and flowering occurred under autonomous mechanism. we suggest c.murale as a suitable model for investigation of physiological and biochemical mechanisms of growth and developmental processes. key words: catalase, chenopodium murale l., germination, gibberelic acid, flowering, peroxidase, superoxide dismutase introduction ! chenopodium murale l. belongs to the family chenopodiaceae, genus chenopodium. ecotype 197 is characterized by c u m m i n g (1967) as a facultative long-day annual and an early flowering species. it is sensitive to photoperiodic flowering induction as early as at the phase of the 1st pair of leaves (p a v l o v á et al., 1989). 10 cycles of continuous light (24 h light) are necessary for photoperiodic flowering induction, regardless of plant age (p a v l o v á et al., 1989). intact c. murale plants, cultured in vitro on optimal media composition and under adequate photoperiodic regime, produces seeds in 18 weeks. gibberellins (gas) are very potent plant growth regulators. gas plays an important role in stimulation of seed germination (b e w l e y , 1997). they are efficient in breaking seed dormancy, by their ability to overcome the requirements for environmental factors, and in accelerating germination in non-dormant seeds (b e w l e y & 10th sfses • 17-20 june 2010, vlasina lake1 (1-2) • december 2010: 71-75 biologica nyssana 1 (1-2) december 2010: 71-75 mitrović, b. et al. chenopodium rubrum l. as a model plant… 72 b l a c k , 1982). ga3 also induces or promotes flowering, being particularly effective in long-day species (e v a n s et al., 1999; ž i v a n o v i ć et al., 1995; m i t r o v i ć et al., 2000; m i t r o v i ć et al., 2000; m i t r o v i ć et al., 2003). continuous production and removal of reactive oxygen species (ros) is, besides as a phenomenon with negative consequences (damage of cell membranes and organelles), linked with a signaling role in plant developmental processes. antioxidative enzymes, catalase (cat), superoxide dismutase (sod) and peroxidase (pod) are engaged in the scavenging of ros (dat et al., 2000) and therefore participate in regulation of plant growth and development (m i t r o v i ć , 2007) as well as in the protection against pathogens or abiotic stress (h e n d r y & c r a w f o r d , 1994). this is a review of our previously reported data that describe the effects of gibberellic acid in regulation of c. murale seed germination and flowering, as two key processes in plant ontogenesis. results and disscusion seed germination seed germination starts with imbibition, and ends with radicule protrusion. it can be divided in three phases: imbibition (rapid initial water uptake – physical process characteristic also for dead seeds), the plato phase (small change of water content but high metabolic activity) and further water uptake coinciding with radicule protrusion and growth (g i b a et al., 2004). plato phase, as the most important in regulation of germination, involves the activation of specific enzymes at the appropriate time and regulation of their activity (r i l e y , 1987). it was supposed that accumulation of reactive oxygen species (ros), during seed imbibition, leads to germination (b a i l l y , 2004). therefore, antioxidant enzymes have been considered to be of particular importance for the completion of germination. sequential expression of antioxidative enzymes and their importance during germination in different plant species was shown (d u č i ć et al., 2003/4; b a i l l y et al., 2000; p u n t a r u l o et al., 1991; b o g d a n o v i ć et al., 2008). gibberellic acid delayed and synchronized c. murale germination and showed similar effect on protein content and activities and isoenzyme pattern of antioxidative enzymes (fig. 1, fig. 2) (b o g d a n o v i ć et al., 2008). this suggests that ga3 and ros, thorough activities of antioxidative enzymes, participate in the same signaling pathway in germination. protein concentration increased at the time of c.murale ridicule protrusion (b o g d a n o v i ć et al., 2008), connected with release from storage and de novo synthesis of building and regulatory proteins in the emerging seedlings (r o b e r t s , 1972). fig. 1. c. murale germination (%) on distilled water or ga3 (160 μm) solution. data points are averages of four replicates of 50 seeds each. modified from bogdanović et al., 2008 in dry seeds and during early imbibition catalase (cat) and superoxide dismutase (sod) activities showed no changes, while peroxidase (pod) activity was under the level of delectability (fig. 2). cat activity increased during radicule protrusion and early seedling development, by appearance of a new cat isoform. pick in sod activity coincided with radicule protrusion and early seedling development. based on the specific inhibition of sod activity by kcn and h2o2, we showed that c.murale seeds/seedlings contain only mn sod form. pod activity appeared prior to or simultaneously with radicule protrusion and increased during early seedling growth, presented with two basic isoforms (b o g d a n o v i ć et al., 2008). specific role of pods in regulation of final phases of germination and early seedling growth was suggested in many species (s c h o p f e r et al., 2001; d u č i ć et al., 2003/4; p r o d a n o v i ć et al., 2007; b o g d a n o v i ć et al., 2008). pods have a very important role in physiological processes in plants (g a s p a r et al. 1991), but their exact relationship to developmental events is often obscure by their extensive polymorphism in a single plants species. changes in cat, sod and pod activities could be “the markers” of different phases of germination. decrease in sod activity (h2o2 producing enzyme), increase in cat and appearance of pod activity (h2o2 consuming enzymes) coincide with early seedling development in c. murale. these data, accompanied with similar findings concerning seed germination in different plant species, suggest that decrease in h2o2 level biologica nyssana 1 (1-2) december 2010: 71-75 mitrović, b. et al. chenopodium rubrum l. as a model plant… 73 might be involved in regulation of this process (bogdanović et al., 2008). fig. 2. catalase, superoxide-dismutase and peroxidase activities [mg g-1 (d.w.)] during c. murale seed germination on distilled water or ga3 (160 μm) solution. modified from bogdanović et al., 2008 flowering transition from vegetative to reproductive phase of development is controlled by genetical (autonomous) and ecological (photoperiod and/or temperature) factors. autonomous control that depends on plant age is the basic control level. induced or photoperiodic control, alone or in combination with temperature, is the second level of flowering control that stimulates or inhibits genetically determined flowering. in c.murale plants photoperiodic sensitivity is age-dependant. it shows oscillatory changes with aging (expressed by number of leaves) (p a v l o v á et al., 1989; m i t r o v i ć et al., 2000). 10 cycles of continuous light (24 h light) are necessary for photoperiodic flower induction, regardless of plant age (p a v l o v á et al., 1989; m i t r o v i ć et al., 2000). glucose and ga3 have cumulative stimulatory effect on c.murale flowering in vitro under inductive photoperiodic conditions, probably affecting flower development, rather than flower initiation (tab. 1). under photoperiod noninductive for flowering neither glucose nor ga3 were able to compensate for c.murale photoperiodic requirements for flowering (mitrović et al., 2000). the photoperiodic control is loosened with aging in some plants (perilla nankensis, rudbeckia bicolor) (b e r n i e r et al., 1981; c h a i l a k h y a n , 1988). exposure to darkness cancels photoperiodic control, so the flowering is controlled only by the autonomous mechanism (c h a i l a k h y a n , 1988; m i t r o v i ć et al., 2003). some long-day and shortday plants produces flowers under continuous darkness (f i f e and price, 1953; sugino, 1957; inouye et al., 1964; t a k i m o t o , 1960; m i t r o v i ć et al., 2003; c v e t i ć et al., 2004). in 5 – 7 month old long-day plant rudbeckia bicolor flowering occurred after exposure to darkness (c h a i l a k h y a n , 1988). in 2 months old c. murale plants, exposed to continuous darkness for 10 days, the addition of ga3 in culture medium was necessary for transition to flowering (tab. 1) (mitrović et al., 2003). ga3 alone was not able to compensate for c. murale photoperiodic demands for flowering. so we suggest that ga3 affected flower development, rather than flower initiation. c.murale plants partly loose demands for specific day length with aging, so transferring to darkness cancels photoperiodic control and flowering occurs under autonomous mechanism (m i t r o v i ć et al., 2003). conclusion this is a review of our work on model plant chenopodium murale. our results improved the knowledge of the role of gibberellic acid in two key processes during ontogenesis germination and flowering. ga3 delay and synchronize germination, while it stimulates flowering. changes in activities and isoforms of antioxidative enzymes could be “the markers” of different phases of seed germination. intact c.murale plants have the ability to be induced for flowering in total darkness in vitro. acknowledgments. this work was supported by a grant (№. 143043) from the ministry of science and technological development, republic of serbia. biologica nyssana 1 (1-2) december 2010: 71-75 mitrović, b. et al. chenopodium rubrum l. as a model plant… 74 references bailly, c. 2004: active oxygen species and antioxidants in seed biology. seed science research, 14: 93-107. bailly, c., benamar, a., corbineau, f., côme, d. 2000: antioxidant systems in sunflower (helianthus annuus l.) seeds as affected by priming. seed science research, 10: 35-42,. bernier, g., kinet, j-m., sachs, r.m. 1981: age and flower initiation. in: the physiology of flowering i: 106-115, crc press., boca raton. bewley, j.d., black, m. 1994: seeds. physiology of development and germination. 2nd ed. plenum press, new york. bewley, j.d. 1997: seed germination and dormancy. plant cell, 9: 1055-1066. bogdanović, j., radotić, k., mitrović, a. 2008: changes in activities of antioxidant enzymes during chenopodium murale seed germination. biologia plantarum, 52: 396-400. chailakhyan, m.ch. 1988: regulyatsiya tsveteniya u vysshikh rastenii. [regulation of flowering in higher plants] – nauka, moscow. [in russ.] cumming, b.g. 1967: early flowering plants. in: will, f.h., wessels, n.k., cromwell, t.y. (eds.): methods in developmental biology: 277299, new york. cvetić, t., budimir, s., grubišić, d. 2004: in vitro flowering of dark-grown centaurium pulchellum. archives of biological sciences, 56: 21-22. dat, j., vandenabeele, s., vranova, e., van montagu, m., inze,  d., van breusegem, f. 2000: dual action of the active oxygen species during plant stress responses. cellular and molecular life sciences, 57: 779-795. dučić, t., lirić-rajlić, i., mitrović, a., radotić, k. 2003/4: activities of antioxidant systems during germination of chenopodium rubrum seeds. biologia plantarum, 47: 527-533. evans, l.t. 1999: gibberellins and flowering in long day plants, with special reference to lolium temulentum. australian journal of plant physiology, 26: 1-8. gaspar, t., penel, c., hagege, d., grepin, h. 1991: peroxidases in plant growth, differentiation and developmental processes. in: lobarewski, j., grepin, h., penel, c., gaspar, t., editors. biochemical, molecular and physiological aspects of plant peroxidases. pp. 249-80. university m. curie-sklodowska, lublin and university of geneva, geneva. giba, z., grubišić, d., konjević, r. 2004: nitric oxide and seed germination. in: jose r. magalhaes, rana p. singh & leonidas p. pasos (eds.): nitric oxide signaling in higher plants: 239-275, studium press, llc, houston, usa. hendry, g.a.f., crawford, r.m.m. 1994: oxigen and envirnmental stress in plants – an overview. proceedings of the royal society of edinbourgh, 102b: 1-10. inouye, j., tashima, y., katayama t. 1964: flower initiation in total darkness in a long day plant, triticum aestivum. plant cell physiology, 5: 355-358. mitrović, a., živanović, b., ćulafić, lj. 2000: the effects of photoperiod, glucose and gibberellic table 1. flowering of c. murale plants in vitro. plants were induced for flowering at different age (expressed by number of leaves) by exposure to 10 days of continuous light or 10 days of continuous darkness; dcl – days of continuous light (24 h light), dcd – days of continuous darkness (24 h darkness), sd – short day (8/16 h light/darkness). modified from mitrović et al., 2000 and mitrović et al., 2003. photoperiodic conditions ga3 (mg dm3) flowering (%) 10 dcl in the phase of development of 1st pair of leaves 12 sd + 10d cl +27 sd 0 17 1 40 5 43 10 dcl in the phase of development of 2nd pair of leaves 29 sd +10 dcl + 30 sd 0 0 5 13 10 dcl in the phase of development of 3rd pair of leaves 42 sd + 10 dcl + 30 sd 0 18 5 67 10 dcl in the phase of development of 4th pair of leaves 60 sd +10 dcl + 30 sd 1 60 10 dcd in the phase of development of 4th pair of leaves 60 sd + 10 d cd + 30 sd 0 0 1 42 5 65 biologica nyssana 1 (1-2) december 2010: 71-75 mitrović, b. et al. chenopodium rubrum l. as a model plant… 75 acid on growth in vitro and flowering of chenopodium murale. biologi plantarum, 43: 173-177. mitrović, a., živanović, b., ćulafić, lj. 2003: effect of darkness on growth and flowering of chenopodium rubrum and c. murale plants in vitro. biologia plantarum, 46: 471-474. mitrović, a., 2007: physiological and biochemical characteristics of vegetative and reproductive development in vitro of photoperiodic sensitive plant chenopodium rubrum l. phd thesis. faculty of biology, university of belgrade, belgrade. pavlová, l., součková, d., ullman, j., krekule, j. 1989: the transition to reproductive phase in chenopodium murale l. ecotype 197 – early flowering long-day plant. biologia plantarum, 31: 386-391. prodanović, o., prodanović, r., bogdanović, j., mitrović, a., milosavić, n., radotić, k. 2007: antioxidative enzymes during germination of two lines of serbian spruce [picea omorika (panč.) purkynĕ]. archives of biological sciences, 59: 209-216. puntarulo, s., sanchez, r.a., boveris, a. 1988: hydrogen peroxide metabolism in soybean embryonic axes at the onset of germination. plant physiology, 86: 626-630. puntarulo, s., galleano, m., sanchez, r.a., boveris, a. 1991: superoxide anion and hydrogen peroxide metabolism in soybean embryonic axes during germination. biochimica biophysica acta, 1074: 277-283. riley, j.m. 1987: gibberellic acid for fruit set and seed germination. california rare fruit growers journal, 19: 10-12. roberts, e.h. 1972: oxidative processes and the control of seed germination. in: heydecker, w. (ed): seed ecology: 189-218, butterworts, london. schopfer, p., plachy, c., frahry, g. 2001: release of reactive oxygen intermediates (superoxide radicals, hydrogen peroxide, and hydroxyl radicals) and peroxidase in germinating radish seeds controlled by light, gibberellin, and abscisic acid. plant physiology, 125: 1591-1602. takimoto, a. 1960: effect of sucrose on flower initiation of pharbitis nil in aseptic culture. plant cell physiology, 1: 241-246. živanović, b., ćulafić, lj., filipović, a. 1995: the effects of hormones and saccharides on growth and flowering of green and herbicides-treated chenopodium rubrum l. plants. biologia plantarum, 37: 257-264. microsoft word bn020201 ljupkovic bojic biologica nyssana 2 (2) december 2011: 00-00 ljupković r.b. et al.. removal cu(ii) ions from water... 85 original article ! removal cu(ii) ions from water using sulphuric acid treated lagenaria vulgaris shell (cucurbitaceae) radomir b. ljupković, jelena mitrović, miljana radović, miloš kostić, danijela bojić, dragana-linda mitić-stojanović, aleksandar lj. bojić department of chemistry, faculty of sciences and mathematics, university of niš * e-mail: bojica@pmf.ni.ac.rs abstract: ljupković, r.b., mitrović, j., radović, m., kostić, m., bojić, d., mitić-stojanović, d-l., bojić, a.lj.: removal cu(ii) ions from water using sulphuric acid treated lagenaria vulgaris shell (cucurbitaceae), biologica nyssana, 2 (2), december 2011: 85-89. removal of cu(ii) ions from water solutions by sulphuric acid treated lagenaria vulgaris shell (cclvb) was studied. batch experiments were done by shaking a fixed mass of biosorbent (1.0 g) with 250 cm3 of 50.0 mg dm–3 cu(ii) solutions, at ph ranged from 2 up to 6. metal concentration in the filtrates as well as in the initial solution was determined by flame atomic absorption spectrometry. results show that efficiency of cu(ii) ions uptake by sulphuric acid treated lagenaria vulgaris shell is significantly greater than raw lagenaria vulgaris biosorbent. in addition, there is no significant effect of initial ph of solution on cu(ii) ions uptake by cclvb and obtained biosorbent can be applied in a wide range of ph. key words: biosorption, lagenaria vulgaris, cu(ii), ph, kinetics introduction ! rapid industrialization throughout the world has generated huge volumes of wastes containing toxic materials, such as heavy metals, dyes, phenols and surfactants. the presence of heavy metals in wastewaters and surface waters is major concern of the public health and the environment. the increased use of metals and chemicals in the industrial processes have resulted in the generation of large quantities of aqueous effluents that contain high levels of metals, creating serious environmental disposal problems (y u et al., 2000). the main sources of copper pollution are metal cleaning and plating baths, pulp, paper board mills, wood pulp production, the fertilizer industry, etc. (d u n d a r et al., 2009). copper usually occurs in nature as oxides and sulfides. in acidic environments, a free aqueous cu2+ ion dominates. at ph 6–8, the predominant species are cu2+, cu(oh)2, cuhco3 +, cuco3 and cuoh +, while at ph > 10 the major species are cu(oh)3− and cu(oh)4 2−. copper may be found as a contaminant in food, especially shellfish, liver, mushroom, nuts and chocolate. copper is essential to human life and health, but in large amount it is toxic as well. excess copper in the human body can cause stomach and intestinal distress such as nausea, vomiting, diarrhea and stomach cramps (n u h o g l u et al., 2002). various methods have been used to remove heavy metals from wastewater, such as reduction and precipitation, coagulation, flotation, adsorption on activated carbon, ion exchange, reverse osmosis and electrodialysis (d a n g et al., 2009). some of these processes are expensive, or have other disadvantages, such as incomplete removal of metals, limited tolerance to ph change, moderate or no metal selectivity, dependence of metal concentration in wastewater, and production of toxic 2 (2) • december 2011: 85-89 biologica nyssana 2 (2) december 2011: 85-89 ljupković r.b. et al. removal cu(ii) ions from water… 86 sludge or other waste products that also need disposal (a n t u n e s et al., 2003). in recent years, biosorption has been suggested as cheaper as and more effective than many chemical or physico-chemical technologies. (krishnani et al., 2008) biosorption can be defined as the ability of biological materials to accumulate heavy metals from wastewater through metabolically mediated or physico-chemical pathways of uptake (u c u n et al., 2002). various biomasses are used as potential sorbents, such as fungi, bacteria, algae, agricultural waste and waste from food industries, wood sawdust and bark, in their natural form or in chemically or physically modified form (b a s s o et al., 2002). lignocellulosic fiber is an unconventional low-cost sorbent that has been examined for potential use in removing heavy metals. however, the ion exchange or adsorption capacity of lignocellulosic fiber is lower than that of other sorbents. as a result, attempts have been made to modify various lignocellulosic fibers with different chemicals (m i n et al., 2004). in this study, lagenaria vulgaris was used as a potential biosorbent for removal of cu(ii) ions from water. biosorbent was prepared by sulphuric acid treatment of plant shell to improve sorption characteristics. materials and methods preparation of biosorbent lagenaria vulgaris (cucurbitaceae family) is a creeping, hardy plant. it is found growing mainly on alluvial sandy soil and red loam, on flat areas and moderate slopes, and in higher-lying areas, and needs light and warmth. the outer shell is hard and ligneous, covering the spongy white pith characterized by its bitter taste. the experiments in this study were carried out using the shell of lagenaria vulgaris grown in the southeastern area of serbia without irrigation and fertilization, at an altitude of 200 m. raw lagenaria vulgaris shell was air dried, washed with distilled water and grounded. grounded biomass was rinsed with 0.3 m hno3 for a 24 h. acid treated material was washed with a deionised water to remove excess of acid and stirred on a magnetic stirrer with 0.1 m naoh for a 30 min. neutralized biomass was washed with deionized water and dried in a oven on 55±50c. obtained biosorbent was fractionized using standard sieves (endecott, england) and particles with size from 0.8 up to 1.25 mm are used in this study. this material is designated as alvb (m i t i c s t o j a n o v i c et al., 2011). prepared biosorbent was soaked with concentrated sulphuric acid for a 2 h. temperature of mixture was held on 40oc. after treatment, obtained black powder was washed with deionised water, and 0.1 m naoh was added in small portions until ph reached 7-7.5. then, biomass was washed with deionized water, filtered and dried in an oven on 55±5oc. result of this process is the most similar to thermal carbonization, and we call it „cold carbonization“. obtained material is designated cclvb. batch experiments the batch experiments are conducted by stirring a fixed mass of biosorbent (1.0 g) with 250 cm3 of 50.0 mg dm–3 cu(ii) into glass beaker. the initial ph of solutions was adjusted using hno3 or naoh (0.01, 0.1m) in range from 2 up to 6. the mixture was agitated for 240 min to reach equilibrium state and filtered through filter paper (wathman no2). samples (5 cm3) were taken at the beginning (before adding the sorbent) and at predetermined time intervals (1, 2, 5, 10, 20, 40, 60, 90, 120 and 240 min) for the residual cu(ii) ion concentration in the solution. to preserve aliquots, 0.1 ml of concentrated hno3 was added. metal concentration in the filtrates as well as in the initial solution was determined by flame atomic absorption spectroscopy (model aanalyst 300, perkin elmer, usa). determine of phpzc point of zero charge was determined by ph drift method (y a n g et al., 2004; a z i z et al., 2009) with some modifications for lvb. as inert electrolyte was used 0.01m kno3. the ph of test solutions were adjusted in range between 2 and 10 using 0.01m hno3 and 0.01 m koh. a 0.2 g of biosorbents were added to 50 ml of test solutions into stopped glass tubes and equilibrated for 24 h. the final ph (phf) was measured after 24 h and plotted against the initial ph (phi). the ph at which the curve crosses line phi = phf was taken as phpzc. ph was measured by sension3 (hach, usa), which was calibrated before every measure. results and disscusion effect of contact time to determine the effect of contact time on copper biosorption, 250 ml of cu(ii) ions solution, concentration 50.0 mg dm–3, was treated with 1.0 g of l. vulgaris shell biomass at 25±0.5oc and initial biologica nyssana 2 (2) december 2011: 85-89 ljupković r.b. et al. removal cu(ii) ions from water… 87 ph 5.0. the residual concentration of cu(ii) ions was determined after 1, 2, 5, 10, 20, 40, 60, 90, 120 and 240 min. results are shown on figure 1. figure 1. effect of contact time on cu(ii) ions biosorption removal (ccu(ii) = 50 mg dm –3, phi 5.0, clvb = 4.0 g dm –3, t = 25±0.5oc) results show that sorption of copper ions onto the biosorbents was very fast, as the equilibrium state was reached in less than 10 min for cclvb. at the end of the treatment, after 240 min of contact time, cu(ii) ions removal efficiency is near 100% for cclvb. sulphuric acid treatment improves sorption characteristics of l. vulgaris shell biomass, because unmodified biosorbent reaches equilibrium within about 30 min with removal efficiency near 90% at the end of treatment (240 min). pseudo-second order model was used to describe the kinetics of adsorption of cu(ii) onto cclvb. the kinetic data were analyzed using linear form of pseudo-second order relation (h o and m c k a y , 1999): t qqkq t eet 11 2 2 += where qt and qe (mg g -1) are amounts of cu(ii) ions adsorbed on cclvb at time t and at equilibrium, and k2 (min g mg -1) is the rate constant of process. as can be seen from figure 2 and table 1, straight line with correlation coefficient 0.999 show that kinetics of adsorption of cu(ii) on cclvb can be good fitted by pseudo-second order kinetic model. kinetic data for alvb are also good fitted by pseudo-second order kinetic model. in accordance with the pseudosecond order mechanism, the rate of cu(ii) sorption process appear to be controlled by chemical process or chemisorption involving valence forces through sharing or exchange of electrons between sorbent and sorbate. figure 2. pseudo-second order kinetic model for cu(ii) ions sorption on cclvb (ccu(ii) = 50 mg dm–3, phi 5.0, clvb = 4.0 g dm –3, t = 25±0.5oc) point of zero charge the ph at which the sorbent surface charge takes a zero value is defined as point of zero charge (phpzc). at this ph, the charge of the positive surface sites is equal to that of the negative ones. at ph values of solution higher than phpzc, sorbent surface is negatively charged and could better interact with metal positive species while at ph values lower than phpzc, solid surface is positively charged and could interact with negative species. table 1. pseudo-second order kinetic model parameters for cu(ii) ions sorption process on cclvb material c0 (mg dm –3) k (min g mg-1) qe,exp (mg g -1) qe,cal (mg g -1) r2 cclvb 50 0.8102 10.90 11.11 0.999 alvb 50 0.0404 7.97 7.70 0.999 biologica nyssana 2 (2) december 2011: 85-89 ljupković r.b. et al. removal cu(ii) ions from water… 88 2 4 6 8 10 2 4 6 8 10 phf phi figure 3. determination of phpzc using dirft method results in figure 3, show that phpzc of sulphuric acid treated lvb is 4.95. it is much more acidic in comparison with starting material alvb, with phpzc value 6.10. heaving in mind applied chemical treatment, increased content of acid groups in lvb structure, probably sulfonic group, is expected. effect of initial ph to determine the effect of initial ph on cu(ii) ions removal, experiments were conducted at ph 2, 3, 4, 5 and 6, adjusted with hno3 or naoh. 1.0 g of cclvb was added to 250 ml of 50 mg dm–3 cu(ii) ions solutions. the mixture was agitated for 240 min and aliquots were taken at predefined time intervals. results are shown in figure 3. 2 3 4 5 6 4 6 8 10 12 q e (m g g1 ) ph figure 4. effect of initial ph on cu(ii) ions removal by cclvb (ccu(ii) = 50 mg dm–3, phi 5.0, clvb = 4.0 g dm–3, t = 25±0.5oc) it is well known that ph has a great importance for cation sorption by biosorbents because it influences chemical speciation of the metal ions in solution and also on the ionization of chemically active sites on the sorbent (b a s c i et al., 2004; g o y a l et al., 2008; i f t i k h a r et al., 2009; d a n g et al., 2009). in contrast, results in figure 4. shown that there is a small effect of initial ph of metal solution on sorption of cu(ii) ions onto cclvb. in ph range from 3 up to 6 almost there is no effect of ph on cu(ii) removal efficiency. only at the initial ph 2 the efficiency of cu(ii) removal is slightly smaller, for about 10%. uptake of cu(ii) by cclvb was favored by ph increasing from 2 to 3 because of different reasons, as in the case of many other biosorbents. the lower sorption of divalent metals observed at low ph values has been attributed to following factors: (a) repulsion between positive charge of the sorbent and free metal cations; (b) competition between h+ and free metal cations for the sorbent active sites; (c) lower formation of complexes with metal ions due to protonation of surface functional groups, and (d) combination of several of these factors (f i o l and v i l l a e s c u s a , 2008; a z i z et al., 2009; i f t i k h a r et al., 2009). authors m i t i ć s t o j a n o v i ć et al. (2011) were shown that metal ions sorption by untreated l. vulgaris biomass is very affected by initial ph of metal solution. they found that sorption efficiency significantly rise with increasing of ph in range from 2 to 5, with maximum at ph 5, which is closely related with obtained phpzc value of 6.10 for alvb. however, for sulphuric acid threated lvb results are not completely consistent with observed phpzc, with value 4.95 (figure 3). this could mean that mechanism of cu(ii) ions sorption by cclvb is not strictly of ionexchange type. lewis base – lewis acid interactions between sorbent surface functional groups and metal ions may play a significant role in the copper ions removal by sulphuric acid treated l. vulgaris shell biomass. results can be, also, explained by presence of acidic –so3 – groups on surface of cclvb, which cannot be protonated on investigated ph values. in addition, sulphuric acid treatment changes the physical and chemical properties of biosorbent, which results in increase of efficiency for metal ions removal. it can be supposed that conc. h2so4 initiated bond cleavage, leading to dehydration and elimination reactions that release volatile products such as water, acetic acid, methanol and other chemical substances (g e r c e l et al., 2007). this is followed by partially aromaticity and recombination of species to form a stronger cross-linked solid (k h a l e d et al., 2008). o z e r et al. (2007) supposed that sulphuric acid reacts with hydroxyl groups in the lignin to form sulphuric esters as a biologica nyssana 2 (2) december 2011: 85-89 ljupković r.b. et al. removal cu(ii) ions from water… 89 non-ionic functional group, which may complex cations. obviously, mechanism of biosorption process is rather complex and involve many different processes: chemisorption, complexation, ion exchange, chelation, physical adsorption, which do not depend of ph on the same way (s u d et al., 2008). conclusion sulphuric acid treated lagenaria vulgaris shell is found to be very efficient biosorbent for cu(ii) ions removal from water. biosorption process was very fast, reaching maximum efficiency (almost 100% of cu(ii) ions was removed from solution) in the first 10 minutes. sorption kinetic was found to be best-fitted pseudo-second order model, which indicates that process is mostly based on chemisorption. biosorption on cclvb is not significantly dependent of initial ph and this material could be used for treatment of acidic wastewater loaded with copper. acknowledgements. authors would like to acknowledge for financial support to the ministry of education and science of the republic of serbia (project tr34008). references antunes, w.m., luna, a.s., henriques, c.a., da costa, a.c.a., 2003: an evaluation of copper biosorption by a brown seaweed under optimized conditiones, electronic journal of biotechnology, 6: 174-184. aziz, a., ouali, m.s., elandaloussi, e.h., de menorval, l.c. lindheimer, m., 2009: chemicaly modified olive stone: a low cost sorbent for heavy metals and basic dyes removal from aqueous solutions, journal of hazardous materials, 163: 441-447. basci, n., kocadagistan, e., kocadagistan, b., 2004: biosorptiion of copper (ii) from aqueous solutions by wheat shell, desalination, 164: 135-140. basso, m.c., gerrela, e.g., cukierman, a.l., 2002: lignocellulosic materials as a potential biosorbents of trace toxic metals from wastewater, industrial and engineering chemistry research, 41: 3580-3585. dang, v.b.h., doan, h.d., dang-vu, t., lohi, a., 2009: equilibrium and kinetics of biosorption of cadmium (ii) and copper (ii) ions by wheat straw, bioresource technology, 100: 211-219. dundar, m., nuhoglu, c., nuhoglu, y. 2008: biosorption of cu(ii) ions onto the litter oo natural trembling poplar forest, journal of hazardous materials, 151: 86-95. fiol, n., villaescusa, i., 2009: determination of sorbent zero charge: usefulnes in sorption studies, environmental chemistry letters, 7: 79-84. yu b., zhang, y., shukla, a., shzkla, s.s., dorris, k.l., 2000: the removal of heavy metal from aqueous solutions by sawdust adsorption-removal of copper, journal of hazardous materials b, 80: 33-42. gercel, o., ozcan, a., ozcan, a.s., gercel, h.f., 2007: preparation of activated carbon from a renewable bioplant of euphorbia rigida by h2so4 activation and its adsorption behaviour in aqueous solutions, applied surface chemistry, 253: 4843-4852. goyal p., sharma p., srivastava s., srivastava m.m., 2008: saraca indica leaf powder for decontamination of pb: removal, recovery, adsorbent characterization and equilibrium modeling. journal of environmental sciences and technology, 5(1): 27-34. ho, y.s., mckay, g., 1999: pseudo-second order model for sorption processes, process biochemistry, 34: 451-465. iftikhar a.r., bhatti h.n., hanif m.a., nadeem r., 2009: kinetic and thermodynamic aspects of cu(ii) and cr(iii) removal fromaqueous solutions using rosewaste biomass, journal of hazardous materials, 161: 941–947. khaled, a., el nemr, a., el-sikaily, a., abdelwahab, o., 2008: removal of direct n blue-106 from artificial textile dye effluent using activated carbon from orange peel, journal of hazardous materials, doi:10.10161.j.hazmat.2008.09.122. krishnani, k., meng, x., christodoulatos, c., boddu, v.m., 2008: biosorption of nine different heavy metals onto biomatrix from rice husk, journal of hazardous materials, 53: 1222-1234 min, s.h., han, j.s., shin, e.w., park, j.k., 2004: improvement of cadmium ion removal by base treatement of juniper fiber, water research, 38: 12891295. mitic-stojanovic, d.l., zarubica, a., purenovic, m., bojic, d., andjelkovic, t., bojic, a., 2011: biosorptive removal of pb2+, cd2+ and zn2+ ions from water by lagenaria vulgaris shell, water sa, 37: 303-312. nuhoglu y., malkoc e., gurses a., canpolat n., 2002: the removal of copper(ii) from aqueous solution by ulothrix zonata, bioresources technology, 85: 331333. ozer, d., dursun, g., ozer, a., 2007: methylen blue adsorption from aqueous solution by dehidrated peanut hull, journal of hazardous materials, 144: 171-179. sud d., mahajan g., kaur m.p., 2008: agricultural waste material as potential adsorbent for sequestering heavy metal ions from aqueous solutions – a review. bioresource technology, 99: 6017-6027. ucun, h., aksakal, o., yildiz, e., 2009: copper (ii) and zinc (ii) biosorption on pinus syvestris l., journal of hazardous materials, 1040-1045. yang, y., chun, y., sheng, g., huang, m., 2004: phdependence of pesticide adsorption by wheat-residuederived black carbon, langmuir, 20: 6736-6741. microsoft word bn020203 milosevic et al biologica nyssana 2 (2) december 2011: 123-128 milošević dj. et al. checklist of the family chironomidae… 123 original article ! checklist of the family chironomidae (diptera) of southern morava river basin, serbia djuradj milošević1, vladica simić2, ivan todosijević1, milica stojković1 1university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia 2university of kragujevac, faculty of sciences and mathematics, institute of biology and ecology, radoja domanovića 14, 34000 kragujevac, serbia * e-mail: djuradj@pmf.ni.ac.rs abstract: milošević, dj., simić, v., todosijević, i., stojković, m.: checklist of the family chironomidae (diptera) of southern morava river basin, serbia. biologica nyssana, 2 (2), december 2011: 123-128. this paper presents the first checklist of the family chironomidae (diptera) in serbia, based on literature citations in two periods (1981/82 and 2010/11) and the unpublished data as well as material examined by the authors. our survey ranges over the central, eastern, southern and southeastern parts of serbia (southern morava river basin). we have listed 110 taxa distributed in following five subfamilies: chironominae (39), diamesinae (3), orthocladiinae (48), prodiamesinae (2) and tanypodynae (16). key words: checklist, chironomidae, serbia, southern morava river introduction ! chironomidae represent the group of flies in the suborder nematocera (diptera). this group is usually the most abundant macroinvertebrate group in number of species and individuals, encountered in the majority of fresh-water aquatic habitats. the fauna of family chironomidae currently consists of 11 described subfamilies and nominally, 22 tribes with 339 genera counting 4147 (sub)species. (f e r r i n g t o n , 2008). the family chironomidae plays the most important role in detritus processing in freshwater ecosystems, recycling natural and introduced organic matter (h i r a b a y a s h i & w o t t o n , 1998; j o n e s & g r e y , 2004). also, they present an important part of the food chain (a r m i t a g e et al., 1995). considering the characteristics mentioned above, chironomidae presents a proper candidate for the water quality assessment. regardless their importance in aquatic ecosystems, little information on qualitative composition of the chironomid fauna in serbia is available at the moment. this is particularly true for lotic systems in central, eastern, southern and southeastern parts of serbia, where research on this topic has not been conducted for 29 years (1981 2010). the first investigation was those of j a n k o v i ć (1985), who sampled across the entire drainage basin of the southern morava river, serbia, in 1981. recently, m i l o š e v i ć et al. (2012) repeated the survey of j a n k o v i ć (1985) over the same range, with the aim of examining the changes in the southern morava river basin. according to the investigations of the authors mentioned above, as well as unpublished data examined by author from the southern morava range, we presented the first chironomid check list of central, eastern, southern and southeastern parts of serbia. the checklist should contribute to the research on this unexplored group of insects in 2 (2) • december 2011: 123-128 biologica nyssana 2 (2) december 2011: 123-128 milošević dj. et al. checklist of the family chironomidae… 124 serbia which have often been proved as useful in the water quality assessment. materials and methods the southern morava river, also known as the binačka morava, has its source in the skopska crna gora mountain, macedonia, and flows roughly towards the north. at km 49 it joins up with the preševska moravica and at km 295 it flows into the morava, which itself eventually flows into the black sea. the southern morava river has a catchment area of 15,469 km2 of which 14,372 km2 (92.91%) are situated in serbia and 1,097 km2 (7.09%) in bulgaria, through this river’s right-hand tributary, the river nišava. the southern morava has 157 tributaries most of which dry out during summer. larger, permanent left-hand tributaries are the jablanica, veternica, toplica and pusta reka rivers. right-hand tributaries are the vlasina, nišava (the longest) and sokobanjska moravica rivers (g a v r i l o v i c & d u k i c , 2002). we studied the chironomid faunal composition once in the each season of 2010 at 28 localities, chosen across the southern morava range to include different stream orders and a variety of habitats (fig. 1). specimens were identified up to the genusor species-level with established keys (s m i t h & d i s t l e r , 1981, w i e d e r h o l m , 1983, p i l l o t , 1984a, 1984b, 2009, v a l l e n d u u k & p i l l o t , 2007). the taxonomy used here is that of s p i e s & s æ t h e r (2004), with adjustments following h. m. p i l l o t (pers. comm.). our survey ranges over the central, eastern, southern and southeastern parts of serbia (m a r k o v i c , 1970). figure 1. sampling localities over the southern morava river. squares indicate localities studied in 1981 and circles indicate localities studied in 2010 biologica nyssana 2 (2) december 2011: 123-128 milošević dj. et al. checklist of the family chironomidae… 125 results we have listed 110 taxa distributed into the following five subfamilies: chironominae (39), diamesinae (3), orthocladiinae (48), prodiamesinae (2) and tanypodynae (16). the review of taxa subfamily chironominae 1. beckidia zabolotzkyi (goetghebuer 1938) site:bob, 2. chernovskiia orbicus (townes 1945) site:bim, sta 3. chironomus sp. site:grd, mez, sta, bob, vet, 2 ,3, 6, 7, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 25, 26, 27 4. cladotanytarsus sp. site: bim, mez, bre, jab, top, 1, 3, 4, 5, 6, 9, 13, 15, 16, 17, 18, 19, 20, 21, 26 5. cryptochironomus sp. site: bim, grd, mez, bre, jab, 1, 2, 3, 5, 6, 7, 9, 10, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 25, 26, 27 6. demicryptochironomus vulneratus (zetterstedt 1838) site: bim, bob, 2, 5, 6, 7, 8, 9, 12, 13, 21 7. dicrotendipes nervosus (staeger 1839) site: 10, 15, 16, 17, 19, 20, 23, 26 8. dicrotendipes notatus (meigen 1818) site: 23 9. endochironomus albipennis (meigen 1830) site: 14, 26 10. endochironomus dispar (meigen, 1830) site: 12 11. glyptotendipes cauliginellus (kieffer 1913) site: vlh, 13 12. harnischia fuscimanus kieffer 1921 site: bob, 1, 10, 13, 18, 19, 21, 27 13. microchironomus tener (kieffer, 1918) site: 19 14. micropsectra bidentata goetghebuer 1921 site: 4, 5, 6, 11, 12, 14, 16, 22, 23, 28 15. micropsectra sp. site: put, 4, 5, 6, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, 22, 23, 25, 27 16. microtendipes pedellus (de geer 1776) site:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 13, 14, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 27 17. parachironomus frequens (johannsen 1905) site: 4 18. paracladopelma laminatum (kieffer 1921) site: 1, 2, 3, 4, 5, 6, 8, 9, 10, 13, 14, 19, 20, 21, 22, 23, 25, 26, 27, 28 19. paracladopelma nigritulum (goetghebuer 1942) site: mpv, 10, 12, 14, 17, 18 20. paralauterborniella nigrohalteralis (malloch 1915) site: 1, 2, 16, 17 21. paratanytarsus austriacus (kieffer 1924) site: 19 22. paratanytarsus dissimilis (johannsen 1905) site: bre, put, vet, 1, 10, 13, 16, 17, 18, 20, 25, 26, 27 23. paratendipes albimanus (meigen 1818) site: grd, 1, 2, 4, 5, 6, 7, 8, 9, 11, 13, 14, 16, 17, 18, 19, 20, 21, 22, 23, 25, 26, 27, 28 24. phaenopsectra sp. site: grd, 10, 11, 12, 13, 14, 16, 17, 19, 20, 21, 25, 26, 27 25. polypedilum albicorne (meigen 1838) site: 1, 4, 7, 12, 13, 14, 17, 18, 19, 21, 22, 23, 24, 25, 27, 28 26. polypedilum convictum (walker 1856) site: mez, bre, sta, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 27. polypedilum cultellatum goetghebuer 1931 site: 13, 25 28. polypedilum laetum (meigen 1818) site: miv, 1, 2, 3, 4, 5, 7, 8, 9, 10, 12, 13, 14, 16, 19, 20, 21, 25, 27, 28 29. polypedilum pedestre (meigen 1830) site: grd, mez, bre, 3, 7, 11, 12, 13, 17, 18, 19, 20, 23, 25, 27, 28 30. polypedilum scalaenum (schrank 1803) site: bim, miv, mpv, grd, mez, bre, sta, bob, 1, 2, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 biologica nyssana 2 (2) december 2011: 123-128 milošević dj. et al. checklist of the family chironomidae… 126 31. polypedilum uncinatum goetghebuer 1921 site: 5, 12 32. pseudochironomus prasinatus (staeger 1839) site: sta 33. rheotanytarsus sp. site: jab, sta, 1, 2, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 16, 17, 18, 19, 20, 21, 22, 23, 25, 26, 27 34. saetheria reissi jackson 1977 site: 2, 6, 10, 13, 17, 18, 19, 20, 21 35. saetheria sp. site: mpv, bre, jab, 1 36. stempellinella brevis (edwards 1929) site: 11, 12, 28 37. stictochironomus maculipennis (meigen 1818) site: 5, 6, 10, 11, 12, 26, 28 38. stictochironomus pictulus (meigen 1830) site: 1, 4, 7, 14, 16, 17, 18, 20, 21, 22, 25 39. tanytarsus sp. site: grd, mez, bre, jab, sta, bob, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 subfamily diamesinae 40. diamesa sp. site: miv, 1, 2, 4, 8, 9, 13, 24, 28 41. potthastia gaedii (meigen 1838) site: 1, 2, 3, 4, 5, 6, 7, 14, 20, 22, 23, 25, 27 42. potthastia longimanus kieffer 1922 site: 1, 3, 4, 5, 6, 7, 9, 12, 13, 14, 16, 17, 18, 19, 20, 21, 22, 23, 25, 26, 27, 28 subfamily orthocladiinae 43. brillia flavifrons (johannsen 1905) site: 9, 10, 13, 17, 18, 21, 23, 27 44. brillia bifida (kieffer 1909) site: 1, 2, 4, 5, 6, 11, 12, 14, 16, 19, 24, 26, 28 45. bryophaenocladius subvernalis (edwards 1929) site: grd 46. cardiocladius fuscus kieffer 1924 site: 3, 8, 13, 18, 19, 21, 27 47. corynoneura celeripes winnertz 1852 site: grd, mez 48. corynoneura lobata edwards 1924 site: 2, 4, 5, 6, 7, 12, 14, 28 49. corynoneura scutellata winnertz 1846 site: grd, mez, 27 50. cricotopus triannulatus agg. sensu moller pillot 1984 site: miv, vlh, grd, mez, bre, sta, vet, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 17, 18, 19, 21, 22, 23, 25, 26, 27, 28 51. cricotopus bicinctus (meigen 1818) site:vlh, grd, mez, bre, jab, sta, bob, vet, top, 1, 5, 6, 8, 9, 10, 13, 14, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 52. cricotopus gr. sylvestris sensu hirvenoja 1973 site: bre, 10 53. cricotopus tremulus (linnaeus 1758) site: vlh, bre, jab, vet, 3, 4, 13, 24, 25 54. cricotopus trifascia edwards 1929 site: mez, jab, 3, 8, 13, 14, 17, 20, 21, 24, 25, 27 55. epoicocladius ephemerae (kieffer 1924) site: 1, 2, 3, 4, 5, 6, 7, 9, 11, 12, 14, 16, 28 56. eukiefferiella brevicalcar (kieffer 1911) site: mor, 4, 5, 6, 8, 12, 20, 28 57. eukiefferiella claripennis (lundbeck 1898) site: bre, 22 58. eukiefferiella clypeata (kieffer 1923) site: 4, 5, 8, 9, 10, 12, 14, 18, 19, 26, 27, 28 59. eukiefferiella gracei (edwards 1929) site: miv, 1, 2, 3, 6, 7, 8, 10, 11, 13, 14, 17, 18, 19, 25, 27 60. eukiefferiella ilkleyensis (edwards 1929) site: 1, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 17, 18, 19, 20, 21, 23, 24, 25, 26, 27 61. eukiefferiella lobifera goetghebuer 1934 site: vlh, bre, 2, 13, 17, 20, 21, 23, 25, 26, 27 62. eukiefferiella minor (edwards 1929) site: 4 63. eukiefferiella sp. site: miv 64. heleniella ornaticollis (edwards 1929) site: 3, 4, 5, 6, 12, 28 biologica nyssana 2 (2) december 2011: 123-128 milošević dj. et al. checklist of the family chironomidae… 127 65. hydrobaenus sp. site: mor, miv, bre, jab 66. limnophyes sp. site: grd, 2, 4, 5, 8, 13, 14, 16, 19, 27 67. nanocladius bicolor (zetterstedt 1838) site: 6, 7, 16, 26 68. nanocladius rectinervis (kieffer 1911) site: mez, bre, jab, top, 4, 5, 8, 10, 13, 14, 17, 18, 20, 24, 25, 26, 28 69. orthocladius (euorthocladius) sp. site: miv, vlh, mez, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 70. orthocladius (orthocladius) sp. site: miv, bre, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 71. orthocladius frigidus (zetterstedt 1838) site: 2, 3, 4, 5, 6, 8, 11, 12, 13, 14 72. orthocladius rivulorum kieffer 1909 site: 2, 3, 4, 5, 6, 7, 8, 13, 14, 16 73. paracladius conversus (walker 1856) site: 1, 2, 3, 6, 9, 16, 18, 19, 21, 25, 27 74. paracricotopus niger (kieffer 1913) site: 4, 22 75. parakiefferiella gracillima (kieffer 1922) site: mor 76. parametriocnemus stylatus (spaerck 1923) site: 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, 26, 27, 28 77. paratrichocladius rufiventris (meigen 1830) site: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 78. paratrissocladius excerptus (walker 1856) site: 2, 3, 6, 7, 11, 12, 14, 16, 23, 28 79. psectrocladius calcaratus (edwards 1929) site: mez, 1, 6 80. pseudosmittia danconai (marcuzzi 1947) site: vlh, top 81. rheocricotopus chalybeatus (edwards 1929) site: 1, 8, 10, 13, 15, 16, 17, 18, 19, 20, 21, 23, 25, 26, 27 82. rheocricotopus effusus (walker 1856) site: jab, 2, 4, 5, 6, 12, 23, 28 83. rheocricotopus fuscipes (kieffer 1909) site: 3, 4, 5, 6, 7, 9, 11, 12, 14, 22, 24, 28 84. rheosmittia spinicornis (brundin 1956) site: 5, 11, 28 85. smittia sp. site: bre, 20 86. synorthocladius semivirens (kieffer 1909) site: mor, 1, 2, 4, 5, 6, 7, 8, 9, 12, 13, 14, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 27 87. thienemanniella clavicornis (kieffer 1911) site: 2, 3, 4, 5, 6, 9, 11, 12, 14, 16, 20, 21, 24, 25, 27, 28 88. thienemanniella majuscula (edwards 1924) site: jab, 1, 3, 8, 10, 13, 17, 18, 19, 21, 22, 23, 26 89. tvetenia bavarica (goetghebuer 1934) site: 4, 5, 6, 7, 11, 12, 14, 22, 28 90. tvetenia calvescens (edwards 1929) site: 1, 2, 3, 8, 9, 10, 13, 16, 17, 18, 19, 20, 21, 23, 24, 25, 27 91. tvetenia discoloripes (goetghebuer & thienemann 1936) site: 2, 3, 4, 5, 6, 10, 11, 12, 17, 19, 23, 25, 28 subfamily prodiamesinae 92. odontomesa fulva (kieffer 1919) site: 2, 3, 4, 6, 7, 11, 12 93. prodiamesa olivacea (meigen 1818) site: grd, 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15 ,16 ,17 ,18 ,19 , 20, 21, 22, 23, 24, 25, 27, 28 subfamily tanypodinae 94. ablabesmyia longistyla fittkau 1962 site: mez, 10, 16, 20 95. ablabesmyia phatta (egger 1864) site: 18 96. anatopynia plumipes (fries 1823) site: 4 97. apsectrotanypus trifascipennis (zetterstedt 1838) site: 4, 6, 11, 14, 23 biologica nyssana 2 (2) december 2011: 123-128 milošević dj. et al. checklist of the family chironomidae… 128 98. arctopelopia barbitarsis (zetterstedt 1850) site: 5, 12 99. conchapelopia melanops (meigen 1818) site: mez, jab, sta, put, 1, 2, 3, 5, 6, 7, 9, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, 24, 25, 26, 27 100. krenopelopia sp. site: 1, 4 101. larsia curticalcar (kieffer 1918) site: mez, bre, jab 102. macropelopia adaucta kieffer 1916 site: 6, 22, 23 103. macropelopia nebulosa (meigen 1804) site: 2, 3, 4, 5, 6, 7, 8, 10, 12, 14, 15, 18, 23, 27, 28 104. nilotanypus dubius (meigen 1804) site: 1, 2, 4, 5, 6, 7, 8, 9, 14, 16, 22, 24, 25, 28 105. procladius sp. site: grd, 2, 3, 4, 6, 7, 10, 12, 13, 14, 16, 17, 18, 19, 20, 21, 22, 23, 25, 26, 27 106. rheopelopia sp. site: 1, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 107. tanypus punctipennis meigen 1818 site: grd, sta, bob, 19, 27 108. thienemannimyia sp. site: mez, jab, sta, put, 4, 5, 6, 12, 22, 28 109. trissopelopia flavida kieffer 1923 site: mez, sta 110. zavrelimyia sp. site: 7 acknowledgements. this work was supported by grant # 043002 ‘biosensing technologies and global system for long-term research and integrated management of ecosystems’ by the ministry of science of the republic of serbia. we want to thank h. m. pillot (tilburg, the netherlands) for the great support and useful advice about this work. references armitage, p. d., cranston, p., & pinder, l., 1995: the chironomidae: biology and ecology of nonbiting midges. chapman & hall, london. ferrington, l. c. 2008: global diversity of nonbiting midges (chironomidae; insecta-diptera) in freshwater. hydrobiologia 595: 447-455 gavrilovic, l. & dukic, d. 2002: reke srbije. zavod za udžbenike i nastavna sredstva, beograd. hirabayashi, k. & wotton, r. s. 1998: organic matter processing by chironomid larvae (diptera: chironomidae). hydrobiologia 382: 151-159. jankovic, m. 1985: the investigation of the chironomid fauna in the south morava and its components rivers. proceedings on the fauna of sr serbia 3: 71-110. jones, r. i. & grey, j. 2004: stable isotope analysis of chironomid larvae from some finnish forest lakes indicates dietary contribution from biogenic methane. boreal environment research 9: 17-24. markovic, j., 1970: geografske oblasti socijalisticke federativne republike jugoslavije. – zavod za izdavanje udžbenika i nastavna sredstva srbije, beograd. milošević, dj., simić, v., stojković, m., živić, i., 2012: chironomid faunal composition represented by taxonomic distinctness index reveals environmental change in a lotic system over three decades. hydrobiologia 683: 62-82 pillot, h. k. m. m. 1984a: de larven der nederlandse chironomiae (diptera). 1a: inleiding, tanypodinae en chironomini. st. e.i.s. nederland, leiden. pillot, h. k. m. m. 1984b: de larven der nederlandse chironomiae (diptera). 1b: orthocladiinae sensu lato. st.e.i.s nederland, leiden. pillot, h. k. m. m. 2009: chironomidae larvae of the netherlands and adjacent lowlands: biology and ecology of the chironomini. knnv publishing, zeist. smith, d. l. & distler, d. a. 1981: recovery of a benthic macroinvertebrate community following a toxic chemical discharge in a sandy plains stream. the southwestern naturalist 25: 547-551. spies, m. & sæther, o. a. 2004: notes and recommendations on taxonomy and nomenclature of chironomidae (diptera). zootaxa 752: 1-90. vallenduuk, h. j. & pillot, h. k. m. m. 2007: chironomidae larvae of the netherlands and adjacent lowlands: general ecology and tanypodinae. knnv publishing, zeist. wiederholm, t. 1983: chironomidae of the holarctic region: keys and diagnoses. publishing house of the swedish research councils, stockholm. microsoft word 0301_calic-dragosavac_radojevic biologica nyssana 1 (1-2) december 2010: 49-55 ćalić-dragosavac, d., radojević, lj. improvement of maturation… 49 original article ! improvement of maturation and conversion of horse chestnut androgenic embryos dušica ćalić-dragosavac*, ljiljana radojević department of plant physiology, institute for biological research, "siniša stanković'', bulevar despota stefana 142, belgrade, serbia * e-mail: calic@ibiss.bg.ac.rs abstract: ćalić-dragosavac, d., radojević, lj.: improvement of maturation and conversion of horse chestnut androgenic embryos. biologica nyssana, 1 (1-2), december 2010: 49-55. horse chestnut (aesculus hippocastanum l., hippocastanaceae) is a relict species of the tertiary flora and endemit of balkan peninsula. it has enormous horticultular and medical important. horse chestnut trees are native to the balkan peninsula, but grow as ornamental trees in parks and avenues throughout the northern hemisphere. because of the slow and difficult reproduction of great importance to be fast and cheap in vitro multiplication. possible solution is regenerated by androgenesis. microspore culture has been used in recent years as a tool for producing haploid plants in a varyety of higher plants, but the low frequencies of microspore-derived plants restrict the use of the technique in plant breeding. key words: aesculus hippocastanum, anther culture, maturation, ploidy level, suspension culture introduction ! horse chestnut showed slow and difficult reproduction in nature conditions. possible solution of this problem is regeneration by androgenesis in vitro. androgenesis in vitro has been used in recent years as a tool for producing haploid plants in a varyety of higher plants, but the low frequencies of microspore-derived plants restrict the use of the technique in plant breeding. regeneration in the genus aesculus via androgenesis has been demonstrated for a. hippocastanum (r a d o j e v i ć , 1978; ć a l i ć et al., 2003), a. carnea (r a d o j e v i ć et al., 1989; m a r i n k o v i ć & r a d o j e v i ć , 1992) and a. flava (ćalić et al., 2005; ćalić-dragosavac et al., 2010). androgenesis provides a large number of embryos at defined stages of development, and allows alterations of the embryonic environment through manipulations of culture conditions. regeneration of this species via androgenesis provides a means of propagation and a model system for conducting physiological and biochemical studies. although such embryos may appear “morphologically” mature, they do not perform well during postembryogenic growth without the imposition of a drying period. improvement of embryo quality can be achieved through the application of osmotic stress, which is an important factor for directing embryo development and maturation both in vivo and in vitro (c a p u a n a & d e b e r g , 1997; t r o c h et al., 2009). generation of horse chestnut somatic embryos is commonly achieved by transferring embryogenic tissue onto an activated charcoal (ac), abscisic acid (aba), polyethylene glycol (peg) and manitol-containing maturation media (c a p u a n a & d e b e r g , 1997). 10th sfses • 17-20 june 2010, vlasina lake1 (1-2) • december 2010: 49-55 biologica nyssana 1 (1-2) december 2010: 49-55 ćalić-dragosavac, d., radojević, lj. improvement of maturation… 50 activated charcoal is commonly used in tissue culture media to darken the immediate media surroundings and to absorb inhibitory or toxic substances and plant growth regulators (m o s h k o v et al., 2008). charcoal has been used in all stages of somatic embryogenesis to increase initiation frequencies of pinus taeda l. (p u l l m a n & j o n s o n , 2002), induce embryogenic tissue on vegetative shoot apices of mature trees of pinus patula (m a l a b a d i & v a n s t a d e n , 2005), improve yield and quality of somatic embryos during maturation (c a p u a n a & d e b e r g , 1997; c a r a w a y & m e r k l e , 1997; l i et al., 1997, 1998; g r o l l et al., 2002; p u l l m a n et al., 2005; l e l u w a l t e r et al., 2006), and most frequently during germination (v o o k o v á & k o r m u t ’ á k , 2001; s a l a j et al., 2004; a n d r a d e & m e r k l e , 2005). aba is commonly used at various concentrations to improve somatic embryo development of abies species prior to plantlet regeneration (s a l a j o v á et al., 1996; v o o k o v á & k o r m u t ’ á k , 2001). abscisic acid has been found to stimulate the production of cotyledonary embryos and regulate the course of embryo maturation (b e c w a r et al., 1987; b o u l a y et al., 1988; d u n s t a n et al., 1988; v o n a r n o l d & h a k m a n 1988; r o b e r t s et al., 1990a). the ability of aba to inhibit precocious germination has facilitated the development of procedures for mass propagation of spuce emblings (r o b e r t s et al., 1990b; w e b s t e r et al., 1990). osmotic treatments have been used to influence embryo maturation in many gymnosperms and angiosperms (c a p u a n a & d e b e r g h , 1997). aba induction of desiccation tolerance may be linked to accumulation of late embryogenesis – abundant proteins that may protect tissues (d u r e et al., 1989). the effect of peg mimics the naturally occurring water stress on seeds during the late stages of maturation. water stress caused by peg and increased concentrations of aba are essential for somatic embryo development to accumulation of storage compounds and inhibit precocious germination (s t r a s o l l a & y e u n g , 2003). peg enhanced somatic embryo maturation in several species, including picea glauca (a t t r e e et al., 1990, 1991, 1992, 1995), abies numidica (v o o k o v á & k o r m u t ’ á k , 2002) and abies hybrids (s a l a j & s a l a j , 2003; s a l a j et al., 2004). the combined application of aba and peg, a non-plasmolyzing osmoticum, has become a routine method for stimulating embryo maturation (a t t r e e & f o w k e , 1993). the aim of this research was to study influence of activated charcoal (ac), abscisic acid (aba) and polyethylene glycol (peg) on the maturation and conversion of horse chestnut microspore-derived embryo for the diversity protection and conservation. material and methods ! closed flower buds (4-5 mm long) used in the experiments were obtained from 100 years old aesculus hippocastanum l. tree growing in the botanical garden "jevremovac" of the belgrade university. the buds were surface sterilized with 95 % and 70 % ethanol. basal medium (bm) contained murashige and skoog (1962; ms) mineral salts, 2 % sucrose and was supplemented with the following (mg l-1): panthotehenic acid (10), nicotinic acid (5), vitamin b1 (2), adenine sulphate (2), myo-inositol (100) and casein-hydrolysate (200). uninuclear microspores culture was established on induction ms liquid (l) medium. induction l medium contained bm with 2,4-d dichlorphenoxyacetic acid (2,4-d) and kinetin (kin), 1.0 mg l-1 of each. about 100 anthers with uninucleate microspores per erlenmeyer flask with filter (50 μm) and 100 cm3 l medium for androgenic induction were cultivated. isolation and culture of microspores the anthers were macerated in a glass petri dish with scalpel through a 50-μm metal sieve in l induction medium. the microspores collected on the surface of the 50-μm sieve were carefully washed with same medium. the microspore suspension were subcultured every 30 days and refreshed with liquid l medium. after 2 months, a microspore suspension was plated by bergmann technique (1960) on a solid medium with reduced concentration of 2,4-d (0.01 mg l-1) and same concentration of kin. after medium for multiplication embryos were cultured on media for embryo maturation supplemented with ac, aba and peg 4000. various concentrations (0.1; 0.5 and 1%) of activated charcoal (ac) were tested. aba (2.5; 10.0 and 25.0 mg l-1) alone, as well as in combination with 1 % ac was investigated. also, influence of peg (5, 25 and 50 g l-1) and combinations peg (5 and 50 g l-1) with ac (1 g l-1) on maturation of androgenic embryos were studied. we used thirteen different maturation media which are presented in figures 1-4. subculturing was done every one month and the maturation phase took 3 months. cultures were monitored at the time of sub-culturing and the mature embryos were biologica nyssana 1 (1-2) december 2010: 49-55 ćalić-dragosavac, d., radojević, lj. improvement of maturation… 51 transferred to hormone and supplement free germination medium. all media were sterilized by autoclaving at 0.9 x 105 kpa and 1140c for 25 min. androgenic embryos were grown at 25 ± 1 0c and a 16-h photoperiod with irradiance of 33 45 μmol m-2s-1 produced by cool white fluorescent tubes. embryos on maturation media were investigated after 3 months. three characteristics were evaluated: 1) shoot elongation; 2) radicle development and 3) conversion into the plants. determination of ploidy level nuclear suspension from androgenic embryos in the cotyledonary stage of development were prepared. young leaf material of horse chestnut was used as control. plant material was macerated with a sharp razor blade in a ice-cold neutral buffer, and placed in plastic petri dishes. neutral dna buffer (ph 7) with 15 mm hepes, 1 mm edta, 80 mm kcl, 20 mm nacl, 0,5 mm spermine, 300 mm sucrose, 0.2 % triton x-100, 15 mm dte (dithiothreitol) and 2 mg l-1 dapi was used (modified by de laat and blaas, 1984). after maceration, the buffered mixture (ca. 2 ml), was passed through a nylon filter of 30 μm mesh size, stained with dapi, and analysed in a flow cytometer. fluorescence levels were determined by a photomultiplier and coverted in voltage pulses that were processed with pc. ploidy level of androgenic embryos was evaluated by flow citometry, using a pas ii cytometer (partec gmbh), equipped with a high pressure mercury lamp (osram hbo 100 w/2) and using the excitation filters ug-1, bg-31, kg-1 and tk-420 and emission filters tk560 and gg435. statistics and repetition sixty androgenic embryos per medium were analysed. three repetitions were performed per each medium. the total number of pollen grains analyzed for each studied medium was 180. the results were assessed using the variation analysis. results were tested according snk (student newman keuls) test (significance level α= 0.05) for determination statistical significant differencesis among treatments. results androgenesis of horse chestnut micospores was induced in liquid and solid ms (murashige and skoog, 1962) medium with 2,4-d acid and kin (1.0 mg l-1 of each). light and scanning microscopy confirmed that androgenic embryos of horse chestnut grown in suspension formed by direct division of microspores. after transfer to basal medium (bm), androgenic embryos showed asynchronous development. great numbers of these embryos were irregular, showed hypertrophy and had abnormal cotyledons or lacked a hypocotyl. percentage germination of androgenic embryos was followed after 3 months of growing on different maturation media. the best results of germination of horse chestnut androgenic embryos which maturated on media supplemented with ac (99%), alone, and in combination with peg (100%) were observed in microspore culture (fig. 1 and 4). with increasing concentration of ac increased the percentage of embryo germination from 92 to 99% after 3 months in suspension culture. root shoot w hole plants control ac 1 g/l ac 5g/l ac 10g/l 0 20 40 60 80 100 % control ac 1 g/l ac 5g/l ac 10g/l fig. 1. effect of ac on maturation microsporederived embryos, after three months root shoot w hole plants control aba 2.5 g/l aba 10 g/l aba 20g/l 0 10 20 30 40 50 60 70 % control aba 2.5 g/l aba 10 g/l aba 20g/l figure 2. effect of aba on microspore-derived embryos, after three months also, the greatest number of androgenic plants originating from microspore (18 %) culture was formed on the medium with supplemented with biologica nyssana 1 (1-2) december 2010: 49-55 ćalić-dragosavac, d., radojević, lj. improvement of maturation… 52 1 % ac (fig. 1). also, cultures on media containing 1 % ac showed better maturation, germination; shoot elongation and conversion into the plants (fig. 1). however, the number of germinated embryos with increasing concentration of aba (from 2.5 to 20 mg l-1) in media was decreased (fig. 2). lowest germination percentages, 37 % and 39 % in microspore culture were obtained on maturation media with aba 20 mg l-1 alone and in combination with ac 1g l-1. root shoot w hole plants control peg 5 g/l peg 25 g/l peg 50 g/l 0 10 20 30 40 50 60 70 80 90 % control peg 5 g/l peg 25 g/l peg 50 g/l figure 3. effect of peg on microspore-derived embryos, after three months root shoot w hole plants control peg 5 g/l +ac 1g/l peg 50 g/ + ac 1g/l 0 20 40 60 80 100 % control peg 5 g/l +ac 1g/l peg 50 g/ + ac 1g/l figure 4. effect of peg and ac on maturation microspore-derived embryos, after three months aba in concentration of 2.5 mg l-1 had a positive effect on shoot (23 %) formation. peg in maturation media improved androgenic embryo germination and conversion into the plants. androgenic embryos on media containing peg (50 g l-1), in combination with ac (1 g l-1) showed rapid development of cotyledonary stage embryos and lowered percentage of abnormal structures. increasing peg from 5 to 50 g l-1 in the maturation media improved germination of androgenic embryos originating from microspore culture from 62 to 87 %. concentration of ac 1 g l-1 in maturation medium with 5 and 50 g l-1 of peg had a further beneficial effect on germination of embryos derived from microspore (79-100 %) culture. the addition of ac and peg to the maturation medium significantly increased the percentage of androgenic embryos forming shoots and increased the conversion frequencies in microspore (5-13 %; fig. 4) culture. cytogenetic analysis of androgenic embryos originating from anther and microspore culture was done after a first generation of regenerants (after 3 months) and after 3 years of subculturing. all androgenic embryos after 3 months of maturation treatments from microspore culture were haploid. however, only 10 % androgenic microspore embryos retained haploidity, while 10.5 % were diploid, 73.5 % tetraploid and 6 % octaploid (fig. 5) after 3 years of subculturing. discussion some of the horse chestnut androgenic embryos were irregular, hypertrophic and had abnormal cotyledons or lacked hypocotyls. this phenomenon is frequently observed in somatic embryos of trees (perez et al., 1986; capuana and deberg, 1997). therefore, addition of different concentrations of ac, aba or peg in media with horse chestnut androgenic embryos has overcome this problem. our results about influence of activated charcoal on the in vitro androgenesis in correlated with the results of özkum çiner and tipirdamaz (2002). the effect of activated charcoal has been attributed to the absorption of inhibitory substances (abscisic acid, phenolics) from the medium (thomas, 2008). in the current study, the best results of germination, maturation and conversion androgenic embryos into the plants were achieved on medium with 1 % activated charcoal which is in accordance with the results pullman et al. (2005). also, aba and water stress may keep an embryo in a maturation state by encouraging development and preventing germination. abscisic acid has been found to stimulate the production of cotyledonary embryos with normal morphology and to regulate the course of horse chestnut somatic embryos (capuana and deberg, 1997; troch et al., 2009) as well as american chestnut (robichaud et al., 2004). biologica nyssana 1 (1-2) december 2010: 49-55 ćalić-dragosavac, d., radojević, lj. improvement of maturation… 53 significant accumulation of biomass and storage products in the cotyledons of chestnut embryos may not be critical for germination under in vitro conditions. in fact, capuana and deberg (1997) and troch et al. (2009) noted that the highest conversion frequencies were obtained with embryos possessing thin cotyledons, while embryos bearing large, thick cotyledons, resembling those of mature zygotic embryos, did not regenerate plants all. aba also promotes the development of globular embryos in embryogenic cultures of horse chestnut as well as in spruce (von arnold and hakman, 1988). peg-derived horse chestnut embryos were also less aberrant than control embryos as well as somatic horse chestnut embryos (troch et al., 2009) and picea glauca embryos (attree et al., 1991). our results that peg increased germination of horse chestnut androgenic embryos are in correlation with results attree et al. (1992) and capuana and deberg (1997). attree et al. (1995) published that drying was essential for subsequent normal growth of picea glauca somatic embryos following maturation on peg. the ploidy level of regenerated embryos and plantlets at different growth stages was determined using flow cytometry and chromosome counts. diploid, tetraploid and octaploid plants were present among the horse chestnut regenerants. this could be a result of spontaneous chromosome doubling occurring during androgenesis. these results suggested that ploidy level significantly increased during long-term cultures (geier, 1991). in contrary of androgenic embryos, somatic embryos of horse chestnut did not show major genetic changes (troch et al., 2009). however, further experiments by this author are necessary to determine whether horse chestnut somatic embryos are genetically stable during long-term cultures. conclusion to the best of our knowledge, this is the first report about improvement of maturation and conversion of horse chestnut androgenic embryo for the diversity protection and conservation. acknowledgement this work was supported by the ministry of science and technological development, serbia, grant n0. 143026. references andrade, g.m., merkle, s.a. 2005: enhancement of american chestnut somatic seedling production. plant cell reports, 24: 326–334. attree, s.m., tautorus, t.e., dunstan, d.j., fowke, l.c. 1990: somatic embryo maturation, germination, and soil establishment of plants of black and white spruce (picea mariana and picea glauca). canadian journal of botany, 68: 2583-2589. attree, s.m., moore, d., sawhney, v.k., fowke, l.c. 1991: enhanced maturation and desiccation figure 5. flow cytometry histograms of haploid (a), diploid (b), tetraploid (c) and octaploid (d) androgenic embryos biologica nyssana 1 (1-2) december 2010: 49-55 ćalić-dragosavac, d., radojević, lj. improvement of maturation… 54 tolerance of white spruce (picea glauca (moench) voss) somatic embryos: effects of a non-plasmolyzing water stress and abscisic acid. annals of botany, 68: 519-525. attree, s.m., pomeroy, m.k., fowke, l.c. 1992: manipulation of conditions for the culture of somatic embryos of white spruce for improved triacylglycerol biosynthesis and desiccation tolerance. planta, 187: 395-404. attree, s.m., fowke, l.c. 1993: embryogeny of gymnosperms: advances in synthetic seed technology of conifers. plant cell tissue and organ culture, 35: 1–35. attree, s.m., pomeroy, m.k., fowke, l.c. 1995: development of white spruce (picea glauca (moench) somatic embryos during culture with abscisic acid and osmoticum, and their tolerance to drying and frozen storage. journal of experimental botany, 46: 433-439. becwar, m.r., noland, t.l., wann, s.r. 1987: a method for quantification of the level of somatic embryogenesis among norway spruce callus lines. plant cell reports, 6: 35-38. bergmann, l. 1960: growth and division of single cell of higher plants in vitro. journal of genetic and physiology, 43: 841-851. boulay, m.p., gupta, p.k., krogstrup, p., durzan, d.j. 1988: development of somatic embryos from cell suspension cultures of norway spruce (picea abies karst). plant cell reports, 7: 134137. capuana, m., debergh, p.c. 1997: improvement of the maturation and germination of horse chestnut somatic embryos. plant cell tissue and organ culture, 48: 23-29. ćalić, d., zdravković-korać, s., jevremović, s., guć-šćekić, m., radojević, lj. 2003: efficient haploid induction in microspore suspension culture of aesculus hippocastanum and karyotype analysis. biologia plantarum, 47: 289292. ćalić, d., zdravković-korać s., radojević, lj. 2005: plant regeneration in anther culture of yellow buck (aesculus flava marshall.). in: proc. cost 843, ”quality enhancement of plant production through tissue culture”, stara lesna, slovakia, pp. 183-185. ćalic-dragosavac, d., zdravković-korać, s., devrnja n., radojević lj., vinterhalter, b. 2010: maturation of aesculus flava (marshall) androgenic embryos. in: proc. international scientific conference forest ecosystems and climate changes. belgrade, pp. 55-59. carraway, d.t., merkle, s.a. 1997: plantlet regeneration from somatic embryos of american chestnut. canadian journal of forest research, 27: 1805–1812. de laat, a.m.m., blaas, j 1984: flow-cytometric characterization and sorting of plant chromosomes. theor. appl. genet. 67: 463-467. dunstan, d.i., bekkaoui, f., pilon, m., fowke, l.c., abrams, s.r. 1988: effects of abscisic acid and analogues on the maturation of white spruce (picea glauca) somatic embryos. plant science, 58: 77-84. dure, i.i.l., crouch, m., harada, j., ho, t.h.d., mundy, j., quatrano, r., thomas, t., sung, z.r. 1989: common amino acid sequence domains among the lea proteins of higher plants. plant molecular biology, 12: 475-486. geier, t. 1991: chromosome variability in callus produced plants. in: harding j, singh f, mol jnm (eds), 79–106: genetics and breeding of ornamental species. kluwer, the netherlands. groll, j., gray, v.m., mycock, d.j. 2002: development of cassava (manihot esculenta crantz.) somatic embryos during culture with abscisic acid and activated charcoal. journal of plant physiology, 159: 437–443. lelu-walter, m.a., bernier-cardou, m., klimaszewska, k. 2006: simplified and improved somatic embryogenesis for clonal propagation of pinus pinaster (ait.). plant cell reports, 25: 767–776. li, x.y., huang, f.h., gbur, e.e. 1997: polyethylene glycol promoted development of somatic embryos in loblolly pine (pinus taeda l.). in vitro cell development biology-plant, 33: 184–189. li, x.y., huang, f.h., murphy, j.b., gbur, e.e. 1998: polyethylene glycol and maltose enhance somatic embryo maturation in loblolly pine (pinus taeda l.). in vitro cell development biology-plant,, 34:22–26. malabadi, r.b., van staden, j. 2005: somatic embryogenesis from vegetative shoot apices of mature trees of pinus patula. tree physiology, 25: 11–16. marinković, n., radojević, lj. 1992: the influence of bud length, age of the tree and culture media on androgenesis induction in aesculus carnea hayne anther culture. plant cell tissue and organ culture, 31: 51-59. moshkov, i.e., novikova, g.v., hall, m.a., george, e.f. 2008: plant growth regulators iii: gibberellins, ethylene, abscisic acid, their analogues and inhibitors; miscellaneous compounds. in: george ef, hall ma, de klerk gj (eds). plant propagation by tissue culture, 3rd edn, 1: 227–282, springer, dordrecht, the netherlands. biologica nyssana 1 (1-2) december 2010: 49-55 ćalić-dragosavac, d., radojević, lj. improvement of maturation… 55 murashige, t., skoog, f. 1962: a revised medium for rapid growth and bioassays with tobacco tissue cultures. physiologia plantarum, 15: 473497. özkum çiner, d., tipirdamaz, r. 2002: the effect of cold treatment and charcoal on the in vitro androgenesis of pepper (capsicum annum l.). turkish journal of botany, 26: 131-139. pérez, c., rodriquez, a., rodriquez, r., tamés, s. 1986: regulation of asexual embryogenesis in filbert cotyledonary nodes. morphological variability. plant science, 45: 59-64. pullman, g.s., johnson, s. 2002: somatic embryogenesis in loblolly pine (pinus taeda l.): improving culture initiation rates. annals of forest science, 59: 663–668. pullman, g.s., gupta, p.k., timmis, r., carpenter, c., kreitinger, m., welty, e. 2005: improved norway spuce somatic embryo development throught the use of abscisic acid combined with activated carbon. plant cell reports, 24: 271279. radojević, lj. 1978: in vitro induction androgenic plantlets in aesculus hippocastanum l. protoplasma, 96: 369-374. radojević, lj., đorđević, n., tucić, b. 1989: in vitro induction of pollen embryos and plantlets in aesculus carnea hayne through anther culture. plant cell tissue and organ culture, 17: 21-26. robichaud, r.l., lessard, v.c., merkle, s.a. 2004: treatments affecting maturation and germination of american chestnut somatic embryos. journal of plant physiology, 161: 957-969. roberts, d.r., flinn, b.s., webb, d.t., webster, f.b., sutton, b.c.s. 1990a: abscisic acid and indole -3-butyric acid regulation of maturation and accumulation of storage proteins in somatic embryos of interior spruce. physiology plantarum, 78: 355-360. roberts, d.r., sutton, b.c.s., flinn, b.s. 1990b: synchronous and high frequency germination of interior spruce somatic embryos following partial drying at high relative humidity. canadian journal of botany, 60: 1086-1090. salaj, t., salaj, j. 2003: somatic embryo formation on mature abies alba x abies cephalonica zygotic embryo explants. biologia plantarum, 47: 7-11. salaj, t., matúšová, r., salaj, j. 2004: the effect of carbohydrates and polyethylene glycol on somatic embryo maturation in hybrid fir abies alba × abies numidica. acta biologia cracoviensia ser. botany, 46: 159–167. salajová, t., jásik, j., kormuť'ák, a., salaj, j., hakman, i. 1996: embryogenic culture initiation and somatic embryo development in hybrid firs (abies alba × abies cephalonica, and abies alba × abies numidica). plant cell reports, 15: 527–530. stasolla, c., yeung, e.c. 2003: recent advances in conifer somatic embryogenesis: improving somatic embryo quality. plant cell tissue and organ culture, 74:15–35. thomas, t.d. 2008: the role of activated charcoal in plant tissue culture. biotechnology and advances, 26: 618-631. troch, v., werbrouck, s., geelen, d., van labeke, m.c. 2009: optimization of horse chestnut (aesculus hippocastanum l.) somatic embryo conversion. plant cell tissue and organ culture, 98: 115-123. von arnold, s., hakman, l. 1988: regulation of somatic embryo development in picea abies by abscisic acid (aba). journal of plant physiology, 132: 164-169. vooková, b., kormut'ák, a. 2001: effect of sucrose concentration, charcoal, and indole-3 butyric acid on germination of abies numidica somatic embryos. biologia plantarum, 44: 181–184. webster, f.b., roberts, d.r., mcinnis, s.m., sutton, b.c.s. 1990: propagation of interior spruce by somatic embryogenesis. canadian journal of forest research, 20: 1759-1765. đokić et al. 2023, biologica nyssana 14(1) 14 (1) june 2023: 57-63 doi: 10.5281/zenodo.8027182 impact of cuscuta spp. seed size variability on machine operation during seed finishing of natural alfalfa seeds original article dragoslav đokić faculty of agriculture, university of niš, kosančićeva 4, kruševac, serbi djdragosla01@gmail.com (corresponding author) violeta oro institute for plant protection and environment, 11000 belgrade, teodora drajzera 9, serbia ratibor štrbanović institute for plant protection and environment, 11000 belgrade, teodora drajzera 9, serbia dobrivoj poštić institute for plant protection and environment, 11000 belgrade, teodora drajzera 9, serbia jasmina milenković institute for forage crops, 37251 globoder-kruševac, serbia jasmina knežević faculty of agriculture, university of priština kosovska mitrovica, lešak 38219, kopaonička bb, serbia rade stanisavljević institute for plant protection and environment, 11000 belgrade, teodora drajzera 9, serbia stanisavljevirade@gmail.com (corresponding author) received: december 05, 2022 revised: march 02, 2023 accepted: march 07, 2023 abstract: the alfalfa seeds intended for marketing must not contain any seeds of the dodder (cuscuta spp.), because it is a quarantine weed in serbia and the world. cuscuta spp. are frequently found in natural alfalfa seeds; nevertheless and because of this, the seed must be processed, i.e. cleaned of weeds. the paper presents a study of seed size variability of cuscuta spp. of four seed lots of natural alfalfa seeds originating from banat, serbia. from each of the four examined seed lots of natural alfalfa seeds, a difference in seed size was determined for 30 seeds of cuscuta spp. per lot. the difference in seed length of cuscuta spp. was 371 µm, and the difference in seed width was 330 µm. the coefficient of variation varied from cv=8.356% to 12.00% for seed width; and from cv=9.383% to 10.69% for seed length. seed size of cuscuta spp. did not significantly affect the efficiency of magnetic separator work in extracting cuscuta spp. seeds from natural alfalfa seeds. key words: cuscuta spp., variability, seed width and seed length, seed processing apstrakt: uticaj varijabilnosti veličine semena cuscuta spp. na rad mašina pri doradi naturalnog semena lucerke seme viline kosice (cuscuta spp.) je karantinski korov u srbiji i svetu i zato se u semenu lucerke koje je namenjeno prodaji, ne sme naći seme korova cuscuta spp. međutim, seme cuscuta spp. se relativno često može naći u naturalnom semenu lucerke i zbog toga se seme mora doraditi, odnosno očistiti od korova. u radu je prikazano ispitivanje varijabilnosti veličine semena cuscuta spp. koja je izdvojena iz naturalnog semena četiri partije semena lucerke poreklom iz banata, srbija. na po 30 semena cuscuta spp., koja su izdvojena iz svake od četiri ispitivane partije naturalnog semena lucerke, uvrđena je varijabilnost za veličini semena. razlika po dužini semena cuscuta spp. je bila 371 µm, a po širini 330 µm. izraženo kroz varijabilnost, koeficijent varijacije se kretao od cv=8.356 do 12.0% za širinu semena i od cv=9.383 do 10.69% za dužinu semena. veličina semena cuscuta spp. nije uticala značajno na efikasnost rada magnetnog separatora prilikom izdvajanja iz naturalnog semena lucerke ključne reči: cuscuta spp., varijabilnost, širina i dužina semena, proces dorade introduction dodder (cuscuta spp.) belongs to the genus convolvulaceae which includes about 200 mostly parasitic species (costea et al., 2015 a,b). it is a parasitic plant that causes significant economic damage around the world (dawson et al., 1994; costea & tardif, 2006; sarić-krsmanović et al., 2020). it is especially harmful in alfalfa seed crops (dawson et al., 1994; sarić-krsmanović et al., 2020). dodder seed causes damage by extracting nutrients from the parent plant; thus, the parent plant weakens, becomes exhausted and lags behind in development. about ten species of cuscuta spp. were detected in serbia in the 1960s and 1970s, and they can cause significant damage to agricultural crops (stojanović et al., 1973), with a particularly large problem in alfalfa (mijatović & stojanović, 1968). in agronomic practice, it is considered that it is best to control cuscuta spp. in the seed crop before harvesting. nevertheless, it happens that alfalfa seed crops often contain cuscuta spp. according to janjic et al. (2005), in serbia, protection from cuscuta spp. began with weed control in crops that had © 2023 đokić et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 57 already been infected. however, a better approach is to apply preventive measures that should be the basic way of weed control. the properties of the dodder seed, and above all, its vitality in the soil, enables the weeded parcels to become a source of further infection during the next ten or more years. therefore, the basic strategy in the fight must be to prevent infestation of parcels (janjić et al., 2005). regardless of the numerous ways of testing and experience in biology and agronomic practice to study its characteristics and control to this day, it is widespread and causes significant damage in the world (abdel-khalik, 2006; córdoba et al., 2021) and serbia (sarić-krsmanović et al., 2022). due to the close resemblance in size between cuscuta spp. and alfalfa seeds, the presence of cuscuta spp. in the seed crop not only directly affects seed production but also presents substantial challenges for seed finishing. the dispersal of cuscuta spp. in the alfalfa seed-growing region is another major issue. also, a significant issue is the ability of cuscuta spp. seeds to retain germination in the soil for decades and germinate again after alfalfa resowing (jayasuriya et al., 2008; olszewski et al., 2020). especially this was a problem for seed production in the eighties of the last century (čuturilo & nikolić, 1986). it is necessary to study the advantages of extracting the seeds of cuscuta sp. from natural alfalfa seeds on a laboratory machine laboratory magnetic separator by german manufacturer emceka gompper and compare it with the efficiency of extracting cuscuta sp. seeds in a conventional way. further studies of weeds cuscuta spp. in serbia have given some solutions in some aspects, but the question of cuscuta spp. is not close to being fully solved (sarić-krsmanović et al., 2020; 2022). it should be added that the seeds of cuscuta spp. in alfalfa seed are considered quarantine weeds which means that such seed cannot be placed on the market gazette of sfry, 1987 (47/87), gazette of the republic of serbia, 45 2005, and supplement to 2021 which is in line with ista rules (ista 2021), which mainly deals with quality and seed trade in europe. the aim of this research was to extract cuscuta spp. seeds from four seed lots of natural alfalfa seed, and examine its variability in seed size. then, to determine whether the size of cuscuta spp seeds affects the efficiency of seed finishing. additionally, to contrast the effectiveness of cuscuta spp. seed separation from natural alfalfa seeds on a laboratory magnetic separator made by german company emceka gompper with the effectiveness of cuscuta spp. seed separation on equipment used in the industrial-standard process of processing natural alfalfa seeds. materials and methods seeds of four seed lots of alfalfa were collected from the banat region (the part of serbia where alfalfa for seed production is grown the most). the seed lots were selected on the basis of a test sample, according to which the seed lots (i to iv) of natural alfalfa seed had approximately the same amount of quarantine weed cuscuta spp. the analyzes were performed in an accredited laboratory for seed quality control, institute of plant protection and environment belgrade. a precise electronic scale was used to measure the samples. the seed samples for analysis weighed 5 g (working) samples in accordance with the regulations of the law on seeds and planting material. all conditions related to the method of production, processing, use, circulation, import and testing are prescribed seeds of agricultural plants (gazette of the sfry no. 47, 1987 and official gazette of the republic of serbia no. 45, 2005). the quality of alfalfa seeds should correspond to the regulation on the quality of seeds of agricultural plants, which is harmonized with the international regulation on the seed quality ista 2021. subsample i: a sample of 5 grams of natural alfalfa seed was obtained from each seed lot. the length and width of the dodder seeds from this weed were measured using the random selection method on a sample of 30 seeds. the length and width of the seeds were examined with an olympus bh-2 microscope. for each seed lot, the average, lowest and highest value (min. and max.), standard deviation (stdev), coefficient of variation (cv%), and standard error of mean were determined to determine the variation in seed size of cuscuta spp. (sem). for each seed lot, the correlation coefficients (r) between cuscuta spp. seeds’ length and width were computed. subsample iia: from each seed lot, 1 kg of seeds was divided into four parts (repetitions) and cleaned on a laboratory magnetic separator. subsample iib: in the seed processing facility of the company „herba” d.o.o. belgrade, the seed lots are processed in the traditional industrial manner, also in four repetitions. all seed batches of natural alfalfa were processed using a selector from the cimbria brand, model delta super 102. this selector features three shaker shoes with two 625 mm x 800 mm sieves each, for a total of six 3 m2 sieves. i shaker shoe has two sieves with round holes of 2.25 mm and 2.25 mm, ii shaker shoe has two sieves with round openings of 1.4 mm and 1.4 mm, and iii shaker shoe has two sieves with rectangular openings of 0.7 mm and 0.5 mm (#-wire). sieves with the following 58 biologica nyssana ● 14 (1) june 2023: 57-63 đokić et al. ● impact of cuscuta spp. seed size variability on machine operation during seed finishing of natural alfalfa seeds biologica nyssana ● 14 (1) june 2023: 57-63 đokić et al. ● impact of cuscuta spp. seed size variability on machine operation during seed finishing of natural alfalfa seeds is the passage through the magnetic separator, where the seeds of the dodder are separated, as well as other harmful weeds and impurities that are undesirable in alfalfa seeds. separation of weeds, especially weeds with wrinkled and unsmooth seeds and other impurities, on this machine is done by electromagnetic force. a certain quantity of steel powder with water is used for the technological process of weed separation on a magnetic separator. between the results of the separation of cuscuta spp. seeds, obtained by industrial and laboratory processing, the correlation coefficient (r) was calculated for each seed lot of natural alfalfa seeds. statistical analysis was done via the freeware software package minitab 16. results and discussion in our experiments, dodder seed, isolated from natural seed lots, showed variability in terms of average seed size (tab. 1). the highest average length and width of seeds of cuscuta spp. was in the third seed lot (1,361 µm and 1,197 µm), which is 371 µm more in length, and 330 µm more in width, compared to the seed lot with the smallest seed (lot iv: length 990 µm and width 867 µm). perforations are used in practice to remove cuscuta spp. quarantine weeds from alfalfa: 1.85 mm, 2.00 mm, and 2.25 mm. other seeds (like lolium sp.) are typically sieved using sieves with openings smaller than 4.5 mm. the sieves are arranged in size order from top to bottom, with larger opening sieves at the top and smaller opening sieves at the bottom. seeds are divided into length and width using rectangular and round sieves, respectively. based on the results of the purity test of the natural seed sampled (determination of the type of inert substances, the presence of weeds and quarantine weeds cuscuta sp., rumex sp., ambrosia sp. and other impurities). depending on the level of purity of the natural seed achieved after the primary seed finishing on the cimbria selector, a subsequent step of seed processing on the cimbria trier model hsr 4020 rl may be required. a total of two segments, one for round seed and the other for long seed, make up the mantle, which is about 600 mm in diameter and 2,000 mm long. a mantle with alveoli with a diameter of less than 2 millimetres was employed in this instance of completing alfalfa seeds. a cimbria gravity table can be used in the seed processing procedure if necessary. the last, final phase of alfalfa seed processing 59 table 1. the seed size variability of cuscuta spp. detected in four seed lots of natural alfalfa seed cuscuta spp. seeds (serial number) seed dimensions of cuscuta spp. detected in a seed lots of natural alfalfa seeds (μm) i ii iii iv length width length width length width length width 1 1053 951 1275 879 1256 1156 1189 1025 2 1062 988 1283 1157 1325 1265 956 845 3 971 1109 1495 1530 1256 1253 899 802 4 1254 1086 1210 1169 1152 989 1075 895 5 1225 1114 1340 1156 1155 1156 1172 956 6 1102 978 1380 1155 1456 1356 856 802 7 1221 1121 1434 1423 1352 1302 848 832 8 1489 1178 1340 1049 1299 1255 1025 1002 9 1164 1014 1183 1127 1158 1199 975 887 10 1206 1066 1196 1073 1522 1325 887 799 11 1381 1114 1298 1181 1632 1025 926 802 12 1094 1026 1564 1346 1452 1152 1175 1011 13 1145 1128 1090 971 1289 1136 956 789 14 1178 1078 1116 1098 1325 1256 875 803 15 1301 1082 1278 1151 1256 1302 1125 1033 16 1236 1027 1198 1140 1488 1235 963 805 17 1341 1341 1313 1051 1502 1156 1023 921 18 1131 1131 1309 1155 1558 1235 985 875 đokić et al. ● impact of cuscuta spp. seed size variability on machine operation during seed finishing of natural alfalfa seeds biologica nyssana ● 14 (1) june 2023: 57-63 60 cuscuta spp. seeds (serial number) seed dimensions of cuscuta spp. detected in a seed lots of natural alfalfa seeds (μm) i ii iii iv length width length width length width length width 19 1143 1143 1211 1068 1365 1099 885 756 20 1208 1208 1619 1398 1236 1156 1025 901 21 1078 1078 1201 1030 1320 1299 1125 1023 22 1347 1347 1330 1272 1398 1005 982 789 23 1214 1214 1193 971 1299 1125 976 821 24 1379 1379 1488 1084 1365 1233 1087 902 25 1242 1242 1211 1063 1455 1302 965 842 26 1314 1314 1269 1163 1266 1235 875 702 27 1347 1347 1448 1172 1566 1099 1023 825 28 1231 1231 1373 1196 1488 1263 986 756 29 1306 1021 1188 1051 1259 1198 831 799 30 1221 956 1626 1221 1368 1022 1025 1002 average 1219 1134 1315 1150 1361 1197 990 867 min 971 951 1090 879 1152 989 831 702 max 1489 1379 1626 1530 1632 1356 1189 1033 stdev 115.2 123.1 140.6 138.0 127.7 99.99 100.7 92.59 cv (%) 9.447 10.86 10.69 12.00 9.383 8.356 10.17 10.68 sem 13.97 14.93 17.05 16.74 15.48 12.13 12.21 11.23 for the dodder seed width, the highest variability was in the second seed lot (cv=12.00%), and the lowest was in the third seed lot (cv=8.356%). the variability of seed size that occurred in different seed lots can be statistically considered low (hadživuković, 1991). ho & costea (2018) stated significantly higher variability for the seed size of cuscuta spp. however, this study was done at different geographical distances and was different from the region where our experiment had been placed. within the genus convolvulaceae, the seed size of cuscuta spp. is most affected by the species (costea et al., 2015a,b). the following physical properties are considered to be the most important for seed finishing: humidity, shape, dimensions, sphericity, mass of 1,000 seeds, volume and porosity, volume-hectolitre mass, density, static and dynamic angle of internal friction (free fall angle), and external condition seed surfaces (copeland & mcdonald, 2004; black et al., 2006; babić & babić, 2012; baskakov et al., 2018). in our trials, all seed lots showed a positive correlation between seed length and width, whereas three seed lots (i, ii and iv) had a very high correlation between these features (p≤0.001). in seed lot iii, seed length and width did not correlate significantly (p≥0.05). also, positive correlative interdependence was high in all seed lots (p≤0.001) (tab. 2). healthy alfalfa seeds have a smooth surface, and after mixing the seeds with steel powder and water in a certain proportion, the steel powder does not stay on that surface. in contrast, the dodder seed has a grain that is porous, and steel powder remains on it, which allows it to separate from alfalfa seeds (fig. 1, fig. 2). table 2. simple correlations (r) between length and width of cuscuta spp. seeds (n=30) seed dimensions of cuscuta spp. originating from lots of natural alfalfa seed (μm) i ii iii iv length width length width length width length width r=0.579*** r=0.672*** r=0.046 ns r=0.818*** * p≤0.05, ** p≤0.01, *** p≤0.001, ns – not significant p≥0.05 đokić et al. ● impact of cuscuta spp. seed size variability on machine operation during seed finishing of natural alfalfa seeds the electromagnetic cleaning machine is used for the final cleaning of alfalfa seeds from the seed of weeds. the quality of weed cleaning on this machine depends on the correct ratio of water and magnetic seed cleaning powder and the amount of seed passed through the magnetized cleaning rollers (đokić & stanisavljević, 2012; uhlarik et al., 2018; đokić et al., 2020). in our experiment, a simple correlation coefficient between the length and width of cuscuta spp. seeds fig. 1. laboratory magnetic separator german manufacturer emceka gompper was very significant (r=0.784; tab. 3). biologica nyssana ● 14 (1) june 2023: 57-63 61 fig. 2. seed of cuscuta spp. table 3. simple correlations (r) between length and width of cuscuta spp. seeds separated from all seed lots of natural seed (n=120) length width r=0.784*** ***, p≤0.001 the principle of operation of electromagnetic separators (trifolin machine-detector) is based on electromagnetic action. they are intended for separating weed seeds with wrinkled and unsmooth surfaces and other impurities. the success of removing the dodder from alfalfa seeds also depends on the method of applying the appropriate metal powder (milošević et al., 1996). electromagnetic seed cleaning machines are very important due to their high-quality cleaning performance. such a high quality of cleanliness cannot be achieved by pneumatic cleaning, triers or selector screens (kozlov, 2013.). the seed of cuscuta spp. was completely isolated from all seed lots of alfalfa seeds in our laboratory tests. also, the seed size did not affect the separation efficiency (tab. 4). table 4. efficiency (%) of separating cuscuta spp. seeds on a laboratory magnetic separator seed size cuscuta spp. (length d μm and width w μm) separated from seed lots of natural seed of alfalfa i ii iii iv l w l w l w l w 100a 100a 100a 100a 100a 100a 100a 100a a tukey’s test (p≤0.05) for length, a tukey’s test (p≤0.0) for width in seed finishing practice, the assumption is that laboratory and industrial processing of alfalfa seeds should be equally or similarly effective in removing cuscuta spp. from natural alfalfa seeds. table 5. simple correlations (r) between the isolation of cuscuta spp. from four seed lots of natural alfalfa seeds in laboratory conditions and in conventional alfalfa seed processing (n=4). seed lots of natural alfalfa seed i ii iii iv r=0.984** r=0.931* r=0.999*** r=0.966** * p≤0.05, ** p≤0.01, *** p≤0.001, ns – not significant (p≥0.05) 62 biologica nyssana ● 14 (1) june 2023: 57-63 in our study, there was a positive and highly correlated relationship (p≤0.05 to p≤0.001) between the efficiency of magnetic separators used in the laboratory and those used in industry (tab. 5), which is in agreement with the results of lin et al. (2022). conclusion an average sample of thirty seeds of cuscuta spp. taken from four seed lots, differed by 371 μm in length and by 330 μm in width. dodder seed isolated from all four seed lots showed variability for width from cv=8.356 (seed from the third lot) to cv=12.00% (seed from the second lot). between the length of the seeds of cuscuta spp. and width was a positive correlation, but at a different level of statistical significance (p≥0.05 to p≤0.001). the influence of cuscuta spp. seed size in the process of its separating from natural alfalfa seeds was not significant. there was a positive and significant correlation (p≤0.05 to p≤0.001) between the separation of cuscuta spp. seeds by laboratory and industrial magnetic separator. according to the law on seed material, processed alfalfa seeds must not contain any seeds of quarantined weed cuscuta spp, regardless of the type and dimensions of cuscuta spp. with electromagnetic cleaning machines, the seeds of the cuscuta spp. must be completely removed from the alfalfa seeds that are being processed. acknowledgements. research was financed by the ministry of education, science and technological development, republic of serbia (contract reg. no. 45103-68/2022-14/200383, 451-03-68/2022-14/ 200010, 451-03-68/2022-14, 451-03-9/2022-14/200133) references abdel-khalik, k.n. (2006). seed morphology of cuscuta l. (convolvulaceae) in egypt and its systematic significance. feddes repert, 117, 217– 224. babić, m. & babić, ljiljana (2012). sušenje i skladištenje (2nd edition). srbija, novi sad: univerzitet u novom sadu, poljoprivredni fakultet. баскаков/baskakov, и/i., карпенко/ karpenko, р/r., & оробинский/orobinskij, в/v. (2018). зерноочистителъные машины и элеваторное оборудование производства ооо „воронежселъмаш”. россия, воронеж: фгбоу во „воронежский государственный аграрный университет имени императора петра i”. black, m., bewley, j., & halmer, p. (2006). the encyclopedia of seed science, technology and uses. uk, wallingford. copeland, l. & mcdonald, m. (2004). seed drying. massachusetts, norwell: seed science and technology. córdoba, e.m., fernández-aparicio, m., gonzález-verdejo, c.i., lópez-grau, c., del valle muñoz-muñoz, m., & nadal, s. (2021). search for resistant genotypes to cuscuta campestris infection in two legume species, vicia sativa and vicia ervilia. plants, 10, 738. https://doi. org/ 10.3390/plants10040738. costea m. & tardif f.j. (2006). the biology of canadian weeds. 133. cuscuta campestris yunck., c. gronovii willd. ex schult., c. umbrosa beyr. ex hook., c. epithymum (l.) l. and c. epilinum weihe. canadian journal of plant science, 86(1), 293–316. costea, m., garcia, m.a., baute k., & stefanović s. (2015a). entangled evolutionary history of cuscuta pentagona clade: a story involving hybridization and darwin in the galapagos. taxon, 64(6), 1225–1242. costea, m., garcía, m.a., & stefanović, s. (2015b). a phylogenetically based infrageneric classification of the parasitic plant genus cuscuta (dodders, convolvulaceae). systematic botany, 40(1), 269285. čuturilo, s. & nikolić, b. (1986). korovi lucerke i njihovo suzbijanje. srbija, beograd: nolit. dawson, j.h., musselman, lj., wolswinkel, p., & dörr i. (1994). biology and control of cuscuta. revolution weed science, 6, 265–317. đokić, d. & stanisavljević, r. (2012). possibility of improving seed processing of red clover (trifolium pratense l.) and alfalfa (medicago sativa l.). international conference on bioscience: biotechnology and biodiversity step in the future. the fourth joint uns psu conference, novi sad, serbia, 18-20 june 2012. book of proceedings., 135–148. đokić, d., stanisavljević, r., milenković, j., koprivica, r., knežević, j., vuković, a., & terzić, d. (2020). effectiveness of the process of cleaning natural alfalfa (medicago sativa l.) and red clover (trifolium l.) seeds. journal on processing and energy in agriculture, 24(1), 9-12. gazette of sfry, 1987: (47/87). gazette of the republic of serbia, 45 2005. hadživuković, s. (1991). statistički metodi s primenom u poljoprivrednim i biološkim istraživanjima (2ed edition). srbija, novi sad: poljoprivredni fakultet. đokić et al. ● impact of cuscuta spp. seed size variability on machine operation during seed finishing of natural alfalfa seeds ho, a. & costea, m. (2018). diversity, evolution and taxonomic significance of fruit in cuscuta (dodder, convolvulaceae); the evolutionary advantages of indehiscence. perspectives in plant ecology, evolution and systematics, 32, 1–17. ista (international seed testing association). 2021. rules for testing seeds. switzerland, zurich. janjić, v., marisavljević, d., & pavlović, d. (2005). vilina kosica i mogućnost suzbijanja. biljni lekar, 5, 590-595. jayasuriya, k.m., baskin, j.m., geneve, r.l., baskin, c.c., & chien, c.t. (2008). physical dormancy in seeds of the holoparasitic angiosperm cuscuta australis (convolvulaceae, cuscuteae): dormancy-breaking requirements, anatomy of the water gap and sensitivity cycling. annals of botany, 102(1), 39–48. козлов/kozlov, в/v. (2013). пневномагнитная сепарация. совершенствование процесса сепарации мелкосеменных кулътур. deutschland, saarbrücken: lap lambert academic publishing. lin, x., zhu, j., huang, p., tian, l., & chen, b. (2022). design and test of an automatic husking and peeling machine for fresh lotus seeds. manufacturing technology, 22, 319-326. mijatović, k. & stojanović, d. (1968). nova forma viline kosice, cuscuta trifolii bab. zaštita bilja, 100101, 285-288. milošević, m., marjanović, j., vujaković, m., polovina r., & pataki, i. (1996). vilina kosica u lucerki i mogućnost njenog uklanjanja iz semena. zbornik radova. viii jugoslovenski simpozijum o krmnom bilju sa međunarodnim učešćem, 26, 175179. olszewski, m., dilliott, m., garcia-ruiz, i., bendarvandi, b., & costea, m. (2020). cuscuta seeds: diversity and evolution, value for systematics/ identification and exploration of allometric relationships. plos one, 15(6), e0234627. sarić-krsmanović, m., uludag, a., bozić, d., radivojević, l., gajić-umiljendić, j., & vrbničanin, s. (2020). the effect of glyphosate on anatomical and physiological features of alfalfa infested with field dodder (cuscuta campestris yunck.). journal of agricultural sciences -tarım bilimleri dergisi, 26, 181-189. sarić-krsmanović, m., zagorchev, l., gajić umiljendić, j., rajković, m., radivojević, l., teofanova, d., božić, d., & vrbničanin, s. (2022). variability in early seed development of 26 populations of cuscuta campestris yunck.: the significance of host, seed age, morphological trait, light, temperature, and genetic variance. agronomy, 12, 559. stojanović, d., mijatović, k., & borić, b. (1973). identifikacija vrsta rasprostranjenih na teritoriji sr srbije. biljni lekar, 18(3-4), 95-100. uhlarik, a., popov, s., karagić, đ., ponjičan, o., & turan, j. (2018). alfalfa seed cleaning using a magnetic separator. journal on processing and energy in agriculture, 22(4), 192-195. đokić et al. ● impact of cuscuta spp. seed size variability on machine operation during seed finishing of natural alfalfa seeds biologica nyssana ● 14 (1) june 2023: 57-63 63 teofilovski 2021, biologica nyssana 12(1) 12 (1) september 2021: 01-10 doi: 10.5281/zenodo.5522951 new floristic data in north macedonia with a first record of allium melanantherum pančić original article aco teofilovski public enterprise nacionalni šumi, pero nakov bb, 1000 skopje, north macedonia acoteofilovski@hotmail.com (corresponding author) received: december 08, 2020 revised: february 10, 2021 accepted: february 17, 2021 abstract: the author discloses new data referring to chorology and habitats of 12 vascular plants in north macedonia of which the balkan endemic allium melanantherum pančić, recorded in the subalpine belt of belasica mt., is reported for the first time in the flora of the country. new locality or localities are presented for the following rare or doubtfully known taxa: asplenium cuneifolium viv., athyrium distentifolium opiz, centaurea scabiosa subsp. spinulosa (spreng.) arcang., chaerophyllum bulbosum l., crepis baldaccii halácsy, dioscorea balcanica košanin, epilobium alpestre (jacq.) krock., eriophorum vaginatum l., euphorbia peplus l., orchis quadripunctata cirillo ex ten. and thelypteris palustris schott. literature data (if present) regarding the chorology of each of the treated taxa in the country are also provided and presented. key words: allium melanantherum, flora, species, distribution, north macedonia apstract: novi floristički podaci za severnu makedoniju sa prvim nalazom vrste allium melanantherum pančić autor otkriva nove podatke vezane za horologiju i staništa 12 vaskularnih biljaka severne makedonije, od kojih je balkanski endemit allium melanantherum pančić, pronađen u subalpskom pojasu planine belasica, po prvi put zabeležen u flori zemlje. nov lokalitet ili lokaliteti su predstavljeni za sledeće retke ili malo poznate taksone: asplenium cuneifolium viv., athyrium distentifolium opiz, centaurea scabiosa subsp. spinulosa (spreng.) arcang., chaerophyllum bulbosum l., crepis baldaccii halácsy, dioscorea balcanica košanin, epilobium alpestre (jacq.) krock., eriophorum vaginatum l., euphorbia peplus l., orchis quadripunctata cirillo ex ten. i thelypteris palustris schott. literaturni podaci (ukoliko postoje) vezani za horologiju svakog obrađenog taksona u zemlji su takođe obezbeđeni i predstavljeni. ključne reči: allium melanantherum, flora, vrste, rasprostranjenje, severna makedonija introduction with more than 3000 species, the vascular flora of north macedonia is among the richest in europe, particularly in regards to its relatively small area of 25713 km2. owing to the researches lasting more than 17 decades, a large amount of floristic data is already published making the flora of this country relatively well studied. however, the chorology of some taxa and taxonomic groups as well as the floristic composition of some parts of the country are still insufficiently known and numerous important data are continuously published. during the enthusiastic field work and performing some applicative projects in the recent years, the author had an opportunity to conduct certain floristic researches in many regions of north macedonia. thereby, an abundant herbarium material belonging to various taxonomic groups was collected and studied. in this work new data concerning some selected taxa are presented aiming to improve the knowledge for their chorology and ecology in the country and in their overall range of distribution. © 2021 teofilovski. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 1 materials and methods during the field work appropriate plant specimens were collected and herbarized. they are stored in the private herbarium of the author, provided with labels containing data regarding the locality, habitat, size and condition of the population. appropriate photographs of live plants are also taken. the plants were identified according to the flora of the republic of macedonia (micevski, 1985, 2001, 2005), flora europaea (tutin & al., 1964-1980) and some other regional floras and monographic works. relevant literature was checked in order to provide the existing data for each of the treated taxa. maps of distribution of most of the presented taxa are also given. results and discussion allium melanantherum pančić (amaryllidaceae) (fig. 1) belasica mt.: sečena skala, subalpine meadow, silicate, 1700 m, 41°20’29.28”n, 22°53’18.94”e, 18.8.2018, leg. a. teofilovski & d. mandzukovski, det. a. teofilovski & z. nikolov. this is a first record of this species in north macedonia. only a small population of no more than 30 individuals was found, spreading on an area of ca. 200 m2. the species grows on a dry subalpine meadow on siliceous geological substrate, accompanied with the following herbaceous plants and shrubs: allium flavum l., asperula aristata l. f., euphorbia barrelieri savi, festuca sp., genista carinalis griseb., hypericum olympicum l., rosa sp., scabiosa triniifolia friv., vaccinium vitis-idaea l., etc. allium melanantherum is a rare balkan endemic plant, which, beside in north macedonia is distributed also in: kosovo part of šar mountain (šutman, gora), several localities in e & se serbia, c & sw parts of bulgaria, and nc & ne floristic regions of greece (see anačkov, 2009; assyov et al., 2012; dimopoulos et al., 2013). having in consideration such distribution, its finding on the territory of north macedonia is not a surprise. this allium is considered generally a high-mountain species of open habitats, although in bulgaria it occurs also in the medium mountain belt, between 900 and 2400 m, on meadows, forest clearings and shrubby 2 fig. 1. allium melanantherum: a inflorescence, b – bulb, c habitat (belasica mt., sečena skala, photo a. teofilovski), d distribution in north macedonia biologica nyssana ● 12 (1) september 2021: 1-10 teofilovski ● new floristic data in north macedonia with a first record of allium melanantherum pančić 3 places (stojanov et al., 1966). similarly, in serbia it grows on high mountain xerophytic or mesophytic meadows and in greece between 1500 and 1900 m, in meadows and rocky places (andersson, 1991; anačkov, 2009). the cited authors do not provide data on the geological substrate of the species habitat. description: bulbs 1-1-5 cm in diameter; outer tunics membranous, with fine, close, parallel fibres separating at the top. stem 13-40 cm. leaves 1-3, up to 20 cm x 2 mm, filiform, sheathing the lower ¼-½ of the stem; margins scabrid. spathe 2-valved; valves unequal, one 0.5-2 cm, the other 1-6 cm, persistent, longer than the pedicels, narrowly ovate or lanceolate at base, abruptly contracted above into a filiform appendage. umbel fastigiate, 3to 25-flowered, with a few bulbils; pedicels 1-2.5 mm, unequal. perianth cylindrical; segments 8-10 x 1-2 mm, pink, with red mid-vein, very narrowly oblong, obtuse. stamens included; filaments c. 6 mm; anthers blackish; pollen yellow. capsule 4 mm (stearn, 1980). allium melanantherum belongs to allium sect. codonoprasum reichenb. (stearn 1981, anačkov 2009). it is easy distinguishable from the other representatives of this section occurring in the balkan peninsula (a. carinatum l., a. flavum l., a. oleraceum l., a. paniculatum s. lato, etc.) by the longer (7-10 mm) and obtuse perigon segments, stamens included in the perigon and dark blue (almost blackish) anthers. asplenium cuneifolium viv. (aspleniaceae) (fig. 2) kavadarci: majdan village, between the road to kruša and majdan river, serpentine, 1020 m, 41°9’10.53”n, 21°55’56.89”e, 5.9.2014, 8.10.2014, leg, det. a. teofilovski. a serpentinophytic species of a. adiantum-nigrum complex with insufficiently known distribution in north macedonia. micevski (1985) in the flora of s.r. macedonia reported this species only from the serpentine area nw of skopje, thereby questioning the previous reports from dudica mt. (konjsko) (bornmüller, 1928) and belasica mt. (mokrievo) (stojanov 1921). recently, it was reported also from nidže mt. (dobro pole) (teofilovski, 2011). on the locality near majdan village this species occurs rather sparse on an area of several hectares, in pinus nigra forests and forest openings, on serpentine geological substrate. athyrium distentifolium opiz (athyriaceae) (fig. 3) šar mountain: otunje village, ginov kamen, near silicate rocks, 1632 m, 42°4’35.68”n, 20°59’19.73”e, 15.11.2017, leg, det. a. teofilovski; belasica mt.: tromegje, 1760 m, between large siliceous stones, 41°20’26.48”n, 22°55’34.06”e, 18.8.2018, leg. a. teofilovski & d. mandzukovski, det. a. teofilovski. a widely distributed species in the north hemisphere but very rare in the balkan peninsula biologica nyssana ● 12 (1) september 2021: 1-10 teofilovski ● new floristic data in north macedonia with a first record of allium melanantherum pančić fig. 2. asplenium cuneifolium: a (kavadarci, majdan village, photo a. teofilovski); b distribution in north macedonia, solid circles herbarium data of the author, rings – data from literature fig. 3. distribution of athyrium distentifolium in north macedonia, solid circles herbarium data of the author, rings – data from literature 4 (croatia, montenegro, romania, bulgaria, north macedonia) (christenhusz et raab-straube, 2013). in north macedonia following two localities were previously known, both of them referring also to siliceous substrate: jablanica mt. (vevčani) and pelister (golemo ezero) (micevski, 1985). the recorded population on belasica mt. is only 200 m distant from the state border with greece and thus the presence of this species could be expected also on the territory of this country. centaurea scabiosa subsp. spinulosa (spreng.) arcang. (asteraceae) (fig. 4) delčevo: 2.6 km ne of zvegor village, meadow, 1015 m, 41°58’30.18”n, 22°50’12.49”e, 26.6.2020, leg. a. teofilovski & d. mandzukovski, det. a. teofilovski. this subspecies was previously known only from the following localities in the northern part of the country: vodno mt. (drenkovski, 1969) and suva gora mt. (lukovica, sedlarevo) (teofilovski, 2011). the only other report of c. scabiosa l. refers to the vicinity of strumica (rudski, 1943), but it is unclear to which infraspecific taxon might it refer as another two subspecies [subsp. scabiosa and subsp. fritschii (hayek) hayek] also occur in this part of balkan peninsula (greuter, 2006 +; dimopoulos et al., 2013). chaerophyllum bulbosum l. (apiaceae) (fig. 5) kičevo: prostranje village, meadow, 940 m, 41°21’30.42”n, 20°59’43.92”e, 22.6.2020, leg, det. a. teofilovski; makedonski brod: near the road to suvodol, 550 m, 41°30’40.43”n, 21°13’45.09”e, 23.6.2020, leg, det. a. teofilovski; kratovo: nw-n of grizilevci village, roadside, 1000-1100 m, 42°2’49.31”n, 22°8’36.54”e, 24.6.2017, leg, det. a. teofilovski; vinica: osojnica river, alder forest, 785 m, 41°47’49.09”n, 22°40’33.59”e, 18.6.2019, leg, det. a. teofilovski; selska river, alder forest, 820 m, 41°47’4.82”n, 22°40’12.95”e, 18.6.2019, observ. a. teofilovski; pehčevo: near the road to ramna reka, 1010 m, 41°43’40.37”n, 22°55’28.46”e, 12.6.2019, leg, det. a. teofilovski; berovo: 2.8 km e of the dam, waste place, 1260 biologica nyssana ● 12 (1) september 2021: 1-10 teofilovski ● new floristic data in north macedonia with a first record of allium melanantherum pančić fig. 4. centaurea scabiosa subsp. spinulosa: a capitulas (delčevo, zvegor village, photo. a. teofilovski); b distribution in north macedonia, solid circles herbarium data of the author, rings – data from literature fig. 5. chaerophyllum bulbosum: a (kratovo, grizilevci village, photo. a. teoflovski); b distribution in north macedonia, solid circles herbarium data of the author, rings – data from literature m, 41°40’5.06”n, 22°55’38.89”e, 28.6.2020, leg, det. a. teofilovski; toward prevedena, dump place, 41°37’13.35”n, 22°50’47.92”e, 1020 m, 2.8.2020, leg, det. a. teofilovski. the following few chorological data regarding this species are given in the flora of the republic of macedonia: kriva palanka (urumov 1923), between tetovo and gostivar, and few localities in the lower part of radika river basin (volkovija, jovan bigorski, bituše) (micevski 2005). the new localities indicate a more frequent occurrence of this species, though the abundance of almost all documented populations is rather low. crepis baldaccii halácsy (asteraceae) (fig. 6) bistra mt.: izvor village, near the road to gorni lopušnik, limestone rocks, 1340 m, 41°30’3.25”n, 20°45’5.24”e, 29.7 & 13.8.2020, leg, det. a. teofilovski; jablanica mt.: strižak, 1865 m, limestone rocks, 41°17’43”n, 20°32’14”e, 14.9.2016. leg, det. a. teofilovski; vevčanska lokva, limestone rocks, 1835 m, 41°14’32.08”n, 20°32’21.55”e, 23.8.2016, leg, det. a. teofilovski; čuma, 1700-1950 m, limestone rocks, 41°13’32.71”n, 20°31’50.09”e, 19.7.2017, observ. a. teofilovski (photo.); labuniški bačila, 41°16’5.53”n, 20°32’5.47”e, limestone rocks, 1900 m, 17.8.2017, observ. a. teofilovski (photo.); podgorečki bačila, 1780 m, 17.8.2017, observ. a. teofilovski (photo.). a balkan endemic species, which geographical range, beside north macedonia, include the mountains of n, c & s albania, e kosovo and nw greece (gajić, 1975; barina et al., 2017; dimopoulos et al., 2013). in north macedonia, previously it was known only from the peak of crn kamen on jablanica mt. (černjavski, 1943, sub c. baldaccii subsp. albanica jáv.), while the report from šar mountain (ljuboten) (c. baldaccii subsp. albanica) (teofilovski, 2015) is incorrect, based on specimens actually belonging to the closely related c. macedonica kitanov. according to the author’s field observation, c. baldaccii is a frequent species in the subalpine belt of jablanica mt. and seems to be very rare on bistra mt. it grows in cervices of limestone rocks while near podgorečki bačila probably also on silicate ones. curiously, despite its relatively wide occurrence on macedonian part of jablanica mt., so far it is not reported from the part of this mountain belonging to albania (kitanov 1948, barina & al. 2017). within c. baldaccii two subspecies (besides the typical one) are so far described subsp. albanica jáv. from n albania and subsp. carpini greuter from nw greece (jávorka et al., 1926; greuter, 1975). however, having in consideration the variations of the morphologic characteristic within the recorded populations on jablanica and bistra mts., both subspecies seem cannot be easily distinguished from the typical c. baldaccii. similar comments are also noted by kamari (1991) in the treatment of the genus crepis l. in the “mountain flora of greece”. dioscorea balcanica košanin (dioscoreaceae) (fig. 7) jablanica mt.: lukovo village, above the road to debar, 710 m, 41°22’0.29”n, 20°36’10.64”e, 11.7.2016, leg, det. a. teofilovski; modrič village, near the road to debar, 41°22’21.86”n, 20°35’45.68”e, 620 m, 15.6.2020, leg. a. teofilovski & z. nikolov, det. a. teofilovski; drenok river gorge, 41°23’43.08”n, 20°34’43.46”e, 694 m, 16.6.2020, leg. a. 5 biologica nyssana ● 12 (1) september 2021: 1-10 teofilovski ● new floristic data in north macedonia with a first record of allium melanantherum pančić fig. 6. crepis baldaccii: a plant with details of fruits and capitula (bistra mt., road to gorni lopušnik, photo. a. teofilovski); b distribution in north macedonia, solid circles herbarium data of the author, rings – data from literature teofilovski & z. nikolov, det. a. teofilovski; globočica hydropower plant, near the road to debar, 41°23’59.53”n, 20°35’0.93”e, 595 m, 15.6.2020, leg. a. teofilovski & z. nikolov, det. a. teofilovski. in the flora of north macedonia this w balkan endemic plant was reported only from a single locality on jablanica mt. (lukovo village) (rizovski, 1977), which represents the southernmost point of the species range. after the publication of this first record, no additional data regarding the chorology or ecology of this species in the country has been disclosed. the new localities extends the known finding site near lukovo village for 5 km toward north, to drenok river and globočica hydropower plant. the species thrives usually on shady places in thermo-mesophylous deciduous forests and their margins, on carbonate geological substrate. at the bottom of the drenok river gorge, it occurs within a relic forest community in which the following species participate: acer campestre l., acer obtusatum willd., acer pseudoplatanus l., aesculus hyppocastanum l., fraxinus excelsior l., fraxinus ornus l., hedera helix l., juglans regia l. (juv.), ostrya carpinifolia scop., sambucus nigra l., etc. epilobium alpestre (jacq.) krock. (onagraceae) (fig. 8) stogovo mt.: 3.9 km nw of ehloec village, wet places near mountain stream, 1670 m, 41°26’6.54”n, 20°43’56.71”e, 30.7.2020, leg, det. a. teofilovski. this epilobium has a wide distribution in c & s europe and part of sw asia, but becoming rare toward southernmost parts of europe and the balkan peninsula. among the countries neighboring north macedonia it is known only from bulgaria (balkan mts., rila mt., vitoša mt.) and serbia (kopaonik mt., golija mt., zlatibor mt., rakoš, kosovo), while doubtfully present in albania and missing from the flora of greece (diklić, 1973; dimopoulos et al., 2013; barina & al., 2018; vladimirov, 2019). the only previous record of this species in north macedonia originates from the upper mountain belt of bistra mt. (careva češma) (micevski, 2001). eriophorum vaginatum l. (cyperaceae) (fig. 9) maleševo mountains: čengino kale, wet place, 41°43’15.09”n, 23°1’43.81”e, 1690 m, leg. a. teofilovski & d. mandzukovski, det. a. teofilovski. a wetland species with a wide eurasian distribution but quiet rare in the flora of north macedonia. it was previously known from a few rather old reports, lacking any recent confirmation: jakupica mt. [salakovo ezero (košanin, 1911), pepelak (bornmüller, 1928)] and šar mountain (lukovo pole) (horvat, 1953). 6 biologica nyssana ● 12 (1) september 2021: 1-10 teofilovski ● new floristic data in north macedonia with a first record of allium melanantherum pančić fig. 7. dioscorea balcanica, male specimen (jablanica mt., modrič village, photo a. teofilovski) fig. 8. epilobium alpestre: a (stogovo mt., photo. a. teofilovski); b distribution oin north macedonia, solid circle herbarium data of the author, ring – data from literature euphorbia peplus l. (euphorbiaceae) (fig. 10) kičevo: 0.7 km sw of the city center, near a fence and on a concrete pavement, 41°30’27.20”n, 20°57’17.68”e, 22.6.2020, leg, det. a. teofilovski; tetovo: blagoja toska str., in concrete planters and between concrete blocks, 42°0’48.23”n, 20°58’48.74”e, 28.9.2020, leg, det. a. teofilovski. in the floristic literature this rare annual spurge is quoted only from two localities in the vicinity of dojran [dub mt. (cirimotić, 1958), nikolić (čarni et al., 2014)] and an unspecified locality between veles and gevgelija (nikolovski & cirimotić, 1958). its occurrence was not confirmed in the flora of the republic of macedonia (micevski, 1998). the observed populations in the urban areas of kičevo and tetovo are rather small, inhabiting ruderal habitats associated with various concrete constructions. orchis quadripunctata cirillo ex ten. (orchidaceae) (fig. 11) makedonski brod: zdunje village, near the bridge, thermophilous forest, limestone, 595 m, 41°46’31.68”n, 21°9’17.08”e, 12.5.2016, leg, det. a. teofilovski. the recorded population which consists of a few tenths of individuals, grows on shallow soil in a sparse thermophilous forest of quercus trojana webb. this north-mediterranean species was known only from a single old report referring to the hilly area near radobil village (prilep) (bornmüller 1928). the new record confirms the poorly known presence of this orchid in the country flora, thereby, extending its general range of distribution more deeply toward the continental part of the balkan peninsula. among the countries neighboring north macedonia it occurs only in albania (frequent in the southern part and scattered elsewhere) and greece (in all floristic regions) (dimopulos et al., 2013; barina et al., 2017). thelypteris palustris schott (thelypteridaceae) (fig. 12) tetovo: jančiste village, alder forest, 400 m, 42°3’23.36”n, 21°7’12.13”e, 3.8.2018, leg, det. a. teofilovski; ohrid: belčišta village, sini viroj, alder forest, 41°18’51.65”n, 20°48’57.68”e, 23.6.2020, leg, det. a. teofilovski. a wetland fern distributed throughout much of the north hemisphere but only with a rare occurrence in the country. in the flora of the republic of 7 biologica nyssana ● 12 (1) september 2021: 1-10 teofilovski ● new floristic data in north macedonia with a first record of allium melanantherum pančić fig. 9. eriophorum vaginatum: a habitat with a detail of a basal part of inflorescence (maleševo mountains, čengino kale, photo. a. teofilovski); b distribution in north macedonia, solid circles herbarium data of the author, rings – data from literature fig. 10. distribution of euphorbia peplus in north macedonia, solid circles herbarium data of the author, rings – data from literature, ring in brackets – insufficiently specified locality from literature macedonia this species is reported from alder forests or wet habitats on three localities: strumica (bansko), gostivar (vranovci) and kožuf mt. (visoka čuka) (micevski, 1985). its actual presence on the cited localities needs to be checked as such habitats are frequently being disturbed by the adverse human activities. conclusions during the recent floristic researches new data regarding the chorology and habitats of 11 rare or poorly known plants and one species new for the country flora are recognized and presented in this work. the balkan endemic allium melanantherum is recorded for the first time in the flora of north macedonia, in the subalpine belt of belasica mt. (sečena skala). considering its occurrence in e & se serbia, c & sw parts of bulgaria, n greece and kosovo part of šar mountain, the finding on the territory of north macedonia was expected in some extend. another one balkan endemic crepis baldaccii, previously known only from an old literature report from jablanica mt. (crn vrv), is confirmed on several new localities of the same mountain with addition of a first record on bistra mt. a previous report from šar mountain (ljuboten) is revised and dismissed as an error. orchis quadripunctata, previously poorly known only from an almost one-century old literature data referring to radobil village (kavadarci), is confirmed for the country flora with its finding near zdunje village (makedonski brod). new locality or localities accompanied with data regarding the habitats, are added for eight rare plants which were previously known only from one or few localities: asplenium cuneifolium kavadarci (majdan village), athyrium distentifolium – šar mountain (otunje village), belasica mt. (tromegje), centaurea scabiosa subsp. spinulosa – delčevo (zvegor village), dioscorea balcanica jablanica mt. (modrič village, drenok river, globočica), epilobium alpestre stogovo mt. (ehloec village), eriophorum vaginatum – maleševo mountains (čengino kale), euphorbia peplus tetovo, kičevo, thelypteris palustris – tetovo (jančište village), ohrid (belčišta village). the new localities of chaerophyllum bulbosum from the vicinity of kratovo, pehčevo, berovo, kičevo and makedonski brod, indicate its wider distribution in north macedonia then it was previously considered. references anačkov, g. 2009: taksonomija i horologija roda allium l. 1754 (amaryllidales, alliaceae) u srbiji. phd thesis, pp. 1-253. univerzitet u novom sadu, prirodno-matematički fakultet. biologica nyssana ● 12 (1) september 2021: 1-10 teofilovski ● new floristic data in north macedonia with a first record of allium melanantherum pančić fig. 11. orchis quadripunctata: a flowers (makedonski brod, zdunje village, photo. a. teofilovski); b distribution in north macedonia, solid circle herbarium data of the author, ring – data from literature fig. 12. distribution of thelypteris palustris in north macedonia, solid circles herbarium data of the author, rings – data from literature 8 andersson, i.a. 1991: allium l. in: strid. a, tan, k. (eds.), mountain flora of greece, 2: 701-714, edinburgh university press. assyov, b., petrova, a., dimitrov, d., vassilev, r. 2012: conspectus of the bulgarian vascular flora. distribution maps and floristic elements. ed. 4, bulgarian biodiversity foundation, sofia. barina, z., mullaj, a., pifkó, d., somogyi, g., meco, m., rakaj, m. 2017: distribution maps. in: barina, z. (ed.): distribution atlas of vascular plants in albania. hungarian natural history museum, budapest, 47-445. barina, z., somogyi, g., pifkó, d, rakaj, m. 2018: checklist of vascular plants of albania. phytotaxa, 378: 1 – 339. bornmüller, j. 1928: beiträge zur flora macedoniens, 3. botanische jahrbücher fur systematik, 61: 1-136. čarni, a., matevski, v., šilic, u., ćušterevska, r. 2014: early spring ephemeral therophytic nonnitrophilous grasslands as a habitat of various species of romulea in the southern balkans. acta botanica croatica, 73(1):107-129. černjavski, p. 1943: prilog poznavanju flore šire okoline ohridskog ezera. ohridski zbornik, 35(2): 11-18. christenhusz, m, raab-straube, e. von 2013: polypodiopsida. – in: euro+med plantbase the information resource for euro-mediterranean plant diversity. (accessed 10.11.2020) cirimotić, j. 1958: prilog poznavanju flore planine duba kod dojranskog jezera. godišnik na šumarskiot institut, skopje, 3: 175-210. diklić, n. 1973: oenotheraceae wettst. in josifović, m. (ed.): flora sr srbije, 5: 8-35. srpska akademija nauka i umetnosti. odeljenje prirodnomatematičkih nauka. beograd. dimopoulos, p., raus, th., bergmeier, e., constantinidis, th., iatrou, g., kokkini, s., strid, a, tzanoudakis, d. 2013. vascular plants of greece: an annotated checklist. englera, 31: 1-372. drenkovski, r. 1969: pregled na florata na vodnenska planina. godišen zbornik, pirodnomatematički fakultet biologija, skopje, 21: 163184. gajić, m. 1975: asteraceae dumortier in: josifović, m. (ed.). flora sr srbije. 7: 1-465. srpska akademija nauka i umetnosti. beograd. greuter, w. 1975: qusquiliae floristicae graecae, candollea, 30: 321-330. greuter, w. (2006+): compositae (pro parte majore). – in: greuter, w, raab-straube, e. von (ed.): compositae. euro+med plantbase the information resource for euro-mediterranean plant diversity. (accessed 15.11.2020) horvat, i. 1953: prilog poznavanju raširenja nekih planinskih biljaka u jugoistočnoj evropi. godišnjak biološkog instituta u sarajevu, 5(1-2): 199-218. jávorka, s., degen, a., gayer g., soó, r., trautmann, r., zahn, k.h. 1926: additamenta ad floram albaniae reisebericht, vii. anthophyta. in: teleki p & csiki e. (eds.). a magyar tudomanyos akademia kiadasa, pp. 74-89, 219-346, budapest. kamari, g. 1991: crepis l. in: strid, a, tan, k. (eds.), mountain flora of greece, 2. pp. 576-595, edinburgh. kitanov, b.p. 1948: prinos kon izučuvanje na florata na istočna albanija. godišen zbornik, filozovski fakultet na univerzitetot skopje, 1: 176-213. košanin, n. 1911: vegetacija planine jakupice u makedoniji. glasnik srpske kraljevske akademije, 85: 184-242. micevski k. 1985: flora na sr makedonija, 1(1). makedonska akademija na naukite i umetnostite, skopje, 1-152. micevski k. 1995: flora na republika makedonija, 1(3). makedonska akademija na naukite i umetnostite, skopje, 398-778. micevski k. 1998: flora na republika makedonija, 1(4). makedonska akademija na naukite i umetnostite, skopje, 779-1113. micevski, k. 2001: flora na republika makedonija, 1(5). makedonska akademija na naukite i umetnostite, skopje, 1121-1430. micevski k. 2005: flora na republika makedonija, 1(6). makedonska akademija na naukite i umetnostite, skopje, 1433-1715. nikolovski, t., cirimotić, j. 1958: karakteristika na krajbrežnite rastitelni grupacii po srednoto i dolnoto tečenie na r. vardar (poteg titov velesgevgelija). godišnik na šumarskiot institut, skopje, 3: 119-149. rizovski, r. 1977: beitrag zur kenntnis die verbreitung von dioscorea balcanica kos. glasnik republičkog zavoda za zaštitu prirode, titograd, 10: 101–104. rudski, i. 1943: prilog poznavanju flore okoline strumice. ohridski zbornik, 35(2): 205-238. stearn, w.t. 1980: allium l. u tutin, t.g., heywood, biologica nyssana ● 12 (1) september 2021: 1-10 teofilovski ● new floristic data in north macedonia with a first record of allium melanantherum pančić 9 v.h., burges, n.a., moore, d.m., valentine, d.h., walters, s.m., webb, d.a. (eds.), flora europaea 5: 49-69. cambridge university press, cambridge. stearn, w.t. 1981: the genus allium in the balkan peninsula. botanicshe jahrbücher für systematik, pflanzengeschichte und pflanzehgeography, 102(14): 201-213. berliner 300-jahr-feier-symposium. stuttgart. stojanov, n. 1921: floristički materijali ot belasica. godišnik na sofiski univerzitet, 15-16: 1-133. stojanov, n., stefanov, b, kitanov, b. 1966: flora na blgaria. ed. 4, vol. 1. nauka & izkustvo, sofia. teofilovski, a. 2011: prilozi za florata na republika makedonija. private edition of the author, skopje, 1-142. teofilovski, a. 2015. in vladimirov, v., dane, f., matevski, v, tan, k. (eds.). new floristic records in the balkans: 27. phytologia balcanica, 21(2): 207212. tutin, t. g., heywood, v. h., burges, n. a., moore, d. m., valentine, d. h., walters, s. m, webb, d. a. (eds.), 1964-1980: flora europaea 1-5. the university press, cambridge. urumov, i.k. 1923: beiträge zur flora of belomorska trakija. izdanie na blgarskata akademia na naukite, 28(13): 1-107. vladimirov, v. 2019: reports 132-134. in: vladimirov, v., aybeke, m, tan, k. (eds.), new floristic records in the balkans: 40. phytologia balcanica, 25(3): 295–335. biologica nyssana ● 12 (1) september 2021: 1-10 teofilovski ● new floristic data in north macedonia with a first record of allium melanantherum pančić 10 sevindik et al. 2021, biologica nyssana 12(2) 12 (2) december 2021: 131-139 doi: 10.5281/zenodo.5759841 assessment of phylogenetic relationships of some worldwide-cultivated cotton genotypes (gossypium hirsutum l.) by using issr and rapd markers original article emre sevindik faculty of agriculture, department of agricultural biotechnology, adnan menderes university, south campus, cakmar, aydin, turkey ph.d-emre@hotmail.com (corresponding author) muhammed ebrar çayir faculty of agriculture, department of agricultural biotechnology, adnan menderes university, south campus, cakmar, aydin, turkey tülay emrebaş ankara university, faculty of agriculture, department of field crops, turkey ertuğrul filiz duzce university, department of crop and animal production, cilimli vocational school, 81750, cilimli, duzce, turkey received: june 09, 2021 revised: september 07, 2021 accepted: october 03, 2021 abstract: cotton belongs to the genus of the malvaceae family and presents an economically and biologically highly valuable multi-purpose crop grown as a source of fiber, food and feed worldwide. in this study, phylogenetic relationships of cotton genotypes of the thirteen countries (twelve gossypium hirsutum and one gossypium barbadense (giza 75 egypt) as outgroup) taken from the directorate of nazilli cotton research institute genetic stock were analyzed by using rapd and issr markers. rapd–pcr analysis obtained total of 53 dna bands with the highest number of bands for opa-02 and opa-13 primers, no bands for ope-08 primer and rate of polymorphism of 90.56%. issr –pcr analysis showed a total of 81 dna bands, where the highest number of bands was obtained with ubc-811, ubc-834 and ubc836 primers, no bands with ubc-853 primer and rate of polymorphism of 77.77%. phylogenetic tree was obtained by using the upgma algorithm and the tree consisted of two large clades. analysis of the results suggests that rapd and issr markers are useful tools to demonstrate phylogenetic relationships between cotton genotypes. key words: gossypium hirsutum, rapd, issr, molecular markers, phylogenetic apstrakt: utvrđivanje filogenetskih odnosa nekih genotipova pamuka (gossypium hirsutum l.) kultivisanih širom sveta korišćenjem issr i rapd markera pamuk pripada rodu gossypium iz familije malvaceae i predstavlja ekonomski i biološki veoma vredan višenamenski usev koji se gaji kao izvor vlakana i hrane širom sveta. u ovom istraživanju, analizirani su filogenetski odnosi genotipova pamuka iz trinaest različitih zemalja (dvanaest gossypium hirsutum i jedna gossypium barbadense (giza 75 egypt) kao zasebna grupa) uzetih iz banke genotipova nazili instituta za istraživanje pamuka, a proučavani su korišćenjem rapd i issr markera. rapd–pcr analizom dobijeno je ukupno 53 dnk traka uz najveći broj traka sa opa-02 i opa-13 prajmerima, nije bilo trake za ope-08 prajmer, a stopa polimorfizma iznosila je 90.56%. issr –pcr analiza pokazala je ukupno 81 dnk traka, gde je najveći broj traka dobijen sa ubc-811, ubc-834 i ubc-836 prajmerima, nije bilo traka za ubc-853 prajmer, a stopa polimorfizma iznosila je 77.77%. filogenetsko drvo je dobijeno korišćenjem upgma algoritma i sadržalo je dve velike klade. analiza rezultata sugeriše da rapd i issr marker predstavljaju korisne alate za demonstriranje filogenetskih odnosa između genotipova pamuka. ključne reči: gossypium hirsutum, rapd, issr, molekularni markeri, filogenetski introduction the malvaceae family consists of 243 genera with 4,225 known species, and contains various economically valuable species such as gossypium (cotton), corchorus (jute), hibiscus, alcea, theobroma cacao (cacao), abelmoschus esculentus (okra) and malva in tropical and subtropical regions (pravin et al., 2018; ben-simchon et al., 2019; wang et al., 2020). the main spreading center of these family members is south america. besides, it spreads everywhere except for the very cold regions of the earth (uzunhisarcıklı & vural, 2009). the members of the family malvaceae include herbs, © 2021 sevindik et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 131 subshrubs, shrubs, trees and even woody climbers (ţîţei & teleuță, 2018). in addition, the malvaceae family has mucilage, fixed oils and essential oils and is used for medicinal purposes. plants of this family are used publicly for the treatment of respiratory and digestive system irritations and inflammations as expectorant, bronchodilator, diuretic and for the treatment of gastric ulcers (erarslan & koçyiğit, 2019). cotton belongs to the gossypium genus of the malvaceae family and contains 51 species, among which 45 are diploid (2n = 26) and 6 are allotetraploid (2n = 52) (elçi & hançer, 2016). in addition, this genus includes only four cultivated cottons that are morphologically distinct and geographically widespread, namely: g. arboreum l. and g. herbaceum l. known as the old world african-asian cottons, g. barbadense l. (sea island cotton) and g. hirsutum l. and or others known as upland cotton (ibrahim et al. 2007). eighty-seven percent of cotton growth area in the world occurs in developing countries (nix et al., 2017). cotton is an economically and biologically highly valuable multipurpose crop grown as a source of fiber, food and feed worldwide. the cottons of gossypium hirsutum l. and gossypium barbadense l., are leading natural fiber crops as well as oilseed crops important to bioenergy, feed and food industries (zhang et al. 2015; noman et al., 2016; bilval et al., 2017). the primary product of the cotton plant is mohair, which covers the seeds in the seed or cocoon. this mohair has been used for thousands of years to dress the people of ancient india, asia, america and africa (abdellatif et al., 2012). molecular markers have developed a wide range of applications in the field of molecular biology, which may include phylogenetic study, genetic diversity, evolution, ecology, population genetics, and the study of complex genomic properties in both plants and animals (atasagun et al., 2018; raza et al., 2019). genetic diversity can be estimated from pedigree analysis or from multivariate analysis carried out on a large number of plant attributes (sharma et al., 2018). dna markers offer a convenient way to study polymorphisms, taxonomy, physiology, embryology, genetic engineering and their use in product development (kesawat & kumar, 2009; shaheen et al., 2013). pcr (polymerase chain reaction) based molecular markers such as issr, rapd, ssr, and aflp were found to be useful in various applications of genetic variation and plant breeding (dongre et al., 2004; cui et al., 2017). for the use in detecting genetic variation, the rapd (random amplified polymorphic dna) marker has a number of advantages such as technical simplicity, speed of testing, minimum dna requirements, and low costs. in addition, no prior knowledge about the sequence under investigation is required. rapd markers have been used to analyze genetic relations and diversity in plant populations and cultivars (ghasemi et al., 2014; saha et al., 2019; akinro et al., 2019). issr (inter simple sequence repeats) technique is a pcr based technique, reported by ztetkiewicz et al. (1994), which can quickly show the differences of individuals with close relationships (jafari et al., 2019). this marker is based on determining the random distribution of dna nucleotides in plant genomes, independent of chromosome regions. issr markers are usually dominant markers, so it is difficult to determine whether individuals are heterozygous. this technique is much more sensitive and reproducible than rapd technique (gülşen & roose, 2001; vijayan, 2005; güngör et al., 2020). in this study, genetic diversity of cotton genotypes of the thirteen countries (twelve gossypium hirsutum and one gossypium barbadense as outgroup) taken from the directorate of nazilli cotton research institute genetic stock was analyzed by using rapd and issr markers. materials and methods plant materials and genomic dna isolation the cotton varieties of the thirteen countries 132 biologica nyssana ● 12 (2) december 2021: 131-139 sevindik et al. ● assessment of phylogenetic relationships of some worldwide-cultivated cotton genotypes (gossypium hirsutum l.) by using issr and rapd markers fig. 1. three-week appearance of thirteen different cotton varieties after planting fig. 2. genotypes of gdna isolation gel image 133 were obtained from nazilli directorate of cotton research institute and this study was carried out in the biotechnology laboratory of the directorate. in addition, the 13 cotton varieties were planted in two repeats and three seeds (fig. 1). the seedlings were grown for about three weeks until they had four leaves. those who did not germinate to yield four leaves were waited or replanted until they grown four leaves. the cottons, which were sown in three seeds and two repeats, were diluted into the best growing single plant after they had four leaves. two or threedays old leaves were collected by observing the selected plants every day. after being washed with pure water and dried, the leaf samples were stored at -86 ºc for genomic dna isolation. power plant dna isolation kit (qiagen) was used for genomic dna isolation from the specimens. the obtained genomic dna samples were made ready for use and stored at -20 ºc. for preparation of the gdna (genomic dna) gel image, the mixture was prepared by using 1 µl gdna, 8 µl dh20 and 1 µl loading dye. then 1% agarose gel electrophoresis was prepared and the samples were loaded. gdna gel images of the 13 cotton varieties are shown in fig. 2. the purity of the genomic dna was assessed with a nanodrop spectrophotometer (implen nanophotometer biologica nyssana ● 12 (2) december 2021: 131-139 sevindik et al. ● assessment of phylogenetic relationships of some worldwide-cultivated cotton genotypes (gossypium hirsutum l.) by using issr and rapd markers table 1. primers used in the rapd-pcr reactions and their tm degrees primers dna sequences(5’-3’) tm (°c) amplifications pcr protocol (35 cycles) opa-02 5' tgccgagctg 3' 34 °c + 94 oc/2 min 94 oc/1 min 32-34 oc/1 min 72 oc/1 min 72 oc/10 min opa-05 5' aggggtcttg 3' 32 °c + opa-13 5' cagcacccac 3' 34 °c + opa-15 5' ttccgaaccc 3' 32 °c + opa-16 5' agccagcgaa 3' 32 °c + opa-18 5'aggtgaccgt 3' 32 °c + opa-20 5' gttgcgatcc 3' 32 °c + ope-08 5' tcaccacggt 3' 32 °c opa-07 5’-gaaacgggtg-3’ 32 °c + opa-03 5’-agtcagccac-3’ 32 °c + table 2. primers used in the issr-pcr reactions and their tm degrees issr primers dna sequences(5’-3’) tm (°c) amplifications pcr protocol (35 cycles) ubc-831 5’-ctctctctctctctctt-3’ 50 oc + 94 oc/1 min 94 oc/1 min 48-53 oc/1 min 72 oc/1 min 72 oc/10 min ubc-830 5’-tgtgtgtgtgtgtgtgg-3’ 52 oc + ubc-807 5'-agagagagagagagagt-3' 50 oc + ubc-819 5' gtgtgtgtgtgtgtgta -3 50 oc + ubc-808 5’-agagagagagagagagc-3’ 52 oc + ubc-836 5'-agagagagagagagagya-3’ 52 oc + ubc-856 5'–acacacacacacacacya-3’ 52 oc + ubc-853 5' tctctctctctctctcrt -3' 52 oc ubc-855 5'-acacacacacacacacyt-3' 52 oc + ubc-810 5' -gagagagagagagagat-3' 50 oc + ubc-826 5’-acacacacacacacacc-3’ 52 oc + ubc-811 5’-gagagagagagagagac-3’ 53 oc + ubc-834 5’-agagagagagagagayt-3’ 52 oc + ubc-873 5’-gacagacagacagaca-3’ 48 oc + 134 p330). blanking was applied with the pd7 solution we used in the last stage of the dna isolation kit. the amount of dna was determined by taking 1 µl of each sample and reading of the od260 and od280 nm in a nanodrop spectrophotometer. the purity amount of dna was determined by od260/od280 ratio. according to nanodrop spectrophotometer measurements after isolation, the lowest dna amount was obtained from brown egyptian and the highest dna amount was obtained from ujchi 2 uzbek. according to od260/od280 ratios which are the indications of purity, the highest value was obtained from haridost and the lowest value was obtained from bulgarian 73. pcr amplifications rapd and issr primers chosen for pcr amplifications are shown in tab. 1 and tab. 2. ready mixes were used as an alternative to manual pcr loadings. amplification was carried out by adding 2 μl genomic dna, 1 μl primers and 5 μl of master mix (cat. no: rp02-ii-400, rp02-ii-2000 0.75u of taq dna polymerase, reaction buffer, 2mm mgcl2, 250µm dntps and enzyme stabilizer) and 17 μl dh2o into pcr tube. amplification products were analyzed by electrophoresis on 1% agarose gel buffered with 1.0x tae (tris-acetate-edta), stained with ethidium bromide and photographed under ultraviolet light. as a results of rapd-pcr, some of the gel images were shown in fig. 3a, fig. 3b, fig. 3c and fig. 3d. data and phylogenetic analysis following the pcr analysis, dna bands were scored as “1” in the presence of dna, “0” in the absence of dna and a ‘’?’’ for missing data. genetic relationship of the some gossypium genotypes used in the study was analyzed using paup 4.0b10 (swofford, 2001), and the genetic distance matrix between the populations was calculated by drawing the upgma (unweighted pair group method) phylogenetic tree in the same program in accordance with the arithmetic mean of the pedigree trees. the phylogenetic trees were evaluated with bootstrap test on 10,000 resamplings (felsenstein, 1985) and these values were added to the upgma tree. results and discussion molecular markers developed over the last 30 years have an important role in breeding programs in agriculture (kalkışım et al., 2016). dna markers based on pcr are versatile tools in different aspects of genomic studies. it is possible to analyze closely related genera to evaluate phylogenic and genetic relationships using these markers (zarei et al., 2017). as a result of rapd-pcr analysis, a total of 53 bands were obtained. of these, 48 were polymorphic and the rate of polymorphism was about 90.56%. most of the bands were obtained from opa-02 and opa-13 primers and no band was obtained from ope-08 primer. phylogenetic tree and genetic distances between genotypes were calculated using the paup 4.0b10 analysis program. according to the paup analysis, the closest genetic distance was 0.06 (tam c66 16 els and sc-2079), while 1.0 was the farthest genetic distance (arcota-129 and ziroatkar-81, biologica nyssana ● 12 (2) december 2021: 131-139 sevindik et al. ● assessment of phylogenetic relationships of some worldwide-cultivated cotton genotypes (gossypium hirsutum l.) by using issr and rapd markers fig. 3. gel image of rapd-pcr bands amplified with opa-02 (a) and opa-13 (b). gel image of issr-pcr bands amplified with ubc-807 (c) and ubc-836 (d) 135 biologica nyssana ● 12 (2) december 2021: 131-139 sevindik et al. ● assessment of phylogenetic relationships of some worldwide-cultivated cotton genotypes (gossypium hirsutum l.) by using issr and rapd markers bulgar 73 and ziroatkar-81) (tab. 3). the upgma tree constructed using the paup 4.0b10 phylogenetic analysis program consists of the two large clades (fig. 4). clade 1 was subdivided into multiple subclades. subclade a1, consisted of tam c66-16 els, sc-2079, tropical 225, azgr-7711, fibermax 832, eva and giza-75 (gossypium barbadense) genotypes. subclade a2, consisted of brown egyptian, arcota-129 and bulgar 73 genotypes and this subclade has a 53% bootstrap value. clade 2 consisted of ujchi 2 uzbek, ziroatkar-81 and haridost table 3. pairwise genetic distance matrix obtained from rapd primers genotypes 1 2 3 4 5 6 7 8 9 10 11 12 13 tam c66 16 els 0.14 0.37 0.21 0.40 0.40 0.14 0.25 0.22 0.58 0.58 0.42 0.06 tropical 225 7 0.35 0.10 0.60 0.60 0.17 0.14 0.22 0.64 0.55 0.47 0.21 brown egyptian 15 14 0.32 0.20 0.20 0.37 0.42 0.20 0.58 0.55 0.47 0.40 azgr-7711 10 5 13 0.36 0.27 0.10 0.08 0.34 0.64 0.52 0.33 0.27 arcota-129 2 3 1 4 0.09 0.40 0.60 0.54 0.60 1.00 0.54 0.60 bulgar 73 2 3 1 3 1 0.40 0.60 0.63 0.60 1.00 0.45 0.60 eva 7 8 15 5 2 2 0.10 0.32 0.64 0.52 0.47 0.17 fibermax 832 12 7 17 4 3 3 5 0.32 0.64 0.52 0.47 0.23 giza 75 9 10 8 16 6 7 13 13 0.47 0.58 0.40 0.20 ujchi 2 uzbek 10 11 10 11 3 3 11 11 8 0.30 0.23 0.58 ziroatkar-81 21 20 20 19 5 5 19 19 21 4 0.19 0.55 haridost 9 10 10 9 6 5 10 10 11 3 4 0.38 sc-2079 3 10 16 13 3 3 8 11 8 10 20 8 fig. 4. the upgma tree generated using rapd data genotypes genotypes with bootstrap value of 83%. as a result of issr-pcr analysis, a total of 81 bands were obtained. of these, 63 were polymorphic and the rate of polymorphism was 77.77%. most of the bands were obtained from ubc-811, ubc834 and ubc-836 primers and no band was obtained from ubc-853 primer. according to the paup analysis, the closest genetic distances were 0.00 (ujchi 2 uzbek and ziroatkar-81, haridost and ziroatkar-81), while the farthest genetic distances were 0.77 (arcota-129 and fibermax 832, arcota-129 and sc2079, azgr-7711 and arcota-129) (tab. 4). the upgma tree constructed using the paup 4.0b10 phylogenetic analysis program consists of two large clades (fig. 5). clade one was subdivided into multiple subclades. subclade a1 consisted of tam c66 16 els, eva, sc-2079, tropical 225, azgr-7711 and fibermax 832 genotypes. subclade a2 is composed of brown egyptian and giza-75 (gossypium barbadense) genotypes. this subclade has 81% bootstrap value. clade two was subdivided into multiple subclades and this subclade has a 73% bootstrap value. subclade b1 is composed of arcota-129, ujchi 2 uzbek and bulgar 73 genotypes.subclade b2 is composed of ziroatkar-81 and haridost genotypes. in our study, both rapd and issr results showed that tam c66 16 els, eva, sc-2079, tropical 225, azgr-7711 and fibermax 832 genotypes belong to the same group. ujchi 2 uzbek, ziroatkar-81 and haridost genotypes appeared in the same group. in addition, these three genotypes have been geographically supported. in rapd results, bulgar 73, arcota-129 and brown egyptian appeared 136 biologica nyssana ● 12 (2) december 2021: 131-139 sevindik et al. ● assessment of phylogenetic relationships of some worldwide-cultivated cotton genotypes (gossypium hirsutum l.) by using issr and rapd markers table 4. pairwise genetic distance matrix obtained from issr primers genotypes 1 2 3 4 5 6 7 8 9 10 11 12 13 tam c66 16 els 0.07 0.23 0.11 0.66 0.55 0.04 0.08 0.28 0.57 0.24 0.31 0.06 tropical 225 6 0.28 0.13 0.66 0.60 0.07 0.13 0.28 0.71 0.27 0.36 0.08 brown egyptian 18 22 0.20 0.44 0.53 0.24 0.22 0.20 0.28 0.32 0.31 0.24 azgr-7711 9 11 16 0.77 0.53 0.11 0.07 0.33 0.14 0.19 0.25 0.07 arcota-129 6 6 4 7 0.33 0.66 0.77 0.44 0.00 0.44 0.44 0.77 bulgar 73 36 39 35 35 3 0.53 0.52 0.60 0.00 0.38 0.33 0.56 eva 4 6 19 9 6 35 0.08 0.32 0.57 0.25 0.32 0.06 fibermax 832 7 11 17 6 7 34 7 0.32 0.42 0.20 0.27 0.07 giza 75 22 22 16 26 4 39 25 25 0.51 0.40 0.41 0.32 ujchi 2 uzbek 4 5 2 1 0 0 4 3 4 0.00 0.00 0.57 ziroatkar-81 19 21 25 15 4 25 20 16 31 0 0.09 0.23 haridost 24 28 4 20 4 22 25 21 32 0 7 0.32 sc-2079 5 7 19 6 7 37 5 6 25 4 18 25 fig. 5. the upgma tree generated using issr data 137 biologica nyssana ● 12 (2) december 2021: 131-139 sevindik et al. ● assessment of phylogenetic relationships of some worldwide-cultivated cotton genotypes (gossypium hirsutum l.) by using issr and rapd markers together, while issr results showed only brown egyptian genotype in a separate group. in our study, both rapd and issr results were consistent with each other. in previous studies, hossain et al. (2017) in bangladesh, determined the genetic diversity of the 4 varieties of the gossypium hirsutum through the rapd technique. five rapd primers were used in the study; they yielded 53 bands in total and determined the polymorphism rate to be 90.56%. maleia et al. (2010) determined the genetic difference between african and american varieties and inbred cotton lines (gossypium hirsutum l. race latifolium h.) through rapd molecular markers. 24 rapd primers were used in the study; 166 bands were obtained in total, and the rate of polymorphism was determined to be 90.96%. consequently, the use of rapd molecular markers has been effective for genetic differentiation in cotton varieties and genotypes. surgun et al. (2012) determined the identification and genetic diversity of some turkish cotton (gossypium hirsutum l.) genotypes through rapd-pcr analysis. in the study, genetic difference of nine cotton genotypes was determined through rapd-pcr technique. among the genotypes, a total of 42 rapd primers were used to detect polymorphism; assessable results were obtained from 34 of these primers, and a total of 319 rapd-pcr bands were obtained. the rate of polymorphism among the studied genotypes was determined as 18.1% and genetic similarity between any 2 genotypes ranged from 90.2% to 96.5%. the results revealed that genotypes can be separated at the molecular level with the help of rapd markers. in the study conducted by khan et al. (2000), 45 rapd primers were used in 35 gossypium species in total containing 31 species, 3 subspecies and 1 interspecific hybrid. in the study, a total of 579 dna bands were obtained and the rate of polymorphism was 99.8%. the results obtained in the study provide good correspondence to the taxonomic sections recognized within the genus gossypium. these results show that the rapd technique produces reliable results to create the phylogenetic history of the gossypium genus. in a study carried out in turkey, şahin et al. (2020) determined the genetic relationship between 30 cultivated cotton varieties (gossypium hirsutum l.) through issr-pcr method. 24 issr primers were used to determine polymorphism in 30 cotton varieties; while creating a total of 41 bands from the primers, it was determined that an average of 22.3 of these bands were polymorphic. the polymorphic information content values for issr primers ranged from 0.19 to 0.68 and it was found as 0.49 in average. as a result of the study, they concluded that issr molecular marker technique can be successfully applied in the studies of determining genetic diversity in cotton; in addition, due to its high polymorphism and reproducibility, it can be successfully applied in many laboratories with limited laboratory facilities. bilval et al. (2017) analyzed genetic variation among nine cotton (gossypium) genotypes by using rapd, issr and ssr markers. in the study, 25 rapd, 22 issr and 24 ssr primers were used. the number of polymorphic amplicons was found to be 156 (rapd), 142 (issr) and 24 (ssr) accounting for polymorphism of 91.22% (rapd), 67.94% (issr) and 72.72% (ssr), respectively. as a result of their work, they explained the suitability and reliability of the molecular markers (rapd, issr and ssr) to predict genetic diversity and genetic relationships between cotton genotypes. bardak and bolek (2012) used 5 issr and 39 ssr primers to determine the genetic relationship between diploid and tetraploid 25 cotton genotypes (gossypium spp.) cultivated in different regions of the world. they found that 155 (89.60%) out of 173 alleles obtained as a result of the study were polymorphic. the average number of alleles per ssr and issr markers was 3.93, ranging from 1 to 8 alleles. genetic diversity ranged from 0.04 to 0.58 among all the genotypes inspected. this ratio was 0.04-0.23 within g. hirsutum and 0.07-0.26 within g. barbadense species. as a result of the study, the use of wild cotton species has been suggested to increase the diversity in the gene pool and it has been stated that it will be useful in the selection of the desired properties. in conclusion, rapd and issr markers were used in this study to investigate the genetic diversity of some worldwide cultivated cotton genotypes. the results of the present research have confirmed that these molecular markers can detect polymorphism among gossypium hirsutum genotypes, predict if they are related, and identify genotype-specific rapd and issr markers. according to the results obtained with rapd markers, polymorphism was found to be high and this rate is 90.56 and 77.77% respectively. in addition, the findings obtained from this study are thought to contribute to breeding programs, development of germplasm sources and future marker studies. references abdellatif, k.f., khidr, y.a., el-mansy, y.m., ellawendey, m.m., soliman, y.a. 2012: molecular diversity of egyptian cotton (gossypium barbadense l.) and its relation to varietal development. journal of crop science and biotechnology, 15(2): 93-99. biologica nyssana ● 12 (2) december 2021: 131-139 138 akinro, l.a., adesoye, a.i., fasola, t.r. 2019: genetic diversity in cola acuminata and cola nitida using rapd primers. genetika, 51(1): 227-236. atasagun, b., aksoy, a., gürcan, k. 2018: genetic diversity in astragalus argaeus, a critically endangered species from turkey. biologia, 73(10): 927-936. bardak, a., bölek, y. 2012: genetic diversity of diploid and tetraploid cottons determined by ssr and issr markers.turkish journal of field crops, 17(2): 139-144. ben-simchon, e., sapir, e., vaknin, y., shelef, o. 2019: malvaceae spp. leaves as a novel crop for food. international journal of agriculture forestry and life sciences, 3(2): 279-286. bilval, b.b., vadodariya, k.v., rajkumar, b.k., lahane, g.r. 2017: genetic diversity of parents using rapd, issr and ssr molecular markers in upland cotton (gossypium hirsutum l.). bulletin environment, pharmacology and life science, 6: 51-57. cui, c., li, y., liu, y., li, x., luo, s., zhang, z., wu, r., liang, g., sun, j., peng, j., tian, p. 2017: determination of genetic diversity among saccharina germplasm using issr and rapd markers. comptes rendus biologies, 340(2): 76-86. dongre, a., parkhi, v., gahukar, s. 2004: characterization of cotton (gossypium hirsutum) germplasm by issr, rapd markers and agronomic values. indian journal of biotechnology, 3: 388393. elçi e., hançer t. 2016: molecular characterization and germination analysis of cotton (gossypium hirsutum l.) genotypes under water deficit irrigation. turkish journal of agricultural research, 3(2): 122-129. erarslan, z.b., koçyiğit, m. 2019: the important taxonomic characteristics of the family malvaceae and the herbarium specimens in iste. turkish journal of bioscience and collections, 3(1): 1-7. felsenstein, j. 1985: confidence limits on phylogenies: an approach using the bootstrap. evolution, 39: 783-791. ghasemi, a.r., golparvar, a.r., isfahani, m.n. 2014: analysis of genetic diversity of sugar beet genotypes using random amplified polymorphic dna marker. genetika, 46(3): 975-984. gülşen, o., roose, m.l. 2001: limonlarda genetik çeşitlilik, bazı turunçgillerle akrabalık derecelerinin dna markırlarının kullanılarak belirlenmesi. bahçe, 30(1-2): 53-63 güngör, h., akbudak, m.a., filiz, e., yüce, i̇., dumlupınar, z. 2020: genetic diversity in sodium azide (nan3) induced barley mutants using issr markers. derim, 37(1): 27-32. hossain, a., afroz, m., sultana, s.s., alam, s.s. 2017: karyotype and rapd diversity in four varieties of gossypium hirsutum l. cytologia, 82(5): 535-541. jafari, s.h., sepehry, a., soltanloo, h., karimian a.a. 2019: genetic differentiation between bitter and sweet asafetida plants using issr markers. molecular biology reports, 46(1): 1069-1078. ibrahim, r.i.h., azuma, j.i.,sakamoto, m. 2007: pcr-rflp analysis of the whole chloroplast dna from three cultivated species of cotton (gossypium l.). euphytica, 156(1-2): 47-56. kalkışım, o., okcu, m., okcu, z., karabulut, b., yildirim, n., agar, g. 2016: relationships among some pears genotypes (pyrus communis l.) based on issr and rapd analysis. erwerbs-obstbau, 58(4): 259-264. kesawat, m.s., kumar, b.d. 2009: molecular markers: it’s application in crop improvement. journal of crop science and biotechnology, 12(4): 169-181. khan, s.a., hussain, d., askari, e., stewart, j.m., malik, k.a., zafar, y. 2000: molecular phylogeny of gossypium species by dna fingerprinting. theoretical and applied genetics, 101(5): 931-938. maleia, m.p., goncalves-vidigal, m.c., gonela, a., lacanallo, g.f., moiana, l.d., chamuene, a., sousa, l.l., darben, l.m. 2010: genetic divergence among african and american cotton (gossypium hirsutum l. race latifolium h.) cultivars and inbred lines through random amplification of polymorphic dna (rapd) markers. african journal of biotechnology, 9(50): 8539-8548. nix, a., paull, c., colgrave, m. 2017: flavonoid profile of the cotton plant, gossypium hirsutum: a review. plants, 6(4): 43. noman, a., bashir, r., aqeel, m., anwer, s., iftikhar, w., zainab, m., zafar, s., khan, s., islam, w., adnan, m. 2016: success of transgenic cotton (gossypium hirsutum l.): fiction or reality? cogent food & agriculture, 2(1): 1207844. pravin, p., shrikant, s., venkataraman, b.k. 2018: species relationships among wild and cultivated abelmoschus medik., (malvaceae) species as reveled by molecular markers. international sevindik et al. ● assessment of phylogenetic relationships of some worldwide-cultivated cotton genotypes (gossypium hirsutum l.) by using issr and rapd markers 139 biologica nyssana ● 12 (2) december 2021: 131-139 sevindik et al. ● assessment of phylogenetic relationships of some worldwide-cultivated cotton genotypes (gossypium hirsutum l.) by using issr and rapd markers journal of life sciences, 6(1): 49-59. raza, a., mehmood, s.s., ashraf, f., khan, r.s.a. 2019: genetic diversity analysis of brassica species using pcr-based ssr markers. gesunde pflanzen, 71(1): 1-7. saha, s., dhar, t.n., ghosh, p., dey, t. 2019: molecular characterization of sesame germplasms of west bengal, india using rapd markers. acta biologica szegediensis, 63(1): 15-24. shaheen, t., zafar, y., stewart, j.m., rahman, m.u. 2013: development of short gssrs in g. arboreum and their utilization in phylogenetic studies. turkish journal of agriculture and forestry, 37(3): 288-299. sharma, s., kumar, p., gambhir, g., kumar, r., srivastava, d.k. 2018: assessment of genetic diversity in lettuce (lactuca sativa l.) germplasm using rapd markers. 3 biotech, 8(1): 9. surgun, y., çöl, b., bürün, b. 2012: genetic diversity and identification of some turkish cotton genotypes (gossypium hirsutum l.) by rapd-pcr analysis. turkish journal of biology, 36(2): 143150. swofford, d.l. 2001: version 4.0 b10. paup*. phylogenetic analysis using parsimony (* and other methods). version 4. sinauer associates, sunderland, massachusetts, usa. şahin, c.b., i̇şler, n., rustamova, v. 2020: characterization of some cotton varieties using issr markers. ksu journal of agriculture and nature, 23(1): 108-116. ţîţei, v., teleuță, a. 2018: introduction and economical value of some species of the malvaceae family in the republic of moldova. in “agriculture for life, life for agriculture” conference proceedings, sciendo, 1(1): 126-133. uzunhisarcıklı, m.e., vural, m. 2009: taxonomy and iucn categories of two alcea l. (malvaceae) species cited in the data deficient (dd) category. biodicon, 2(2): 90-95. vijayan, k. 2005: inter simple sequence repeat (issr) polymorphism and its application in mulberry genome analysis. international journal of industrial entomology, 10(2): 79-86. wang, d., fan, w., guo, x., wu, k., zhou, s., chen, z., danyang, l., wang, k., zhu, y., zhou, y. 2020: magendb: a functional genomics hub for malvaceae plants. nucleic acids research, 48(1): 1076-1084. zarei, a., erfani-moghadam, j., mozaffari, m. 2017: phylogenetic analysis among some pome fruit trees of rosaceae family using rapd markers. biotechnology & biotechnological equipment, 31(2): 289-298. zhang, m., rong, y., lee, m.k., zhang, y., stelly, d.m., zhang, h.b. 2015: phylogenetic analysis of gossypium l. using restriction fragment length polymorphism of repeated sequences. molecular genetics and genomics, 290(5): 1859-1872. zietkiewicz, e., rafalski, a., labuda, d. 1994: genome fingerprinting by simple sequence repeat (ssr)-anchored polymerase chain reaction amplification. genomics, 20:176183. microsoft word bn-oa-0201-08 stojkovic m_et_al biologica nyssana 2 (1) september 2011: 59-66 stojković, m. et al. ichthyological integral indices, the history of development… 59 original article ! ichthyological integral indices, the history of development and possible application on rivers in serbia milica stojković*1, djuradj milosević1, vladica simić2 1university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia 2university of kragujevac, faculty of sciences and mathematics, institute of biology and ecology, radoja domanovića 12, 34000 kragujevac, serbia * e-mail: milicastojkovic83@gmail.com abstract: stojković, m., milošević, đ., simić, v.: ichthyological integral indices, the history of development and possible application on rivers in serbia. biologica nyssana, 2 (1), september 2011: 59-66. based on a literature review, the different approaches in the water quality assessment using fish communities in freshwaters are summarized. fish assemblage indicators, developed throughout the world, were reviewed and the main differences in methodologies, number of metrics and values are summarized. we have drawn attention to the methods used for designing a fish-based index with a particular focus on original developments in north america and its adaptations in many different regions and habitat types. the main obstacles for ecological assessment are scarce ecological information and the problem with the classification. the lack of knowledge is especially true for species assemblages in the relatively unexplored river basins of europe, e.g. the balkans peninsular. key words: biotic indices, fish, multimetric approach, water quality assessment introduction ! monitoring the ecological status of aquatic systems is in use by water resource managers worldwide. historically, aquatic systems were assessed primarily through chemical measuring, providing a snapshot of water quality conditions. the anthropogenic capacity to alter the natural world usually overcomes our ability to assess the impacts of these alterations on the biological inhabitants within the water ecosystems (s i m o n , 1999). fish are an unavoidable element for such assessments because of their biological and socioeconomic status. fish are considered as a reliable indicators in water quality assessment for various reasons: fish are present in most surface waters; the identification of species is relatively easy; the sensitivity to disturbance is well known for many species; as well as their responses to environmental stressors; different species represent distinct trophic levels; fish occupy a variety of habitats in rivers; diminished growth and recruitment are easily assessed and reflect exposure to stress (fame consortium, 2004). however, there are a few disadvantages: manpower needs a three person crew is required to effectively and safely sample fish communities; migration of fish may provide misleading data; overlapping the effects of overfishing with pollution effects may lead to erroneous conclusions (g r a b a r k i e w i c z & d a v i s , 2008; s i m i ć & s i m i ć , 2009) various fish-based indices have been developed worldwide for assessing the ecological status of rivers. most of them include a reference condition approach and appropriate biological variables or metrics (n o b l e & c o w s , 2007; r o s e t et al., 2007), describing the fish assemblage 2 (1) • september 2011: 59-66 biologica nyssana 2 (1) september 2011: 59-66 stojković, m. et al. ichthyological integral indices, the history of development… 60 attributes and quantifying the impact of anthropogenic activities on the biota. the index of biotic integrity (ibi) was initially developed by dr. james karr for the purpose of evaluating and describing the condition of small warm water streams in central illinois and indiana (k a r r , 1981). as the ibi became widely used, different versions were developed for different regions and ecosystems. new versions, generally, retained most of the original metrics but the some of them have been modified for application in a particular region or type of stream. the aim of this paper is to compare different approaches for assessing the ecological condition using fish communities. likewise, we want to propose the most applicable metrics for rivers in serbia. material and methods the comparison was made between different methods used for designing a fish-based index according to a literature review from a period 19812009. the different approaches, used in north america, according to k a r r (1981), l e o n a r d & o r t h (1986), m o y l e et al. (1986), h u g h e s & g a m m o n (1987), m i l l e r et al. (1988), s t e e d m a n (1988), s i m o n (1991), l y o n s (1992), s i m o n & l y o n s (1995), l y o n s et al. (1996), o v e r t o n (2001), t e e l s et al. (2004), barbour et al. (2002), n i e m e l a & f e i s t (2000), were described. in addition, different modifications of ibi applied for water quality assessment in europe (b e l l i a r d et al., 1999; b r e i n e et al., 2004; a n g e r m e i e r & d a v i d e a n u , 2004; fame consortium, 2004; l e n h a r d t et al., 2009) were compared. the original version had 12 metrics that refer to fish species richness and composition, number and abundance of species, trophic organization and function, reproductive behavior, fish abundance, and condition of individual fish (tab. 1). k a r r (1981) proposed a rating system for each metric based upon the degree of deviation (5 (none to slight), 3 (moderately) and 1 (significantly)) from the appropriate ecoregional reference conditions. the metrics were scored and summed to arrive at an index ranging from 60 (best) to 12 (worst). metrics, which are defined in original version of ibi (k a r r , 1981) and envisaged for illinois area, have been changed for application in many different regions worldwide. in every other version (k a r r , 1981; l e o n a r d & o r t h , 1986; m o y l e et al., 1986; h u g h e s & g a m m o n , 1987; m i l l e r et al., 1988; s t e e d m a n , 1988; s i m o n , 1991; l y o n s , 1992; s i m o n & l y o n s , 1995; l y o n s et al., 1996; o v e r t o n , 2001; t e e l s et al., 2004; b a r b o u r et. al., 2002; n i e m e l a & f e i s t , 2000), list of metrics has been modified according to features of target region (tab. 1). some of them are commonly used such as: total number of fish species, % individuals as omnivores, % individuals with anomalies; but most of them was adjusted according to assemblage of investigated area (n o b l e & c o w s , 2007). results and discussion according to the metrics from the list above and its variations, it can be concluded that each of them assess the same aspect of functional community but in a different way. before making a choice which metrics are more appropriate, it should be drawn attention to their applicability in the investigated area. the potential metrics have to be changed in a predictive manner according to anthropogenic influence. likewise, they have to be sensitive to stressors and to provide the response that can be discriminated from natural variation (n o b l e et al., 2007). the utilization of the fish-based indices in europe is less widespread than in the united states of america. recently, scientists in france, belgium and romania have endeavored to adjust ibi for usage in their own countries (v i d a l , 2008). b e l l i a r d et al. (1999) have suggested 10 metrics for water quality assessment in france, applying them on the seine river. in belgium, from the total of 28 candidate metrics, nine had been selected (breine et al., 2004). in romania, angermeier & davideanu (2004) made distinction between hilly and montane region, and have chosen 7 metrics for each region to include in preliminary multimetric indices (pmis). k a r r ’ s (1981) original ibi was also adapted in serbia. this version related to lentic system, analyzed changes in the fish assemblage compared to sediment deposition in the reservoir. it contains 10 suitable metrics (l e n h a r d t et al., 2009). in each of these four versions, scoring system was used but with different scores applied. b e l l i a r d et al. (1999) kept the original scoring system (k a r r , 1981) using score values: 5, 3, 1 for slightly, moderately and the most impacted, respectively. in the second case, ibi score of an investigated site is the mean of scores for all metrics, and varies between 0 and 5. in dependence of the final ibi score, a given site will be classified in an appropriate water class according to water framework directive (b r e i n e et al., 2004). values for metric scoring classes in romania were assigned for each metric at each site as 3, 2, 1 for biologica nyssana 2 (1) september 2011: 59-66 stojković, m. et al. ichthyological integral indices, the history of development… 61 table 1. metrics of the original ibi and other versions used for designing ibi in different regions category metrics of the original ibi (karr, 1981) version of metrics used in different regions species richness total number of species 1. total number of native species (karr et al., 1986, leonard & orth, 1986, moyle et al., 1986), hughes & gammon, 1987, miller et al., 1988, steedman, 1988, simon, 1991, lyons, 1992, simon & lyons, 1995, lyons et al., 1996, teels et al., 2004) 2. salmonid age classes (moyle et al., 1986, hughes & gammon, 1987) species composition number of darter species 1. number of darter and sculpin species (steedman, 1988) 2. number of sculpin species (hughes & gammon, 1987) 3. number of benthic species (karr et al., 1986, leonard & orth, 1986, moyle et al., 1986, hughes & gammon, 1987, miller et al., 1988, steedman, 1988, simon, 1991, lyons, 1992, simon & lyons, 1995, lyons et al., 1996) 4. number of darter species, excluding tolerant species (gatz & harig, 1993) number of sunfish species 1. number of sunfish species, including genus micropterus (karr et al., 1986, simon, 1991, hoefs and boyle, 1992, lyons, 1992) 2. number of cyprinid species (hoefs and boyle, 1992) 3. number of sunfish and trout species (steedman, 1988) 4. number of water column species (miller et al., 1988, oberdorff & hughes, 1992) 5. number of headwater species (simon, 1991) number of sucker species 1. number of sucker and cyprinid species (hoefs and boyle, 1992) 2. number of sucker and catfish species (steedman, 1988) 3. number of minnow species (hughes & gammon, 1987, simon, 1991, hoefs and boyle, 1992) tolerance guilds number of intolerant species 1. number of amphibian species (moyle et al., 1986) 2. % individuals as brook trout (langdon, 1989) 3. number of trout species (moyle et al., 1986, simon, 1991) 4. number of sensitive species (karr et al., 1986, leonard & orth, 1986, moyle et al., 1986), hughes & gammon, 1987, miller et al., 1988, steedman, 1988, simon, 1991, lyons, 1992, simon & lyons, 1995, lyons et al., 1996) % individuals as green sunfish 1. % individuals as common carp (hughes & gammon, 1987) 2. % tolerant species (karr et al., 1986, leonard & orth ,1986, moyle et al., 1986), hughes & gammon, 1987, miller et al., 1988, steedman, 1988, simon, 1991, lyons, 1992, simon & lyons, 1995, lyons et al., 1996) 3. % introduced species (hughes & gammon, 1987) 4. % individuals as common roach (oberdorff & hughes, 1992) trophic guilds % individuals as omnivores 1. % individuals as omnivores and herbivores (overton, 2001) 2. % individuals as generalists (karr et al., 1986, leonard & orth, 1986, moyle et al., 1986), hughes & gammon, 1987, miller et al., 1988, steedman, 1988, simon, 1991, lyons, 1992, simon & lyons, 1995, lyons et al., 1996) % individuals as insectivorous cyprinids 1. % individuals as insectivorous (hughes and gammon, 1987, simon, 1991, miller et al., 1988, lyons, 1992, hoefs and boyle, 1992, oberdorff and hughes, 1992) 2. % specialized insectivores (leonard & orth ,1986) 3. % individuals as benthic insectivorous (teels et al., 2004) % individuals as piscivorous 1. % catchable salmonids karr et al., 1986, leonard & orth ,1986, moyle et al., 1986), hughes & gammon, 1987, miller et al., 1988, steedman, 1988, simon, 1991, lyons, 1992, simon & lyons, 1995, lyons et al., 1996) 2. % pioneering species (karr et al., 1986, leonard & orth ,1986, moyle et al., 1986), hughes & gammon, 1987, miller et al., 1988, steedman, 1988, simon, 1991, lyons, 1992, simon & lyons, 1995, lyons et al., 1996) biologica nyssana 2 (1) september 2011: 59-66 stojković, m. et al. ichthyological integral indices, the history of development… 62 abundance number of individuals 1. density of individuals (karr et al., 1986, leonard & orth ,1986, moyle et al., 1986), hughes & gammon, 1987, miller et al., 1988, steedman, 1988, simon, 1991, lyons, 1992, simon & lyons, 1995, lyons et al., 1996) 2. biomass of fish (hughes and gammon, 1987) reproduction and condition % individuals as hybrids 1. % introduced species (karr et al., 1986, leonard & orth ,1986, moyle et al., 1986), hughes & gammon, 1987, miller et al., 1988, steedman, 1988, simon, 1991, lyons, 1992, simon & lyons, 1995, lyons et al., 1996) 2. % simple lithophils (simon, 1991, lyons, 1992, hoefs and boyle, 1992) 3. number of late maturing species (teels et al., 2004) 4. percentage of species with multiple age groups (ncdehnr, 1997) % individuals with anomalies 1. percent of individuals with heavy infestation of cysts of the parasite neascus (steedman, 1988) slightly, moderately and the most impacted, respectively (a n g e r m e i e r & d a v i d e a n u , 2004). finally, in serbia, for each metric, data were sorted into quartiles, according to l o n g & w a l k e r (2005). a site had score of 1 where the observed value for the metric fell below the first quartile threshold. a score of 5 was attributed to those sites for which the observation occurred above the fourth quartile. sites with the observation values falling within the second and third quartiles scored 3. european fish index (efi) has been developed as a result of european union project, named fame (development, evaluation and implementation of the standardized fish-based assessment method for the ecological status of european rivers). generally, efi is based on a predictive model that derives reference condition for the each site and quantifies the deviation between predicted and observed condition of the fish fauna (fame consortium, 2004). efi employs 10 metrics (table ii.) and their response to human pressures has been already known. theoretical values are predicted for each metric using environmental variables by means of a multilinear regression model. in order to quantify a level of degradation, the residuals, calculated as difference between observed and predicted metric values, are used. the ecological status is expressed as an index ranging from 1 (high ecological status) to 0 (bad ecological status). it has been recommended that efi should not be applied in areas where a fish fauna significantly deviates from those of the tested regions e.g. rivers of the south-eastern part of europe (fame consortium, 2004). in order to adjust ibi for application on rivers in serbia, a great amount of ecological information is required for each species, which is the base for their classification in ecological guilds. the election of metrics is essential to make ibi strong enough to represent the condition of the river ecosystems in serbia. we have suggested the preliminary list of metrics (table iii.) which can be potentially used in serbia. the total number of species represents a reliable indicator of the target population condition. healthy ecosystem contains a great number of species (k a r r et al., 1986). however, invasive and introduced species should be considered as a separate metric, because of their negative influence on the status of natural populations. according to fame (fame consortium, 2004), metrics, which refer to tolerance guilds, are represented as a number of intolerant species and a number of tolerant species. in addition, fame proposed to employ metrics related to reproductive requirements: relative abundance of lithophilic species and density of the phytophilic species. the evaluation of these characteristics is based on the fact that the increase of habitat degradation reduces the possibility of finding a specific substrate for spawning. the percentage of species with multiple age groups simultaneously may assess suitability of habitat conditions for reproduction and the degree of reproductive success. from the parameters related to the assessment of trophic structure, the percentage of: omnivorous individuals; specialized insectivores; obligate piscivores can be applied as the appropriate metrics. this recommendation can made a difference between species specialized for the certain type of food from those within a wide spectrum. each species has its own specific requirements according to the habitat type (reophilic, limnophilic and euritop). according to k a r r (1981), species are divided in two groups: the species which lives nearby the bottom and the species which lives in the water column (percidae, centrarchidae, respectively). this kind of division, biologica nyssana 2 (1) september 2011: 59-66 stojković, m. et al. ichthyological integral indices, the history of development… 63 table 2. metrics used by european fish index (efi) and their response to anthropogenic influence selected metrics response to anthropogenic influence 1. density of the insectivorous species decrease 2. density of the omnivorous species increase 3. density of the phytophilic species increase 4. relative abundance of lithophilic species decrease 5. number of benthic species decrease 6. number of rheophilic species decrease 7. relative number of intolerant species decrease 8. relative number of tolerant species increase 9. number of species migrating over long distances decrease 10. number of potamodromous species decrease table 3. preliminary list of metrics for application on rivers in serbia selected metrics response to anthropogenic influence 1. total number of native species decrease 2. total number of alien species increase 3. number of intolerant species decrease 4. number of tolerant species increase 5. % individuals as omnivores increase 6. % individuals as specialized insectivorous decrease 7. % individuals as obligate piscivorous decrease 8. number of rheophilic species decrease 9. number of euritop species increase 10. percentage of species with multiple age groups decrease 11. % individuals with anomalies increase beside habitat type, takes trophic requirements into account and introduces the problem of stratification in shallow waters. therefore, it would be recommented more suitable metrics such as: number of rheophilic species (specialized of habitat type), and number of euritop species (tolerant of habitat type). metrics related to migration guilds are not acceptable for applying on rivers in serbia, because there are very few species which can be classified as diadromous (acipenseridae and clupeidae). the total number of individuals, as a rule, should be reduced with increasing the environmental degradation. in some instances, although the environmental conditions are degraded, the number of individuals increase in term of abundance. increased abundance of individual species is an outcome of their tolerance to the changes in the environment. percentage of diseased fish, the presence of tumors and other abnormalities, in most cases has low value or it is absent, but may have a great importance for identifying areas with high concentration of toxic substances. conclusion classification of fish into ecological guilds is a prerequisite for the development of an ibi. regional modifications of ibi are based on a different metrics which are employed. choice of metrics requires the great ecological knowledge of the local fish population. the chosen list of metrics should be suitable for the target fish assemblage and strong enough to detect changes in functioning biologica nyssana 2 (1) september 2011: 59-66 stojković, m. et al. ichthyological integral indices, the history of development… 64 community. formulation of an index, such as index of biotic integrity in serbia, based on fish assemblages, and its affiliation to the already existing balkan biotic index (s i m i c & s i m i c , 1999), based on macrozoobenthos, may contribute to the development of a new approach in the environmental condition assessment. such a comprehensive assessment, including a majority of freshwater biota, may lead to the formulation of an advanced strategy for conservation of ecosystem health. acknowledgments. this work was supported by ministry of education and science, serbia, grant no.043002. references angermeier, p.l. and davideanu, g. 2004: using fish communities to assess streams in romania: initial development of an index of biotic integrity. hydrobiologia, 511: 65-78. barbour, m.t., gerritsen, j., snyder, b.d., stribling, j.b. 2002: rapid bioassessment protocols for use in streams and wadeable rivers: periphyton, benthic, macroinvertebrates and fish. environmental protection agency, united states. belliard, j.,berrebi dit thomas, r., monnie d. 1999: fish communities and river alteration in the seine basin and nearby coastal streams. hydrobiologia 400: 155-166. breine, j., simoens, i., goethals, p., quataert, p., ercken, d., van liefferinghe, c. and belpaire, c. 2004: a fish-based index of biotic integrity for upstream brooks in flanders (belgium ). hydrobiologia 522:133-148. fame consortium 2004: manual for the application of the european fish index-efi. a fish-based method to assess the ecological status of european rivers in support of water framework directive. gatz, a. j. jr. and harig, a. l. 1993: decline in the index of biotic integrity of delaware run, ohio, over 50 years. ohio journal of science 93(4): 95100. grabarkiewicz, j.d. and davis, w.s. 2008: an introduction to freshwater fishes as biological indicators. environmental protection agency, united states. hoefs, n.j. and boyle, t.p. 1992: contribution of fish community metrics to the index of biotic integrity in two ozark rivers. ecological indicators, 1: 283-303. hughes, r.m. and gammon, j.r. 1987: longitudinal changes in fish assemblages and water quality in the willamette river, oregon. transactions of the american fisheries society 116(2):196-209. karr, j. r. 1981: assessment of biotic integrity using fish communities. fisheries 6(6): 21–27. karr, j. r., faush, k. d., angermeier, p. l., yant, p. r. and schlosser i. j. 1986: assessing biological integrity in running waters: a methods and its rationale. illinois natural history survey. special publication 5, urbana illinois. langdon, r. w. 1989: the development of fish populationbased biocriteria in vermont. in simon, t.p., holst, l.l. and shepard, l.j. (ed.), proceedings of the first national workshop on biocriteria 12-25, u. s. epa, region 5, chicago. lenhardt, m., markovic, g. and gacic, z. 2009: decline in the index of biotic integrity of the fish assemblage as a response to reservoir aging. water resource manage 23: 1713-1723. leonard, p.m. and orth, d.j. 1986: application and testing of an index of biotic integrity in small, coolwater streams. transactions of the american fisheries society 115:40114. long, j. m. and walker, d. j. 2005: small scale application and assessment of an index of biotic integrity for large boreal river. hydrobiologia 544:177-187. lyons, j. 1992: using the index of biotic integrity (ibi) to measure environmental quality in warmwater streams of wisconsin. general technical report, nc-149. u.s. department of agriculture, forest service, st. paul, minnesota. lyons, j., l. wang, and t.d. simonson. 1996: development and validation of an index of biotic integrity for coldwater streams in wisconsin. north american journal of fisheries management 16:241256. miller, d.l.,leonard, p.m.,hughes, r.m.,karr, j.r.,moyle, p.b.,schrader, l.h., thompson, b.a.,daniel, r.a.,fausch, k.d.,fitzhugh, g.a.,gammon, j.r.,halliwell, d.b., angermeier, p.l. and orth, d.j. 1988: regional applications of an index of biotic integrity for use in water resource management. fisheries 13(5):12-20. moyle, p.b., brown, l.r. and herbold, b. 1986: final report on development and preliminary tests of indices of biotic integrity for california. final report to the u.s. environmental protection agency, environmental research laboratory, corvallis, oregon. niemela, s. and feist, m. 2000: index of biotic integrity (ibi). guidance for coolwater rivers and streams of the st. croix river basin in minnesota. st paul, minnesota. noble, r.a.a. and cows, i.g. 2007: assessing the health of european rivers using functional biologica nyssana 2 (1) september 2011: 59-66 stojković, m. et al. ichthyological integral indices, the history of development… 65 ecological guilds of fish communities: standardizing species classification and approaches to metric selection. fisheries management and ecology 14: 381-392. north carolina department of environment, health and natural resources (ncdehnr). 1997: standard operating procedures for biological monitoring. environmental sciences branch. biological assessment group. north carolina. 52 pp. oberdorff, t. and hughes, r.m. 1992: modification of an index of biotic integrity based on fish assemblages to characterize rivers of the seine basin. hydrobiologia 228: 117-130. overton, j.r. 2001: standard operating procedures. stream fish community assessment and fish tissue. department of environment and natural resources, north carolina. roset, n., grenouillet, g., goffaux, d., pont, d. and kestemont, p. 2007: a review of existing fish assemblage indicators and methodologies. fisheries management and ecology 14: 393– 405. simic , v. and simic , s. 1999: use of the river macrozoobenthos of serbia to formulate a biotic index. hydrobiologia 416: 51-64. simic, s. and simic, v. 2009: ekologija kopnenih voda. beograd, bioloski fakultet; kragujevac, prirodno matematicki fakultet. zemun. 295 p. simon, t.p. 1991: development of ecoregion expectations for the index of biotic integrity (ibi) central corn belt plain. u.s. environmental protection agency, region v, chicago, illinois. epa 905/9-91/025. simon, t. p. 1999: assessing the sustainability and biological integrity of water resources using fish communities.crc press, boca ration, florida. simon, t.p. and lyons, j. 1995: application of the index of biotic integrity to evaluate water resource integrity in freshwater ecosystems. in: davis, w.s. and simon, t.p (ed.), biological assessment and criteria: tools for water resource planning and decision making 245-262, lewis publishers, bocaraton, florida. steedman, r.j. 1988: modification and assessment of an index of biotic integrity to quantify stream quality in southern ontario. canadian journal of fisheries and aquatic science 45:492-501. teels, b.m., mazati, l.e. and rewa, c.a. 2004: using an ibi to assess effectiveness of mitigation measures to perlace loss of wetland-stream ecosystem. wetlands 24: 375-384. vidal, l.b. 2008: fish as ecological indicators in mediterranean freshwater ecosystems. ph.d. thesis. institute of aquatic ecology and department of environmental science. university of girona. biologica nyssana 2 (1) september 2011: 59-66 stojković, m. et al. ichthyological integral indices, the history of development… 66 microsoft word 0104_stesevic_petrovic biologica nyssana 1 (1-2) december 2010: 35-42 stešević, d., petrović, d.. preliminary list of plant invaders… 35 original article ! preliminary list of plant invaders in montenegro danijela stešević, danka petrović faculty of natural sciences and mathematics, university of montenegro, džordža vašingtona bb, 81 000 podgorica, montenegro * e-mail: denist@t-com.me abstract: stešević, d., petrović, d.: preliminary list of plant invaders in montenegro. biologica nyssana, 1 (1-2), december 2010: 35-42. due to the fact that invasive alien species (ias) are considered to be the second cause of global biodiversity loss after direct habitat destruction and have adverse environmental, economic and social impacts from the local level upwards, in last decades investigations of alien flora of montenegro are intensified. in this paper we are presenting a preliminary list of ias, with the aim to provide a basic data on ias in montenegro, to enable future monitoring and to draw attention on the problems which expansion of ias is bringing with itself. the list consists of 50 plant taxa species and supspecies level. key words: invasive alien plants, montenegro abbrevations: iasinvasive alien species, pphanaerophytes, chchamaephytes, h hemicriptophytes, ttherophytes, ggeophytes, namnorth america, samsouth america, cam & samcentral and south america, asasia, afrafrica, eas-euroasia. introduction ! unlike some worldwide regions where invasive ecology is well developed, in montenegro the interest for this a rather new field in ecology, that study a human mediate transfer of organisms to areas outside their natural dispersal range and the consequences of such transfer, has appeared in last few years: s t e š e v i ć & j o v a n o v i ć (2005, 2008), s t e š e v i ć (2005), steš ević & j o g a n (2006, 2007), t o m o v i ć & s t e š e v i ć (2007), s t e š e v i ć et al. (2009). due to the concept of r i c h a r d s o n et al. (2000), later supplemented by p y š e k et al. (2004), richardson & pyšek (2004), plants taxa in a given area whose presence there is due to intentional or unintentational human involvement, or which have arrived there without the help of people from an area in which they are alien are known as alien plants (exotic, non-native, non-indigenous). invasive plants are naturalized plants that produce reproductive offspring often in very large numbers, at considerable distance from parent plants (approximate scales: > 100m; <50 years for taxa spreading by seeds and other propagules; > 6m/3 years for taxa spreading by roots, rhizomes, stolons, or creeping stem) and thus have a potential to spread over considerable area. on global level invasive species are recognized as a major factor of environmental change, and are considered one of the most important causes of biodiversity loss worldwide (w a l k e r & s t e f f e n , 1997; m c n e e l y , 1999, 2001, issg 2000, millennium ecosystem assessment 2005 etc.). these species have invaded and affected native biota in virtually every ecosystem type on earth and ecological cost is the irretrievable loss of native species and ecosystems. it is estimated that the direct economic cost of alien invasive species runs into many billions of dollars annually (issg 2000). beside mentioned impacts, invasive alien plants sometimes have a negative impact on humans health, such as ambrosia artemisiifolia, heracleum mantegazzianum etc. 10th sfses • 17-20 june 2010, vlasina lake1 (1-2) • december 2010: 35-42 biologica nyssana 1 (1-2) december 2010: 35-42 stešević, d., petrović, d.. preliminary list of plant invaders… 36 considering habitat types, many invasive species seem to do best in urban und urban-fringe environments where long histories of human disturbance have created vacant niches and abundant bare ground. cities also tend to be the focal points of the global economy and the entry points for many invasives (k o w a r i k 1990). pathways, as the process by which alien species are introduced from one location to another are different and it is possible to distinguish six ways: i) deliberate release, ii) escape (from gardens, aquaculture), iii) contamination, iv) stowaway, v) corridor (transportation infrastructure), vi) unaided from neighboring countries (the natural spread of an alien species from another region where it is not native) (h u l m e et al. 2008). compared to the other pathways ornamental horticulture is therefore the main pathway for plant invasions worldwide (h u l m e et al. 2008), although its importance compared to other pathways has found the attention of ecologists rather late (m a c k 1991; r e i c h a r d & w h i t e 2001; m a c k & e r n e b e r g 2002). according to j e n k i n s (1996); l e v i n e & d ' a n t o n i o (2003) an increasing amount of species inadvertently introduced into areas outside their natural range is directly linked with increases in trade. due to specific history, long period of isolation, low level of industrial and economic development, bad road infrastructure as well as international commerce and travel, montenegro for a long time hesitated to introduction of alien species. but, in last last decade fast urban, trade and tourism development facilitated their spread. although alien flora of montenegro has never been systematically explored, first records of some alien species dates from the beginning of xix century (p a n č i ć , 1875, p a n t o c z e k , 1874 etc.). these historical notes consist only of information about the presence of a species, so population size or ecological impact of its spreading on natural communities was not documented. during the field excursions conducted in last decade, some interesting results in monitoring of selected alien species are obtained. in this paper we are presenting these results and giving an overview of a species that should be considered as invasive in montenegro. in addition we are giving taxonomic, biologic and geographic analysis of invasive alien flora. material and methods ! preliminary check list of invasive alien species (ias) is mainly created according to authors own field observations, while the list of alien species is extracted from: r o h l e n a (1942), p u l e v i ć (2005) and s t e š e v i ć et al. (2008). invasiveness of species is estimated according to r i c h a r d s o n et al. (2000). life forms follows raunkier’s system (r a u n k i e r , 1943, e l l e n b e r g et al., 1967), while origin of species is given according to p i g n a t t i (1982), pyšek et al. (2002), t u t i n et al. (1964-1980, 1993). results and disscusion due to the to the sheme proposed by r i c h a r d s o n et al. (2000), p y š e k et al. (2004), p y š e k & r i c h a r d s o n (2004), 50 taxa of alien plants of montenegro can be classified as invasive. the list of ias is given in table 1, while one taxa from the list carpobrotus edulis is for the first time reported for alien flora of montenegro. first finding of this species dates from 2006, when rather poor population was noted near the side shore in bijela (boka kotorka bay). in the meantime species is recorded on numerous locations near the coast, where compose rather dense patches. the list of ias of montenegro consists of 50 species and subspecies belonging to 36 genera and 20 families. majority of families (18), genera (31) and species/subspecies (42) belongs to dicotyledons, while other representative belongs to monocotyledons. it is not surprising that two of the largest families worldwide poaceae and asteraceae (h e y w o o d , 1993) provide the highest number of invasive alien species in montenegro: asteracee (16), poaceae (7) (tab.2). similar to the world’s list of leading invaders families (see c r a w l e y , 1987, w e b e r , 1997, p y š e k , 1998) fabaceae and brassicaceae are near to the top. due to c r o n q u i s t (1981) asteraceae is considered as one of the evolutionarily most advanced families. possessing a number of features advantageous in the invasion process, e.g. high reproductive rate, specialized dispersal structures, diversity of metabolic products providing protection from grazing, high level of apomixes etc. (h e y w o o d , 1989, p y š e k , 1997). similarly, successful dispersal mechanisms in poaceae and fabaceae, together with a highly evolved inflorescence in the former, and an ability to fix atmospheric nitrogen as well as remarkably successful pollination system in the latter may serve to explain why these families among the world’s leading invaders. advanced features that might be considered as a clue for successful invasion are: high reproductive rate, long biologica nyssana 1 (1-2) december 2010: 35-42 stešević, d., petrović, d.. preliminary list of plant invaders… 37 table 1. list of ias in montenegro: taxonomy, biology, origin, first record and habitat type. legend: p phanaerophytes, chchamaephytes, hhemicriptophytes, ttherophytes, ggeophytes, namnorth america, samsouth america, cam & samcentral and south america, asasia, afrafrica, easeuroasia. taxon family life form origin 1st record/author habitat type acer negundo l. aceraceae p nam 2005 (steševič&jovanović) riverine forest, ruderalis/mainly waste places ailanthus altissima (mill.) swingle simaroubaceae p as 1971 (popović & sterniša) ruderalis/widespread, vegetation near the road sides, railways, waste places etc., shrubland of carpinus orientalis, rocklands alcea rosea l. malvaceae h as 1900 (baldacci) ruderalis/vegetation near the road sides, railways, waste places, etc. amaranthus hybridus l. amaranthaceae t nam 2005 (steševič&jovanović) city lawns, ruderalis/vegetation near the road sides, waste placeas, cultivated soils amaranthus retroflexus l. amaranthaceae t nam 1875 (pančić) ruderalis/wate places, vegetation near the road sides, cultivated land , city lawns … ambrosia artemisiifolia l. asteraceae t nam 2009 (stešević) ruderalis/vegetation near the road sides, gravely river banks, city lawns amorpha fruticosa l. fabaceae p nam 1973 (pulević) river banks, sandy dunes artemisia verlotiorum lamotte asteraceae g as 2006 (stešević & jogan) ruderalis/ vegetation near the road sides, railways, waste places …. asclepias syriaca l. asclepiadaceae g as 2005 (steševič&jovanović) wet meadows aster squamatus (spreng.) hieron asteraceae t cam, sam 2004 (hadžiablahović) ruderalis/ vegetation near the road sides and railways, waste places.. broussonetia papyrifera (l.) vent. moraceae p as 2005 (steševič&jovanović) ruderalis/ waste places, vegetation near the road sides, roofs … bidens subalternans dc. asteraceae t sam 1993 (trinajstić) ruderalis/ waste places, vegetation near the road sides, railways etc. bidens frondosa l. asteraceae t nam 2005 (steševič&jovanović) river banks, rudelralis carpobrotus edulis (l.) n.e.br. aizoaceae ch afr 2006 stešević (leg.) ruderalis/vegetation near road side, costal rocks commelina communis l. commelinaceae h as 1997 (karaman) ruderalis/ waste places, city lawns chamomilla suaveolens (pursh.) rydb. asteraceae t as 1986 (vasić) ruderalis, vegetation near the road sides conyza albida willd. asteraceae t sam 2005 (steševič&jovanović) ruderalis/ widespread conyza bonariensis (l.) cronq. asteraceae t sam 2005 (steševič&jovanović) ruderalis/ widespread conyza canadensis (l.) cronq. asteraceae t nam 1874 (pantoczek) ruderalis/ widespread cuscuta caesattiana bertol. cuscutaceae t nam 1949 (černjavski et al.) ruderalis, (sub)mediterranean shrublands and rocklands cuscuta campestris yuncker. cuscutaceae t nam 1997 (karaman) ruderalis, (sub)mediterranean shrublands and rocklands datura stramonium l. solanaceae t nam 1875 (pančić) ruderalis, widespread eleusine indica (l.) gaertn. poaceae t afr 1959/1960 (hodak) ruderalis/ vegetation near the road sides, trampled habitats, city lawns etc. eleusine tristachya (lam.) lam. poaceae t sam 1998 (lakušić) ruderalis/ trampled habitats, city lawns erigeron annuus (l.) pers. asteraceae t nam 1972 stanković-tomić ruderalis/widespread, seminatiral habitats, meadows, shurbalnd erigeron annuus subsp. serpentionalis asteraceae t nam 2005 (steševič&jovanović) ruderalis/widespread, seminatiral habitats, meadows, shurbalnd euphorbia maculata l. euphorbiaceae t nam 1979 (obradović & budak) ruderalis/ trampled habitats euphorbia prostrata aiton. euphorbiaceae t nam 1984 (pulević) ruderalis/trampled habitats galinsoga parviflora cav. asteraceae t nam 1968 (blečić) ruderalis, widespread helianthus xlaetiflorus pers. asteraceae g nam 1980 (obradović) ruderalis/waste places, river banks biologica nyssana 1 (1-2) december 2010: 35-42 stešević, d., petrović, d.. preliminary list of plant invaders… 38 helianthus tuberosus l. asteraceae g nam 2005 (steševič&jovanović) ruderalis/waste places, river banks impatiens parviflora dc. balsaminaceae t as 2010 (steševič & drescher) river bank lepidium virginiacum l. brassicaceae t sam 1936 (rohlena) ruderalis/ vegetation near the road sides and railways, roofs medicago sativa l. fabaceae h as 1874 (pantoczek) widespread oenothera biennis l. onagraceae h nam 1976 (pulević) ruderalis oenothera glazioviana micheli onagraceae h nam 2008 (rakaj & krzysztof) sandy dunes, ruderalis oenothera fallax renner et rostanski onagraceae h nam 2008 (rakaj & krzysztof) sandy dunes, ruderalis opuntia vulgaris mill. cactaceae h nam 1930 (in pulević 2005) mediterannean rocklands, vegetation near the roadside paspalum dilatatum poir. poaceae h sam 1986 (ilijanić & topić) ruderalis,/trampled habitats, lawns, wet meadows paspalum paspaloides (michx.) schribn. poaceae h cam, sam 1949 (černjavski et al.) ruderalis, city lawns, wet meadows reynoutria japonica houtt. polygonaceae g as 2005 (stešević&jovanović) natural forests, river banks, ruderalis/vegetation near the road sites robinia pseudacacia l. fabaceae p nam 1911 (rohlena) natural forests, river banks, ruderalis/vegetation near the road sites solanum elaeagnifolium cav. solanaceae h sam 2003 (hadžiablahović et al.) ruderalis, wet meadows sorghum halepense (l.) pers. poaceae h eas 1874 (pantoczek) widespread sporobolus poirettii (r.et s.) hitche poaceae h nam 1998 (niketić in greuter) ruderalis, vegetation near the road sides, city lawns sporobolus vaginiiflorus (torrey) wood poaceae t nam 2006 (stešević & jogan) ruderalis/ vegetation near the road sides, city lawns tamarix dalmatica baum. tamaricaceae p afr 1942 (rohlena) river banks, sandy dunes veronica persica poir. scrophulariaceae t as 1900 (baldacci) widespred xanthium strumarium l. italicum (moretti) d.löve asteraceae t nam 1900 (horak) ruderalis, sandy dunes xanthium spinosum l. asteraceae t sam 1874 (pantoczek) ruderalis, widespread viability of seeds or c4 photosynthetic pathways like in amaranthaceae (c r o n q u i s t , 1981, h e y w o o d , 1989). majority of families (13) are represented with only one species (tab. 2). genera represented with highest number of ias are conyza (3) oenothera (3), erigeron (2), euphorbia (2), amaranthus (2), bidens (2), xanthium (2), eleusine (2), paspalum (2), sporobolus (2). up to now only two subspecies of erigeron annuus are recorded in the flora of montenegro: ssp. annuua and ssp. serpentionalis, but considering the fact that third ssp. strigosus is reported for the flora of neighboring countries (croatian i k o l i ć (2010), serbiaj o v a n o v i ć (1994)) we expect to find it soon, so taxonomic spectrum of genera will look a bit different. as it was expected considering life forms therophytes shows predominance with 26 species fig. 1. they are followed by hemicriptophytes (12), than phanaerophytes (6), geophytes (5) and chamaephytes (1). hydrophytes are not present at all. feature that are enabling therophytes to become very successful invaders are short life cycle (annuals), high reproductive rate, easily dispersed seeds etc. due to its native range, exactly half of ias originates from north america (25), while second dominant are asian (11) (fig.2). these species were mainly introduced as ornamental plants (robinia pseudoacacia, acer negundo, ailanthus altissima, reynoutria japonica, etc.) and very soon they started to escape into the wild, invading native ecosystems with disastrous results and become invasive species. from the other side, greatly improved transport that enables traders to move goods from distant destinations provided ideal opportunities for the accidental introduction of ais (paspalum spp., eleusine spp.). considering the patterns of habitat invasion, our results are similar to c h y t r ý et al. (2008). despite large differences in species, it is possible to notice regularity in the patterns of habitat invasion. a general trend is: in harsh climatic conditions and nutrient-poor habitats, invasion levels are low. alien biologica nyssana 1 (1-2) december 2010: 35-42 stešević, d., petrović, d.. preliminary list of plant invaders… 39 plants tend to thrive in nutrient-rich and man-made habitats. mountains, cliffs, bogs, dry grasslands and coniferous woodlands tend to resist alien invasion, while coastal and riverine habitats, where nutrient availability and disturbance can be high, are more prone to invasion by alien plants. human made habitats such as farmland and urban landscapes also facilitate the spread of alien plants. table 2. taxomonic spectrum of families of ias in montenegro family no of taxa % asteraceae 16 32 poaceae 7 14 fabaceae 3 6 brassicaceae 3 6 onagraceae 3 6 amaranthaceae 2 4 cuscutaceae 2 4 euphorbiaceae 2 4 aceraceae 1 2.0 aizoaceae 1 2.0 asclepiadaceae 1 2.0 balsaminaceae 1 2.0 cactaceae 1 2.0 commelinaceae 1 2.0 malvaceae 1 2.0 moraceae 1 2.0 polygonaceae 1 2.0 scrophulariaceae 1 2.0 simaroubaceae 1 2.0 solanaceae 1 2.0 tamaricaceae 1 2.0 due to the favorable climate and higher level of disturbance mediterranean part of the country hosted significantly higher number of invasive species than continental-mountainous. ias typical for mediterranean part of the country are: carpobrotus edulis, opuntia vulgaris, oenothera glazioviana, o. fallax, conyza bonariensis, c. albida, aster squamatus, asclepias syriaca, acer negundo, amaranthus hybridus, lepidium virginicum, bidens subalternans, helianthus tuberosus, h. laetiflorus, xanthium spinosum, eleusine indica, e. tristachya, sporobolus poiretti, s. vaginiiflorus, commelina communis, solanum elaeagnifloium, tamarix dalmatica, amorpha fruticosa, paspalum paspaloides, p. dilatatum. up to now only one species (impatiens parviflora) has distribution restricted only to continental part of the country (riverine forests). this is explained by its ecological preferences of species: very susceptible to water stress and a shade tolerant, mostly found at 5-40% relative daylight, prefer nitrogen rich stands, with ph range between 4.5 and 7.6 (c o m b e , 1956). fig. 1. structure of invasive alien flora with respect to life forms. 52% 24% 12% 10% 2% t h p g ch fig. 2. structure of invasive alien flora with respect to origin. 50% 16% 4% 22% 6% 2% nam sam sam&cam as afr eas other species, such as robinia pseudoacacia, erigeron annuus, amaranthus retroflexus, veronica persica, xanthium strumarium ssp. italicum, ailanthus altissima, reynoutria japonica are distributed in both part of the country. considering ailanthus and reynoutria, at the beginning of our field study we noticed that first species mainly invaded mediterannean part of the country, while the second one is restricted to continental part. but during the last year both species started to move its borders. along the roadsides ailanthus moved towards the north and reynoutria along roadsides and riverbanks towards the south. success of this “movement” is strongly supported by increase of a disturbance. as it can be seen in table 1, majority of ias are for the first time recorded in our flora in last few decades, but we can’t exactly state when these species were really introduced, by which pathways and when expansion of its populations has really started. with this study we intended to do a zero stage for a future monitoring of ias, but also to draw attention to the problems which expansion of ias is bringing with itself. biologica nyssana 1 (1-2) december 2010: 35-42 stešević, d., petrović, d.. preliminary list of plant invaders… 40 majority of species listed above are also recognized as invasive in our first neighboring mediterranean country croatia (b o r š i ć , 2008). conclusion preliminary list of ias in montenegro consists of 50 flowering plants belonging to 36 genera, 20 families and 2 classes. carpobrotus edulis is for the first time reported for montenegro. the highest number of ias has families asteraceae and poaceae, while between genera dominant are conyza and oenothera. in the spectrum of life forms therophytes are prevailing, while due to its native range, majority of ias has north american and asian origin. considering the patterns of habitat invasion, highly disturbed, man made and nutrient rich habits are more prone to invasion by alien plants than natural and nutrient-poor habitats. due to the favorable climate and higher level of disturbance mediterranean part of the country is more favorable for plant invaders than continentalmountainous. the aim of this study was to provide a basic data on ias in montenegro, to enable future monitoring and to draw attention on the problems which expansion of ias is bringing with itself. references baldacci, a. 1900: controbutio la conoscenza della flora montenegrino-albanese. memoria letta ala r. accademia delle scienze dell'instituto di bologna, p.1-43. blečić, v., tatić, b. & krasnići f. 1968: kratak prilog flori jugoslavije. bulletin de l'l institit et du jardin botaniques de l'université de begrad, 1965/1966, 3(1-4): 227-232. boršić, i, milović, m., dujmović, i., bogdanović, s., cigić, p., rešetnik, i, nikolić, t., mitić b. 2008: preliminary check list of invasive alien plant species (ias) in croatia, natura croatica, 17(2): 55-71. crawley, m.j. 1987: what makes community invasible? in: gray, a.j. crawley m.j., edwards, p.j. (eds), colonization, succession and stability, pp. 429-454, blackwell, oxford. chytrý, m., maskell, l.c., pino, j. 2008: habitat invasions by alien plants: a quantitative comparison among mediterranean, subcontinental and oceanic regions of europe. journal of applied ecology, 45: 448-458. coombe, d.e. 1956: impatiens parviflora dc. journal of ecology 44: 701-713 cronquist, a. 1981: a integrated system of classification of flowering plants, columbia university press, new york. černjavski p., grebenščikov o. & pavlović z. 1949: o vegetaciji i flori skadarkog područja, glasnik prirodnjackog muzeja srpske zemlje, ser. b (1, 2): 4-91. ellenberg, h. & d. mueller-dombois 1967: a key to raunkiaer plant life forms with revised subdivisions, ber. geob. inst. eth rübel, 37: 56-73. greuter w. & raus t. 1998: med-checklist notulae 17, wildenowia 28: 163-174. hadžiablahović, s., karaman v., bulić z. 2003: solanum elaeagnifolium cav, a new neotophyta in the flora of montenegro, 2nd congress of ecologists of the republic of macedonia with international partitipation, ohrid, pp. 145. hadžiablahović, s. 2006: floristic and chorological additions to the vascular flora of montenegro. in: pešić, v. & hadžiablahović, s. (eds), proceedings of the symposium, ii international symposium of ecologists of montenegro, p. 93101. heywood, v.h. 1989: patterns, extents and modes of invasions by terrestrial plants, in: drake, j.a. et al. (eds): biological invasions, a global persepctive, wiley, chichester, p. 31-60 heywood, v.h. 1993. flowering plants of the world, oxford university press, ny hodak n. 1959/1960: nalazište tropske vrste eleusine indica gaertn. u flori jugoslavije.acta botanica croatica, 28-29: 65-67 horak 1900: zweiter beitrag zur flora montenegro’s, österraichische botanische zeitschrift, 50: 156-164 & 208-212 hulme, p., bacher, s., kenis, m. 2008: grasping at the routes of biological invasions: a framework for integrating pathways into policy. journal of applied ecology. 45(2):403-414. ilijanić, lj. & topić, j. 1986: paspalum dilatatum poiret., a new adventitious plant in the flora of yugoslavia. acta botanica croatica, 45: 141144. invasive species specialist group 2005: iucn guidelines for the prevention of biodiversity loss caused by alien invasive species, iucn, gland, switzerland jovanović, s. 1994: ekološka studija ruderalne flore i vegetacije beograda, biološki fakultet univerziteta u beogradu. karaman, v. 1997: flora istočnog dela bokokotorskog zaliva, msc thesis, faculty of biology, university in belgrade biologica nyssana 1 (1-2) december 2010: 35-42 stešević, d., petrović, d.. preliminary list of plant invaders… 41 kowarik, i. 1990: some responses of flora and vegetation to urbanization in central europe, in sukopp et al. (eds.): urban ecology, p, 45-74, spb academic publ, haque levine, j.m., d’antonio, c.m. 2003: forecasting biological invasions wuth increase international trade. conservation biology 17: 322-326 mack, r.n. 1991: the commercial seed trade: an early disperser of weeds in the united states. economic botany 45, 257-273 mack r.n., erneberg m. 2002: the united states naturalized flora: largely the product of deliberate introductions. annals of the missouri botanical garden 89, 176-189 mcneely, j.a. 1999: the great reshuffling: how alien species help feed the global economy. in: sandlund o.t. et al. (eds.) invasive species and biodiversity management, pp. 11-31, kluwer mcneely, j. 2001: invasive species: a costly catastrophe for native biodiversity, land use and water resources research, 2: 1–10 millennium ecosystem assessment 2005: ecosystems and human well-being: biodiversity synthesis, world resources institute, washington, dc, 86 pp. nikolić, t. 2010. flora croatica database, on-line: (http://hirc.botanica.hr/fcd). department of botany, faculty of science, univeristy of zagreb obradović, m. & budak, v. 1979: prolog flori herceg-novog. boka, 10(2): 107-121 obradović, m. 1980: prilog adventivnoj flori okoline herceg-novog, boka, 12: 203-212. pantoczek, j. 1874: adnotationes ad floram et faunam hercegovinae, crnagorae et dalmatiae, verhanndlunen des vereins fuer naturkunde zu presburg, 2: 1-143. pančić, j. 1875: elenchus plantarum vascularium quas aestate 1873 in crna gora legit dr. j pančić, pp. 1-106. pignatii, s. (1982): flora d’italia 1-3, edagricole. popović, d. & sterniša a. 1971: flora i vegetacija hercegnovskog područja. herceg novi. pulević, v. (1973): prilog flori crne gore, glas. republ. zavoda zast. prirode-prirodnjackog muzeja titograd, 6: 77-83. pulević, v. 1976: neke nove i rijetke biljke u flori cre gore, glasnik republičkog zavoda za zaštitu prirode-prirodnjačkog muzeja titograd, 9: 99-102. pulević, v. 1984. euphorbia prostrata aiton, nova adventivna vrsta za flori jugoslavije. drugi kongres o korovima, osijek: 113-117. pulević, v. 2005: građa za vaskularnu floru crne gore, posebno izdanje republičkog zavoda za zaštitu prirode crne gore, podgorica. pp. 218. pyšek, p. 1997: compositae as invadersbetter than the others? preslia 62: 9-22. pyšek, p. 1998: is there a taxonomic pattern to plant invasions? oikos 82: 282-294. pyšek p., sádlo j., & mandák b. 2002: catalogue of alien plants of the czech republic. preslia, 74: 97-186. pyšek, p., richardson, d.m., rejmánek, m., webster, g., williamson, m. & kirschner, j. 2004: alien plants in check lists and floras: towards better communication between taxonomist and ecologist. taxon, 53: 131-143. rakaj, m. & krzysztof, r. 2008: species from the genus oenothera l., from costal part of albania and montenegro, natura montenegrina 8(3): 163-171. raunkier, c. 1934: life forms of plants and statistical plant geography. clarendon press, oxford. reichard s.h., white p. 2001: horticulture as a pathway of invasive plant introductions in the united states. bioscience 51, 103-113. richardson, d.m., pyšek, p. 2004. what is an invasive species?crop protection compendium. cab international, wallingford ((http:/www.cabicompendium.org/cpc) richardson, d.m., pyšek, p., rejmánek, m., barbour m.b., panetta, f.d., west, c.j. 2000: naturalization and invasion of alien plants: concept and definitions, diversity and distributions, 6: 93-107. rohlena, j. 1912: fünfter beitrag zur flora montenegro, sitzungsberichte der könig. böchemischen gesellschaft der wissenschaften in prag, 1-143. rohlena, j.1936: elfter beitrag zur flora von montenegro und (macedonien), sitzungsberichte der könig. böchemischen gesellschaft der wissenschaften in prag, 1:21, prag rohlena, j. 1942: conspectus florae montenegrinae, preslia 20-21. stanković-tomić, k. 1972: flora lovćena ii, zbornik filosofskog fakulteta u prištini 8, 1-50. stešević, d. 2005: contribution to the knowledge on the invasive species in the flora of montenegro. in brunel, s. (ed), proceedings from international workshop "invasive plants in mediterranean type regions of the world", meze. stešević, d. & jovanović s. 2005: contribution to the knowledge of non indigenous flora of montenegro. in: terzić, s. (ed.), proceedings of the workshop devoted to 25th anniversary of the faculty of sciences and mathematics, p. 65-78, university of montenegro. biologica nyssana 1 (1-2) december 2010: 35-42 stešević, d., petrović, d.. preliminary list of plant invaders… 42 stešević, d. & jogan, n. 2006: two new neophytes in the flora of montenegro: artemisia verlotiorum and sporobolus vaginiflorus, natura montenegrina, 5: 173-175. stešević, d. & jogan, n. 2007a: additions to the flora of montenegro: setaria verticilliformis dumort., setaria viridis (l.) pb. subsp. pycnocoma (steud.) tzvel., impatiens balsamina l. and catalpa bignoniodes walt., natura montenegrina, 6: 153-160. stešević, d. jovanović, s. 2008: flora of the city of podgorica, montenegro (taxonomic analysis), archives of biological sciences, 60 (2): 245-253 stešević, d., bubanja, n., vuksanović, s., petrović, d. bulić, z. & biberdžić, v. 2008: contribution to the flora of montenegro, natura montenegrina, 7(2): 605-631. stešević, d., jovanović, s. & šćepanović, s. 2009: flora of the city of podgorica, montenegro a chorologic structrure, and comparative analysis with the floras of roma, patras and thessaloniki", archives of biological sciences, 61 (2): 307-315. stešević, d. 2009: ekološko-fitogeografska studija flora šireg urbanog područja podgorice, phd thesis, biološki fakultet, univerzitet u beogradu. stešević, d. drescher, a. 2010: additions to the flora of montenegro, natura montenegrina 9(3), in press. trinajstić, i. 1993: bidens subalternans dc. u neofitskoj flori hrvatske. acta botanica croatica, 52: 107-112. tomović, i. & stešević, d. 2007: duchesnea indica (andr.) focke, new alien species in the flora of montenegro, natura montenegrina, 6: 161-163. tutin, t.g., heywood, v., burges, n.a., moore, d.m., valentine, d.h., walters, s.m.& webb, d.a. 1964-1980: flora europaea 1-5, university press, cambridge. tutin, t.g., heywood, v., burges, n.a., moore, d.m., valentine, d.h., walters, s.m.& webb, d.a. 1993: flora europaea 1, university press, cambridge. vasić, o. 1986: chamomilla suaveolens (pursh) rydb. 1916 (asterales, asteraceae)nova vrsta u flori crne gore.vii kongres biologa jugoslavijerez. ref. (budva), p. 203 walker b.h., steffen w. 1997: an overview of the implications of global change for natural and managed terrestrial ecosystems. conservation ecology 1, http://www.consecol.org/vol1/iss2/art2/index.ht ml (online journal) weber, e.f. 1997: the alien flora of europe: a taxonomic and biogeographic overview? journal of vegetation science, 8: 565-572. microsoft word bn-oa-0201-02 pehadjieva k biologica nyssana 2 (1) september 2011: 19-28 pechadjieva k. distribution of calthion palustris tüxen 1937… 19 original article ! distibution of calthion palustris tüxen 1937 in eninska river basin, central stara planina mountain kalina pachedjieva* sofia university “st. kliment ohridski” –faculty of biology, department of ecology and environmental protection, 8 dragan tzankov blvd, 1164 sofia, bulgaria * e-mail: kalina.pachedjieva@biofac.uni-sofia.bg abstract: pachedjieva, k.: distibution of calthion palustris tüxen 1937 in eninska river basin, central stara planina mountain, biologica nyssana, 2 (1), september 2011: 19-28. during a phytocoenological investigation of eninska river basin in central stara planina mountain the wetland vegetation proved to be an element of calthion alliance. this conclusion is made on the basis of cluster analysis applied by the computer program syn-tax and a detailed review of species composition of the analyzed relevés with the respective comparisons. the alliance is presented by the central european ass. filipendulo ulmariae-menthetum longifoliae. the balcan influence over the association is apparent from the balcan endemic species angelica pančičii and dactylorhiza incarnata. the association is dependent on water sufficiency and unifies mentha longifolia grasslands on nutrient soils with slightly acidic to slightly basic reaction. it is anthropogenically influenced. some stands form mentha longifolia and urtica dioica community. key words: calthion, central stara planina mountain, cluster analysis introduction ! vegetation of calthion palustris tüxen 1937 alliance has been profoundly investigated in europe by many authors (s ý k o r a , 1982, r a n d j e l o v i ć & z l a t k o v i ć , 1994, h á j k o v á et al., 2006, h á j k o v á , 2008 and others). during the last decade the studies on this type of vegetation in bulgaria according to the principles of braunblanquet phytosociological school also have increased and generalized by tzonev et al. (2009). still, there are gaps in this field especially in the conditions of continuously changing environment. the present research is a part of a phytocoenological investigation of eninska river basin situated on the southern slopes of central stara planina mountain. different types of forest and grass communities form the vegetation cover of the region. the present paper deals only with hygro and mesohygrophyte tall-herb coenosis (wetlands) distributed along and around the river and its tributaries. the aim of the investigation is put in this context: clearing up the syntaxonomy and habitat affiliation of these communities as referred in the available papers and investigations (m u c i n a , 1997; r o d w e l l et al., 2002; h á j e k et al., 2008; t z o n e v et al., 2009 and others). materials and methods object of investigation. eninska river basin covers about 7 000 ha area on the southern slopes of central stara planina mountain (fig. 1). the altitude range it takes is from 500 m a.s.l. (the lowest point) to 1450 m a.s.l. (at the main ridge). shipchenski anticlinorium is the basic geological 2 (1) • september 2011: 19-28 biologica nyssana 2 (1) september 2011: 19-28 pechadjieva k. distribution of calthion palustris tüxen 1937… 20 structure in the region. it is presented by paleozoic granitoids in the core and calcareous rocks in the mantle of triassaic, jurassic and early cretaceous age (j o r d a n o v a et al., 2002). the topographic forms are very rough because of the steep slopes and the deeply indented gorges and tributaries of the river which in its low stream forms the almost vertical and narrow eninsko zhdrelo (enina gorge). warm summer and cold winter characterize the climate of this region complimented by the following features big annual amplitude of temperature, spring-summer and winter maximum of precipitations and yearly stable snow cover (v e l e v , 2002). stara planina mountain is an example of a mountain variant of temperatecontinental climate – colder and damper. temperature decreases and wind, precipitation and clouds increasewith an increase of altitude. figure 1. geographical position of the studied area soil diversity in the region is presented mainly by brown forest soils (eutric cambisols according to fao clasiification) taking predominantly slopes at higher than 700 – 800 m a.s.l. altitudes (n i n o v , 2002). there are different types of vegetation in the investigated region normally distributed along the altitudinal gradient – temperate heathlands and grasslands at lower or higher altitude (festuco-brometea, molinioarrhenatheretea), temperate broadleaved forests and scrubs (querco-fagetea), chasmophytic vegetation and synanthropic vegetation in relation with anthropogenic pressure (m u c i n a , 1997). the basin includes two protected areas within its borders the natural reserve “kamenshtitsa” and the protected site “eninsko zhdrelo” (enina gorge). it is also a part of a nature 2000 protected zone (central balkan buffer). methods of investigation. the methods of the investigation follow the principles of the braunblanquet phytosociological school (w e s t h o f f & m a a r e l , 1973; m u e l l e r -d o m b o i s & e l l e n b e r g ; 1974 and others). the terrain work was carried out during the period from 2003 to 2006. the investigated vegetation is presented by 15 relevés made in presentable and comparatively homogeneous sites according to the requirements of the methodology (k e n t & c o k e r , 1992) at the wet lowest points of the relief. 1 relevé is removed from the analysis as transitional and complex. a cluster analysis is applied using the computer program syn-tax (p o d a n i , 2000). average linkage method (upgma) is used and floristic similarity among relevés is evaluated according to horn’s index. the diagnostic species for the revealed vegetation groups are determined after a detailed review and comparisons with the referred literature. the species determination and taxonomical nomenclature is according to flora of bulgaria (y o r d a n o v (ed.), 1963–1989, k o z h u h a r o v (ed.), 1995) and the field guide to the vascular plants in bulgaria (a n d r e e v et al., 2002). floral elements are determined according to a s s y o v (2006). raunkiaer’s life forms are also indicated (g o r u i s h i n a , 1979). the nomenclature of syntaxa and habitat diversity is according to r o d w e l l et al. (2002). results and discussion the cluster analysis resulted in the differentiation of two comparatively heterogeneous groups, both belonging to calthion palustris tüxen 1937 alliance from molinio-arrhenateretea tüxen 1937 class (fig. 2, tab. 1). the floristic similarity between relevés in the first group consisting of 9 relevés is of 35%. according to some authors (t h e u r i l l a t & m a t t h e y , 1987, t z o n e v , 2002) this is enough in order for the group to be considered one association. dominant species are mentha longifolia and deshampsia caespitosa and co-dominants are filipendula ulmaria and caltha palustris in some relevés. constant species with constancy above iii are lathyrus pratensis, filipendula ulmaria, caltha palustris, carex hirta (tab. 1). these together with ranunculus repens, biologica nyssana 2 (1) september 2011: 19-28 pechadjieva k. distribution of calthion palustris tüxen 1937… 21 myosotis scorpioides, scirpus sylvaticus, juncus effusus, lysimachia nummularia (constancy ii and i) form the characteristic species complex of ass. filipendulo ulmariae-menthetum longifoliae zlinská 1989 described in slovakia (h á j k o v á et al., 2008). calthion vegetation is comparatively well studied in central europe. great diversity at association and subassociation level was registered for slovakia (according to h á j k o v á et al., 2008) where 14 associations were distinguished in calthion alliance. six of them had been described earlier in western carpathians (h á j k o v á et h á j e k , 2005). these are cirsietum rivularis nowiński 1927, chaerophyllo hirsuti-calthetum palustris balátová-tuláčková 1985, angelico sylvestris-cirsietum palustris darimont ex balátová-tuláčková 1973, scirpetum silvatici ralski 1931, angelico sylvestris-cirsietum oleracei tüxen 1937 and scirpo silvatici-cirsietum cani balátová-tuláčková 1973. angelico-cirsietum oleracei, cirsietum rivularis and scirpetum sylvatici were recorded for hungaria (b o r h i d i , 2003) and romanian calthion vegetation (s a n d a et al., 1999). ass. angelico sylvestris-cirsietum oleracei shows some resemblance to calthion communities from central stara planina mountain – it develops on nutrient-rich, mostly alluvial, alkaline soils and forms similar physiognomy with dominant species mentha longifolia and filipendula ulmaria but is distributed at lower altitudes in areas with calciumrich bedrock. besides most of the constant species are absent in central stara planina mountain. ass. scirpetum silvatici has most common distribution in central and southeastern europe. these species poor wet grasslands dominated by scirpus silvaticus and developing on nutrient-rich acidic to alkaline soils (h á j k o v á et al., 2008) are recorded by h á j e k et al. (2005) for vitosha mountain from bulgaria. the central european origin of calthion vegetation is indirectly confirmed by r a n d j e l o v i ć & z l a t k o v i ć (2010). they treat calthion vegetation of vlasina plateau and record three associations equiseto-scirpetum silvaticae šegulja 1974, polygono-scirpetum silvaticae schwick. 1944 and the locally distributed brachythecio-menthetum longifoliae v. randj. 2001. although the euro-asian, boreal and holarctic elements prevail in the phytogeographical specters of the three associations the balkan influence is discernible by balkan endemic species in the composition of polygono-scirpetum silvaticae and brachythecio-menthetum longifoliae. in synecological respect calthion vegetation from vlasina plateau is similar to the same in central stara planina mountain – these are waterlogged wet grasslands on acidic soils with well-developed humus horizons distributed in the subalpine belt. there are more considerable differences in the constant species at association level with calthion communities from central stara planina mountain. it could be concluded that the major group of calthion communities (9 relevés) in eninska river basin shows great resemblance in ecology and species diversity with ass. filipendulo ulmariaementhetum longifolie zlinská 1989 described in slovakia (h á j k o v á et al., 2008). this association unifies grasslands dominated by mentha longifolia on nutrient-rich soils in small alluvia with slightly acidic to slightly basic groundwater reaction as described by h á j k o v á (2008) for the slovakian molinio-arrhenatheretea vegetation. here, it is marked by almost the same complex of constant species. in the context of the habitat differentiation of wet grasslands in bulgaria made by h á j e k et al. (2008) the discussed vegetation group is most likely to be a part of the sub-montane waterlogged grasslands on alkaline soils of calthion wetland vegetation. it also resembles the intermittently deschampsion alliance with diagnostic species alopecurus pratensis, carex otrubae, carex spicata, juncus compressus, potentilla reptans. but most of the diagnostic species of that alliance are either not present or occur with low constancies in the discussed group. the exposed arguments give reason to consider “our” calthion group as an element of the central european filipendulo ulmariae-menthetum longifoliae. in central stara planina mountain however this association is normally modified by the presence of angelica pančičii and dactylorhiza incarnata as balkan floral elements. thus in the present case it could be considered as a balkan variant of the central european association. it takes stands on silicate bedrock at 1200 – 1450 m a.s.l. and predominantly southern slopes of 15–30 degrees inclination. moss layer is poorly presented. the association has comparatively heterogeneous horizontal structure – mosaics are character feature (fig. 3). this is caused by the irregularity of the environmental conditions typical for this type of vegetation. being located on the watersides of the river and its tributaries it is dependent on water sufficiency. actually, mentha longifolia forms different types of communities. s ý k o r a (1982) discusses the syntaxonomy and synecology of juncomenthetum longifoliae lohmeyer 1953 and caricetum vulpinae nowinsky 1927 distributed from the northern part of europe through central to southeastern europe. the author distinguishes conglomerates in these associations on the basis of ecological variations and differences obtained by biologica nyssana 2 (1) september 2011: 19-28 pechadjieva k. distribution of calthion palustris tüxen 1937… 22 figure 2. classification dendrogram of the wetland relevés from eninska river basin made in the computer program syn-tax (podani 2002) figure 3. the heterogeneous structure gives a characteristic appearance of calthion vegetation biologica nyssana 2 (1) september 2011: 19-28 pechadjieva k. distribution of calthion palustris tüxen 1937… 23 table 1. phytosociological table of calthion palustris tüxen 1937 communities in eninska river basin, central stara planina mountain releve number 10 3 5 8 9 4 1 2 6 c on st an cy 11 7 c on st an cy relevé area (m2) 100 100 100 100 100 100 100 100 100 100 100 altitude (m) 1450 1400 1400 1200 1400 1400 1430 1400 1400 1000 1200 aspect s s s s s w s s s e s slope (degrees) 30 30 15 10 15 25 30 20 15 30 15 cover total (%) 100 100 100 100 100 100 100 100 100 100 100 ass. filipendulo ulmariae menthetum longifoliae zlinská 1989 mentha longifolia & urtica dioica com. lathyrus pratensis + + + + + + iv + iii carex hirta + + + + + iii + iii filipendula ulmaria 1 + + 3 3 iii + iii caltha palustris + + 1 2 iii ranunculus repens + + + ii myosotis scorpioides + + ii + iii scirpus sylvaticus + i juncus effusus + i lysimachia nummularia + i mentha longifolia + 2 4 + iii 2 3 v urtica dioica + + + + iii 2 2 v calthion palustris tüxen 1937 deschampsia caespitosa + 1 1 1 3 4 + iv 1 iii equisetum palustre 2 2 2 2 + + iv dactylorhiza saccifera + + + + + + + iv galium palustre + + 1 + + + iv hypericum tetrapterum + + + + + iii + + v ranunculus acris + + + + iii potentilla erecta + + + + iii dactylorhiza incarnata + + + ii 1 iii carex echinata + + + ii + iii molinietalia w. koch 1926 prunella vulgaris + + + + + + + iv + + v carex ovalis + + 1 + + + + iv juncus articulatus + + + ii + iii trifolium pratense + + ii + iii angelica sylvestris + i 2 iii mulgedio-aconitetea hadač et klika in klika 1948 angelica pancicii 1 1 + 1 + + + 2 v alchemilla glabra 1 1 + + + + + + v geum rivale + + + + + + iv molinio-arrhenatheretea tüxen 1937 juncus compressus + 1 ii poa palustris + 2 ii blysmus compressus 1 i carex otrubae + i + iii cynosurus cristatus + i potentilla reptans + i glyceria plicata + i dactylis glomerata + iii rumex crispus + iii galio urticetea passarge ex kopecký 1969 and epilobietea angustifoliae tx. & preising ex von rochow 1951 rubus idaeus + + + + iii rumex alpinus + + ii fragaria vesca + + ii + iii biologica nyssana 2 (1) september 2011: 19-28 pechadjieva k. distribution of calthion palustris tüxen 1937… 24 salix caprea + + ii + iii geum urbanum + i stachys sylvatica + i + + v nardo-callunetea preising 1949, festuco-brometea braun-blanq. et tüxen ex soó 1947, querco-fagetea braun-blanq. et vlieger in vlieger 1937 potentilla recta gr. + i stellaria graminea + i + iii bruckenthalia spiculifolia + i chamaespartium sagittale + i lerchenfeldia flexuosa + i festuca dalmatica + i hypericum perforatum + i lotus corniculatus + i cruciata laevipes + i + iii verbascum longifolium ssp. pannosum + i heracleum sibiricum + i carex sylvatica + i calystegia sepium + i dryopteris filix-mas + i calamagrostis arundinacea + iii digitalis grandiflora + iii clinopodium vulgare + iii geranium rotundifolium + iii acer platanoides + iii others athyrium filix-femina 1 + + + + + iv mentha spicata 2 + 3 1 3 iii inula helenium + + + 1 iii + iii veronica anagallis-aquatica + + + + iii epilobium nutans + + + ii + iii stachys alpina + + ii ranunculus ophioglossifolius + + ii marchantia polymorpha + + ii veronica beccabunga + + ii galium album + i carduus nutans + i + iii senecio rupestris + i glyceria maxima 1 i lapsana communis + i rumex sanguineus + i bromus secalinus + i glyceria fluitans + i carex flava + i eleocharis palustris + i cicerbita alpina + i sagina procumbens + i epilobium alpestre + i arctium lappa 1 2 v equisetum arvense + iii telekia speciosa + iii torilis japonica + iii vicia cracca + iii elymus caninus 1 iii geranium phaeum + iii pyrola rotundifolia + iii biologica nyssana 2 (1) september 2011: 19-28 pechadjieva k. distribution of calthion palustris tüxen 1937… 25 principle component analysis. the described in the present paper association resembles mentha longifolia and hypericum tetrapterum conglomerate in species composition. the last is based on relevés from former czechoslovakia at rather lower altitudes (350 – 800 m a.s.l.) and is notable with its open character. as it was already underlined much more resemblance could be found with the described calthion association equiseto-scirpetum sylvaticae šegulja 1974, subass. deschapsietosum caespitosae randjelović & zlatković 1994 in neighbouring serbia (r a n d j e l o v i ć & z l a t k o v i ć , 1994). again, these comparisons confirm the balkan modification of the accepted central european association. the second group presented in the table and dendrogram (tab. 1, fig. 2) consisting of 2 relevés belongs to the same alliance calthion but is not given a concrete syntaxonomical rank as it is highly influenced by antropophyte elements. thus it is determined as mentha longifolia and urtica dioica community. it indicates habitat degradation and is somehow a “return” in succession. in this community mentha longifolia (constancy v) could be considered as a characteristic species of the nitrophylous alliance agropyro–rumicion crispi nordh. 1940 (t z o n e v , 2009, r a n d j e l o v i ć & z l a t k o v i ć , 2010) together with rumex crispus (constancy iii) and urtica dioica (constancy v). most of the character species for calthion alliance are presented in this community (tab. 1) and it could not be affiliated to agropyro-rumicion crispi at present. future investigations will clarify its succession status. in accordance with the discussion hitherto the following classification scheme for the hygroand mesohygrophyte vegetation in eninska river basin could be proposed: class molinio-arrhenatheretea tüxen 1937 order molinietalia w. koch 1926 alliance calthion palustris tüxen 1937 ass. filipendulo ulmariae-menthetum longifoliae zlinská 1989 mentha longifolia and urtica dioica community in addition, geoelements and raunkiaer’s life forms for the species are indicated. the holarctic group of elements is well presented in calthion communities in the region by the boreal and subboreal geoelement respectively 17% and 15%. it is followed by the palearctic group including euroasian (12%) and eurosiberian elements (9%). european elements are 9%. this distribution corresponds with the central-european origin of calthion vegetation habitat conditions (climate, relief) – lowand mid-mountain. the sub-european group is presented mainly by euromediterranean elements (10%) which together with submediterranean elements (8%) indicates the south-european position of the treated vegetation. kosmopolitan species are 11%. in the biological specter the hemicryptophytes prevail – 52%, followed by the cryptophytes (helophytes) – 35%. the chamaephytes represent 2%. closeness with forest belt is apparent by phanaerophytes which take 4%. the percentage of therophytes, not typical for this kind of vegetation, is comparatively high – 7%. some of them are anthropophytes (arctium lappa, carduus nutans, lapsana communis). conclusions phytosociological analysis showed distribution of calthion alliance in eninska river basin, central stara planina mountain. the alliance is presented by ass. filipendulo ulmariaementhetum longifoliae and mentha longifolia and urtica dioica community. the described association is the optimal wetland vegetation in the subalpine belt of the region. calthion is an azonal vegetation and its structure is determined by nutrient availability, water regime and management practices (f l i e r v o e t & w e r g e r , 1985). being close to the timber-line it is influenced by mulgedioaconitetea hadač et klika in klika 1948 class. the anthropogenic pressure in the region has also influenced the species composition and the condition of calthion communities. this is marked by galio-urticetea passarge ex kopecký 1969 and epilobietea angustifoliae tüxen & preising ex von rochow 1951 species. the presented analysis is based only on species composition of the described communities. additional investigations of this type of vegetation and enlarging the region, for example shipchenska stara planina mountain, are necessary in order to clarify the status and ecological patterns of these communities. acknowledgements. the author is sincerely grateful to dr rossen tzonev for the help with syntaxonomical analysis and interpreting the results. thanks are also extended to evgenia angelova for the corrections of the english text. this research was supported by the national science fund of the ministry of education, science and youth of bulgaria within the project bg051po0013.3.04/41. biologica nyssana 2 (1) september 2011: 19-28 pechadjieva k. distribution of calthion palustris tüxen 1937… 26 references андреев, н, анчев, м., кожухаров, с., маркова, м., пеев, д., петрова, а. 1992: определител на висшите растения в българия (field guide to the vascular plants in bulgaria). издателство „наука и изкуство”, софия. 788 стр. (in bulgarian) велев, ст. 2002. атмосферно налягане и вятър (atmospheric pressure and wind). в: копралев, и. (ред.) география на българия. физическа и социално-икономическа география. форком, софия: 149-150 (in bulgarian) горышина, т. 1979. экология растений (plant ecology). „высшая школа”, москва. 368 стр. (in russian) йорданов, д. (ред.). 1963–1989. флора на народна република българия (flora of the people’s republic of bulgaria). t. 1-9. с., издателство на бан (in bulgarian) йорданова, м., велев, ст., дреновски, и. 2002: старопланинска област (stara planina mountain region). в: копралев, и. (ред.) география на българия. физическа и социално-икономическа география. форком, софия: 401–402 (in bulgarian) кожухаров, с. (ред.). 1995. флора на република българия (flora of republic of bulgaria). т. 10. софия, издателство на бан. 428 с. (in bulgarian) нинов, н. 2002. почви (soils). в: копралев, и. (ред.) география на българия. физическа и социално-икономическа география (geography of bulgaria. physical and socio-economical geography). форком, софия: 277–311 (in bulgarian) цонев, р. 2002: флора и растителност на средна дунавска равнина между долините на реките вит и студена (flora and vegetation of the middle danubian plain between the valleys of vit and studena rivers). дисертация. биологически факултет, су „св. климент охридски” (in bulgarian, unpublished) assyov, b., petrova, a. (edit.). 2006: conspectus of the bulgarian vascular flora. bulgarian biodiversity foundation. 454 p. borhidi a., 2003. magyarország növénytársulásai (plant communities of hungary). akadé miai kiadó, budapest. 610 p. (in hungarian) fliervoet, l., werger m. 1985. vegetation structure and microclimate of three dutch calthion palustris communities under different climatic conditions. vegetatio, 59: (1/3): 159-169 hájek, m., hájková, p., apostolova, i. 2008. new plant associations from bulgarian mires. phytologia balcanica, 14 (3): 377-399 hájek, m., hájková, p., sopotlieva, d., apostolova, i., velev, n. 2008: the balkan wet grassland vegetation: a prerequisite to better understanding of european habitat diversity. plant ecology, 195: 197-213 hájek, m., tzonev, r., hájková, p., ganeva, a., apostolova, i. 2005. plant communities of the subalpine mires and springs in mt vitosha. phytologia balcanica, 11(2): 193-205 hájková, p., 2008. calthion palustris tüxen 1937 in: janišová m. et. al., travinnobylinná vegetácia slovenska – elektronický expertný systém na identifikáciu syntaxónov. botanický ústav sav, bratislava, pp. 158-160. neprešlo jazykovou úpravou hájková, p., hájek, m. 2005. diversity of calthion wet meadows in the western part of flysch carpathians: regional classification based on national formal definitions. thaiszia – journal of botany, košice, 15: 85-116 hájková, p., hájek, m., apostolova, i. 2006: diversity of wetland vegetation in the bulgarian high mountains, main gradients and contextdependence of the ph role. plant ecology 184: 111-130 kent, m., coker, p. 1992. vegetation description and analysis a practical approach. john wiley and sons, london. 363 p. mucina, l., 1997: conspectus of classes of european vegetation. folia geobotanica et phytotaxonomica, 32:117–172 mueller-dombois, d., ellenberg, h. 1974. aims and methods of vegetation ecology. wiley, london: 547 p. podani, j. 2002: syn-tax-pc. computer programs for multivariate data analysis in ecology and systematics. scientia publishing, budapest: 53 p. ranđelović, v., zlatković, b. 2010. vegetacija dolinskih livada vlasinske visoravni. in ranđelović, v., zlatković, b.: flora i vegetacija vlasinske visoravni. prirodno-matematički fakultet, univerzitet u nišu. 238-259 ranđelović, v., zlatković, b. 1993/94: vegetacija sveze calthion tx. 1936 u jugoistočnoj srbiji. ekologija, vol. 28-29 (1-2): 19–32 rodwell, j. s., schaminée, j. h. j., mucina, l., pignatti, s., dring, j., moss, d., 2002: the diversity of european vegetation. an overview of phytosociological alliances and their relationships to eunis habitats. national reference centre for agriculture, nature and fisheries, wageningen, nl sanda, v., popescu, a., arçus, m. 1999. revizia critică a comunităţilor de plante din romănia (critical revision on the plant communities in biologica nyssana 2 (1) september 2011: 19-28 pechadjieva k. distribution of calthion palustris tüxen 1937… 27 romania). editura “tilia press international”, constanţa. 143 p. (in romanian) sýkora, k. v. 1982. syntaxonomic status of the junco-menthetum longifoliae lohmeyer 1953, the junco-menthetum rotundifoliae oberdorfer (1952) 1957 and the carucetum vulpinae nowinski 1927. acta botanica neerlandica 31: 391-416 theurillat, j., matthey, e. 1987: le vallon de l’allondon. promenade botanique. introduction a la phytosociologie. edit. des conserv. et jard. botaniq. de la ville de geneve. 196 p. tzonev, r. 2009. plant communities, habitats and ecological changes in the vegetation on the territory of three protected areas along the danube river in: ivanova, d. (ed.), proc. fourth balkan bot. cong., sofia 2006. pp. 321-331. publishing house bulg. acad. sci., sofia tzonev, r., dimitrov, m., rousakova, v. 2009: syntaxa according to the braun-blanquet approach in bulgaria. phytol. balcanica, 15(2): 209–233 westhoff, v., van der maarel., e. 1973. the braunblanquet approach. in: whittaker, r. (ed.). handbook of vegetation science. part 5. ordination and classification of communities. junk, the hague, the netherlands: 617-726 biologica nyssana 2 (1) september 2011: 19-28 pechadjieva k. distribution of calthion palustris tüxen 1937… 28 microsoft word 0403_mihajilov-krstev_et_al biologica nyssana 1 (1-2) december 2010: 95-98 mihajilov-krstev et al. antimicrobial activity of satureja l. essential oils… 95 original article ! antimicrobial activity of satureja l. essential oils against phytopathogenic bacteria erwinia amylovora tatjana mihajilov-krstev1, dragan radnović2, dušanka kitić3 1 university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia 2 faculty of sciences and mathematics, university of novi sad, serbia 3 faculty of medicine, university of niš, serbia * e-mail: nis_mikrobi@yhoo.com abstract: mihajilov-krstev, t., radnović, d., kitić, d.: antimicrobial activity of satureja l. essential oils against phytopathogenic bacteria erwinia amylovora. biologica nyssana, 1 (1-2), december 2010: 95-98. in this paper, antimicrobial activity of s. kitaibelii wierzb. ex heuff., s. montana ssp. montana l., s. adamovicii šilić and s. fukarekii šilić has been investigated. during the testing, two methods were used: disc-diffusion and broth micro-dilution. the results showed high sensitivity of this bacteria to all four essential oils. the essential oil of s. montana ssp. montana exhibited the largest inhibition zone (25 mm), while s. adamovicii showed the highest inhibitory and bactericidal activity (mic=mbc=0.09 μlml-1). all investigated oils showed the same values for mic and mbc, which means that the oils posses bactericidal effect at very low concentrations and they could be used as non-harmful source of bactericides. key words: genus satureja l., essential oils, antimicrobial activity, erwinia amylovora introduction ! erwinia is a genus of enterobacteriaceae bacteria containing mostly plant pathogenic species which was named for the first phytobacteriologist, erwin smith. a well-known member of this genus is the species e. amylovara, which causes fireblight on apple, pear, and other rosaceous crops. this species produce enzymes that hydrolyze pectin between individual plant cells. this causes the cells to separate, a disease plant pathologists term plant rot. fireblight is a systemic disease. the term "fireblight" describes the appearance of the disease, which can make affected areas appear blackened, shrunken and cracked, as though scorched by fire. sprays of the antibiotics, streptomycin or terramycin can prevent new infections (f a l k e n s t e i n et al., 1998). during the last several decades, natural products with antimicrobial effect are investigated in order to eliminate the use of synthetic antibiotics which cause the resistance of microorganisms and can exhibit side effects to human health. aromatic plants are known for a very long time and they are used in phytotherapy and food preservation (b u r t , 2004). among the aromatic plant species, genus satureja l. occupies a special position. essential oils of satureja l. species showed very significant antimicrobial activity against various species of bacteria and fungi (c i a n i et al., 2000; a z a z et al., 2002; s a h i n et al., 2003; c h o r i a n o p o u l o s et al., 2004; b e ž i ć et al., 2005; s k o č i b u š i ć & b e ž i ć , 2004, 2006; a d i g u z e l et al., 2007; r a z z a g h i -a b y a n e h et al., 2008). in this paper, disc-diffusion and broth microdilution method were used to investigate antibacterial effect of essential oils of four species of satureja l. genus against phytopathogenic bacteria e. amylovora. according to our knowledge, these are the first investigations of essential oil’s effect against this bacterium. considering these 10th sfses • 17-20 june 2010, vlasina lake1 (1-2) • december 2010: 95-98 biologica nyssana 1 (1-2) december 2010: 95-98 mihajilov-krstev et al. antimicrobial activity of satureja l. essential oils… 96 facts, the results of the present study can find application in the pomology as harmless and natural bactericidal agents. material and methods ! plant material the aerial parts of wild growing plant materials of eight satureja species were collected during the beginning of the flowering stage in serbia (s. kitaibelii wierzb. ex heuff.), montenegro (s. monatana l.) and f.y.r.o.m. (s. fukarekii šilić and s. adamovicii šilić). voucher specimens were deposited in the herbarium at the faculty of natural sceince and mathematics of the university of novi sad. extraction of the essential oils air-drying of the plant was performed in a shady place at room temperature for 10 days. dried aerial parts (100 g) of plants, were cut and subjected to the hydro-distillation for 3 h, using clevengertype apparatus. the resulting essential oils were dried over anhydrous sodium sulfate and stored at 4°c. antimicrobial screening the antimicrobial activities of the tested essential oils were evaluated using disc-diffusion and broth micro-well dilution method. all tests were performed in triplicate with two growth controls: ethanol (negative control) and streptomycin (positive control). microbial strain. laboratory control strain erwinia amylovora ncppb 595. disc-diffusion assay. antimicrobial tests were carried out by disc-diffusion method using 100 μl of suspension (containing 2.0 x 108 cfu/ml of bacteria) spread on mueller-hinton agar (mha, torlak) in sterilized petri dishes (90 mm in diameter). the discs (6 mm in diameter, himedia laboratories pvt. limited) were impregnated with 15 μl of oil dilution (2 %, 5 % and 10 %) and placed on the inoculated agar. the inoculated plates were kept at 4 0c for 2 h and incubated at 37 0c for 24h. antimicrobial activity was evaluated by measuring the zone of inhibition against the test bacterial strain. micro-well dilution assay. the inocula of the bacterial strain was prepared from overnight broth culture and suspension was adjusted to 0.5 mcfarland standard turbidity (corresponding to 107108 cfu ml-1) (consensus standard by the nccls). ethanol was used to dissolve the essential oils and then diluted to the highest concentration (500 μl ml-1). a serial doubling dilutions of the oils were prepared in a 96/well microtiter plate over the range of 50.000.02μl ml-1 in inoculated nutrient broth (the final concentration in each well adjusted to 2.0 x 106 cfu ml-1). the plates were incubated for 24 h at 370c. the microbial growth was determined by absorbance at 620nm using the universal microplate reader (thermolabsystems, multiskan ex, software for multiscan ver.2.6.). the highest dilution without growth is the minimum inhibitory concentration – mic. to determine mbc, broth was taken from each well and inoculated in mueller hinton agar (mha) for 24 h at 37 0c. the mbc is defined as the lowest concentration of the essential oil at which inoculated microorganism was 99.9 % killed. statistical analysis of data. analysis of variance (anova) was used to determine the significance (p≤0.05) of the data obtained in all experiments. results and discussion the dominant components of satureja l. essential oils were monoterpenes carvacrol and thymol, p-cymene, limonene and also monoterpenoid alcohols borneol and linallole. considering sesquiterpenes, the most ambudant were β-caryophyllene, caryophyllene–oxide and spathulenol (p a v l o v i ć et al., 1987; s l a v k o v s k a et al., 2001; a z a z et al., 2002; s a h i n et al., 2003; c h o r i a n o p o u l o s et al., 2004; b e ž i ć et al., 2005; s k o č i b u š i ć & b e ž i ć , 2004; a d i g u z e l et al., 2007; r a z z a g h i -a b y a n e h et al., 2008).according to the results of cited authors rich in carvacrol was essential oil of the species satureja montana ssp. montana l. (16.1 – 52.4 %) (p a v l o v i ć et al., 1987; s l a v k o v s k a et al., 2001; s k o č i b u š i ć et al., 2004). monoterpenoidal alcohol borneol was present in high content in the satureja fukarekii šilić (55.0 %) essential oil (p a v l o v i ć et al., 1987). significantly smaller content of borneol was present in the satureja kitaibelii wierzb. ex heuff. (9.8 %) oil, where dominant compounds were the precursors of carvacrol p–cymene (20.9 %) and limonene (16.0 %) (s l a v k o v s k a et al., 2001). in the oil of the species satureja adamovicii, p-cymol was present (40.0 %) together with 1,8-cineole and limonene (35.0 %) (p a v l o v i ć et al., 1987). the results of disc-diffusion testing showed that all tested oils (0.75 μl/disc) ehibited very significant antimicrobial activity against e. amylovora. the oils showed higher activity than the biologica nyssana 1 (1-2) december 2010: 95-98 mihajilov-krstev et al. antimicrobial activity of satureja l. essential oils… 97 tested reference antibiotic streptomycin (38 mm, 30 μl of the active substance/disc). especially high activity showed the oil isolated from s. montana ssp. montana with inhibition zone of 25 mm (fig. 1). fig.1. mean diameter of inhibition zone for satureja essential oils and streptomycin (30 μg/disc) against erwinia amylovora, obtained by disc-diffusion method the results of broth micro-well dilution method confirmed high essential oil activity of four satureja l. species (fig. 2). their mic/mbc values were in the range from 0.09 – 0.18 μl ml-1, which is very good considering the values of the reference antibiotic, streptomycin (mic/mbc = 8.0/16.0 μl ml-1). fig. 2. the mic/mbc values (μl ml-1) of the four satureja essential oils and streptomycin against erwinia amylovora by the broth microdilution method phenolic compound carvacrol, monoterpene limonene and alcoholes linalool and borneol are mostly responsible for the obtained high antimicrobial activity. the most possible mechanism is sinergistic effect of sesquiterpenes like β-caryophyllene and caryophyllene–oxide with other active compounds. beside this, it is wellknown that some compounds do not possess antimicrobial activity, but can enhance the activity of some other antimicrobial compounds by incorporating into the membrane bilayer and enabling the passage of the active compounds. one of these components is p-cymene, which is constituent of allmost all investigated species of the genus satureja l. (lamber et al., 2001; arfa ben et al., 2006). conclusion essential oils of four species from the genus satureja l. showed high antibacterial activity against e. amylowora. that can be explained by high content of monoterpenes and sesquiterpenes, which are well known and many times proved as antimicrobials. these results demonstrated that essential oils of the investigated species could be used as a natural potential antimicrobial agents in prevention and treatment of "fireblight". references adiguzel, a., ozer, h., kilic, h. and cetin, b. 2007: screening of antimicrobial activity of essential oil and methanol extract of satureja hortensis on foodborne bacteria and fungi. chez j food sci., 25: 81-89. arfa ben, a., combes, s., preziosi-belloy, l., gontard, n. and chalier, p. 2006: antimicrobial activity of carvacrol related to its chemical structure. letters in apllied microbiology, 43: 149-154. azaz, d., demirci, f., satil, f., kurcouglu, m. and baser, k.h. 2002: antibacterial activity of some satureja. z naturforsch, 57: 817-821. baser, k.h.c., ozek, t., kirimer, n. and tumen, g. 2004: a comparative study of the essential oils of wild and cultivated satureja hortensis l. jeor, 16: 584-589. bežić, n., skočibušić, m. and dunkić, v. 2005: phytochemical composition and antimicrobial activity of satureja montana l. and satureja cuneifolia ten. essential oils. acta bot croat., 64: 313-322. burt, s. 2004: essential oils: their antibacterial properties and potential applications in food – a 0 2 4 6 8 10 12 14 16 satureja montana satureja kitaibelii satureja fukarekii satureja adamovicii streptomycin μlm 1 0 5 10 15 20 25 30 35 40 satureja montana satureja kitaibelii satureja fukarekii satureja adamovicii streptomycin zone of inhibition in mm biologica nyssana 1 (1-2) december 2010: 95-98 mihajilov-krstev et al. antimicrobial activity of satureja l. essential oils… 98 review. international journal of food microbiology, 94: 223-253. chorianopoulos, n., kalpoutzakis, e., aligiannis, n., mitaku, s., nychas, g.j. and haroutounian, s.a. 2004: essential oils of satureja, origanum, and thymus species: chemical composition and antibacterial activities against foodborne pathogens. j agric food chem., 52: 8261-8267. ciani, m., menghini, l., mariani, f., pagiotii, r., menghini, a. and fatichenti, f. 2000: antimicrobial properties of essential oil of satureja montana l., on pathogenic and spoilage yeasts. biotechnology letters, 22: 1007-1010. dorman, h.j.d. and deans, s.g. 2000: antimicrobial agents from plants: antibacterial activity of plant volatile oils. journal of applied microbiology, 88: 308-316. falkenstein, h., bellemann, p., walter, s. zeller, w., geider, k. 1988: identification of erwinia amylovora, the fireblight pathogen, by colony hybridization with dna from plasmid pea29. applied and environmental microbiology, 54(11): 2798-2802. lamber, r.j.w., skandamis, p.j., coote, g. and nychas, j.e. 2001: a stady of the minimum inhibitory concentration and mode of action of oregano essential oil, thymol and carvacrol. journal of applied microbiology, 91: 453-462. nccls – national committee for clinical laboratory standards. 2000: performance standards for anti-microbial susceptibility testing: eleventh informational supplement. document m100-s11. national committee for clinical laboratory standard, wayne, pa, usa. pavlović, s., živanović, p., jančić, r., todorović, b., ševarda, a.l., kuznjecova, g.a. 1987: the qualitative composition of the essential oil in species of genus satureja l. (lamiaceae) distributed in yugoslavia. biosistematika, 13: 19-24. razzaghi-abyaneh, m., shams-ghahfarokhi, m., yoshinari, t., rezaee, m.b., jaimand, k., nagasawa, h., sakuda, s. 2008: inhibitory effects of satureja hortensis l. essential oil on growth and aflatoxin production by aspergillus parasiticus. international journal of food microbiology, 123: 228–233. sahin, f., karaman, i., gulluce, m., oguta, h., sengul, m., adiguzel, a., ozturk, s. and kotan, r. 2003: evaluation of antimicrobial activities of satureja hortensis l. j ethnopharmacol, 87: 6165. skočibušić, m. and bežić, n. 2004: chemical composition and antimicrobial variability of satureja montana l. essential oils produced during ontogenesis. jeor, 16: 387-391. skočibušić, m. and bežić, n. 2006: phytochemical composition and antimicrobial activities of the essential oils from satureja subspicata vis. growing in croatia. food chemistry, 96: 20-28. slavkovska, v., jančić, r., bojović, s., milosavljević, s. and djoković, d. 2001: variability of essential oil of satureja montana l. and satureja kitaibelii wierzb. ex heuff. from the central part of balkan peninsula. phytochemistry, 57: 71-76. jugović et al. 2023, biologica nyssana 14(1) 14 (1) june 2023: 39-46 doi: 10.5281/zenodo.8027130 the frequency and type of mutations in the egfr gene of colorectal carcinoma patients in southeastern serbia original article dragana jugović laboratory for immunology and genetics, center for medical and clinical biochemistry, university clinical center niš, serbia dragana.jugovic@live.com (corresponding author) marija vukelić nikolić faculty of medicine, research center for biomedicine, university of niš, serbia višnja madić department of biology and ecology, faculty of sciences and mathematics, university of niš, serbia aleksandar milićević pathology and pathological anatomy center, university clinical center niš, serbia perica vasiljević department of biology and ecology, faculty of sciences and mathematics, university of niš, serbia received: february 28, 2023 revised: april 28, 2023 accepted: april 28, 2023 abstract: colorectal carcinoma (crc) represents a global health problem. the egfr signaling pathway is very important in the initiation of different human epithelial cancers and plays a key role in colorectal carcinogenesis. considering the role of egfr as a mitogenic inducer, this study aimed to determine the frequency and type of mutations in the egfr gene and its correlation with clinicopathological characteristics of patients with crc on the territory of southeastern serbia. the genomic dna of patients was isolated from formalin-fixed and paraffin-embedded tissues. the presence of mutations in exons 18, 19, 20, and 21 was determined by the real-time pcr method. the frequency of mutations in the egfr gene in crc patients of southeastern serbia was 7.9%. all detected mutations were g719x. no statistically significant correlation was found between mutational status in the egfr gene and the clinicopathological characteristics of patients. key words: colorectal carcinoma, egfr, mutations apstrakt: učestalost i tip mutacija u egfr genu kod pacijenata sa kolorektalnim karcinomom u jugoistočnoj srbiji kolorektalni karcinom (krk) predstavlja globalni zdravstveni problem. egfr signalni put je veoma važan u inicijaciji različitih humanih epitelnih karcinoma i ima ključnu ulogu u kolorektalnoj kancerogenezi. s obzirom na ulogu egfr-a kao mitogenog inducera, ovo istraživanje imalo je za cilj da utvrdi učestalost i tip mutacija u genu za egfr, kao i hjihovu korelaciju sa kliničkopatološkim karakteristikama pacijenata sa krk-om na teritoriji jugoistočne srbije. genomska dnk pacijenata izolovana je iz tkiva fiksiranih u formalinu i ukalupljenih u parafin. prisustvo mutacija u egzonima 18, 19, 20 i 21 određivano je real-time pcr metodom. učestalost mutacija egfr gena kod pacijenata sa krk-om na teritoriji jugoistočne srbije iznosila je 7,9%. sve otkrivene mutacije bile su tipa g719x. nije pronađena statistički značajna korelacija između mutacionog statusa u egfr genu i kliničkopatoloških karakteristika pacijenata. ključne reči: kolorektalni karcinom, egfr, mutacije introduction colorectal carcinoma (crc) is a malignant epithelial tumor that represents one of the major health problems in the world. namely, it is the third most common cancer in men and the second most common cancer in women, accounting for about 11% of all diagnosed cancers (ferlay et al., 2020). usually, it is diagnosed in the older population, however, recent research shows an increased incidence of crc in the younger population as well (campos, 2017; connel et al., 2017). colorectal carcinogenesis is a gradual process where the clonal accumulation of genetic changes results in the loss of cell cycle control, absence of the cells’ natural response to genomic damage, further accumulation of mutations and, finally, in general genomic instability, which represents the basic characteristic of malignant cells (mármol et al., 2017). the main role in the evolution of crc has the dysregulation of several intracellular signaling pathways such as mitogen-activated protein kinase (mapk) and phosphatidylinositol 3 kinase (pi3k)/protein kinase b (akt) signaling pathway activated by epidermal growth factor receptor © 2023 jugović et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 39 (egfr) bounded with the ligand. dysregulation of these pathways leads to the activation of various transcription factors whose disruption can lead to malignant cell transformation and tumor progression through increased cell proliferation, anti-apoptosis, angiogenesis, and invasion (wee et al., 2017; koveitypour et al., 2019). the egfr gene is located on chromosome 7p12– 13. it encodes a 170-kda transmembrane receptor with an intracellular tyrosine kinase domain. it is a member of the erbb family of receptors that has four closely related members: erbb-1bb-1 (her1/ egfr), erbb-2 (her2), erbb-3 (her3), and erbb-4 (her4). all these receptors contain an extracellular ligand-binding domain, a membranespanning region, and a cytoplasmic region which contains a tyrosine kinase (hirsh, 2018). after epidermal growth factor (egf) ligand binding, egfr forms a functionally active dimer (homodimer or heterodimer) that causes phosphorylation of tyrosine kinases in the intracellular domain of egfr. this phosphorylation initiates different complex intracellular signals in the cytoplasm followed by the activation of a variety of transcription factors responsible for controlling multiple cellular functions including proliferation, differentiation, apoptosis, and cell migration (koveitypour et al., 2019). the presence of mutations in some genes involved in these signaling pathways often represents a negative predictive biomarker of response to current therapy (mármol et al., 2017). the principal mechanism of egfr dysregulation in crc is egfr overexpression. protein upregulation is found in 60-80% of crc, making it an adequate target for anti-egfr therapy (porru et al., 2018). nowadays, in addition to the standard chemotherapy, there are two other therapeutic approaches in the treatment of cancer, i.e., administration of tyrosine kinase inhibitors (tki) and administration of monoclonal antibodies (mabs). although the mechanism of these two therapies is similar, i.e., blocking of the egfr autophosphorylation, the outcome of the therapy may differ depending on the mutational status of egfr. to wit, studies showed that the administration of tki failed to improve the outcome of the treatment in crc patients with egfr mutations in the tk domain located in exons 8,19, 20 and 21 (saletti et al., 2015). on the other hand, the study of cho and associates showed that cell lines that express g719s and g724s mutations in exon 18 are sensitive to the cetuximab monoclonal antibody (cho et al., 2014). thus, patients that are carriers of these mutations might benefit from the use of cetuximab, even though it is still mainly used only in the treatment of metastatic crc patients with wild-type (wt) kras. moreover, although somatic mutations are rare in crc, some of them can cause acquired resistance to anti-egfr therapy (montagut et al., 2012; cho et al., 2014). the most common somatic mutations that lead to the oncogene activation of egfr are missense mutations, insertions, or deletions, mainly in the tyrosine kinase (tk) domain (liu et al., 2020). according to the study of metzger and associates, the frequency of mutations in the egfr tk domain of crc patients in europe is 3.8% in exon 21 (codons 847, 836), 2.6% in exon 18 (codons 707, 710, 711, 712, 725), 0.8% in exon 20 (codons 795, 796, 787), and only 0.5% in exon 19 (codon 742) (metzger et al., 2011). namely, egfr mutants bypass the mechanisms that initiate termination of the signal transduction by avoiding receptor endocytosis and degradation (mosesson et al., 2008). studies have indicated that egfr mutations not only act as oncogenic drivers of crc but can also serve as genomic predictors of response to anti-egfr therapy in crc patients (kim et al., 2020). having in mind such an important role of egfr as a mitogen inducer, the aim of this study was to determine the frequency and type of mutations in the tyrosine kinase domain of egfr in starting (t1/ t2) and final (t4) stages of tumor infiltration and its correlation with clinical characteristics of patients with crc on the territory of southeastern serbia. materials and methods this study investigated the mutational status in egfr of 38 crc patients. the analysis was performed on the 5-10 μm thick formalin-fixed paraffin-embedded (ffpe) sample tissue sections of patients with confirmed crc who were referred to the laboratory for immunology and genetics, the center for medical and clinical biochemistry, university clinical center niš, serbia during 20192021. ethical approval has been obtained from the ethics committee of the clinical center, niš, serbia no 24722/6. the following clinical data were collected for each patient: sex, age, tumor location. stage of tumor infiltration, nodal status, and the presence of distant metastases. the stage of the tumor was determined in accordance with tumor-nodes-metastasis (tnm) classificational system recommended by the american joint committee on cancer staging system (ajcc), where t1 and t2 denote starting stages (t1, when tumor invades submucosa, and t2, when tumor invades muscularis propria) and t4 denotes the final stage of tumor infiltration, when tumor perforates visceral peritoneum and/or directly 40 biologica nyssana ● 14 (1) june 2023: 39-46 jugović et al. ● the frequency and type of mutations in the egfr gene of colorectal carcinoma patients in southeastern serbia biologica nyssana ● 14 (1) june 2023: 39-46 jugović et al. ● the frequency and type of mutations in the egfr gene of colorectal carcinoma patients in southeastern serbia 41 following cycling conditions initial denaturation step at 95 °c for 2 min, then running 40 cycles of 95 °c for 10 seconds/58 °c for 60 seconds. the mutation analysis was carried out in relation to the amplification of positive and negative control tests provided by the manufacturer and according to the included protocol. statistical analysis statistical analysis was done using microsoft excel 2010 (microsoft corporation, usa). results of egfr mutational analysis were used as categorical variables (presence or absence of the mutation). comparison between clinical characteristics and mutational status was done using fisher’s exact test or χ2 test where appropriate. statistical significance was accepted if p<0.05. results this study included samples of 38 patients with crc, collected during 2019-2021. there were 24 male and 14 female patients, aged from 42 to 79 years (tab. 1). of all 38 tested patients, 35 of them (92.1%) did not have any detectable mutations in the egfr. egfr mutations (g719x) were observed in exon 18 in 3 (7.9%) patients. the g719x mutation in egfr refers to point mutations that result in substitutions of the glycine at position 719 to other residues, primarily alanine (g719a), cysteine (g719c), and serine (g719s) (fig. 1). no mutations were detected in exons 19, 20, and 21. two out of the three patients with detected mutations have the t2 stage of crc, located in the left colon, and with no metastases in lymph nodes. the third case was a patient with the t4 stage of the tumour, located in the right colon and with metastases in lymph nodes. mutation in egfr (g719x) was detected in one patient with distant metastases in the lungs, one patient with distant metastases in both lungs and liver, and in one patient with distant metastases in the pelvis. there was no statistically significant correlation between the mutational status in the egfr and the clinical characteristics of patients (tab. 1). discussion in serbia, colorectal carcinoma is the third most common cancer, as well as the second most common cause of cancer-related death, right after lung cancer (ferlay et al., 2020). namely, according to the cancer register for republic of serbia, there were even 4646 new crc cases registered in serbia in 2018. more than a thousand of these new crc cases invades other organs or structures (tong et al., 2018). according to the location, the tumors were divided into the left-sided colon (lcc) and rightsided colon (rcc). lcc included splenic flexure, descending colon, sigmoid, and rectum, while the rcc included caecum, ascending colon, hepatic flexure, and transverse colon. dna isolation genomic dna was extracted from the ffpe tumour tissue sections using a qiaamp dna ffpe tissue kit (ce-ivd-marked; qiagen, hilden, ger many), according to the manufacturer’s protocol. the dna quality was determined with 260/280 optical density (od) ratios in all samples, which were stored at -20°c until use. real-time pcr the analysis of the egfr mutation was performed using the „easy® egfr” kit (diatech pharmacogenetics, italy) with a rotorgene 6000-corbett rt-pcr device (diatech pharmacogenetics, italy), where the list detectable mutations were: egfr exon 18: g719s (2155g>a), g719c (2155g>t), g719a (2156g>c) not distinguishable between them; egfr exon 19: e746_a750del (2235_2249del15), e746_a750del (2236_2250del15), l747_p753>s (2240_2257del18), l747_a750>p (2239_2248ttaagagaag>c), e746_s752>v (2237_2255>t), l747_t751del (2240_2254del15), l747_s752del (2239_2256del18), e746_t751>a (2237_2251del15), l747_t751del (2239_2253del15), l747_t751>p (2239_2251>c), l747_e749del (2239_2247del9), e746_e749del (2235_2246del12), l747_p753>q (2239_2258>ca), l747_t751>s (2240_2251del12), e746_s752>a (2237_2254del18), l747_a750>p (2238_2248>gc), e746_s752>d (2238_2255del18), e746_ t751>i (2235_2252>aat), l747_t751>q (2238_2252>gca), e746_t751del (2236_2253del18) not distinguishable between them. egfr exon 20: v769_d770insasv ( 2 3 0 7 _ 2 3 0 8 i n s g c c a g c g t g ) , d 7 7 0 _ n771insg (2310_2311insggt), h773_v774insh (2319_2320inscac) not distinguishable between them, t790m (2369c>t), s768i (2303g>t); egfr exon 21: l858r (2573t>g), l861q (2582t>a). egfr sequence was amplified using a mixture of 10 μl taq premix, 4 μl double distilled (dd) water, 1 μl mutation primers, and 5 μl dna template. each run included at least one amplification of the negative control (dd water) and one amplification of the positive control („easy® egfr” kit positive control). egfr sequence was amplified using the 42 biologica nyssana ● 14 (1) june 2023: 39-46 were patients from southern and eastern serbia, with 640 males, and 371 females. at the same time, the total number of deaths caused by crc was 479, with 397 male and 182 female patients (institute of public health „dr. milan jovanovic batut”, 2020). a key role in colorectal carcinogenesis has the egfr signaling pathway, which is involved in the initiation of human epithelial cancers. the most common pathological changes of egfr are overexpression of egfr protein and egfr mutations that activate the kinase. based on the hypothesis that a monoclonal antibody against egfr can inhibit receptor tyrosine kinase activity and cancer cell proliferation, proposed in 1980 by dr. john mendelsohn and gordon sato, years of studies led to the discovery of anti-egfr treatments for specific carcinomas such as crc and non-small-cell lung cancer (nsclc) (mendelsohn et al., 2015). it has been observed that egfr-directed therapy such as cetuximab was more successful in crc patients with highly expressed egfr than in patients with low egfr expression (liu et al., 2016). many studies evaluated the connection between over-expressed egfr with a variety of carcinomas such as breast cancer (silva rocha et al., 2021), table 1. correlation between egfr mutation status and clinicopathological parameters of crc patients jugović et al. ● the frequency and type of mutations in the egfr gene of colorectal carcinoma patients in southeastern serbia parameters n=38 egfr status n=38 p value n % mutated wt sex male 24 63.2 2 22 0.89 female 14 36.8 1 13 age at diagnosis ≤50 6 15.8 / 6 51-60 6 15.8 / 6 61-70 18 47.4 2 16 ≥71 8 21.0 1 7 location left colon 23 60.5 2 21 0.82 right colon 15 39.5 1 14 tumor extent t1/t2 14 36.8 2 12 0.26 t4 24 63.2 1 23 nodal status without metastasis 14 36.8 2 12 0.26 with metastasis 24 63.2 1 23 distant metastasis liver 19 50.0 / 19 lung 6 15.8 1 5 liver and lung 5 13.2 1 4 other 8 21.0 1 7 wt-wild type fig. 1. g719x mutated sample and amplification plot detected by „easy® egfr” kit biologica nyssana ● 14 (1) june 2023: 39-46 jugović et al. ● the frequency and type of mutations in the egfr gene of colorectal carcinoma patients in southeastern serbia stomach adenocarcinoma (wang et al., 2017), prostate cancer (hashmi et al., 2019), nsclc (tasdemir et al., 2019), head and neck cancer (barnes et al., 2020). in all these cancers, the level of expression was different with a tendency to increase, but in all cases, egfr expression was recognized as an unfavorable prognostic biomarker of the disease. furthermore, many scientists have been researching overexpression and abnormal activation of the egfr in crc patients. these studies have shown the prognostic value of egfr expression visible as the key factor in the invasion and development of metastasis in crc patients (du et al., 2017; del carmen et al., 2020; uhlyarik et al., 2020). in addition, many studies evaluated the frequency of mutations in the egfr gene in a variety of cancers. for example, it has been observed that in nsclc patients mutations of the gene were highly frequent in exons 19 (over 50%) and less frequent in exons 18 and 20 (8.1% and 3.5%) (skřičková et al., 2020). interestingly, nsclc patients with a mutation in exon 19 had higher overall survival (os) rate compared to nsclc patent with mutations in exons 18 and 20 (syrigos et al., 2018). also, a high frequency of mutations was found in glioblastoma multiforme (26.8%), lung adenocarcinoma (14.4%), while a low frequency of mutations was found in breast invasive carcinoma, kidney renal papillary cell carcinoma, and prostate adenocarcinoma (liu et al., 2020). the results of this study showed that of 38 patients only 3 of them had mutations in the kinase domain of the egfr. two out of these three patients had t2 stage of crc, located on the left side of the colon and the absence of metastasis in lymph nodes. similarly to our results, the study of oh and associates showed that the presence of egfr mutations is more frequent in the earlier stages and the absence of lymph node metastasis (oh et al.,2011). moreover, our results showed that all these three patients had the same mutation, i.e., in exon 18 (g719x). ruhe and colleagues showed that one of the three mutations in exon 18 (g719s) identified in the sw48 colon cancer cell line had oncogenic potential (ruhe et al., 2007). at the same time, according to kim and associates, inhibitors of egfr tyrosine kinases, such as cetuximab and panitumumab, efficiently block the growth of the same cell line with the same mutation (kim et al. 2020, cho et al., 2014). having in mind all these, it might be concluded that crc carriers of egfr mutation in exon 18 might benefit from cetuximab or panitumumab applied as first-line therapy. the incidence of mutated egfr in crc patients included in our study was only 7.9%, while its frequency in the korean population was even 22.41%. on the contrary to the population of southeast serbia, their population had mutations only in exon 20 (oh et al., 2011). additionally, the frequency of mutations of in exon 18 (codon 719) in our study was higher than the frequency of mutations in the same exon in the european population (2.6%, codons 707, 710, 711, 712, 725) (metzger et al., 2011). our results are different from the study of phua and associates who did not detect any mutations of egfr in codons 18, 19, and 21 in a group of 45 patients with crc (phua et al. 2015). similarly, the study that included 31 caucasians did not detect any mutations in exons 18 and 19, while only one of them had a missense heterozygous mutation in exon 21 (gly857arg) (moroni et al., 2005). on the other hand, barber and colleagues found only one carrier of g719s out of 293 examined crc patients (0,34%) (barber et al., 2004), while the study of ogino and associates showed that the incidence of this mutation was 3.33% (1/30) (ogino et al., 2005). in romanian population, the frequency of egfr mutation in exon 19 was 5% (3/60), and it was positively correlated with lymphovascular invasion, perineural invasion, tumor budding of all grades as well as with the invasion of tissues in the proximity of the tumor (liver, visceral, parietal pleura, etc.) (ionescu et al., 2022). in our study, we did not identify any mutation in exon 19. however, we noticed a trend of egfr mutation appearance in the left-sided crc (2/3), while the study of randon and associates observed a statistically important correlation between the amplification of egfr and wt ras/braf in leftsided crc (randon et al., 2021). over 60% of crc patients develop metastases in the liver, while the second most common site of metastasis is the lungs (uhlyarik et al., 2020). the presence of distant metastases greatly reduces the five-year survival rate, and the development of these metastases is associated with the mutation of many genes (tanaka et al., 2019, hao et al., 2022). according to the results of our study, the presence of egfr mutation was detected in patients with distant metastases in both lungs and liver, metastasis only in lungs, and metastasis in pelvis. however, contrary to the study of zou and associates that detected a g735c mutation of egfr in one patient with liver metastasis, we found no mutation in patients with metastasis in the liver only (zou et al., 2018). to the best of our knowledge, this is the first report on the evaluation of frequency and type of mutations in the gene and its correlation with clinicopathological characteristics of patients with 43 biologica nyssana ● 14 (1) june 2023: 39-46 jugović et al. ● the frequency and type of mutations in the egfr gene of colorectal carcinoma patients in southeastern serbia garcía, j., bengoechea, o., muñoz-bellvis, l., sayagués, j.m., & abad, m. (2020). prognostic implications of egfr protein expression in sporadic colorectal tumors: correlation with copy number status, mrna levels and mirna regulation. scientific reports, 10, 4662. du, p., xu, b., zhang, d., shao, y., zheng, x., li, x., xiong, y., wu, c., & jiang, j. (2017). hierarchical investigating the predictive value of p53, cox2, egfr, nm23 in the post-operative patients with colorectal carcinoma. oncotarget, 8(1), 954– 966. ferlay, j., ervik, m., lam, f., colombet, m., mery, l., piñeros, m., znaor, a., soerjomataram, i., & bray, f. (2020). global cancer observatory: cancer today. france, lyon: international agency for research on cancer. hao, m., wang, k., ding, y., li, h., liu, y., & ding, l. (2022). which patients are prone to suffer liver metastasis? a review of risk factors of metachronous liver metastasis of colorectal cancer. european journal of medical research, 27(1), 130. hashmi, a.a., hashmi, s.k., irfan, m. asif, h., nisar, l., naeem, m., khan, e.y., baloch, s., & farid, n. (2019). prognostic utility of epidermal growth factor receptor (egfr) expression in prostatic acinar adenocarcinoma. applied cancer research, 39, 2. hirsh, v. (2018) turning egfr mutation-positive non-small-cell lung cancer into a chronic disease: optimal sequential therapy with egfr tyrosine kinase inhibitors. therapeutic advances in medical oncology, 10, 1758834017753338. institut za javno zdravlje srbije „dr milan jovanović batut”. (2018). malignant tumours in republic of serbia. retrieved from https:// w w w . b a t u t . o r g . r s / d o w n l o a d / p u b l i k a c i j e / malignitumori2018.pdf ionescu, a., bilteanu, l., geicu, o.i., iordache, f., stanca, l., pisoschi, a.m., miron, a., serban, a.i., & calu, v. (2022). multivariate risk analysis of ras, braf and egfr mutations allelic frequency and coexistence as colorectal cancer predictive biomarkers. cancers, 14(11), 2792. kim, n., cho, d., kim, h., kim, s., cha, y.j., greulich, h., bass, a., cho, h.s., & cho, j. (2020). colorectal adenocarcinoma-derived egfr mutants are oncogenic and sensitive to egfr-targeted monoclonal antibodies, cetuximab and panitumumab. international journal of cancer, 146(8), 2194–2200. crc on the territory of southeastern serbia. conclusion the incidence of mutations in the egfr in 38 crc patients of the southeastern serbia was relatively low, and there was no significant correlation between egfr mutational status with clinicopathological characteristics. all detected mutations were g719x. however, we did observe a trend of mutations appearing in the lower stages of tumor infiltration, especially on the left side of the colon without the presence of metastases in the lymph nodes. since our study was conducted on relatively small number of patients, more cohort studies are needed. acknowledgements. this work was supported by the ministry of education, science and technological development of the republic of serbia (grant no. 45103-47/2023-01/ 200124). references barber, t.d., vogelstein, b., kinzler, k.w., & velculescu, v.e. (2004). somatic mutations of egfr in colorectal cancers and glioblastomas. new england journal of medicine, 351-2883. barnes, p., yeboah, f.a., zhu, j., saahene, r.o., obirikorang, c., adinortey, m.b., amoani, b., kyei, f., akakpo, p., & awuku, y.a. (2020). prognostic worth of epidermal growth factor receptor (egfr) in patients with head and neck tumors. journal of cancer epidemiology, 5615303. campos, f.g. (2017). colorectal cancer in young adults: a difficult challenge. world journal of gastroenterology, 23, 5041. cho, j., bass, a.j., lawrence, m.s., cibulskis, k. cho, a., lee, s.n., yamauchi, m., wagle, n., pochanard, p., kim, n., park, a.k.j., won, j., hur, h.s., greulich, h., ogino, s., sougnez, c., voet, d., tabernero, j., jimenez, j., baselga, j., gabriel, s. b., lander, e.s., getz, g., eck, m.j., park, w.y., & meyerson, m. (2014). colon cancer-derived oncogenic egfr g724s mutant identified by whole genome sequence analysis is dependent on asymmetric dimerization and sensitive to cetuximab. molecular cancer, 13, 141. connel, l.c., mota, j.m., braghiroli, m.i., & hoffa, p.m. (2017). the rising incidence of younger patients with colorectal cancer: questions about screening, biology and tretmant. current treatment options in oncology, 18(4), 23. del carmen, s., corchete, l.a., gervas, r., rodriguez, a., garcia, m., álcazar, j.a., 44 45 biologica nyssana ● 14 (1) june 2023: 39-46 koveitypour, z., panahi, f., vakilian, m., peymani, m., seyed forootan, f., nasr esfahani, m.h., & ghaedi, k. (2019). signaling pathways involved in colorectal cancer progression. cell & bioscience, 9, 97. liu, h., zhang, b., & sun, z. (2020). spectrum of egfr aberrations and potential clinical implications: insights from integrative pan-cancer analysis. cancer commun (lond), 40(1), 43-59. liu, j., zhou, q., xu, j., wang, j., & zhang, y. (2016). detection of egfr expression in patients with colorectal cancer and the therapeutic effect of cetuximab. journal of b.u.on. : official journal of the balkan union of oncology, 21(1), 95–100. mármol, i., sánchez de diego, c., pradilla, d.a., cerrada, e., & rodriguez yoldi, m.j. (2017). colorectal carcinoma: a general overview and future perspectives in colorectal cancer. international journal of molecular sciences, 18(1), 197. mendelsohn, j., prewett, m., rockwell, p., & goldstein, n.i. (2015). ccr 20th anniversary commentary: a chimeric antibody, c225, inhibits egfr activation and tumor growth. clinical cancer research, 21(2), 227–9. metzger, b., chambeau, l., begon, d.y., faber, c., kayser, j., berchem, g., pauly, m., boniver, j., delvenne, p., dicato, m., & wenner, t. (2011). the human epidermal growth factor receptor (egfr) gene in european patients with advanced colorectal cancer harbors infrequent mutations in its tyrosine kinase domain. bmc medical genetics, 12, 144. montagut, c., dalmases, a., bellosillo, b., crespo, m., pairet, s., iglesias, m., salido, m., gallen, m., marsters, s., tsai, s.p., minoche, a., seshagiri, s., serrano, s., himmelbauer, h., bellmunt, j., rovira, a., settleman, j., bosch, f., & albanell, j. (2012). identification of a mutation in the extracellular domain of the epidermal growth factor receptor conferring cetuximab resistance in colorectal cancer. nature medicine, 18(2), 221–223. moroni, m., veronese, s., benvenuti, s., marrapese, g., sartore-bianchi, a., di nicolantonio, f., gambacorta, m., siena, s., & bardelli, a. (2005). gene copy number for epidermal growth factor receptor (egfr) and clinical response to antiegfr treatment in colorectal cancer: a cohort study. the lancet. oncology, 6(5), 279–286. mosesson y., mills g.b., & yarden, y. (2008). derailed endocytosis: an emerging feature of cancer. nature reviews. cancer, 8(11), 835–850. ogino, s., meyerhardt, j.a., cantor, m., brahmandam, m., clark, j.w., namgyal, c., kawasaki, t., kinsella, k., michelini, a.l., enzinger, p.c., kulke, m.h., ryan, d.p., loda, m., & fuchs, c.s. (2005). molecular alterations in tumors and response to combination chemotherapy with gefitinib for advanced colorectal cancer. clinical cancer research: an official journal of the american association for cancer research, 11(18), 6650–6656. oh, b.y., lee, r.a., chung, s.s., & kim, k.h. (2011). epidermal growth factor receptor mutations in colorectal cancer patients. journal of the korean society of coloproctology, 27(3), 127–132. phua, l.c., ng, h.w., yeo, a.h., chen, e., lo, m.s., cheah, p.y., chan, e.c., koh, p.k., & ho, h.k. (2015). prevalence of kras, braf, pi3k and egfr mutations among asian patients with metastatic colorectal cancer. oncology letters, 10(4), 2519–2526. porru, m., pompili, l., caruso, c., biroccio, a., & leonetti, c. (2018). targeting kras in metastatic colorectal cancer: current strategies and emerging opportunities. journal of experimental & clinical cancer research: cr, 37(1), 57. randon, g., yaeger, r., hechtman, j.f., manca, p., fucà, g., walch, h., lee, j., élez, e., seligmann, j., mussolin, b., pagani, f., germani, m.m., ambrosini, m., rossini, d., ratti, m., salvà, f., richman, s.d., wood, h., nanjangud, g., gloghini, a., milione, m., bardelli, a., de braud, f., morano, f., cremolini, c., & pietrantonio, f. (2021). egfr amplification in metastatic colorectal cancer. journal of the national cancer institute, 113(11), 1561–1569. ruhe, j.e., streit, s., hart, s., wong, c.h., specht, k., knyazev, p., knyazeva, t., tay, l.s., loo, h.l., foo, p., wong, w., pok, s., lim, s.j., ong, h., luo, m., ho, h.k., peng, k., lee, t.c., bezler, m., mann, c., gaertner, s., hoefler, h., iacobelli, s., peter, s., tay, a., brenner, s., venkatesh, b., & ullrich, a. (2007). genetic alterations in the tyrosine kinase transcriptome of human cancer cell lines. cancer research, 67(23), 11368–11376. saletti, p., molinari, f., de dosso, s., & frattini, m. (2015). egfr signaling in colorectal cancer: a clinical perspective. gastrointestinal cancer: targets and therapy, 5, 21-38. silva rocha, f., da silva maués, j.h., brito lins pereira, c.m., moreira-nunes, c.a., & rodriguez burbano, r.m. (2021). analysis of increased egfr and igf-1r signaling and its correlation with socio-epidemiological features and biological profile in breast cancer patients: a 45 jugović et al. ● the frequency and type of mutations in the egfr gene of colorectal carcinoma patients in southeastern serbia 46 study in northern brazil. breast cancer (dove med press), 13, 325-339. skřičková, j., májková, p., barinová, m., bratová, m., pešek, m., svaton, m., kolek, v., fišer, o., koubková, l., benejová, a., venclícek, o., cernovská, m., havel, l., hrnciarik, m., roubec, j., milada, z., opálka, p., krejcí, j., krejcí, d., coupková, h., & merta, z. (2020). epidermal growth factor receptor (egfr) gene mutation testing prior to tyrosine kinase inhibitors (tki) treatment – prospective data from the czech tulung registry. european respiratory journal, 56(64), 1686. syrigos, k., kotteas, i., paraskeva, m., gkiozos, i., boura, p., tsagouli, s., grapsa, d., & charpidou, a. (2018). prognostic value of egfr genotype in egfr-mutant non-small cell lung cancer. european respiratory journal, 52, pa 2852. tanaka, t., kaida, t., yokoi, k., ishii, s., nishizawa, n., kawamata, h., katoh, h., sato, t., nakamura, t., watanabe, m., & yamashita, k. (2019). critical relevance of genomic gains of prl3/egfr/c-myc pathway genes in liver metastasis of colorectal cancer. oncology letters, 17(1), 1257– 1266. tasdemir, s., taheri, s., akalin, h., kontas, o., onal, o., & ozkul, y. (2019). increased egfr mrna expression levels in non-small cell lung cancer. the eurasian journal of medicine, 51(2), 177–185. biologica nyssana ● 14 (1) june 2023: 39-46 tong, g.j., zhang, g.y., liu, j., zheng, z.z., chen, y., niu, p.p., & xu, x.t. (2018). comparison of the eighth version of the american joint committee on cancer manual to the seventh version for colorectal cancer: a retrospective review of our data. world journal of clinical oncology, 9(7), 148–161. uhlyarik, a., piurko, v., papai, z., raso, e., lahm, e., kiss, e., sikter, m., vachaja, j., kenessey, i., & timar, j. (2020). egfr protein expression in kras wild-type metastatic colorectal cancer is another negative predictive factor of the cetuximab therapy. cancers, 12(3), 614. wang, d., wang, b., wang, r., zhang, z., lin, y., huang, g., lin, s., jiang, y., wang, w., wang, l., & huang, q. (2017). high expression of egfr predicts poor survival in patients with resected t3 stage gastric adenocarcinoma and promotes cancer cell survival. oncology letters, 13(5), 3003-3013. wee, p. & wang, z. (2017). epidermal growth factor receptor cell proliferation signaling pathways. cancers, 9(5), 52. zou, s.m., li, w.h., wang, w.m., li, w.b., shi, s.s., ying, j.m., & lyu, n. (2018). the gene mutational discrepancies between primary and paired metastatic colorectal carcinoma detected by next-generation sequencing. journal of cancer research and clinical oncology, 144(11), 2149– 2159. jugović et al. ● the frequency and type of mutations in the egfr gene of colorectal carcinoma patients in southeastern serbia čubrić & crnobrnja-isailović 2022, biologica nyssana 13(1) 13 (1) september 2022: 47-57 doi: 10.5281/zenodo.7375498 a view on human perception of snakes in serbia with special reference to nose-horned viper original article tijana čubrić department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, 18 000 niš, serbia tijana.cubric@pmf.edu.rs (corresponding author) jelka crnobrnja-isailović department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, 18 000 niš, serbia department of evolutionary biology, institute for biological research “siniša stanković”, university of belgrade, despota stefana 142, 11000 belgrade, serbia received: march 05, 2022 revised: september 13, 2022 accepted: september 21, 2022 abstract: snakes are frequently misunderstood and ill-perceived animals. having in mind the frequent killings of snakes near villages in serbia as their habitats coincide with agricultural land, we administered questionnaire to the local inhabitants at several localities to evaluate human perception of the snakes with a special reference to vipera ammodytes. we interviewed 87 individuals where the majority (68%) were men of age 65 and older. we classified responses to eight different topics: attitude, knowledge, awareness, folklore, knowledge about nose-horned vipers, possible threats to the vipers, direct killings and snakebite incidence. younger age groups of the respondents (18-24 and 25-34) had positive attitude and more knowledge about snakes while other age groups were ambivalent. further, gender groups were also ambivalent. almost half (45%) of respondents recognized v. ammodytes, but 36% stated that this viper “jumps and attacks” humans, and majority of respondents (61%) perceived this species as aggressive. regarding these results, we have proposed possible conservation measures. key words: ophiophobia, serbia, survey, snake awareness, folklore, vipera ammodytes apstrakt: pregled ljudskog stava o zmijama u srbiji sa posebnim osvrtom na poskoka zmije su često neshvaćene životinje. imajući u vidu česta ubijanja zmija na selima u srbiji obzirom na to da se staništa ovih životinja nekada nalaze u blizini ili na poljoprivrednim zemljištima, sprovedeno je istraživanje mišljenja o zmijama lokalnih meštana, putem dodele upitnika, na nekoliko lokaliteta kako bi bio procenjen stav prema zmijama sa posebnim osvrtom na vrstu vipera ammodytes. anketirano je 87 osoba od kojih su većina (68%) bili muškarci starosti 65 godina i više. odgovore smo klasifikovali u osam različitih tema: stav, znanje, svest, folklor, znanje o poskoku, moguće pretnje zmijama, direktna ubistva i učestalost zmijskih ujeda. mlađe starosne grupe ispitanika (18-24 i 25-34) imaju pozitivan stav i više znanja o zmijama, dok su ostale starosne grupe ambivalentne. dalje, rodne grupe su takođe bile ambivalentne. skoro polovina (45%) ispitanika prepoznala je vrstu v. ammodytes, ali je 36% izjavilo da ova zmija „skače i napada“ ljude, a većina ispitanika (61%) ovu vrstu doživljava kao agresivnu. u vezi sa ovim rezultatima, predložili smo potencijalne mere zaštite. ključne reči: ofiofobija, srbija, anketa, svest o zmijama, folklor, vipera ammodytes introduction territory of the republic of serbia is inhabited by ten snake species (tomović et al., 2015): seven are non-venomous and belong to family colubridae while three are from venomous snakes’ family viperidae (vipera ammodytes l. and v. berus l. are highly venomous, while v. ursinii b. is mildly venomous). in serbia, snake species are protected by the national law where all of them, except v. ammodytes, are in category “strictly protected” (v. ammodytes is in a lesser category “protected”) (tomović et al., 2015). vipera ammodytes (nosehorned viper) is widely distributed throughout the balkan peninsula (crnobrnja-isailović & haxhiu, 1997), where it have been tradionally persecuted and killed by local inhabitants, illegally traded as a pet, and over-collected for venom extraction (jelić et al., 2013). in serbia, where this viper inhabits area on the south of sava and danube rivers, during a 16 © 2022 čubrić & crnobrnja-isailović. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 47 year period (1993 2008), 9,800 adult nose-horned vipers were collected by local suppliers and sold to the national institute for immunology and virology for venom extraction, where they later died due to improper care (ajtić, 2009). the ban on harvesting nose-horned vipers in serbia was proclaimed in 2009. many of the snake species in serbia can be found near and/or in villages: e.g. dolichophis caspius g., natrix natrix l., zamenis longissimus l., coronella austriaca l. and v. ammodytes, among the other habitats, can be found on road banks, near or in the ruins and in abandoned houses – often in the basements (speybroeck et al., 2016). villagers are frequently encountering and killing snakes, and this persecution, besides the urban development, habitat fragmentation, road killings and pet trade, represents additional strong threat for the snake species (soulsbury & white, 2016; de souza et al., 2018). as snakes could be one of the apex predators in agricultural ecosystems, their eliminations by direct killings can severely damage trophic webs (mills et al., 1993). in regard to these anthropogenically caused threats to both non-venomous and venomous snake species in serbia, the primary goal of this study was to analize data obtained from questionnaires on human attitude, knowledge and awareness related to snakes in serbia and particularly to the nose-horned viper, and to help development of future conservation programs by suggesting conservation measures based on implementation of acquired knowledge. our secondary aim was to educate and possibly mitigate human-snake conflict by distributing leaflets and booklets among the respondents after the conducted survey. leaflets were providing general information about nose-horned viper, instruction for proper reaction in case of venomous snake bite, rules on how to behave in nature, and information about the importance of nose-horned vipers, while booklets were containing photos and cohesive information about non-venomous and venomous snake species in serbia. materials and methods data collection we conducted personal survey using structured anonymous questionnaire in several villages on the territories of svilajnac town (central serbia), protected area “sićevačka gorge” (southeastern serbia), “djerdap” national park (eastern serbia), bosilegrad town (southeasternmost part of serbia), and krupanj town (western serbia) in the year 2016. (fig. 1). we purposefully selected these villages because their inhabitants have been in direct contact with both agricultural land and forested areas which are suitable for snakes, their prey and predators. furthermore, the areas surrounding svilajnac and krupanj are known as localities where the nosehorned vipers were collected for venom extraction in the past (information obtained by previous personal communication and from the press). additionally, all these areas also represent habitats where we have been conducting monitoring of nose-horned viper’s populations. we have questioned any local inhabitant willing to help us and we have obtained consent from the participants for publication of this study and any accompanying photographs. we recorded age and gender of participants. the questionnaire consisted of 20 questions (fig. 2) defined in a simple and concise way as older inhabitants have little or no education. nineteen questions were close-ended with response options of “yes”, “no” or “i don’t know” and “i am not sure”, while one question was open-ended. 48 biologica nyssana ● 13 (1) september 2022: 47-57 čubrić & crnobrnja-isailović ● a view on human perception of snakes in serbia with special reference to nose-horned viper fig. 1. interviewing local villagers data analyses we classified respondents into two gender groups and separately, into following six age groups: 1) 1824, 2) 25-34, 3) 35-44, 4) 45-54, 5) 55-64 and 6) 65 and older. we classified questions into following topics: 1. attitude questions numbered 1, 4, 5, 10, 12 and 14 (fig. 2); positive attitude was defined if the respondents have chosen answer “no” in questions no. 1, 4, 5, 10, 12 and “b” or “v” in question no. 14; negative attitude was defined if they have chosen “yes” for the questions no. 1, 4, 5, 10, “yes” and “i don’t know” for question no. 12, and answer “a” for question no. 14. 2. knowledge questions no. 2, 3, 7, 9, 11 and 13 (fig. 2); demonstrates knowledge if the respondents have chosen answer “yes” in questions no. 2, 7, 9, 11, and “no” for questions 49 biologica nyssana ● 13 (1) september 2022: 47-57 čubrić & crnobrnja-isailović ● a view on human perception of snakes in serbia with special reference to nose-horned viper fig. 2. questionnaire 50 no. 3 and 13; demonstrates lack of knowledge if the respondents have chosen answer “no” in questions no. 2, 7, 9, 11, and “yes” for question no. 3 and “yes” and “i don’t know” for question no. 13. 3. awareness – questions no. 6, 15, 16 and 17; we classified respondents as “aware” if they answered “yes” on three or more questions and not aware otherwise. 4. folklore questions no. 13, 18 and 19. 5. knowledge about nose-horned viper in particular open-ended question no. 8. 6. possible threat to vipers question no. 20. 7. pressure on the snakes’ and in particular nosehorned viper’s local populations from direct killing with fear as possible source of conflict questions no. 1, 4 and 10. 8. snakebite incidence with lethal outcome question no. 11. for the attitude and knowledge, we analyzed answer scores using the non-parametric wilcoxon test with median scores as the depended variable (wilcoxon, 1992; rosner et al., 2006). we used one sample wilcoxon signed rank test to understand median scores for each demographic group (gender and age group) and wilcoxon sum rank test to compare differences in scores. we proposed zero hypotheses as follows: 1. attitude. respondents do not have positive attitude if h0=4 of 6 questions (as we had 6 questions, where median is 3.5; positive answers classified as the defined above), alternative hypothesis h>h0. 2. knowledge. respondents did not give correct biologica nyssana ● 13 (1) september 2022: 47-57 čubrić & crnobrnja-isailović ● a view on human perception of snakes in serbia with special reference to nose-horned viper table 1. positive and negative attitudes towards snakes in general, and nose-horned viper in particular (positive and negative answers for questions numbered 1, 4, 5, 10, 12,14) answers if h0=4 of 6 questions (as we had 6 questions, where median is 3.5; correct answers classified as the defined above), alternative hypothesis h>h0 we considered all results to be significant at p<0.05. all analyses where done by statistica 7.0. for the remaining six topics, we summarized the results of the questionnaire as the percentages of the total number of respondents who selected each response for each question. results demographics as the samples per village were not large enough, we analyzed them in total. we questioned 87 individuals where the majority were men (n=59; 68%) and 32% were women (n=28). most respondents were 65 and older 22% (n=19), followed by 55-64 -18% (n=16), 16% were of age groups 45-54 and 55-64 (n=14), and 14% were of age groups 25-34 and 18-24 (n=12). detailed percentages per every yes/no question per gender group are given in appendix 1, and per age group in appendix 2, while percentages of answers per gender on triple choice questions are given in appendix 3 and per age group in appendix 4. attitude men (n=59) gave 4 of 6 positive answers (median 35; tab. 1) and 2 of 6 negative answers (median 24) while women (n=28) gave 2 positive (median 20; tab. 1) and 4 negative answers (median=8) indicating that both groups have ambivalent attitude (tab. 2). regarding age groups, both group aged 18-24 (n=12) and 25-34 (n=12) had positive attitude positive attitude negative attitude number of respondents median range w p value median range w p value gender males 59 35 24-57 2.2 0.03* 24 2-35 2.2 0.03* females 28 20 5-27 2.2 0.03* 8 1-23 2.20 0.03* age group 18-24 12 8 5-12 1.99 0.04* 4 0-7 2.20 0.03* 25-34 12 9 3-12 1.85 0.06 3 0-9 2.20 0.03* 35-44 14 9.5 6-14 1.85 0.06 4.5 0-8 2.20 0.03* 45-54 14 8 6-14 2.02 0.04* 6 0-8 2.20 0.03* 55-64 16 8 7-14 2.20 0.03* 8 2-9 2.20 0.03* 65 and older 19 11 5-18 2.20 0.03* 8 1-14 2.20 0.03* * denotes p values<0.05 51 biologica nyssana ● 13 (1) september 2022: 47-57 čubrić & crnobrnja-isailović ● a view on human perception of snakes in serbia with special reference to nose-horned viper (median=11, median=8 respectively). knowledge men gave 4 of 6 correct answers (median 43.5; tab. 3) and 2 of 6 incorrect answers (median 15.5) while women gave 3 correct (median 12.5; tab. 3) and 3 incorrect answers (median=15.5) indicating that both groups had the same level of aptitude medium one (tab. 4). regarding age groups, both group aged 18-24 and 25-34 had correct 4 answers, indicating high level of aptitude (median=8.5; median=7.5 respectively, tab. 3, tab. 4) while other 3 groups had medium aptitude (tab. 3, tab. 4): 3544 gave 3 correct and two incorrect answers, and the equal number of correct and incorrect answers on one question (median=8; median=6 respectively; tab. 3); 45-54 gave the equal number of correct and incorrect answers on one question, 4 correct and 1 incorrect answer (median=10.5, median=3.5 respectively); 55-64 gave 4 correct and 2 incorrect answers (median=11, median=5 respectively); 65 and older gave 4 correct and 2 incorrect answers (median=10.5, median=8.5 respectively). awareness 70% of respondents answered that they know that snakes can be important part of ecosystem and useful in rodent populations control (appendix 1); 61% of respondents didn’t know that the nosehorned viper is lethargic snake as they considered it aggressive, while 10% of them didn’t believe in that statement; 57% of villagers didn’t know about the nose-horned viper’s important role in ecosystem, while 79% knew about the usage of v. ammodytes answering with 5 positive answers (median=8; median=9 respectively, tab. 1, tab. 2) while other 3 groups were ambivalent (tab. 1, tab. 2): 3544 gave four positive and two negative answers (median=9.5; median=4.5 respectively; tab. 1), 4554 gave the equal number of positive and negative answers on 2 questions, 3 positive and 1 negative answer (median=8, median=6 respectively), 5564 gave the equal number of positive and negative answers on 3 questions, 2 positive and 1 negative answer (median=8, median=8 respectively), 65 and older gave 3 positive and 3 negative answers table 2. scores for positive and negative attitudes toward snakes in general and nose-horned viper in particular, calculated separately for genders and age groups (questions numbered 1, 4, 5, 10, 12,14;h0=4, h>h0) positive attitude negative attitude w p value w p value gender males 0.31 0.75 1.36 0.17 females 5 0.24 0.10 0.91 age group 18-24 2.02 0.04* 2.20 0.03* 25-34 2.20 0.03* 1.21 0.22 35-44 0.73 0.46 1.82 0.07 45-54 0.07 0.50 2.20 0.3 55-64 1.57 0.11 2.02 0.04* 65 and older 1.75 0.07 1.61 0.10 correct answers incorrect answers number of respondents median range w p value median range w p value gender males 59 43.5 1-59 2.02 0.04* 15.5 0-58 2.20 0.03* females 28 12.5 1-26 2.20 0.02* 15.5 2-27 2.20 0.03* age group 18-24 12 8.5 0-12 2.02 0.04* 3.5 1-12 2.02 0.04* 25-34 12 7.5 0-12 2.02 0.04* 4.5 0-12 2.02 0.04* 35-44 14 8 0-13 2.20 0.03* 6 1-14 2.02 0.04* 45-54 14 10.5 0-14 2.02 0.04* 3.5 0-14 2.02 0.04* 55-64 16 11 0-16 2.02 0.04* 5 0-16 2.02 0.04* 65 and older 19 10.50 2-19 2.02 0.04* 8.5 0.17 2.20 0.03* table 3. correct and incorrect answers on questions related to the knowledge on snakes and nose-horned viper in particular (questions numbered 2,3,7,9,11,13) venom in anti-venom production. therefore, we classified them as not completely aware. folklore 39% of villagers did not think that nose-horned viper is used in human consumption, while 34% did not know the answer (appendix 3); 43% did not think that nose-horned viper’s meat can be used as a medicine, while 51% did not know the answer; 36% of respondents didn’t believe in the folklore premise that v. ammodytes “jumps and attacks” humans, while 30% did not know the answer. knowledge about nose-horned viper 45% of respondents gave correct answer when asked to describe the morphology of v. ammodytes, while 52 biologica nyssana ● 13 (1) september 2022: 47-57 čubrić & crnobrnja-isailović ● a view on human perception of snakes in serbia with special reference to nose-horned viper 36% gave incorrect answers and 20% did not know to describe the snake. in regarding to the animal’s behavior, most common mistakes were in describing this viper as an animal which is very aggressive and which “jumps”, while in regard to morphology of the snake most common mistake was that this species can be uniformly colored (often mentioned brown and red color). gender and age group differences in answers are given in fig. 3 and fig. 4, respectively. possible threat 32% of respondents answered they have heard or know person who had been (in case of svilajnac) or is (in case of krupanj) harvesting the nose-horned vipers for venom supply. direct killing and fear 53% of villagers were afraid of snakes, 51% of them never killed a snake while 84% never killed a nosehorned viper (appendix 1). interestingly, majority of the respondents who are afraid of snakes, never killed a snake (31% from 53%) while majority of the respondents who aren’t afraid of snakes did kill a snake in a past (31% from 47%) (fig. 5). snakebite occurrence only 2 respondents (2%) answered that they knew the person who died due to the snakebite. further observations in order to additionally evaluate human perception of snakes, here we report some further observations. in “sićevačka gorge” protected area, villagers have burned the vegetation subsequent day after we were seen performing morphometric measurements on one nose-horned viper. respondents from one locality (krupanj) stated that some neighbors captured nose-horned vipers, pulled out their fangs, skinned them and wore the skin as bracelets. in the year 2017, (year following our field activities and conducted survey), during our educational lecture in krupanj and field work in svilajnac, two of the inhabitants respectively stated that someone released nose-horned vipers in their village referring to us, which is of course false information. this was the main reason why we have suspended conducting survey further, as it draw’s attention on local snake table 4. scores for correct and incorrect answers on questions related to the knowledge on snakes and nose-horned viper in particular (questions numbered 2,3,7,9,11,13;h0=4, h>h0) correct answers incorrect answers w p value w p value gender males 0.73 0.46 0.31 0.75 females 0.94 0.34 1.15 0.25 age group 18-24 1.99 0.046* 0.20 0.83 25-34 2.20 0.03* 1.78 0.07 35-44 1.36 0.17 1.04 0.29 45-54 2.20 0.06 0.41 0.67 55-64 1.57 0.11 0.27 0.78 65 and older 0.73 0.46 1.78 0.07 fig. 3. knowledge about nose-horned viper among genders 53 biologica nyssana ● 13 (1) september 2022: 47-57 čubrić & crnobrnja-isailović ● a view on human perception of snakes in serbia with special reference to nose-horned viper populations in the areas where local inhabitants were not even aware of their close proximity. discussion in serbia, relatively ambivalent current attitude toward snakes is the result of intermixing tradition with contemporary citizen science and education. in fig. 4. knowledge about nose-horned viper among age groups fig. 5. fear and responses to snake encounters general, human perception of snakes can be positive or negative, ranging from fascination to fear and hatred (moura et al., 2010). reasons for fear or adoration can be various; from innate to learned fear with conjunction of folklore and spiritual beliefs. namely, south slavic believed in protectorancestor spirit which represented the male founder of the family who has been incarnated into a house54 biologica nyssana ● 13 (1) september 2022: 47-57 čubrić & crnobrnja-isailović ● a view on human perception of snakes in serbia with special reference to nose-horned viper snake, the white snake, which was called “house protector” (conrad, 2001). in serbia, in particular, one of traditional beliefs is that this white snake lives in the foundation wall of the house or nearby, where preys on mice, and must not be harmed as it was considered the source of good fortune (conrad, 2001). on the contrary, in some villages in serbia, on lazar’s saturday (christian calendar marking the transition from winter to spring and from death to life), villagers perform acts such as throwing stones, shouting, making loud noise to chase away snakes (moussakova, 2016). among the vlach people in eastern serbia, it is commonly believed that nosehorned viper can jump and follow a person for a long time; also, snake skulls and sheds are there the most often used in magical rituals (crnobrnja-isailović et al., 2015). the causes of ophiophobia are still up to debate; is ophiophobia innate, learned or both factors influence person’s cognitive response (isbell, 2006; van le et al., 2013). regardless the cause, the fear of snakes is most probably a silent stimulus and can easily be triggered and amplified by external causes such are secondary experiences i.e. a scary story (ohman & mineka, 2001). this could be the possible explanation for our finding that younger age groups (age 18-24 and 25-34) have positive attitude towards snakes as they were probably more educated and in our personal communication with them some stated that they learned about snakes more in the nature documentaries. nevertheless, in our sample more people were afraid of snakes than not and, interestingly, the ones who were afraid tend to kill them less and possibly due to their avoidance of being in the proximity of the animal. studies have shown that people with ophiophobia tend to avoid snakes (geer, 1965; lang, 1969). our results have shown that people who are not afraid of snakes tend to kill them more often, probably to protect themselves, or they display excitement seeking behavior (bradshaw et al., 2007), portrayed in movies, articles and documentaries with cheap sensationalism, including even charismatic storytellers whose reckless and attention seeking behavior lead to their death (bradshaw et al., 2007). direct killings and vigorous killings of snakes have also been frequently reported in other countries such are nepal (pandey et al.,2016), jordan (eid et al.,2021), etc. showing a consistency in ill-perceived attitude towards snakes. the same two age groups also gave most accurate answers about snakes. our findings imply that even though education about snakes is important, fear of snakes can be beneficial as causes people to avoid snakes altogether as similar observations in japan suggest (tanaka et al.,1999). even though 45% of respondents gave correct answer when asked to describe the morphology and behavior of v. ammodytes, 36% gave incorrect answers, frequently stating that this viper “jumps and attacks” humans. moreover, most respondents (61%) perceived this snake as aggressive, which is in concordance with previously mentioned folklore. despite this belief, only two respondents in our sample knew a person who died due to the snakebite, which further confirms this viper’s lethargic nature. this is in concordance with report in nikolić (2020) that 164 cases of venomous snakebites in serbia were recorded between 1893 and 2018, and that only four deaths were reported in a ten-year period, although serbia has no systematized information regarding this topic. having in mind unwillingness of some of the local villagers to participate in our study, it is important to reconsider that negative attitude could be more prevalent than described in this study. these inhabitants could decline to answer because they were aware of the potential law penalty for killing the wild animals although they would actually like to kill them; the other possible reason for declining to answer could be the feeling of animosity and distrust towards persons who study the species they are afraid of and/or do not like to see around. therefore, even though most respondents gave negative and neutral answer on question number 12, it is important to interpret the ambivalent attitude with caution. conservation implications despite limited sample size, which is the result of performing the study in small villages, unwillingnes of the inhabitants to participate in the survey and our further suspension of survey because of the possible negative influense on the local snake populations as described above and below, results of our study provided information about attitudes of people in the rural serbia toward local snake species, especially about the most common and persecuted venomous one. additionally, we revealed that, analysing answers on question number 20, even the ban has been proclaimed since 2009, some people have been illegaly harvesting nose-horned vipers (this information was reported to the institute for nature protection of serbia). all these findings encouraged us to recommend a few guidelines for future national snake conservation action plans, originally proposed by dodd on global level (2016) which we broadened and specified for the republic of serbia: a. set the goals. it is important to choose the adequate core audience for education activities (lectures, documentaries, etc). have in mind that people afraid of snakes mainly do not kill them, and that it is important to recognize and understand the reasons which motivate people unafraid of snakes 55 biologica nyssana ● 13 (1) september 2022: 47-57 čubrić & crnobrnja-isailović ● a view on human perception of snakes in serbia with special reference to nose-horned viper to kill them and therefore target the education activities towards them. b. choose the right education campaign, whether that be earned through media, digital media or direct community outreach. the education cannot be left to the income motivated zoos, serpentariums or sensationalized movies, documentaries and news reports. snakes must be documented exclusively in their natural habitats with minimum to no handling in order to not send potentially harmful message. c. it is important to be consistent and regularly engaging with members of local communities. although, if it is recognized that local villagers are not aware of snakes’ presence in their vicinity, it is best not to draw attention to them. our experience is that villagers have burned the vegetation one day after we were seen performing morphometric measurements on one nose-horned viper; additionally, two of the inhabitants respectively stated that someone released nose-horned vipers in their village. in this case, our survey and community outreach were contra productive and this must be taken into consideration. thankfully, answers on questions regarding folklore (questions no. 18. and no. 19.) were mostly negative compared to dangerous threat in form of overexploiting a wild reptile species for purpose of consuming its meat – the case of testudo hermanni in some parts of serbia (crnobrnja-isailović et al., 2015). nevertheless, as the one third of interviewed villagers did not know the answer on question no. 18, while one half did not know the answer on the question 19., it would be the best not to mention it anymore in order to do not suggest to particular people the idea for very dangerous actions. d. education of primary and secondary schoolchildren could be quite beneficial. ballouard et al. (2013) found that young children up to 14 years old, liked snakes and wanted them protected after they were given correct information and interacted with animals. furthermore, similar preliminary children response was also noted by us when giving lectures in the primary school in donji milanovac (near djerdap national park) and in the secondary school in svilajnac. acknowledgements. the fieldwork was kindly funded by the rufford foundation (grants no: 19578-1 and 233922) for tč. tč and jci were also supported by the ministry of education, science and technological development of republic of serbia – contracts no. 173025 (2016-2019), 451-03-68/2020-14/200124 (2020), 451-03-9/202114/200124 (2021) and 451-03-68/2022-14/200124 (2022). jci was additionally funded by the ministry of education, science and technological development of republic of serbia – contracts no. 451-03-68/202014/200007 (2020), 451-03-9/2021-14/200007 (2021)and 451-03-68/2022-14/200007 (2022). references ajtić, r. 2009: nose-horned viper (vipera ammodytes) conservation problems in serbia. protection of nature, 60: 319-326. ballouard, j. m., ajtic, r., balint, h., brito, j. c., crnobrnja-isailović, j., desmonts, d., elmouden, e.h., erdogan, m., feriche m., pleguezuelos, j.m., prokop, p., sanchez, a., santos, x., slimani, t., tomović,lj., usak,m., zuffi, m., bonnet, x. 2013: schoolchildren and one of the most unpopular animals: are they ready to protect snakes? anthrozoös, 26(1): 93-109. bradshaw, c., brook, b., mcmahon, c. 2007: dangers of sensationalizing conservation biology. conservation biology, 21(3): 570-571. crnobrnja-isailović, j., haxhiu, i. 1997: vipera ammodytes. in: gasc, j.p. et al. (ed.), atlas of amphibians and reptiles in europe: 384-385, societas europaea herpetologica and muséum national d’ histoire naturelle, paris. crnobrnja-isailović, j., milojković, d., macura, b. 2015: vodozemci i gmizavci djerdapa/ amphibians and reptiles of djerdap national park: 26-28. donji milanovac, serbia. conrad, j. l. 2001: male mythological beings among the south slavs. folklorica-journal of the slavic, east european, and eurasian folklore association, 6(1): 3-9. de souza, j. c., da silva, r. m., gonçalves, m. p. r., jardim, r. j. d., markwith, s. h. 2018: habitat use, ranching, and human-wildlife conflict within a fragmented landscape in the pantanal, brazil. biological conservation, 217: 349-357. dodd, c. k. (ed.) 2016: reptile ecology and conservation: a handbook of techniques. oxford university press. eid, e., al awaji, m., nasarat, h., alhiyasat, a. 2021: a perceptions and knowledge towards snakes: a study from jordan. herpetological conservation and biology, 16(2): 345-354. isbell, l.a. 2006: snakes as agents of evolutionary change in primate brains. journal of human evolution, 51(1): 1-35. jelić, d., ajtić, r., sterijovski, b., crnobrnjaisailović j., lelo s., tomović, lj. 2013: legal status and assessment of conservation threats to vipers 56 biologica nyssana ● 13 (1) september 2022: 47-57 čubrić & crnobrnja-isailović ● a view on human perception of snakes in serbia with special reference to nose-horned viper (reptilia: squamata: viperidae) of the western and central balkans. herpetological conservation and biology, 8 (3): 764-770. mills, l.s., soulé, m.e., doak, d.f. 1993: the keystone-species concept in ecology and conservation. bioscience, 43(4): 219-224. moura, m.r.d., costa, h.c., sao-pedro, v.d.a., fernandes, v.d., feio, r.n. 2010: the relationship between people and snakes in eastern minas gerais, southeastern brazil. biota neotropica, 10: 133-141. moussakova, e. 2016: the dragon/snake motif in the illuminated old glagolitic manuscripts. slovo, 66: 139-163. nikolić, s. 2020: venomous snakebites in serbia through 125 years: what we do (not) know in comparison with neighboring countries: a literature review. acta medica medianae, 59(4): 95-103. öhman, a., mineka, s. 2001: fears, phobias, and preparedness: toward an evolved module of fear and fear learning. psychological review, 108(3): 483. pandey, d.p., subedi pandey, g., devkota, k., goode, m. 2016: public perceptions of snakes and snakebite management: implications for conservation and human health in southern nepal. journal of ethnobiology and ethnomedicine, 12(1): 1-25. rosner, b., glynn, r.j., lee, m.l.t. 2006: the wilcoxon signed rank test for paired comparisons of clustered data. biometrics, 62(1): 185-192. soulsbury, c.d., white, p.c. 2015: human– wildlife interactions in urban areas: a review of conflicts, benefits and opportunities. wildlife research, 42(7): 541-553. speybroeck, j., beukema, w., bok, b., van der vort, j. 2016: field guide to the amphibians and reptiles of britain and europe. bloomsbury, london. tanaka, h., hayashi, y., nakamura, a. 1999: factors affecting annual incidence of habu bites, and how residents develop and transfer cognition of high risk sites. in: rodda,g.h., sawai, y., chiszar, d., tanaka, h. (ed.), problem snake management: thehabu and brown treesnake: 139–146, cornell university press, ithaka. tomović, lj., kalezić, m., džukić, g. 2015: crvena knjiga faune srbije. ii. gmizavci. univerzitet u beogradu, biološki fakultet & zzps. van le, q., isbell, l.a., matsumoto, j., nguyen, m., hori, e., maior, r.s., tomaz, c., tran, a.h., ono, t., nishijo, h. 2013: pulvinar neurons reveal neurobiological evidence of past selection for rapid detection of snakes. proceedings of the national academy of sciences, 110(47): 19000-19005. wilcoxon, f. 1992:. individual comparisons by ranking methods. in: kotz, s., johnson, n.l. (ed.), breakthroughs in statistics: 196-202, springer, new york. appendices appendix 1. percentage of answers per gender on yes/no questions total in percentage males females question number yes no yes no yes no 1 53 47 30 38 23 9 4 49 51 40 28 9 23 5 54 46 28 40 26 6 6 70 30 51 17 20 13 7 70 30 53 15 17 15 9 59 41 47 21 11 21 10 16 84 15 53 1 31 11 2 98 1 67 1 31 16 43 57 33 34 9 23 17 79 21 51 17 29 3 20 34 66 23 45 11 21 57 biologica nyssana ● 13 (1) september 2022: 47-57 čubrić & crnobrnja-isailović ● a view on human perception of snakes in serbia with special reference to nose-horned viper appendix 2. percentage of answers per age group on yes/no questions 18-24 25-34 35-44 45-54 55-64 65 and older question number yes no yes no yes no yes no yes no yes no 1 6 8 7 7 9 7 8 8 9 9 14 8 4 8 6 1 13 6 10 9 7 9 9 16 6 5 5 9 10 3 9 7 8 8 10 8 11 10 6 8 6 8 6 14 2 14 2 13 6 14 8 7 11 2 8 6 10 6 14 2 14 5 13 9 9 8 6 9 5 8 8 10 6 11 7 11 10 10 1 13 0 14 0 16 5 11 7 11 3 18 11 0 14 0 14 0 16 0 16 0 16 2 20 16 6 8 5 9 8 8 10 6 9 9 5 17 17 9 5 8 6 15 1 14 2 16 2 17 5 20 2 11 3 10 3 13 7 9 8 10 10 11 appendix 3. percentage of answers per gender on triple choice questions question number total male female 2 98 2 0 68 0 0 30 2 0 3 3 92 5 0 67 1 3 25 3 8 45 36 20 37 17 14 8 18 6 12 14 63 23 10 40 17 3 23 6 13 34 36 30 23 29 16 11 7 14 14 3 66 31 2 43 23 1 23 8 15 29 61 10 22 39 3 7 22 3 18 26 39 34 21 28 15 6 11 15 19 7 43 51 3 37 28 3 6 23 appendix 4. percentage of answers per age group on triple choice questions question number 18-24 25-34 35-44 45-54 55-64 65 and older 2 13 1 0 14 0 0 15 1 0 16 0 0 18 0 0 22 0 0 3 0 14 0 0 11 2 0 15 1 1 15 0 0 17 1 2 20 0 8 7 2 5 8 6 0 3 9 3 9 3 3 10 6 2 7 9 6 12 1 9 3 0 8 6 0 11 5 2 10 3 6 9 3 5 15 2 13 7 5 2 5 3 6 5 6 6 6 8 2 7 7 5 6 7 9 14 0 9 5 0 9 5 0 6 10 0 10 6 2 13 3 1 18 2 15 3 10 0 1 13 0 5 9 2 7 8 1 6 8 5 7 13 2 18 3 5 6 3 6 5 5 7 5 5 5 7 7 6 6 3 11 7 19 1 7 6 0 6 8 1 7 8 1 3 11 2 7 9 1 13 8 zlatković et al. 2022, biologica nyssana 13(2) 13 (2) december 2022: 109-118 doi: 10.5281/zenodo.7437240 brometum commutati – a new halophytic plant association in central and south serbia original article ivana zlatković toplica academy of professional studies, department of agricultural and technological studies, prokuplje, serbia gajevicivana@yahoo.com (corresponding author) dragana jenačković gocić department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, niš, serbia svetlana bogdanović toplica academy of professional studies, department of agricultural and technological studies, prokuplje, serbia vladimir ranđelović department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, niš, serbia received: september 13, 2022 revised: october 25, 2022 accepted: november 15, 2022 abstract: during the phytocoenological studies in salt marshes of central and south serbia data were collected from 168 stands. after the data were entered into the vegetation database turboveg, cluster analysis performed in flora software package has shown presence of 13 associations in the study area. at the sites of oblačina and bresničić salt marshes a new plant association was recorded and phytocoenologically described under the name brometum commutati ass. new. continuous monitoring of floristic composition during an entire vegetation season has shown that this association was built by 58 species. in addition to the dominant species bromus commutatus, higher abundance and cover values were recorded for: hordeum geniculatum, puccinellia distans, cerastium dubium and trifolium lappaceum. the total cover value for stands at both localities was 100%. this association occupies a much greater surface area at oblačina salt marsh, where 11 stands were recorded. stands of this association alternate with stands of association puccinellietum limosae soó 1933. the sample also included three phytocoenologically described stands from bresničić salt marsh. although this plant community was recorded at the less halophytic soils, in the syntaxonomic sense it still belongs to the alliance puccinellion limosae soó 1933 due to the floristic similarity with other associations of that alliance. key words: saline habitats, southern serbia, plant community, oblačina salt areas, bresničić salt area apstrakt: brometum commutati nova halofitna asocijacija u centralnoj i južnoj srbiji tokom fitocenoloških istraživanja na slatinama centralne i južne srbije prikupljeni su podaci sa 168 sastojina. nakon unošenja podataka u vegetacijsku bazu turboveg i urađenu klaster analizu u flora softver paketu, na istraživanom području konstantovano je 13 asocijacija. na lokalitetima oblačinska i bresničićka slatina, konstatovana je i fitocenološki opisana nova biljna zajednica imenovana kao brometum commutati ass. nova. kontinuiranim praćenjem florističkog sastava tokom jedne vegetacione sezone, ustanovljeno je da u izgradnji zajednice učestvuje 58 vrsta. pored dominantne vrste, bromus commutatus, većom brojnošću i pokrovnošću individua ističu se: hordeum geniculatum, puccinellia distans, cerastium dubium i trifolium lappaceum. ukupna pokrovnost sastojina na oba lokaliteta iznosi 100%. asocijacija zauzima mnogo veće površine na oblačinskoj slatini, gde je zabeleženo 11 sastojina. sastojine ove asocijacije smenjuju se sa sastojinama acocijacije puccinellietum limosae soó 1933. na bresničićkoj slatini fitocenološki su opisane tri sastojine. iako je ova biljna zajednica zabeležena na zemljištu nižeg saliniteta, u sintaksonomskom smislu ipak pripada svezi puccinellion limosae soó 1933 zbog florističke sličnosti sa drugim asocijacijama pomenute sveze. ključne reči: slatine, južna srbija, biljna zajednica, oblačinska slatina, bresničićka slatina introduction halomorphic (saline) soils are relatively widespread throughout the world and occupy 10% of the earth’s surface (o’leary & glenn, 1994). these are specific types of soil covered by the general terms of salt marsh and dryland halomorphic soils. the main characteristic of saline habitats is saline or halomorphic soil suitable for development of specific flora and vegetation that is adapted to the conditions of increased salt concentration. at the same time, salt marshes are highly endangered habitats that occur in © 2022 zlatković et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 109 14th symposium on the flora of southeastern serbia and neighboring regions fragments, so the ranges of habitat-specific species are also characterized by marked fragmentation (nedeljković et al., 2014). salines are unique ecosystems and often the last refugia for a large number of endemic and relict species. plant associations represented on halomorphic soils are relatively poor in species. species combinations are specific but very diverse, so the classification of halophyte associations is quite complex. distribution of certain vegetation types is closely related to salt content, salt type and depth of soil horizons with higher salt concentration (molnar & borhidi, 2003). globally speaking, salt marsh habitats occupy significant areas and appear naturally on about one million hectares in the world. they are mostly represented within the desert, semidesert, steppe, chernozem and forest-steppe areas (yadav, 2003). in serbia, halomorphic soils are mostly present in vojvodina, while in the southern part of the country saline habitats are a real rarity and are reduced to a few small localities in the valley of rivers južna morava and toplica. they represent specific ecosystems characterized by presence of characteristic flora and vegetation which is more or less specialized to this type of habitats (zlatković et al., 2005). in that region these specific habitats develop in conditions of isolation and high anthropogenic pressure. the first studies of these important areas were carried out by pančić (1884) and petrović (1885). lalinac salt marsh is the best studied locality (niketić, 1995; milosavljević et al., 2002; ranđelović et al., 2007). a significant contribution to research on this type of vegetation in the area of vojvodina and southeastern serbia was made by slavnić (1940, 1948). there are also data on the halophytic vegetation around prokuplje (ranđelović at al., 2000) while in 2005 a detailed study on salt marshes in this part of serbia was published by the institute for nature conservation. the authors presented the results of their research on flora but also on vegetation, which was not previously described in much detail (zlatković et al., 2005). in 2014, a group of authors (zlatković et al., 2014) combined the previous study results with their own research data for all salt marsh areas of central and south serbia. they recognized presence of 333 taxa classified into 176 genera and 46 families. the ecology and ecological affinities of selected halophytes from the salt marsh areas in central and south serbia were presented in a paper from 2019 (zlatković et al., 2019a), while in the same year a new association, allio guttate-limonietum gmelinii, was described from the area of lalinac and lepaja salt marshes (zlatković et al., 2019b). materials and methods study area the study area includes localities in central and south serbia (tab. 1, fig. 1), divided into 3 groups: aleksandrovac salt marsh area, lalinac salt marsh area and the salt marsh fragments in the valley of toplica river (zlatković et al., 2014). the aleksandrovac salt marsh area includes several smaller localities (aleksandrovac, oslare, levosoje, bujanovac and neradovac) with developed halomorphic soils and very interesting flora and vegetation. the largest surface area under halomorphic soils is present at the salt marsh near aleksandrovac. the lalinac salt marsh area is situated at the foothills of mali jastrebac mt. it includes several 110 biologica nyssana ● 13 (2) december 2022: 109-118 zlatković et al. ● brometum commutati – a new halophytic plant association in central and south serbia table 1. overview of study sites with data on geographic position, altitude and general climate characteristics id locality longitude latitude altitude (m) mean annual air temperature (°c) total annual precipitation (mm) 1 levosoje 42°25'50'' 21°44'50'' 400 11.2 553 2 oslare 42°25'39'' 21°43'21'' 405 11.2 553 3 bujanovac 42°27' 21°45' 395 11.2 550 4 aleksandrovac 42°29'20'' 21°54'12'' 401 11.2 540 5 neradovac 42°31'17'' 21°52'56'' 403 11 550 6 lalinac 43°20'42'' 21°44'45'' 200 11.1 633 7 oblačina 43°18'26'' 21°40'54'' 285 11.1 636 8 lepaja 43°17'35'' 21°39'50'' 285 11.1 636 9 suva česma 43°13'58'' 21°30'43'' 257 10.9 655 10 bresničić 43°14'50'' 21°27'10'' 293 10.8 670 biologica nyssana ● 13 (2) december 2022: 109-118 zlatković et al. ● brometum commutati – a new halophytic plant association in central and south serbia 111 salt marsh fragments. lalinac salt marsh is the largest fragment while there are also oblačina and lepaja salt marsh. in addition to these salt marshes, another important fragment is situated near the village bresničić, about 1 km from the bed of river toplica, on its left bank. previously there were also several salt marsh fragments in the area between prokuplje and kuršumlija, in the alluvial plain of toplica. their soils have barely shown any halomorphic characteristics, and the flora and vegetation with halomorphic character were subsequently destroyed. the salt marsh near the village bresničić remains as one of the better preserved salt marsh fragments. the results of phytocoenological studies were fig. 1. map of study area. the main data on geographic position and climate characteristics of study sites are presented in tab. 1 used to determine the diversity of halophyte communities in the researched area and their mutual syntaxonomic relationships. for these purposes, during 2013 year, data were collected from 168 stands with individual surface areas of 10-60 m2. the phytocoenological research of the stands followed the braun-blanquet’s (1951) approach. the plant material necessary for determination was collected from the surrounding stands in order not to disrupt the qualitative and quantitative composition of the studied vegetation plots. the material was herbarized, and the herbarium specimens were stored in the herbarium collection “herbarium moesiacum” of the faculty of sciences and mathematics at the university in niš (hmn). 112 biologica nyssana ● 13 (2) december 2022: 109-118 zlatković et al. ● brometum commutati – a new halophytic plant association in central and south serbia relevant dichotomous keys (tutin et al. (ed.) 19641980; josifović (ed.) 1970-1980) and “флора на нр българия” (јорданов (ед.), 1963-1979, велчев, 1979-1995) were used for determination of plant material. nomenclature of recorded taxa was matched to the euro+med database at the following link http://ww2.bgbm.org/europlusmed/query.asp. the taxa that were not determined to species level were excluded from the analysis, as well as the mosses and lichens. the collected data were imported into the vegetation database turboveg (hennekens & schaminee, 2001). cluster analysis was performed in flora software package in order to define associations and determine the floristic similarities between associations (karadžić & marinković, 2009; karadžić, 2013). results and discussion the results of this research have shown that salt marshes of central and south serbia host 13 associations from three classes: thero-salicornietea tx. in tx. et oberd. 1958, festuco-puccinellietea soó ex vicherek 1973 and molinio-arrhenatheretea tx. they include an association with the species bromus commutatus, which was described for the first time as brometum commutati ass. new (fig. 2, appendix 1). this association was recorded at oblačina and bresničić salt marshes at altitudes of 289-308 m above sea level. vegetation cover was 100% at both sites. in addition to the dominant species bromus commutatus, following species have also shown significant values of cover: hordeum geniculatum, puccinellia distans, cerastium dubium and trifolium lappaceum. due to the grazing intensity and vicinity of cultivated areas, the association also includes a high number of ruderal species. the surface areas under this association are much greater at oblačina salt marsh, where 11 stands were recorded. the sizes of test plots were 30-50 m2. the stands of this association are interspersed with stands of association puccinellietum limosae soó 1933. at the bresničić salt marsh there were three stands for which phytocenological data were collected, with surface area of 20 m2. the species bromus commutatus was also recorded at localities: aleksandrovac, in associations puccinellietum convolutae micevski 1965 and hordeetum hystricis wendelbg. 1943; lalinac, in associations: puccinellietum limosae soó 1933, camphorosmetum monspeliacae micevski 1965, caricetum divisae slavnić 1948, juncetum compressi br.-bl. 1918 ex libb, scorzonero parviflorae-juncetum gerardii (wenzl 1934) wendelberger 1943 and allio guttate-limonietum gmelinii ass. new; and lepaja, in association allio guttate-limonietum gmelinii ass. new. at oblačina salt marsh, in addition to creating the association brometum commutati ass. new, it was also recorded within the following associations: puccinellietum limosae soó 1933, hordeo-caricetum distantis micevski 1957 and caricetum divisae slavnić 1948, while at bresničić salt marsh it was recorded within the associations puccinellietum limosae soó 1933 and caricetum divisae slavnić 1948. however, its presence is not of any significant diagnostic level in any of these associations. micevski (1965) described the association bromo-alopecuretum k. micevski 1957 from the higher, drier habitats of ovče polje as a therophyte community representing the most arid type of meadows at ovče polje. this association belongs to the alliance trifolion resupinati k. micevski 1957 and it is almost void of any halophytic plants. in addition, ranđelović & zlatković (2005) stated in the syntaxonomic overview that at the salt marshes of central and south serbia there is an association bromo-alopecuretum k. micevski 1957, composed of species bromus commutatus and alopecurus fig. 2. photos of brometum commutati ass. new. oblačina salt marsh, july 2013 113 biologica nyssana ● 13 (2) december 2022: 109-118 zlatković et al. ● brometum commutati – a new halophytic plant association in central and south serbia utriculatus, from class molinioarrhenatheretea tx. 1937 and alliance trifolion resupinati k. micevski 1957. however, in a different study several years later, presence of species alopecurus utriculatus was not recorded in stands of this association, while significantly higher number of typical halophytes (hordeum geniculatum, puccinellia distans, podospermum canum) were present, so we believe that this association belongs to the alliance puccinellion limosae soó 1933. in the dendrogram it is situated immediately next to the association puccinellietum limosae soó 1933 (fig. 3). conclusions association brometum commutati ass. new is a particularly interesting and important newly discovered halophytic association. it covers large areas at oblačina salt marsh and smaller areas at bresničić salt marsh. as the salt marsh areas in central and south serbia occupy small areas and experience strong anthropogenic impact, it is necessary to continue with appropriate conservation measures. these areas are important biodiversity centers, which is recognized as the continental salt marshes throughout europe are protected with the network natura 2000 (interpretation manual of european union habitats 1999; piernik et al. 2006). bresničić salt marsh is a protected area of the iv degree under the iucn guidelines (iucn category iv – protected area managed for conservation through management). according to classification of the national legislative, it belongs to the third category of protection, protected areas of local character, and represents a protected habitat. it was also included in the group of rare and fragile habitats (ret/frag (a)) and the list of emerald habitats, as a potential natura 2000 habitat (nedeljković et al., 2014). it is necessary to implement appropriate measures so oblačina salt marsh would be assigned a protection category as well. acknowledgements. this work was supported by the ministry of education, science and technological development fi g. 3 . d en dr og ra m o f h al op hy te v eg et at io n of c en tra l a nd s ou th s er bi a (1 -3 3 p uc ci ne lli et um li m os ae s oó 1 93 3; 3 470 c ar ic et um d iv is ae s la vn ić 1 94 8; 71 -8 3 b ro m et um c om m ut at i as s. n ew ; 84 -9 3 h or de oc ar ic et um d is ta nt is m ic ev sk i 19 57 ; 9 410 9 h or de et um h ys tr ic is w en de lb er ge r 19 43 ; 11 011 8 c am ph or os m et um m on sp el ia ca e m ic ev sk i 1 96 5; 1 19 -1 33 a lli o gu tta te -l im on ie tu m g m el in ii as s. n ew ; 1 34 -1 37 j un ce tu m c om pr es si b r.b l. 19 18 e x li bb . 19 32 ; 13 8 s co rz on er o pa rv ifl or ae -j un ce tu m g er ar di i (w en zl 1 93 4) w en de lb er ge r 19 43 ; 13 914 7 p uc ci ne lli et um c on vo lu ta e m ic ev sk i 19 65 ; 14 815 4 p la nt ag in et um c or on op i g ill ha m 1 95 3; 1 55 -1 57 c yn od et um d ac ty li m én de z 19 83 ; 1 58 c om m un ity w ith s pe ci es d om in an ce t ae ni at he ru m c ap ut -m ed us ae an d b ro m us c om m ut at us ; 1 59 c om m un ity w ith s pe ci es d om in an ce t rif ol iu m fr ag ife ru m ; 1 60 -1 63 c om m un ity w ith s pe ci es d om in an ce t rif ol iu m la pp ac eu m ; 16 416 8 c ry ps ie tu m a cu le at ae w en zl 1 93 4; 1 69 -1 70 h el eo ch lo et um s ch oe no id is t op a 19 39 ) 114 biologica nyssana ● 13 (2) december 2022: 109-118 zlatković et al. ● brometum commutati – a new halophytic plant association in central and south serbia of the republic of serbia (contract number 451-0368/2022-14/200124). references bjelić, m., nešić, lj., ćirić, v. 2014: popravka halomorfnih zemljišta. univerzitet u novom sadu, poljoprivredni fakultet. braun-blanquet, j. 1951: pflanzensoziologie. springer, wien. hennekens, s., schaminĕe, j. 2001: turboveg, a comprehensive data base management system for vegetation data. journal of vegetation science, 12: 589-591. josifović, m. (ed.) 1970–1980: flora of serbia i-x. sanu, belgrade. interpretation manual of european union habitats. 1999. version eur 15/2. jорданов, д., (ed.) 1963-1986: флора на нр българия 1-8. издателство на бан. софия. karadžić, b., marinković, s. 2009: kvantitativna ekologija. insitut za biološka istraživanja “siniša stanković”, beograd. karadžić, b. 2013: flora: a software package for statistical analysis of ecological data. water research and management 3(2): 45-54. micevski, k. 1965: halofitska vegetacija ovčeg polja. acta mus. maced. sci. nat. skopje 10: 67-90. milosavljević, v., randjelović, v., zlatković, b. 2002: vegetacija lalinačke slatine kod niša. 7. simpozijum o flori srbije i susednih područja zbornik rezimea, 47. dimitrovgrad. nedeljković, d., grubač, b., jović, d., nešić, d., branković, s. 2014: zaštićeno stanište “bresničićka slatina”. predlog zaštite. zavod za zaštitu prirodebeograd. o’leary, j.w., glenn, e.p. 1994: global distribution and potential for halophytes. in: squiers v. r., ayoub a. t. (eds.). halophytes as resource for livestock and rehabilitation of degraded lands. tasks for vegetation science 32, kluwer, dordrecht: 7–15. pančić, j. 1884: dodatak flori kneževine srbije. kraljevska srpska državna štamparija. beograd. petrović, s. 1885: dodatak flori okoline niša. kraljevsko-srpska državna štamparija. beograd. piernik, a., nienartowicz, a., hulisz, p. 2006: inland saline habitats in poland and their protection. in: czyż h. (ed.). salt grasslands and coastal meadows, ar szczecin: 31–37. ranđelović, v., amidžić, l., ilić, n. 2000: halofitska vegetacija okoline prokuplja. 6. simpozijum o flori jugoistočne srbije i susednih područja zbornik rezimea, 39. sokobanja. ranđelović, v., zlatković, b., dimitrijević, d. 2007: fitogeografska analiza flore lalinačke slatine. ix simpozijum o flori jugoistočne srbije i susednih područja sa međunarodnim učešćem zbornik rezimea, niš. slavnić, ž. 1940: prilog halofitskoj flori i vegetaciji jugoistočne srbije. glasnik skopskog naučnog društva xxii: 65-77. slavnić, ž. 1948: slatinska vegetacija vojvodine. arhiv za poljoprivredne nauke i tehniku 3: 1–80. tutin t.g., heywood v.h., burges n.a., moore d.m., valentine d.h., walters s.m., webb da (eds) 1964-1980: flora europaea i-v. cambridge university press, london. велчев, в. (ед.) 1982-1989: флора на нр българия 8-9. издателство на бан. софия. yadav, j.s.p. 2003: managing soil health for sustained highproductivity. journal of the indian society of soil science 51: 448-485. zlatković, b., ranđelović, v., amidžić, l. 2005: flora i vegetacija slatina centralne i južne srbije i njihova valorizacija sa aspekta zaštite [flora and vegetation of central and southern serbian salines and its valorization according to conservation aspect]. zavod za zaštitu prirode srbije. [in serbian]. zlatković, i., zlatković, b., ranđelović, v., jenačković, d., amidžić, l. 2014: taxonomical, phytogeographical and ecological analysis of the salt marsh flora of central and southern serbia. biologica nyssana 5(2): 91-102. zlatković, i., jenačković, d., ranđelović, v. 2019a: inland salt areas of southeast serbia: ecological preferences of certain representatives of flora. biologia 74(11): 1425-1440. zlatković, i., jenačković gocić, d., ranđelović, v. 2019b: allio guttate-limonietum gmelinii new halophytic association in south serbia. 13th symposium on the flora of southeastern serbia and neighbouring regions, stara planina, june 20-23, book of abstracts, 68 p. 115 biologica nyssana ● 13 (2) december 2022: 109-118 zlatković et al. ● brometum commutati – a new halophytic plant association in central and south serbia a pp en di x a pp en di x 1. p hy to co en ol og ic al ta bl e of b ro m et um c om m ut at i a ss . n ew l oc al it y o bl ač in a sa lt m ar sh b re sn ić ič s al t m ar sh d at e of r ec or di ng 21 m ay 23 j un 21 m ay 23 j un 6 ju l 27 m ay 25 se p degree of presence su rf ac e (m 2 ) 30 30 50 50 50 30 30 30 30 30 30 20 20 20 c ov er ag e (% ) 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 a lt it ud e (m ) 28 9 29 1 29 1 29 0 30 8 30 5 29 2 29 5 29 2 29 2 29 1 29 9 29 8 29 8 l on gi tu de 43° 18.552' 43° 18.550' 43° 18.432' 43° 18.438' 43° 18.462' 43° 18.450' 43° 18.404' 43° 18.467' 43° 18.474' 43° 18.471' 43° 18.471' 43° 14.862' 43° 14.859' 43° 14.859' l at it ud e 21° 41.078' 21° 41.076' 21° 40.954' 21° 40.952' 21° 40.939' 21° 40.961' 21° 40.989' 21° 40.996' 21° 40.998' 21° 41.005' 21° 41.018' 21° 27.247' 21° 27.250' 21° 27.250' n um be r of s pe ci es 10 7 8 6 13 14 11 15 8 10 6 15 20 7 d ia gn os ti c sp ec ie s of c la ss m ol in io -a rr he na th er et ea b ro m us c om m ut at us s ch ra de r 5 5 5 5 5 5 5 5 5 5 5 5 5 5 v tr ifo liu m la pp ac eu m l . r + 1 1 2 1 r iv ju nc us c om pr es su s ja cq . r r r r r r ii i tr ag op og on p ra te ns is l . + + r r ii i p oa p ra te ns is l . r r r ii r an un cu lu s sa rd ou s c ra nt z r r i a ch ill ea m ill ef ol iu m l . r i tr ifo liu m h yb ri du m l . r i sa lv ia p ra te ns is l . r i r um ex c ri sp us l . r i biologica nyssana ● 13 (2) december 2022: 109-118 zlatković et al. ● brometum commutati – a new halophytic plant association in central and south serbia d ia gn os ti c sp ec ie s of c la ss f es tu co -p uc ci ne lli et ea h or de um g en ic ul at um a ll. 3 3 2 + + + + 1 1 + r v p uc ci ne lli a di st an s (l .) pa rl . 1 1 1 1 1 1 + 1 1 2 4 r v p od os pe rm um c an um c .a . m ey er r r r r + 1 + r iv c ar ex d iv is a h ud so n r r r ii p ho liu ru s pa nn on ic us (h os t) t ri n. r r r r ii ju nc us g er ar di i l oi se l. r i lo tu s te nu is w al ds t. & k it. ex w ill d. r i d ia gn os ti c sp ec ie s of c la ss f es tu co -b ro m et ea tr ifo liu m c am pe st re s ch re be r 3 i tr ifo liu m n ig re sc en s v iv . r i p la nt ag o la nc eo la ta l . r r i p ro sp er o au tu m na le (l .) sp et a r i x er an th em um a nn uu m l . r i d ia gn os ti c sp ec ie s of c la ss p ap av er et ea r ho ea di s m at ri ca ri a ch am om ill a l . r + r r ii m yo so tis a rv en si s (l .) h ill + i c re pi s se to sa h al le r fi l. r i v ic ia p an no ni ca c ra nt z + i a nt he m is a rv en si s l . r i d ia gn os ti c sp ec ie s of c la ss p hr ag m ite te a co m m un is p hr ag m ite s au st ra lis (c av .) st eu d. r i b ol bo sc ho en us m ar iti m us (l .) pa lla r i e le oc ha ri s pa lu st ri s (l .) r oe m er & s ch ul te s r i e le oc ha ri s pa lu st ri s (l .) r i 116 117 biologica nyssana ● 13 (2) december 2022: 109-118 zlatković et al. ● brometum commutati – a new halophytic plant association in central and south serbia d ia gn os ti c sp ec ie s of c la ss a rt em is ie te a vu lg ar is c ic ho ri um in ty bu s l . + i li na ri a vu lg ar is m ill . r i c en ta ur ea s ol st ic ia lis l . r i m el ilo tu s offi ci na lis (l .) l am . r i so nc hu s ar ve ns is l . r i r um ex p at ie nt ia l . r i d ia gn os ti c sp ec ie s of c la ss c ak ile te a m ar iti m ae a tr ip le x pr os tr at a b ou ch er ex d c . r i d ia gn os ti c sp ec ie s of c la ss s is ym br ie te a la ct uc a se rr io la l . r r r + ii h or de um m ur in um l . r i c re pi s pu lc hr a l . r i g er an iu m d is se ct um l . + i g er an iu m m ol le l . r i d ia gn os ti c sp ec ie s of c la ss p ol yg on op oe te a an nu ae c yn od on d ac ty lo n (l .) pe rs . r 1 i p ol yg on um a vi cu la re l . r r r + r r ii i d ia gn os ti c sp ec ie s of c la ss i sö et on an oj un ce te a c er as tiu m d ub iu m (b as t.) o . sc hw ar tz 3 3 1 3 + r 1 iv m en th a pu le gi um l . r r i d ia gn os ti c sp ec ie s of c la ss c he no po di et ea tr ifo liu m e ch in at um b ie b. 1 + + + + ii m ed ic ag o ar ab ic a (l .) h ud . r i a ve na fa tu a l . r i d ia gn os ti c sp ec ie s of c la ss t ri fo lio -g er an ie te a sa ng ui ne i c am pa nu la r ap un cu lu s l . r i biologica nyssana ● 13 (2) december 2022: 109-118 zlatković et al. ● brometum commutati – a new halophytic plant association in central and south serbia d ia gn os ti c sp ec ie s of c la ss h el ia nt he m et ea g ut ta ti tr ifo liu m s tr ia tu m l . r i d ia gn os ti c sp ec ie s of c la ss s ed osc le ra nt he te a c er as tiu m b ra ch yp et al um pe rs . 2 i m ed ic ag o m in im a (l .) l . r i u ns pe ci fie d sp ec ie s ta ra xa cu m o ffi ci na le w eb er r r i b ro m us m ol lis l . r r r i b up le ur um te nu is si m um l . + 1 + i a gr os te m m a gi th ag o l . r i g al iu m te nu is si m um m . b ie b. r i 118 šabanović et al. 2023, biologica nyssana 14(1) 14 (1) june 2023: 7-13 doi: 10.5281/zenodo.8027050 orchis anthropophora (l.) all. (orchidaceae), a confirmed species for the flora of bosnia and herzegovina original article elvedin šabanović international university of brčko district, m. malića and i. džindića bb, brčko 76100, bosnia and herzegovina sabanovic2021@outlook.com (corresponding author) vladan djordjević faculty of biology, institute of botany and botanical garden, university of belgrade, takovska 43, 11000 belgrade, serbia đorđije milanović faculty of forestry, university of banja luka, stepe stepanovića 75a, banja luka 78000, bosnia and herzegovina dragana jenačković gocić department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, 18106 niš, serbia sanida bektić department of biology, faculty of sciences and mathematics, university of tuzla, tuzla, bosnia and herzegovina samira huseinović department of biology, faculty of sciences and mathematics, university of tuzla, tuzla, bosnia and herzegovina vladimir ranđelović† department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, 18106 niš, serbia received: december 09, 2022 revised: april 12, 2023 accepted: april 20, 2023 abstract: orchis anthropophora (l.) all. (orchidaceae) is a mediterranean-atlantic species distributed around the mediterranean region and in central and western europe. during a floristic survey in may 2022, this species was found at the ravno (trebimlja) locality (yh35 10×10 km utm grid cell), which is the first confirmed record of this species on the territory of bosnia and herzegovina. the population was found in rocky grassland, on limestone, at an altitude of 423 m. three individuals of o. anthropophora were found within an area of 100 m². based on the iucn red list categories and criteria, o. anthropophora is estimated as critically endangered in bosnia and herzegovina. key words: orchis anthropophora, orchid, flora, bosnia and herzegovina, balkan peninsula apstrakt: orchis anthropophora (l.) all. (orchidaceae), potvrđena vrsta za floru bosne i hercegovine orchis anthropophora (l.) all. (orchidaceae) je mediteransko-atlantska vrsta rasprostranjena u mediteranskom regionu, centralnoj i zapadnoj evropi. florističkim istraživanjem sprovedenim u maju 2022. godine, ova vrsta je pronađena na lokalitetu ravno (trebimlja) (yh35 10×10 km2 utm kvadrat), što je prvi potvrđeni nalaz ove vrste na području bosne i hercegovine. populacija je pronađena na travnom, karbonatnom kamenjaru, na nadmorskoj visini od 423 m. tri individue vrste o. anthropophora zabeležene su na površini od 100 m2. prema kategorijama i kriterijumima iucn crvene liste, o. anthropophora je procenjena kao krajnje ugrožena vrsta bosne i hercegovine. ključne reči: orchis anthropophora, orhideja, flora, bosna i hercegovina, balkansko poluostrvo introduction orchis tourn. ex l. is a genus in the orchid family (orchidaceae), occurring mainly in europe, north africa, and temperate asia, with the center of its diversity located in the mediterranean region (kretzschmar et al., 2007). this genus once included taxa of the genera anacamptis rich., dactylorhiza neck. ex nevski and gymnadenia r.no. in w.t. aiton (tyteca et al., 2012). molecular analyses have confirmed the polyphyletic status of the genus orchis s.l. and many taxa have been assigned to the expanded genera anacamptis rich. and neotinea rchb.f. (pridgeon et al., 1997; bateman et al., 1997, 2003). the genus orchis is divided into the subgenera orchis s.str. and masculae (kretzschmar et al., 2007). there is also a division of this genus into two genera: orchis, which includes all species and subspecies with a so-called ‘anthropomorphic’ lip (o. militaris group), and androrchis tyteca & klein, which includes all remaining ‘non-anthropomorphic’ species and subspecies (o. mascula group) (tyteca & klein, 2008, 2009). the genus orchis consists of 23 species and © 2023 šabanović et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 7 this paper is dedicated to professor vladimir ranđelović as a sign of gratitude for the provided knowledge, advice, support and the opportunity to be his collaborators. the authors 10 subspecies (wcsp, 2022). in bosnia and herzegovina, 12 taxa have been known until now: orchis anthropophora (l.) all., o. italica poir., o. mascula (l.) l. subsp. mascula, o. mascula (l.) l. subsp. speciosa (mutel) hegi, o. militaris l. subsp. militaris, o. pallens l., o. pauciflora ten., o. provincialis balb. ex lam. & dc, o. purpurea huds. subsp. purpurea, o. quadripunctata cirillo ex ten., o. simia lam. subsp. simia and o. spitzelii saut. ex w.d.j. koch subsp. spitzelii (šabanović et al., 2019, 2021; milanović et al., 2022). however, in the current „checklist of the orchidaceae of bosnia and herzegovina” (šabanović et al., 2021), the status of o. anthropophora in bosnia and herzegovina is marked ‘l’, which means that there are published data but no herbarium or photographic material confirming its finding. this study reports the first confirmed record of orchis anthropophora (l.) all. in bosnia and herzegovina. the objectives were: a) to provide its morphological description; b) to present its currently known distribution; c) to determine its habitat preferences; d) to determine its population size and e) to estimate the iucn threat status of this species in bosnia and herzegovina. materials and methods during a floristic survey conducted in may 2022 in the area of ravno (trebimlja), data were collected on the morphology, distribution, habitat preferences, and population size of orchis anthropophora (l.) all. the taxon was identified according to delforge (2006) and kretzschmar et al. (2007), while the nomenclature followed the world checklist of kew gardens (wcsp 2022). one specimen of o. anthropophora was collected for documentation purposes and deposited in the museum of the franciscan monastery in visoko–herbarium collection of fr. ivo radman. the present morphological description of the species is based on own observations and according to baumann et al. (2006), delforge (2006), kretzschmar et al. (2007) and fay & taylor (2015). the values of the measured traits of the specimen from bosnia and herzegovina are marked with bold numbers. the distribution of o. anthropophora in bosnia and herzegovina was mapped on a grid map with squares of 10×10 km, using the universal transverse mercator (utm) projection, grid zone 33t. geographic coordinates (longitude, latitude) and altitude were recorded using a garmin etrex 30 handheld gps device in the world geodetic system 84 (wgs 84). the boundaries of the regions on the distribution map of this species are given according to čadro et al. (2019). the vegetation units of the site with o. anthropophora were determined according to braun-blanquet (1964), whereas the nomenclature follows mucina et al. (2016). the habitat type was determined according to the eunis habitat classification (http://eunis.eea.europa.eu/). the geological substrate was determined in the field and from a 1:100,000 scale geologic map of the study area. abundance of o. anthropophora was determined by counting the total number of individuals. for the estimation of the threat status of o. anthropophora in bosnia and herzegovina, iucn (2012) red list categories and criteria were applied. results orchis anthropophora (l.) all., fl. pedem. 2: 148 (1785) synonyms: aceras anthropophorum (l.) w.t.aiton, arachnites anthropophorus (l.) f.w.schmidt, himantoglossum anthropophorum (l.) spreng., loroglossum anthropophorum (l.) rich., ophrys 8 biologica nyssana ● 14 (1) june 2023: 7-13 šabanović et al. ● orchis anthropophora (l.) all. (orchidaceae), a confirmed species for the flora of bosnia and herzegovina fig. 1. orchis anthropophora (l.) all. (bosnia and herzegovina, ravno, trebimlja, 09.05.2022, photo e. šabanović) biologica nyssana ● 14 (1) june 2023: 7-13 šabanović et al. ● orchis anthropophora (l.) all. (orchidaceae), a confirmed species for the flora of bosnia and herzegovina 9 anthropophora l., satyrium anthropophorum (l.) pers., serapias anthropophora (l.) jundz. (fig. 1). morphological characteristics perennial plant, 10–36.1–40 (–50) cm high, with two ovoid tubers. the stem is glabrous and cylindrical. there are 5–6–10 leaves, oblong-lanceolate, shiny above, bluish-green, not spotted, distinctly veined, 5–7.9–15 cm long and 0.5–2.4–4 cm wide. basal leaves are in a rosette, the middle leaves are erect, an upper leaf is bract-like, while 1–3 smaller sheathing leaves surround the lower part of the stem. the bracts are yellowish-green to pale green, acute, 5–7 mm long and 1.2–2.3 mm wide, shorter than the ovaries. the inflorescence is elongated, denser at the apex, 5–13.4–20 (–28) cm long, with 42–50 (–90) flowers. the flowers are ‘anthropomorphic’. sepals are oval, 5–8 (–11) mm long and 2.5-5 mm wide, green with reddish margins, forming a dense cap. petals are linear, hidden inside hood, 5–8 mm long and 1–1.7 mm wide, pale green. the lip is 3-lobed, without a spur, 10–12–16 mm long and 2.5–4 mm wide, dirty yellow, greenish yellow to orange with often darker reddish or brownish margins, with two shiny pale bosses at the base forming a nectariferous cup. the lateral lobes are linear and slender, forming the ‘arms’, 7 mm long. the middle lobe is 10 mm long, always longer than the lateral lobes and divided into two strap-shaped halves, forming the ‘legs’, sometimes separated by a tooth. the column is short, obtuse. the ovary is sessile, twisted. taxonomic notes the studied taxon changed its taxonomic position, starting from ophrys anthropophora l. in the broad definition of ophrys l. since then it has been classified in aceras r.br., arachnites f.w.schmidt, himantoglossum spreng., loroglossum rich., orchis l., satyrium l. and serapias l. (fay & taylor, 2015). since 1813, this taxon has been included in the monospecific genus aceras r.br. as aceras anthropophorum (l.) r.br (delforge, 2006). the only distinctive feature in which aceras differs from orchis is the absence of a spur. however, recent molecular studies have shown that this species actually belongs to the genus orchis s.s., excluding species placed in anacamptis rich. and neotinea rchb.f. by bateman et al. (1997). it is sister to the remainder of the genus (bateman et al., 2003) and actually belongs to the orchis militaris group and is one of the earliest differentiated species of the genus orchis. flowering time on may 9, 2022, three specimens of o. anthropophora at the trebimlja site were at peak flowering. this is consistent with the flowering time reported by fay & taylor (2015), who reported that this species flowers from march to july, with an optimal time from april to june. in greece, the peak flowering time of the species is around mid-april (tsiftsis & antonopoulos, 2017). general distribution orchis anthropophora is a mediterranean-atlantic species distributed around the mediterranean region and in central and western europe, occurring from england, belgium and the netherlands in the north to morocco and algeria in the south and from spain and england in the west to cyprus and rhodes in the east (delforge, 2006; kretzschmar et al., 2007; fay & taylor, 2015; wcsp, 2022). it is widespread in western europe, while rare in the eastern and northern parts of its range. according to published sources, it is distributed in albania, algeria, austria, the balearic islands, belgium, corsica, crete, croatia, cyprus, france, germany, great britain, greece, italy, lebanon-syria, montenegro, morocco, the netherlands, portugal, sardinia, sicily, spain, switzerland, tunisia, and turkey (delforge, 2006; kretzschmar et al., 2007; fay & taylor, 2015; tsiftsis & antonopoulos, 2017; wcsp, 2022). distribution in bosnia and herzegovina orchis anthropophora was found in the southern region of bosnia and herzegovina: ravno, trebimlja (near the trebimlja-čepikuće border crossing, on both sides of the road), n 42.8757111˚, e 17.8624806˚, mgrs 33t yh35, 423 m a.s.l., rocky grassland, limestone, 09 may 2022; coll. e. šabanović, det. e. šabanović, v. djordjević [the museum of the franciscan monastery in visoko – herbarium collection of fr. ivo radman 00315; photo documentation: e. šabanović] (fig. 2). the finding of o. anthropophora at the trebimlja locality (near the trebimlja-čepikuće border crossing) is the first confirmed record of this species on the territory of bosnia and herzegovina, representing the first record of this species in mgrs 33t yh35 10×10 km utm grid cell and southern region of bosnia and herzegovina. previously, this species was recorded in bosnia and herzegovina only by protić (1902: 29), who listed the species under the name aceras anthropophora r.br. and found it at the two localities (zelengora – utm cp00 and prijevor – utm cn19; fig. 2). these records have been mentioned for bosnia and herzegovina later by hayek (1933: 403) and šilić (1996: 360), but firstly by beck-mannagetta (1904: 512), who considered these records very doubtful. moreover, this researcher did not collect the plants during his botanical excursion in the mountains in se bosnia 10 biologica nyssana ● 14 (1) june 2023: 7-13 šabanović et al. ● orchis anthropophora (l.) all. (orchidaceae), a confirmed species for the flora of bosnia and herzegovina fig. 2. distribution of orchis anthropophora (l.) all. in bosnia and herzegovina fig. 3. habitat of orchis anthropophora (l.) all. (bosnia and herzegovina, ravno, trebimlja, 09.05.2022, photo e. šabanović) 11 biologica nyssana ● 14 (1) june 2023: 7-13 šabanović et al. ● orchis anthropophora (l.) all. (orchidaceae), a confirmed species for the flora of bosnia and herzegovina (fukarek, 1969), and there is no herbarium evidence or photographic material confirming these findings. since these records are located deep in the dinaric mountains, outside the climatic conditions and habitats suitable for this species, they must be treated as at least very doubtful or even erroneous (fig. 2). habitat and ecology orchis anthropophora was found on the locality trebimlja in submediterranean rocky grassland of the alliance chrysopogono grylli-koelerion splendentis horvatić 1973 within the ordo scorzoneretalia villosae kovačević 1959, partially overgrown by some individuals of fraxinus ornus l. and pistacia terebinthus l (fig. 3). the following accompanying taxa were recorded at the site with o. anthropophora: brachypodium retusum (pers.) p. beauv., koeleria splendens c. presl (aggr.), achnatherum bromoides (l.) p. beauv., micromeria juliana (l.) benth. ex rchb., salvia officinalis l., euphorbia spinosa l., tanacetum cinerariifolium (trevir.) sch. bip., genista sylvestris subsp. dalmatica (bartl.) h. lindb., inula verbascifolia (willd.) hausskn. and others. according to the eunis classification, this habitat type belongs to the eastern submediterranean dry grasslands (e1.55 code; https:// eunis.eea.europa.eu/habitats/997). the population of o. anthropophora was recorded on limestone, under full light regime, at an altitude of 423 m. the habitat preferences of this orchid recorded in bosnia and herzegovina correspond to its habitat preferences and ecological requirements in other parts of its range. namely, many authors pointed out that this species occurs on calcareous substrates, in full sun to mid-shade conditions, in xerophilous short grasslands, garrigue, scrub, forest edges, and more rarely in open forest habitats (delforge, 2006; kretzschmar et al., 2007; fay & taylor, 2015; tsiftsis & antonopoulos, 2017). this species has been found on soils with ph ranging from 5.5 to 8.6, but generally prefers alkaline soils (sundermann, 1980; wallenwein & saad, 2000; vakhrameeva et al., 2008; djordjević & tsiftsis, 2022). the recorded altitudinal range of this species from other countries is between 0 m and 1,700 m (baumann et al., 2006; delforge, 2006; tsiftsis & antonopoulos, 2017). population size only three individuals of o. anthropophora were found within an area of 100 m². therefore, its population size falls within the iucn category of fewer than 50 mature individuals. conservation status orchis anthropophora is protected by the convention on international trade in endangered species of wild fauna and flora (cites). the current status of this species in bosnia and herzegovina is estimated as critically endangered – cr d. the iucn status of the species in other countries is as follows: regionally extinct in cyprus, critically endangered in austria and bavaria, endangered in the united kingdom and luxembourg, vulnerable in switzerland, near threatened in croatia and last concern in spain and france (kull et al., 2016). monitoring of this species is needed to assess potential threats that may affect the species (e.g. road construction and habitat succession) and to monitor population dynamics. acknowledgements. the authors thank two anonymous reviewers and editor for their useful suggestions and comments on a previous version of the manuscript. phd elvedin šabanović was supported by the environmental fund of the federation of bosnia and herzegovina (www. fzofbih.org.ba). vladimir randjelović, phd was supported by the ministry of education, science and technological development of the republic of serbia (grant no. 45103-68/2022-14/200124); vladan djordjević, phd was supported by the ministry of education, science and technological development of the republic of serbia (grant no. 451-03-47/2023-01/200178). references bateman, r.m., pridgeon, a.m., & chase, m.w. (1997). phylogenetics of subtribe orchidinae (orchidoideae, orchidaceae) based on nuclear its sequences. 2. infrageneric relationships and taxonomic revision to achieve monophyly of orchis sensu stricto. lindleyana, 12, 113–141. bateman, r.m., hollingsworth, p.m., preston, j., luo, y.b., pridgeon, a.m., & chase, m.w. (2003). molecular phylogenetics and evolution of orchidinae and selected habenariinae (orchidaceae). botanical journal of the linnean society, 142, 1–40. baumann, h., künkele, s., & lorenz, r. (2006). die orchideen europas. mit angrenzenden gebieten. germany, stuttgart: eugen ulmer kg. beck-mannagetta, g. (1904). flora von bosnien, der herzegowina und des sandžaks noovipazar i. teil. wissenschaftliche mitteilungen aus bosnien und der hercegovina, 9, 407-518. braun-blanquet, j. (1964). pflanzensoziologie. grundzüge der vegetationskunde. springer-verlag, vienna, new york. čadro, s., cherni–čadro, s., marković, m., & žurovec, j. (2019). a reference evapotranspiration map for bosnia and herzegovina. international soil and water conservation research, 7, 89–101. delforge, p. (2006). orchids of europe, north africa 12 biologica nyssana ● 14 (1) june 2023: 7-13 šabanović et al. ● orchis anthropophora (l.) all. (orchidaceae), a confirmed species for the flora of bosnia and herzegovina and the middle east. united kingdom, london: a. & c. black. djordjević, v., & tsiftsis, s. (2022). the role of ecological factors in distribution and abundance of terrestrial orchids. in: mérillon, j.-m., kodja, h. (eds.), orchids phytochemistry, biology and horticulture, reference series in phytochemistry. fundamentals and applications, (pp. 3–72). springer nature switzerland ag. fay, m.f. & taylor, i. (2015). 805. orchis anthropophora: orchidaceae. curtis’s botanical magazine, 32(1), 63–71. fukarek, p. (1969). dosadašnja floristička i vegetacijska istraživanja na području nacionalnog parka „sutjeska.” akademija nauka i umjetnosti bosne i hercegovine, odjeljenje prirodnih i matematičkih nauka, posebna izdanja, 3, 73–90. hayek, a. (1933). prodromus florae peninsulae balcanicae. 3. band monocotyledonae. germany, dahlem bei berlin: verlag des repertoriums. iucn (2012). iucn red list categories and criteria: version 3.1. [2nd ed.]. iucn, gland & cambridge: iucn. kretzschmar, h., eccarius, w., & dietrich, h. (2007). the orchid genera anacamptis, orchis and neotinea. phylogeny, taxonomy, morphology, biology, distribution, ecology and hybridization. (2nd ed.). germany, bürgel: echinomedia verlag. pridgeon, a.m., bateman, r.m., cox, a.v., hapeman, j.r., & chase, m.w. (1997). phylogenetics of subtribe orchidinae (orchidoideae, orchidaceae) based on nuclear its sequences. 1. intergeneric relationships and polyphyly of orchis sensu lato. lindleyana, 12, 89–109. kull, t., selgis, u., pecina, m.v., metsare, m., ilves, a., tali, k., & shefferson, r.p. (2016). factors influencing iucn threat levels to orchids across europe on the basis of national red lists. ecology and evolution, 6(17), 6245–6265. milanović, dj., stupar, v., šabanović, e., djordjević, v., brujić, j., boškailo, a., & ranđelović, v. (2022). on the distribution and conservation status of some rare orchid taxa (orchidaceae) in bosnia and herzegovina (western balkans). hacquetia, 21(2), 327–346. mucina, l., bültmann, h., dierßen, k., theurillat, j.-p., raus, t., čarni, a., šumberová, k., willner, w., dengler, j., gavilán garcía, r., chytrý, m., hájek, m., di pietro, r., iakushenko, d., pallas, j., daniëls, f. j. a., bergmeier, e., santos guerra, a., ermakov, n., valachovič, m., schaminée, j. h. j., lysenko, t., didukh, ya. p., pignatti, s., rodwell, j. s., capelo, j., weber, h. e., solomeshch, a., dimopoulos, p., aguiar, c., freitag, h., hennekens, s. m., & tichý, l. (2016). vegetation of europe: hierarchical floristic classification system of plant, lichen, and algal communities. applied vegetation science, 19, 3–264. protić, ð. (1902). treći prilog k poznavanju flore bosne i hercegovine. glasnik zemaljskog muzeja u bosni i hercegovini, 14, 17–68. sundermann, h. (1980). europäische und mediterrane orchideen. germany, hildesheim: brücke-verlag kurt schmersow. šabanović, e., boškailo, a., šarić, š., bektić, s., & ranđelović, v. (2019). the genus orchis tourn. ex l. and its related genera in the zenicadoboj canton (bosnia and herzegovina): diversity, distribution and conservation. biologica nyssana, 10(2), 143–153. šabanović, e., djordjević, v., milanović, đ., boškailo, a., šarić š., huseinović s., & randjelović, v. (2021). checklist of the orchidaceae of bosnia and herzegovina. phyton-annales rei botanicae, 61, 83–95. šilić, č. (1996). spisak biljnih vrsta (pteridophyta i spermatophyta) za crvenu knjigu bosne i hercegovine. glasnik zemaljskog muzeja bosne i hercegovine, prirodne nauke, nova serija, 31, 323– 367. tsiftsis s. & antonopoulos z. (2017). atlas of the greek orchids, volume i. greece, rethymno: mediterraneo editions. tyteca, d. & klein, e. (2008). genes, morphology and biology – the systematics of orchidinae revisited. journal europäischer orchideen, 40, 501–544. tyteca, d. & klein e. (2009). genes, morphology and biology – the systematics of orchidinae revisited: a reappraisal. journal europäischer orchideen, 41, 473–480. tyteca, d., ceinos, m., gathoye, j.-l., brys, r., & jacquemyn, h. (2012). on the morphological, biological and genetic heterogeneity of the genus orchis (orchidaceae, orchidinae). phytotaxa, 75, 19–32. vakhrameeva, m.g., tatarenko, i.v., varlygina, t.i., torosyan, g.k., & zagulski, m.n. (2008). orchids of russia and adjacent countries (within the borders of the former ussr). liechtenstein, ruggell: a.r.g. gantner verlag. wallenwein, f. & saad, a. (2000). messungen des ph-wertes an den wuchsorten mediterraner orchideen. journal europäischer orchideen, 32, 375–386. wcsp (world checklist of selected plant families). 2022. facilitated by the royal botanic gardens, kew. <http://apps.kew. org/wcsp> [accessed 01 december 2022]. biologica nyssana ● 14 (1) june 2023: 7-13 šabanović et al. ● orchis anthropophora (l.) all. (orchidaceae), a confirmed species for the flora of bosnia and herzegovina 13 uzelac et al. 2022, biologica nyssana 13(2) 13 (2) december 2022: 191-203 doi: 10.5281/zenodo.7476250 structure and chemistry of glandular trichomes of selected micromeria and clinopodium species (lamiaceae): in vitro culture approach original article branka uzelac department of plant physiology, institute for biological research “siniša stanković” national institute of the republic of serbia, university of belgrade, bulevar despota stefana 142, 11060 belgrade, serbia branka@ibiss.bg.ac.rs (corresponding author) snežana budimir department of plant physiology, institute for biological research “siniša stanković” national institute of the republic of serbia, university of belgrade, bulevar despota stefana 142, 11060 belgrade, serbia dragana stojičić department of biology and ecology, faculty of sciences and mathematics, university of niš, višegradska 33, niš, serbia received: november 14, 2022 revised: november 30, 2022 accepted: december 07, 2022 abstract: many of the species belonging to the lamiaceae family are considered aromatic plants due to the presence of glandular trichomes, which have a distinct ability to synthesize, secrete or store large amounts of specialized metabolites that play a crucial role in mediating the plant–environment interactions. these compounds often have marked bioactive properties, rendering a commercial value to the plants that produce them. a number of biological effects have been associated with the main monoterpenoids detected in investigated micromeria spp. and clinopodium spp. essential oils. one alternative for the production of these bioactive metabolites is in vitro plant tissue culture. the present study was initiated to investigate the effects of in vitro culture on the secretion of leaf glandular trichomes, the main structures involved in the essential oil production. the glandular indumentum was studied by means of light microscopy and scanning electron microscopy in an attempt to correlate the phytochemical traits with the glandular trichome morphotypes of selected lamiaceae species. key words: glandular trichomes, histochemistry, lamiaceae, micropropagation, morphology apstrakt: struktura i hemija žlezdanih trihoma odabranih vrsta rodova micromeria i clinopodium (lamiaceae): primena kulture in vitro mnoge vrste familije lamiaceae su označene kao aromatične biljke zahvaljujući prisustvu žlezdanih trihoma, koje odlikuje karakteristična sposobnost da sintetišu, luče ili skladište velike količine specijalizovanih metabolita koji posreduju u interakciji između biljaka i njihove životne sredine. ova jedinjenja često imaju značajna bioaktivna svojstva, koja biljkama koje ih produkuju daju komercijalnu vrednost. veliki broj bioloških efekata dovodi se u vezu sa glavnim monoterpenoidima detektovanim u etarskim uljima ispitivanih vrsta micromeria spp. and clinopodium spp. kultura biljaka in vitro predstavlja jednu od alternativa za proizvodnju ovih bioaktivnih metabolita. prikazano istraživanje je započeto sa ciljem da se ispitaju efekti kulture in vitro na sekreciju žlezdanih trihoma listova, osnovnih struktura uključenih u proizvodnju etarskih ulja. žlezdani pokrivač je proučavan primenom svetlosne i skenirajuće elektronske mikroskopije, u pokušaju da se ustanovi korelacija između fitohemijskih odlika i morfotipova žlezdanih trihoma odabranih biljnih vrsta familije lamiaceae. ključne reči: histohemija, lamiaceae, mikropropagacija, morfologija, žlezdane trihome secretion refers to the complex phenomena of separation of secreted substances from the protoplast and their removal either to the plant surface or into internal spaces, or their accumulation in some compartment of the cell (evert, 2006). the secreted substances are produced by the secretory tissues of diverse structure and topographic position that occur in most vascular plants. glandular trichomes are the most recently evolved secretory structures found on the surface of about 30% of all vascular plants. they vary in their structure, in the chemical composition of the substances they produce and the mode of their secretion, and in their function. many of the species belonging to the lamiaceae © 2022 uzelac et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 191 14th symposium on the flora of southeastern serbia and neighboring regions family are considered aromatic plants due to the presence of glandular trichomes, a valuable source of biologically active volatile compounds. glandular trichomes in lamiaceae can produce a variety of specialized metabolites, among which terpenoids are particularly well represented and have been used by humans in a variety of industries (tissier et al., 2013). glandular trichomes secreting lipophilic substances generally consist of a group of glandular cells at the apex of a stalk of one or more cells in length. the storage compartment of glandular trichomes is part of the glandular cell, or cells, which are metabolically active. the present taxonomy ascribes great value to the structure (shape, cell number) and distribution of trichomes, as well as to the type of the secretion and its storage, and mode of release (giuliani & maleci bini, 2008). lamiaceae members are characterized by a great variety of trichomes. from a functional viewpoint and in accordance with their mode of secretion, the glandular trichomes of lamiaceae species can be classified into two main types, peltate and capitate trichomes (hallahan, 2000; werker, 2000). peltate trichomes have common characteristics in structure and morphology across genera and mostly produce and store biogenic volatile or semi-volatile organic compounds related to plant abiotic or biotic stress responses (corsi & bottega, 1999; turner et al., 2000; machado et al., 2006). they are the main sites of essential oil production and storage, as revealed by their ultrastructure and histochemistry (werker, 1993; turner et al., 2000). peltate trichomes are considered long-term trichomes, given that the accumulation of the secreted material continues during the growth of the organs that bear them (werker, 1993). there are various types of capitate trichomes that differ significantly in both structure and size (werker, 1993; werker et al., 2000), and whose primary function is thought to be the repelling of the pests (lin & wagner, 1994). capitate trichomes are specialized to produce and store a large amount of diterpenes and a wide array of nonvolatile or poorly volatile compounds that are directly exuded onto trichome surface and are particularly active at the early stages of organ development. they are considered short term trichomes because the secretory materials are extruded to the outside soon after their production, and the entire process of secretion is soon terminated (werker, 1993). the plant family lamiaceae, one of the largest among the dicotyledons, comprises more than 7200 species across approximately 240 genera. the vast number of genera and species, especially those placed in subtribe menthinae (tribe menthae, subfamily nepetoideae), are spices and medicinal herbs of great economic importance (brauchler et al., 2010). the genus micromeria benth. comprises more than 70 perennial herbs, sub-shrubs and shrubs, rarely annual herbs, distributed throughout the temperate belt (harley et al., 2004; erhardt et al., 2014). micromeria spp. are more or less aromatic species producing a small quantity of essential oils dominated by various terpene compounds. closely related genus clinopodium l. comprises ca. 100 (braüchler et al., 2010) to 135 (erhardt et al., 2014) perennial herbs, including taxa that were recently transferred from the polyphyletic genus micromeria sect. pseudomelissa based on molecular and morphoanatomical evidence (braüchler et al., 2006). clinopodium species are distributed worldwide, with the mediterranean being the main center of species diversity and sectional diversity and are also aromatic. they produce variable quantities (depending on the sample collection site) of essential oils dominated by oxygenated monoterpenes. essential oils of micromeria and clinopodium species exhibit substantial antimicrobial (marinković et al., 2002; duru et al., 2004; šavikin et al., 2010; vuko et al., 2012) and antioxidant activities (vladimirknežević et al., 2011), thus protecting the plant from pathogen attacks. it is for these properties that the aboveground plant parts of some micromeria and clinopodium species are used for medical, insecticidal, herbicidal, and culinary purposes (duru et al., 2004; šavikin et al., 2010; vladimir-knežević et al., 2015). plant sources are increasingly being exploited for new drug development, and there is a growing interest in validating traditional medicines and herbal remedies (verpoorte, 2000). natural sources of bioactive compounds used as pharmaceuticals, agrochemicals, flavors, fragrances, food additives and biopesticides are often endangered due to increasing industrial demands. plant cell, tissue, and organ cultures have emerged as potential sources of structurally complex and high-value natural products, especially if the plant source material is an overexploited, slow-growing, or low-yielding plant. through the application of various in vitro approaches and strategies, plant cell, tissue, and organ culture techniques permit manipulation of growth and production of medicinally or commercially important plant metabolites in the microenvironment of in vitro cultures, independently of geographical or seasonal variation (isah et al., 2018). a valuable method for the multiplication of selected genotypes and chemotypes of many medicinal and aromatic plants is in vitro propagation from wild-growing plants through axillary shoot formation (fig. 1). the present study was initiated to investigate the effects of in vitro culture on the secretion of leaf 192 biologica nyssana ● 13 (2) december 2022: 191-203 uzelac et al. ● structure and chemistry of glandular trichomes of selected micromeria and clinopodium species (lamiaceae): in vitro culture approach biologica nyssana ● 13 (2) december 2022: 191-203 uzelac et al. ● structure and chemistry of glandular trichomes of selected micromeria and clinopodium species (lamiaceae): in vitro culture approach 193 glandular trichomes, the main structures involved in the essential oil production, in an attempt to correlate the phytochemical traits with the glandular trichome morphotypes of selected lamiaceae species. to that aim, we carried out observations on micromorphology, morphoanatomy, and secretion of the glandular trichomes in shoot cultures of micromeria croatica (pers.) schott, micromeria graeca (l.) benth. ex rchb., clinopodium pulegium (rochel) bräuchler and clinopodium thymifolium (scop.) kuntze. general aspects of plant trichomes the plant trichomes are specialized unior multicellular epidermal projections of diverse form, structure, and functions, which cover most surfaces of most plants (esau, 1953). they display greatly variable morphology depending on the organ and the species, which has often been used in plant classification (wagner, 1991). in lamiaceae, the fig. 1. micropropagation of selected micromeria and clinopodium species. in vitro plantlets of micromeria graeca cultured on plant growth regulator-free medium after 4 weeks of culture (a-c); note numerous axillary shoots and well-developed adventitious roots in c. in vitro plantlet of micromeria croatica (d). axillary bud proliferation of clinopodium pulegium (e). acclimatized c. pulegium plantlets regenerated via axillary shoots (f). type, size and density of trichomes vary within genera, within species, and between different organs of the same plant, but most differences are speciesspecific and can often be of taxonomic significance (bhatt et al., 2010). the physiological functions of trichomes are diverse, as their morphological and mechanical features (size, shape, density, orientation) influence many aspects of plant physiology and ecology (wagner et al., 2004). two general types of trichomes can be discerned: non-glandular trichomes, which mainly differ in their morphology, and glandular (secreting) trichomes, which typically differ in the substances that they secrete. non-glandular trichomes play an important role in mechanical defense against biotic and abiotic stresses, and generally do not produce or secrete phytochemicals. these trichomes are diverse in morphology, anatomy and microstructure. nonglandular trichomes are abundant in the early phases of leaf development, but their density decreases with 194 biologica nyssana ● 13 (2) december 2022: 191-203 uzelac et al. ● structure and chemistry of glandular trichomes of selected micromeria and clinopodium species (lamiaceae): in vitro culture approach leaf maturity (corsi & bottega, 1999; ascensão et al., 1999). glandular trichomes are the specific sites for the biosynthesis, accumulation and excretion of secondary metabolites, i.e. pestor pollinatorinteractive chemicals that are stored or volatilized from the plant surface (keene & wagner, 1985; turner et al., 2000; werker et al., 2000). they not only accumulate and store what is often phytotoxic secretory material in a compartment that is virtually outside the plant body, but also position these compounds as an apparent first line of defense at the surface of the plant (wagner, 1991). glandular trichomes contain cells that are highly specialized for the biosynthesis and secretion of copious amounts of particular secretory products (lange & turner, 2013). glandular trichomes are the most recently evolved secretory structures, and are characteristic feature of many angiosperms (fahn, 1988; lange & turner, 2013). they are suggested to have developed phylogenetically from non-glandular trichomes, because both trichome types are initiated and develop similarly up to the stage of a three-celled primordium, after which the differences between the two types begin to appear (fahn & shimony, 1977). from a functional viewpoint, based on the mode and timing of secretion, glandular trichomes can be classified into two types: long-term glandular trichomes, which gradually accumulate their secretion in a subcuticular space, and short-term glandular trichomes, which start and end their secretion rapidly (werker, 1993). the former corresponds to peltate trichome morphotype, whereas the latter correspond to capitate trichome morphotype. peltate trichomes do not exhibit considerable variations in their basic morphology, although they can differ significantly in their size (ascensão et al., 1999; corsi &bottega, 1999; turner et al., 2000; machado et al., 2006; stojičić et al., 2022). various types of capitate trichomes differ significantly in both structure and size (werker et al., 1985; ascensão & pais, 1998; kolb & müller, 2004; amrehn et al., 2014). apart from morphology, peltate and capitate trichomes also differ in metabolites that they produce. besides volatile or semi-volatile stress-related organic compounds that are secreted and stored in extracellular storage space, capitate trichomes produce a wide array of nonvolatile or poorly volatile compounds that are directly exuded onto trichome surface. trichome types, functions, and distribution in micromeria and clinopodium spp. in vitro the aerial parts of in vitro grown plants of all examined species are covered with indumentum that appears colorless to the naked eye and consists of non-glandular and glandular trichomes. the most conspicuous are numerous sharply pointed, unbranched non-glandular trichomes, which are particularly elongated and abundant on leaf petioles and midribs (fig. 2a). uniseriate non-glandular trichomes with warty surfaces (fig. 2b) are found on both leaf sides and are especially abundant along the margins and veins and at the leaf base (fig. 3a). their morphology and distribution on plants in vitro fig. 2. stem and leaf indumentum of in vitro grown micromeria graeca plantlets (a). cross section of pubescent leaf, showing many non-glandular and glandular trichomes on both leaf surfaces (b) 195 biologica nyssana ● 13 (2) december 2022: 191-203 uzelac et al. ● structure and chemistry of glandular trichomes of selected micromeria and clinopodium species (lamiaceae): in vitro culture approach concentrated on the intervein areas in all examined species (fig. 3). trichome production and maturation is limited to short periods early in leaf development (hülskamp et al., 1994). in all examined in vitro plants, glandular trichomes are initiated very early during leaf ontogeny, with trichome initials and young developing trichomes present already on the youngest leaf primordia (fig. 4). glandular trichomes arise from a single expanding protodermal cell undergoing a periclinal division. peltate and capitate trichomes morphotypes cannot be easily distinguished at their inception, both being first discernible as protruding protodermal cells with an asymmetrical cytoplasmic distribution (fig. 4a). after enlarging and extending above from the leaf surface, the expanded protodermal cell is partitioned by a periclinal, asymmetrical cell division, resulting in the two-celled stage trichome comprised of an apical, more meristematic cell and a basal, more vacuolated cell (fig. 4b). slightly older, three-celled stage trichomes are further partitioned by periclinal cell divisions, separating an apical initial and a stalk cell, atop a vacuolated basal cell (fig. 4c). the apical initial may remain unicellular, as is the case in capitate trichomes of all investigated species or give rise to multicellular secretory head of peltate trichomes, as a result of several rounds of anticlinal divisions, depending on the species. further changes on the ultrastructural level occur in accordance with the chemistry of glandular trichome secretion (ascensão & pais, 1998; turner et al., 2000; giuliani & maleci bini 2008; amrehn et al., 2014; uzelac et al., 2015). the main types of glandular trichomes observed in micropropagated plants are peltate and capitate, which is a characteristic feature of lamiaceae species (ascensão et al., 1999). peltate trichomes are less abundant than capitate trichomes in all examined species. they are present on both leaf surfaces but are more abundant on the abaxial side (fig. 3, fig. 5). two types of capitate trichomes, differing in size and structure, could be distinguished in all examined species: type 1 (c1) and type 2 (c2) capitate trichomes (fig. 3c). both capitate types are found on both abaxial and adaxial surfaces of leaves, on stems and fully correspond to wild-growing plants (kremer et al., 2012; dunkić et al., 2017). the length of these trichomes varies from very short on the leaf lamina, to very long on leaf petioles and stems, which display the densest indumentum of non-glandular trichomes. glandular trichomes are constant features of many plant species and develop over the aerial vegetative and reproductive plant organs without external stimuli (fahn, 1988). although covering all the aboveground organs of in vitro propagated plants, glandular trichomes are particularly abundant on the leaves, where they appear on both sides but at different proportions and are particularly fig. 3. sem micrographs showing distribution and types of trichomes on vegetative and reproductive organs of in vitro grown plants. young, not fully developed leaf of micromeria graeca, with many non-glandular trichomes at the leaf margins and along the veins on the abaxial surface (a). mature glandular trichomes on calyx of clinopodium thymifolium. inset: detail of b, showing c2 trichomes at full secretion (bar=25 μm) (b). abaxial side of fully developed leaf of micromeria croatica. p – peltate trichome; c – capitate trichomes type 1 (c1) and type 2 (c2); ng – nonglandular trichome (c) 196 biologica nyssana ● 13 (2) december 2022: 191-203 uzelac et al. ● structure and chemistry of glandular trichomes of selected micromeria and clinopodium species (lamiaceae): in vitro culture approach on the abaxial side of calices (figs. 2, 3b-c, 5b). on leaves of in vitro grown plants, c1 trichomes were the most abundant trichome type, randomly distributed over the entire surface (predominantly abaxial). c1 trichomes are the most commonly occurring capitate trichome type in lamiaceae, reported in nearly all the species examined (werker et al., 1985; ascensão et al., 1999; mota et al., 2013). they were found in all investigated micromeria and clinopodium sp. plants in vitro, as well as in their wild-growing counterparts (kremer et al., 2012; marin et al., 2013; dunkić et al., 2017; kremer et al., 2021). compared fig. 4. glandular trichome initiation on leaves of in vitro cultured micromeria croatica. a transverse section through shoot apex, showing two youngest pairs of leaf primordia with trichomes at different developmental stages, mostly on the abaxial side. b detail of a. note trichome initial (arrowhead) adjacent to two-celled stage trichome (arrow) on the youngest leaf primordium. c early-stage glandular trichome following periclinal division of protodermal cell, comprising a more vacuolated basal cell (asterisk) and two densely cytoplasmatic cells; later during ontogeny, a smaller proximal meristematic cell (arrow) will give rise to the stalk cell, whereas larger apical cell (double arrow) will give rise to glandular head 197 biologica nyssana ● 13 (2) december 2022: 191-203 uzelac et al. ● structure and chemistry of glandular trichomes of selected micromeria and clinopodium species (lamiaceae): in vitro culture approach fig. 5. peltate trichomes of in vitro grown plants. a-b sem micrographs showing peltate trichomes at different secretory stages: immature, presecretory to early secretory stage trichomes of micromeria graeca (a) and fully mature, secretory stage trichome of clinopodium thymifolium (b). longitudinal section of post secretory stage peltate trichome of micromeria croatica (c). note multicellular secretory head (h), comprising 4 central cells surrounded by 12 peripheral cells arranged in a shield, subtended by unicellular stalk (s) and vacuolated basal cell (b). upper view of glandular head of fully developed peltate trichome of m. graeca, with 4 central cells surrounded by 16 peripheral cells arranged in a shield (d). note cuticular cap (arrowhead) detached from the secretory cell lateral walls. positive nile blue a reaction in the subcuticular space (pink droplet) and head cells (intense blue staining) of clinopodium pulegium (e). secretion in the subcuticular space stained positively (dark violet) for terpenes with nadi reagent in m. croatica (f) biologica nyssana ● 13 (2) december 2022: 191-203 uzelac et al. ● structure and chemistry of glandular trichomes of selected micromeria and clinopodium species (lamiaceae): in vitro culture approach 198 to dominant c1 trichomes, c2 trichomes were less abundant in all examined in vitro plants and occurred more frequently on the adaxial leaf side. morphoanatomy of glandular trichomes in micromeria and clinopodium spp. in vitro in micropropagated plants of all examined species, peltate trichomes at different secretory stages were observed (fig. 5). immature glands with wrinkled surface (fig. 5a), indicative of the close attachment of the cuticle to the upper secretory cells’ walls are predominantly located at the leaf base and closer to the midrib. during gland maturation, the secretory material is gradually secreted from the head cells into developing subcuticular space, formed by detachment and elevation of the cuticle, where the secretion was shown to accumulate in many lamiaceae species (werker, 1993; serrato-valenti et al., 1997; zuzarte et al., 2010). therefore, mature glands appear balloon shaped, and their surface becomes smoother, displaying occasional droplets of secreted material (fig. 5b). peltate trichomes exhibited rather uniform morphology across examined species and consisted of a broad basal cell embedded in the epidermis, a short unicellular stalk with cutinized walls and a round multicellular secretory head (ca. 50-60 μm in diameter, at secretory stage), with a variable number (8-20) of secretory cells, depending on the species (fig. 5c-d). different types of capitate trichomes, differing in stalk length and secretory head structure, have been described (werker et al., 1985; giuliani & maleci bini, 2008). in vitro grown plants of examined micromeria and clinopodium species displayed two types of capitate trichomes, differing in size and structure (fig. 3c). differences in the morphological characteristics of various capitate trichome types reflect different secretory processes within trichomes, and probably distinct functions (ascensão et al., 1999). type 1 capitate trichomes (c1) were positioned at an angle to the leaf surface (fig. 6). they were composed of one basal epidermal cell, short unicellular stalk with cutinized lateral walls and unicellular ellipsoidal head of ca. 20-25 μm in size (fig. 6a). no cuticle elevation was observed, and the secretory product accumulated within the secretory cell. as suggested by fahn (1988), the thickenings of the cuticle on the lateral walls of the neck cell, functioning also as a short stalk, probably prevent the backflow of secreted products into mesophyll tissue. in this way, trichomes remain structurally (and biosynthetically) isolated from the rest of the leaf, and can therefore, unlike leaf, produce large quantities of secretory material that is often phytotoxic (nielsen et al., 1991; werker, 2000). type 2 capitate trichomes (c2) are positioned upright and composed of conical, elongated, and highly vacuolated basal cell, unito bicellular stalk, and unicellular, usually elongated secretory fig. 6. capitate trichomes type 1 on leaves of in vitro grown plants. a-b longitudinal sections of c1 trichomes, showing large basal cell, short unicellular stalk and ellipsoidal unicellular head bent to the surface of micromeria graeca leaf, after staining with sudan iv (a) and nile blue a (b); c-d longitudinal sections of c1 trichomes showing faint staining with nadi reagent of the secretion in the head cell of clinopodium thymifolium (c) and micromeria croatica (d). note intense staining of the stalk cell lateral walls 199 biologica nyssana ● 13 (2) december 2022: 191-203 uzelac et al. ● structure and chemistry of glandular trichomes of selected micromeria and clinopodium species (lamiaceae): in vitro culture approach head of ca. 10 μm in diameter (fig. 7). immature c2 trichomes (fig. 7a, fig. 7d) appear finger-like since elongated head cell is of the same diameter as the stalk cell(s). during maturation, secretory head of c2 trichomes develops spherical subcuticular space where the secretion accumulates (fig. 7b, fig. 7e, fig. 7f), rendering trichome head rounded appearance (fig. 3c). in the post-secretory phase, after cuticle rupture, subcuticular elevation is no longer visible, so that the glandular head of c2 trichomes no longer appears round but regains fig. 7. capitate trichomes type 2 on leaves of in vitro grown plants. longitudinal sections of c2 trichomes, showing vacuolated pyramidal basal cell, subtending cylindrical unicellular stalk and unicellular head, both of approximately same diameters, after staining with sudan iv (a, d), nile blue a (b, e) and nadi reagent (c, f). note secretion accumulated in the subcuticular space of the early (b) and late secretory-stage trichomes (e, f), rich in essential oils (b, e) and terpenoids (f); in post secretory stage trichome (c), following rupture, the cuticle remains firmly attached to the cell wall in the basal part of the secretory cell, but no subcuticular elevation is visible. mg – micromeria graeca, mc – micromeria croatica, cp –clinopodium thymifolium finger-like shape (fig. 7c). type 2 capitate trichomes of in vitro grown plants correspond to those described by werker et al. (1985) for the lamiaceae, and are very similar to those detected in salvia officinalis (corsi & bottega, 1999) or lavandula pedunculata (zuzarte et al., 2010). nevertheless, kremer et al. (2021) distinguished 4 subtypes of capitate trichomes in studied micromeria and clinopodium spp. besides c1 capitate subtype, present in all investigated micromeria and clinopodium species, the authors biologica nyssana ● 13 (2) december 2022: 191-203 uzelac et al. ● structure and chemistry of glandular trichomes of selected micromeria and clinopodium species (lamiaceae): in vitro culture approach 200 observed c2 subtype trichomes (a rounded head cell) only in micromeria species studied, whereas roundish-head c3 and finger-like c4 (an elongated head cell, as narrow as the stalk cells, and only slightly enlarged above) subtypes were observed only in clinopodium taxa. based on these findings, and since the investigated taxa of the genus clinopodium belong to the former micromeria section pseudomelissa, the authors concluded that micromorphological traits also support the recent transfer of micromeria sect. pseudomelissa to the genus clinopodium (kremer et al., 2021). however, these claims were based solely on scanning electron micrographs, which do not always allow precise determination of the cell number or shape. in our study, both sem and light micrographs showed that the appearance of c2 trichomes differed depending on trichome developmental stage and secretory phase, as well as on sectioning plane. capitate trichomes type 2 that we observed in both investigated micromeria species fully corresponded to those observed in clinopodium species. fingerlike capitate trichomes were observed in both taxa, and were indicative of early secretory-stage trichomes, with still developing subcuticular space. as the secretion continued, and more secretory material accumulated in this space, the morphology of the glandular head changed from roundish to spherical, as observed in m. croatica (fig. 3c) as well as in c. thymifolium (fig. 3b) and c. pulegium (fig. 7e). histochemical characterization of secretions of glandular trichomes in micromeria and clinopodium spp. in vitro histochemical studies enable preliminary identification of the classes of bioactive compounds in tissues and cell compartments and are often employed to localize in situ the main classes of secondary metabolites present in plant secretions. together with morphological and structural investigations, histochemical studies provide data on secretory modes and secretions, thus allowing us to postulate a functional role for different glandular trichome types in plant interactions with its abiotic and biotic environment. the main classes of metabolites secreted by glandular trichomes of micropropagated plants were characterized by the histochemical tests in plant exudates in situ (figs. 5-7). in all examined species, peltate trichome secretion contained lipophilic substances, as revealed by sudan iv, and was for the most part composed of essential oil (tab. 1). nile blue a staining for neutral and acidic lipids gave positive reaction in the subcuticular space and to a lesser degree in secretory cells and was particularly intense in clinopodium species (fig. 5e, tab. 1). the presence of terpenoid compounds in this trichome morphotype was evidenced by intense dark-violet staining, with nadi reagent, of the secretory product within subcuticular space of all examined species (fig. 5f). the use of nadi reagent demonstrated the presence of terpenoids (resiniferous acids and essential oils) in the trichome glandular cells or in their secretion in a number of plant species belonging to lamiaceae (corsi & bottega, 1999; ascensão et al., 1999; marin et al., 2013). in all examined species, capitate trichomes type 1 of in vitro grown plants stained weakly positive for lipophilic secretion (fig. 6, tab. 1). on the other hand, type 2 capitate trichomes exhibited much stronger histochemical reactions (fig. 7, tab. 1). lipophilic secretory products accumulated in the subcuticular space of c2 trichomes, and small amounts could also be observed within secretory cells. that the majority of those lipophilic substances were the essential oils was shown by nile blue a staining (fig. 7b, e). nadi reaction resulted in an intense dark-violet staining of the secretory product, thus confirming the presence of terpenoid compounds in type 2 capitate trichomes (fig. 7c, f). although it is generally assumed that the bulk of the essential oil in lamiaceae is produced by peltate trichomes, studies showed that capitate trichomes in some species seem to produce significant amounts of essential oils (ascensão et al., 1999; mota et al., 2013; stojičić et al., 2016). reports on the histochemical detection of different secondary metabolites carried out in plants table 1. histochemical characterization of secretions of leaf glandular trichomes of selected micromeria and clinopodium species chemical compounds micromeria graeca micromeria croatica clinopodium thymifolium clinopodium pulegium p c1 c2 p c1 c2 p c1 c2 p c1 c2 lipids ++ ± + ++ ± + ++ + ± + + ++ essential oils + ± + + ± + ++ + ± ++ + ++ terpenoids ++ ± + ++ ± + ++ + ± ++ + ++ 201 biologica nyssana ● 13 (2) december 2022: 191-203 uzelac et al. ● structure and chemistry of glandular trichomes of selected micromeria and clinopodium species (lamiaceae): in vitro culture approach grown in vitro are scarce (uzelac et al., 2015; stojičić et al., 2016; dantas et al., 2017; tošić et al., 2019). defined environment of in vitro culture provides plants with nearly optimal growth conditions, in contrast to marked variations of growth conditions of wild-growing plants, which may contribute to increased secondary metabolite production in vitro reported for a number of species (fraternale et al., 2003; affonso et al., 2009; tošić et al., 2019). shoot cultures of many medicinal and aromatic plants have been shown to accumulate secondary metabolites to a greater extent compared to natural plants (bassolino et al., 2015; makowczyńska et al., 2016). since in vitro cultured plants are by definition considered as juvenile-stage plants (croteau et al., 1981), the reported quali-quantitative differences in the volatile profile between wild-growing plants and those grown in vitro could be the consequence of different ontological stage of the plants. conclusions in recent decades, considerable research has focused on understanding and exploiting glandular trichomes ability to secrete phytochemicals that might improve plant resistance to pests, modify trichome metabolism towards improving exudate levels and quality traits and allow commercial production of useful compounds. plant cell, tissue and organ culture is a valuable method for the multiplication of selected genotypes and chemotypes, enabling efficient clonal propagation regardless of the season as well as quantitative and qualitative modifications in the production of plant secondary metabolites by different physical and chemical factors. acknowledgements. this research was financially supported by the ministry of education, science and technological development of the republic of serbia through contracts nos. 451-03-68/2022-14/200007 and 451-03-68/2022-14/200124. references affonso, v.r., bizzo, h.r., salguiero lage, c.l., sato, a. 2009: influence of growth regulators in biomass production and volatile profile of in vitro plantlets of thymus vulgaris l. journal of agricultural and food chemistry, 57: 6392–6395. amrehn, e., heller, a., spring, o. 2014: capitate glandular trichomes of helianthus annuus (astearceae): ultrastructure and cytological development. protoplasma, 251: 161-167. ascensão, l., mota, l., castro, m. de m. 1999: glandular trichomes on the leaves and flowers of plectranthus ornatus: morphology, distribution and histochemistry. annals of botany, 84: 437-447. ascensão, l., pais, m.s. 1998: the leaf capitate trichomes of leonotis leonurus: histochemistry, ultrastructure and secretion. annals of botany, 81: 263-271. bassolino, l., giacomelli, e., giovanelli, s., pistelli, l., casetti, a., damonte, g., bisio, a., ruffoni, b. 2015: tissue culture and aromatic profile in salvia dolomitica codd. plant cell, tissue and organ culture, 121: 83-95. bhatt, a., naidoo, y., nicholas, a. 2010: an investigation of the glandular and non-glandular foliar trichomes of orthosiphon labiatus n.e.br. [lamiaceae]. new zealand journal of botany, 48: 153-161. bräuchler, c., meimberg, h., heubl, g. 2006: new names in old world clinopodium – the transfer of the species of micromeria sect. pseudomelissa to clinopodium. taxon, 55: 977-981. braüchler, c., meimberg, h., heubl, g. 2010: molecular phylogeny of menthinae (lamiaceae, nepetoideae, mentheae) – taxonomy, biogeography and conflicts. molecular phylogenetics and evolution, 55: 501–523. corsi, g., bottega, s. 1999: glandular hairs of salvia officinalis: new data on morphology, localization and histochemistry in relation to function. annals of botany, 84: 657–664. croteau, r., felton, m., karp, f., kjonaas, r. 1981: relationship of camphor biosynthesis to leaf development in sage (salvia officinalis). plant physiology, 67: 820–824. dantas, l.a., melo, a.m., pereira, p.s., souza, l.a., filho, s.c.v., silva, f.g. 2017: histochemical screening of leaves compared to in situ and in vitro calluses of solanum aculeatissimum jacq. journal of agricultural science, 9: 80-96. dunkić, v., kremer, d., jurišić grubešić, r., vuković rodríguez, j., ballian, d., bogunić, f., stešević, d., kosalec, i., bezić, n., stabentheiner, e. 2017: micromorphological and phytochemical traits of four clinopodium l. species (lamiaceae). south african journal of botany, 111: 232–241. duru, m.e., öztürk, m., uğur, a., ceylan, ö. 2004: the constituents of essential oil and in vitro antimicrobial activity of micromeria cilicica from turkey. journal of ethnopharmacology, 94: 43-48. erhardt, w., götz, e., bödeker, n., seybold, s. 2014: zander-handwörterbuch der pflanzennamen, 19th ed. aufl. eugen ulmer gmbh und co. stuttgart. 576 p. biologica nyssana ● 13 (2) december 2022: 191-203 uzelac et al. ● structure and chemistry of glandular trichomes of selected micromeria and clinopodium species (lamiaceae): in vitro culture approach 202 esau, k. 1953: plant anatomy. john wiley & sons, new york. evert, r.f. 2006: external secretory structures. in: evert, r.f. (ed.), esau’s plant anatomy: meristem, cells, and tissues of the plant body: their structure, function and development, 3rd ed.: 447-472, john wiley & sons, new york. fahn, a. 1988: secretory tissues in vascular plants. new phytologist, 108: 229-257. fahn, a., shimony, c. 1977: development of the glandular and non‐glandular leaf hairs of avicennia marina (forsskål) vierh. botanical journal of the linnean society, 74: 37-46. fraternale, d., giamperi, l., ricci, d., rocchi, m.b.l., guidi, l., epifano, f., marcotullio, f.c. 2003: the effect of triacontanol on micropropagation and on secretory system of thymus mastichina. plant cell, tissue and organ culture, 74: 87-97. hallahan, d.l. 2000: monoterpenoid biosynthesis in glandular trichomes of labiate plants. in: hallahan, d.l. & gray, j.c. (eds.), advances in botanical research, 31: 77-120, academic press, new york. harley, r.m., atkins, s., budantsev, a.l., cantino, p.d., conn, b.j., grayer, r., harley, m.m., de kok, r., krestovskaja, t., morales, r., paton, a.j., ryding, o., upson, t. 2004: labiatae. in: kubitzki, k. & kadereit, j.w. (eds.), the families and genera of vascular plants, 7: 167275, springer, berlin. hülskamp, m., miséra, s., jürgens, g. 1994: genetic dissection of trichome cell development in arabidopsis. cell, 76: 555-566. isah, t., umar, s., mujib, a., sharma, m.p., rajasekharan, p.e., zafar, n., frukh, a. 2018: secondary metabolism of pharmaceuticals in the plant in vitro cultures: strategies, approaches, and limitations to achieving higher yield. plant cell tissue and organ culture, 132: 239–265. keene, c.k., wagner, g.j. 1985: direct demonstration of duvatrienediol biosynthesis in glandular heads of tobacco trichomes. plant physiology, 79: 1026–1032. kolb, d., müller, m. 2004: light, conventional and environmental scanning electron microscopy of the trichomes of cucurbita pepo subsp. pepo var. styriaca and histochemistry of glandular secretory products. annals of botany, 94: 515-526. kremer, d., stabentheiner, e., bogunić, f., ballian, d., eleftheriadou, e., stešević, d., matevski, v., ranđelović, v., ivanova, d., ruščić, m., dunkić, v. 2021: micromorphological traits of balcanic micromeria and closely related clinopodium species (lamiaceae). plants, 10: 1666. kremer, d., stabentheiner, e., dunkić, v., dragojević müller, i., vujić, l., kosalec, i., ballian, d., bogunić, f., bezić, n. 2012: micromorphological and chemotaxonomical traits of micromeria croatica (pers.) schott. chemistry and biodiversity, 9: 755-768. lange, b.m., turner, g.w. 2013: terpenoid biosynthesis in trichomes – current status and future opportunities. plant biotechnology journal, 11: 2-22. lin, y., wagner, g.j. 1994: surface disposition and stability of pest-interactive, trichome-exuded diterpenes and sucrose esters of tobacco. journal of chemical ecology, 20: 1907–1921. machado, s.r., gregório, e.a., guimarães, e. 2006: ovary peltate trichomes of zeyheria montana (bignoniaceae): developmental ultrastructure and secretion in relation to function. annals of botany, 97: 357–369. makowczyńska, j., sliwinska, e., kalemba, d., piątczak, e., wysokińska, h. 2016: in vitro propagation, dna content and essential oil composition of teucrium scorodonia l. ssp. scorodonia. plant cell, tissue and organ culture, 127: 1-13. marin, m., jasnić, n., ascensão, l. 2013: histochemical, micromorphology and ultrastructural investigation in glandular trichomes of micromeria thymifolia. botanica serbica, 37(1): 49–53. marinković, b., marin, p.d., knežević-vukčević, j., soković, m., brkić, d. 2002: activity of essential oils of three micromeria species (lamiaceae) against micromycetes and bacteria. phytotherapy research, 16: 336-339. mota, l., figueiredo, a.c., pedro, l.g., barroso, j.g., ascensão, l. 2013: glandular trichomes, histochemical localization of secretion, and essential oil composition in plectranthus grandidentatus growing in portugal. flavour and fragrance journal, 28: 393-401. nielsen, m.t., akers, c.p., järlfors, u.e., wagner, g.j., berger, s. 1991: comparative ultrastructural features of secreting and nonsecreting glandular trichomes of two genotypes of nicotiana tabacum l. botanical gazette, 152(1): 13-22. serrato-valenti, g., bisio, a., cornara, l., ciarallo, g. 1997: structural and histochemical investigation of the glandular trichomes of salvia 203 biologica nyssana ● 13 (2) december 2022: 191-203 uzelac et al. ● structure and chemistry of glandular trichomes of selected micromeria and clinopodium species (lamiaceae): in vitro culture approach aurea l. leaves, and chemical analysis of the essential oil. annals of botany, 79: 329-336. stojičić, d., tošić, s., slavkovska, v., zlatković, b., budimir, s., janošević, d., uzelac, b. 2016: glandular trichomes and essential oil characteristics of in vitro propagated micromeria pulegium (rochel) benth. (lamiaceae). planta, 244: 393-404. stojičić, d., tošić, s., stojanović, g., zlatković, b., jovanović, s., budimir, s., uzelac, b. 2022: volatile organic compound composition and glandular trichome characteristics of in vitro propagated clinopodium pulegium (rochel) bräuchler: effect of carbon source. plants, 11: 198. šavikin, p.k., menković, r.n., zdunić, m.g., tasić, r.s., ristić, s.m., stević, r.t., dajićstevanović, p.z. 2010: chemical composition and antimicrobial activity of the essential oils of micromeria thymifolia (scop.) fritsch., m. dalmatica benth., and satureja cuneifolia ten. and its secretory elements. journal of essential oil research, 22: 91-96. tissier, a., sallaud, c., rontein, d. 2013: tobacco trichomes as a platform for terpenoid biosynthesis engineering. in: bach, t., rohmer, m. (eds.), isoprenoid synthesis in plants and microorganisms: 271-283, springer, new york. tošić, s., stojičić, d., slavkovska, v., mihailovkrstev, t., zlatković, b., budimir, s., uzelac, b. 2019: phytochemical composition and biological activities of native and in vitro-propagated micromeria croatica (pers.) schott (lamiaceae). planta, 249: 1365-1377. turner, g.w., gershenzon, j., croteau, r.b. 2000: development of peltate glandular trichomes of peppermint. plant physiology, 124: 665–679. uzelac, b., janošević, d., stojičić, d., budimir, s. 2015: in vitro morphogenesis and secretion of secondary metabolites of nicotiana tabacum tall glandular trichomes. botanica serbica, 39: 103-110. verpoorte, r., van der heijden, r., memelink, j. 2000: engineering the plant cell factory for secondary metabolite production. transgenic research, 9: 323-343. vladimir-knežević, s., blažeković, b., bival štefan, m., alegro, a., kőszegi, t., petrik, j. 2011: antioxidant activities and polyphenolic contents of three selected micromeria species from croatia. molecules, 16: 1454-1470. vladimir-knežević, s., cvijanović, o., blažeković, b., kindl, m., bival štefan, m., domitrović, r. 2015: hepatoprotective effects of micromeria croatica ethanolic extract against ccl4induced liver injury in mice. bmc complementary and alternative medicine, 15: 233. vuko, e., dunkić, v., bezić, n., ruščić, m., kremer, d. 2012: chemical composition and antiphytoviral activity of essential oil of micromeria graeca. natural product communications, 7(9): 1227-1230. wagner, g.j. 1991: secreting glandular trichomes: more than just hairs. plant physiology, 96: 675–679. wagner, g.j., wang. e., shepherd, r.w. 2004: new approaches for studying and exploiting an old protuberance, the plant trichomes. annals of botany, 93: 3–11. werker, e. 1993: function of essential oil-secreting glandular hairs in aromatic plans of the lamiaceae – a review. flavour and fragrance journal, 8: 249– 255. werker, e. 2000: trichome diversity and development. in: hallahan, d.l. & gray, j.c. (eds.), advances in botanical research, 31: 1-35, academic press, new york. werker, e., ravid, u., putievsky, e. 1985: structure of glandular hairs and identification of the main components of their secreted material in some species of the labiatae. israel journal of botany, 34: 31-45. zuzarte, m.r., dinis, a.m., cavaleiro, c., salgueiro, l.r., canhoto, j.m. 2010: trichomes, essential oils and in vitro propagation of lavandula pedunculata (lamiaceae). industrial crops and products, 32: 580-587. stanojević et al. 2022, biologica nyssana 13(2) 13 (2) december 2022: 205-216 doi: 10.5281/zenodo.7476279 comparative analysis of chemical composition and antioxidant activity of essential oil isolated from orange and red marigold (tagetes patula l.) flower petals original article jelena stanojević faculty of technology, university of niš, bulevar oslobođenja 124, 16000 leskovac, serbia jstanojevic@tf.ni.ac.rs (corresponding author) nataša simonović faculty of technology, university of niš, bulevar oslobođenja 124, 16000 leskovac, serbia ljiljana stanojević faculty of technology, university of niš, bulevar oslobođenja 124, 16000 leskovac, serbia zoran ilić faculty of agriculture, university of priština, kopaonička bb, 38219 lešak, serbia aleksandra milenković faculty of technology, university of niš, bulevar oslobođenja 124, 16000 leskovac, serbia jelena zvezdanović faculty of technology, university of niš, bulevar oslobođenja 124, 16000 leskovac, serbia dragan cvetković faculty of technology, university of niš, bulevar oslobođenja 124, 16000 leskovac, serbia received: november 20, 2022 revised: december 12, 2022 accepted: december 15, 2022 abstract: the present study aimed to determine and compare chemical composition and antioxidant activity of essential oils (eos) isolated from marigolds (tagetes patula l.) cultivated in the garden in southeast serbia. the eos were isolated from dry orange and red flower petals by clevenger type hydrodistillation during 2 h by using 1:15 m/v hydromodule. their qualitative composition was determined by gc/ms and quantitative by gc/fid method. the antioxidant activity was determined by using the dpph assay. the most abundant components in the essential oil isolated from orange flower petals were geranyl acetate (36.7%) and (e)-caryophyllene (31.6%) while the one isolated from the red flower petals contained (e)-caryophyllene (69.4%) in the highest percentage. since phototoxic thiophenes were identified in both eos, they should not be used as components in cosmetic products for applications on areas of skin exposed to sunshine. essential oils showed similar antioxidant activity, with orange flower eo being somewhat better. key words: marigold, tagetes patula l. flower petals, thiophenes, gc/ms, antioxidant activity apstrakt: uporedna analiza hemijskog sastava i antioksidativne aktivnosti etarskog ulja izolovanog iz narandžastih i crvenih cvetnih latica baštenske kadife (tagetes patula l.) cilj ovog rada bio je određivanje i poređenje hemijskog sastava i antioksidativne aktivnosti etarskih ulja izolovanih iz baštenske kadife (tagetes patula l.) gajene u jugostočnoj srbiji. etarska ulja su izolovana iz suvih narandžastih i crvenih cvetnih latica clevenger hidrodestilacijom u toku 2 h pri hidromodulu 1:15 m/v. kvalitativni sastav izolovanih etarskih ulja određen je gc/ms metodom a kvantitivni sastav gc/fid metodom. antioksidativna aktivnost određena je dpph testom. najzastupljeniji sastojci u etarskom ulju izolovanom iz narandžastih latica bili su geranil acetat (36.7%) i (e)-kariofilen (31.6%) dok je u etarskom ulju izolovanom iz crvenih latica (e)-kariofilen bio zastupljen u najvećem procentu. s obzirom na to da su fototoksični tiofeni identifikovani u oba etarska ulja, ova etarska ulja ne bi trebalo koristiti kao komponente u kozmetičkim proizvodima namenjenim nezi delova tela izloženih sunčevim zracima. etarska ulja su pokazala slična antioksidativna svojstva, pri čemu je etarsko ulje izolovano iz narandžastih cvetnih latica nešto bolje. ključne reči: baštenska kadifa, tagetes patula l. cvetne latice, tiofeni, gc/ms, antioksidativna aktivnost introduction the life of people and animals on the earth, as members of delicate world ecosystem, is possible due to the most important link of the chain with which they live in symbiosis – plants. people use plants more than 10,000 years. they cultivate them, consume (as the main source of vitamin c), and use as animal feed, as well as for fuel production (parisi et al., 2010). the “dark side” of ethnobotany is reflected in phytodermatosis (dermatitis caused by plants) occurrence. it could be caused by the direct contact with plants or by association with sunlight. skin reactions caused by contact with plants could © 2022 stanojević et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 205 14th symposium on the flora of southeastern serbia and neighboring regions be divided as follows: dermatitis by physical trauma, dermatitis by pharmacological action, ige-mediated dermatitis, dermatitis by irritation, sunlight-induced dermatitis and dermatitis by sensitization. the letter occurs when sensitizing substances are present. among other plant families, these substances are present in ornamental plants such as chrysantemum, daisies, and marigolds from sunflower (asteraceae) family (dos reis, 2010). the genus tagetes consists of annual and perennial herbaceous plants, commonly called marigolds. it is native to the area from south-western america to argentina, with maximum variations occurring in mexico, but also naturalized all over the globe (singh et al., 2015). phytochemical studies of different plant parts revealed the presence of phenylpropanoids, carotenoids (such as lutein, zeaxanthin, neoxanthin plus violaxanthin, β-carotene, lycopene, α-cryptoxanthin, phytoene and phytofluene), flavonoids, thiophenes, and triterpeniods (priyanka et al., 2013; salehi et al., 2018; singh et al., 2020). there are at least fifty-six tagetes species (salehi et al., 2018) and the most widely known are t. erecta (also known as mexican marigold), t. patula (french marigold, dwarf marigold) and t. minuta (black mint or stinking roger). they are mainly studied in the field of agriculture, because of fungicidal, bactericidal, and insecticidal activities (singh et al., 2015; salehi et al., 2018). t. minuta is mainly cultivated for its essential oil, while t. patula and t. erecta have floricultural use. the flowers of african marigolds are yellow to orange and do not include red coloured marigolds, while french marigolds have red, orange and yellow as well as red and orange bicolour patterns flowers (fig. 1). the marigold species are traditionally used as analgesics, antiseptics, carminatives, diuretics, stimulants etc. in mexico; in food seasoning and for insect repellence in usa; and in ceremonial purposes in india, mexico and guatemala. recently, they have been recognised as commercial resources of essential oils and biologically active compounds for potential use as agrochemicals, colorants and nutritional supplements as well as in cosmetics (singh et al., 2015). the aerial parts of marigolds are rich sources of strongly aromatic eo (tagetes oil), mainly used for perfumes production (singh et al., 2020). generally, tagetes essential oils are rich in monoterpene hydrocarbons (such as ocimenes, limonene, etc.) and in acyclic monoterpene ketones (such as tagetone, dihydrotagetone, and tagetenone) which are the primary odorants – fig. 2. there are also sesquiterpene hydrocarbons and oxygenated compounds but in much lower amounts. however, the chemical diversity is quite high (tomova et al., 2005; singh et al., 2015). marigolds are very popular as garden plants in southeastern serbia. however, there is scarce information on the chemical composition and antioxidant properties of their essential oil. therefore, the aim of the present study was to determine the chemical composition of essential oils isolated from red and orange tagetes patula l. flower petals grown in southeastern serbia in order to detect potentially present phototoxic compounds as well as to determine and compare their antioxidant activities. materials and methods plant material the seeds of tagetes patula l. (tagetes patula mix 5436) were purchased in local agricultural pharmacy (producer: semenarna ljubljana, slovenia). plant material was cultivated in the garden in village manastirište (municipality of vlasotince, southeastern serbia; 42.9612° n, 22.1590° e). the orange and red flowers in their full flowering state (july and august 2022) were collected and dried in a shadowy place, at room temperature. before 206 biologica nyssana ● 13 (2) december 2022: 205-216 stanojević et al. ● comparative analysis of chemical composition and antioxidant activity of essential oil isolated from orange and red marigold (tagetes patula l.) flower petals fig. 1. the aerial parts of tagetes patula l. (a, c) with dry orange (b) and red (d) flower petals used in this study biologica nyssana ● 13 (2) december 2022: 205-216 stanojević et al. ● comparative analysis of chemical composition and antioxidant activity of essential oil isolated from orange and red marigold (tagetes patula l.) flower petals 207 the analysis, the dried flower petals were separated from the capitula and ground in an electric grinder (moulinex multi moulinette 3 in 1, 500 w). chemicals and reagents the following chemicals and reagents were used in this study: ethanol, 96% (centrochem, zemun, serbia); 1,1-diphenyl-2-picrylhydrazyl (dpph radical), butylated hydroxytoluene (bht); alkane standard solution c8-c20 (~40 mg/l each, in hexane), alkane standard solution c21-c40; (e)-caryophyllene (≥98.0%, sum of enantiomers); β-pinene (≥99%); γ-terpinene (97%); linalool (97%) (sigma chemical company, st. louis, mo, usa); terpinen-4-ol (97%, j&k scientific ltd., beijing, china); hplc grade hexane (≥95%, fisher scientific, uk), and redistilled water. isolation of essential oil the essential oils (eos) were isolated by clevenger type hydrodistillation from dry flower petals during 2 h by using 1:15 m/v hydromodule. the volatile compounds were collected in hexane (1 ml) added fig. 2. chemical structures of the primary odorants present in tagetes eos in a graduated tube of the apparatus. the hexane solution was separated from the water phase and evaporated at room temperature. the residual essential oil was measured gravimetrically and stored at 4 °c until analysed. the yield of the essential oils was determined in g per 100 grams of the extracted plant material (g/100 g p.m.). gas chromatography-mass spectrometry (gc/ ms) and gas chromatography-flame ionization detection (gc/fid) analyses gc/ms analysis was performed on agilent technologies 7890b gas chromatograph, equipped with nonpolar, silica capillary column, hp-5ms (5% diphenyland 95% dimethyl-polysiloxane, 30 m × 0.25 mm, 0.25 μm film thickness; agilent technologies, santa clara, ca, usa) and coupled with inert, selective 5977a mass detector of the same company. the essential oils were dissolved in diethyl ether in concentrations of ~19 mg/ml (ofeo) and ~33 mg/ml (rfeo). one μl of the solution prepared was injected to the gc column through a split/splitless inlet set at 220 °c in 20:1 208 biologica nyssana ● 13 (2) december 2022: 205-216 stanojević et al. ● comparative analysis of chemical composition and antioxidant activity of essential oil isolated from orange and red marigold (tagetes patula l.) flower petals split mode. helium was used as the carrier gas, at a constant flow rate of 1 ml/min. the oven temperature increased from 60 °c to 246 °c at the rate of 3 °c/ min. temperatures of the msd transfer line, ion source and quadrupole mass analyzer were set at 300 °c, 230 °c and 150 °c, respectively. the ionization voltage was 70 ev and mass range m/z 41-415. gc/fid analysis was carried out under identical experimental conditions as gc/ms. the flows of the carrier gas (he), make up gas (n2), fuel gas (h2), and oxidizing gas (air) were 1, 25, 30, and 400 ml/ min, respectively. the temperature of the flameionization detector (fid) was set at 300 °c. data processing was performed using msd chemstation, masshunter qualitative analysis and amdis_32 softwares (agilent technologies, usa). retention indices of the components from the analyzed samples were experimentally determined using a homologous series of n-alkanes from c8-c20 and c21-c40 as standards. essential oil constituents identification was based on the comparison of their retention indices (riexp) with those available in literature (adams, 2007) (rilit); their mass spectra with those of authentic standard as well as with those from willey 6, nist2011 and rtlpest3 libraries (ms) and wherever possible, by co-injection with an authentic standard (co-i). quantification was done by an external standard method according to the procedure described by sparkman et al. (2011). the standards used were in the concentration ranges as follows: β-pinene (0.1252 mg/ml), linalool (1.67-15 mg/ml), γ-terpinene (0.75-5 mg/ml), terpinen-4-ol (0.0625-1 mg/ml) and (e)-caryophyllene (0.0625-1 mg/ml). the response factor (rf) for each standard used was calculated as follows: rf=areastd/ssd where areastd is the peak area of the analyte standard and cstd is the concentration of the standard used. the values of the mean response factors for the 5-points calibration curves of each standard used were: 8.39×106; 2.45×107; 3.02×107; 8.36×106; and 6.83×106 for β-pinene, γ-terpinene, linalool, terpinen-4-ol, and (e)-caryophyllene, respectively. then, the mean value of response factors (rfmean=1.56×10 7 for rfeo and 1.51×107 for ofeo) was calculated and used for quantification according to the following formula: cx=areax/rfmean where cx is the concentration of an analyte in the sample (in mg/ml of eo), areax is the peak area of the analyte in the sample and rfmean is the mean response factor of the standards used (sparkman et al., 2011). the calculated concentrations of the β-pinene, and γ-terpinene in rfeo were: 0.02 mg/ml and 0.03 mg/ ml respectively, while the concentrations of linalool, terpinen-4-ol, and (e)-caryophyllene were: 0.16 mg/ ml; 0.15 mg/ml; and 45.79 mg/ml in rfeo and 0.20 mg/ml; 0.11 mg/ml, and 12.12 mg/ml in ofeo, respectively. finally, the content of each component in the sample expressed in % was normalized to get percents according to the formula: c (%)=(cx/σcx)x100 where cx is the concentration of each component in the sample and ʃcx is the total concentration of all components in the sample. dpph assay the ability of the essential oils to scavenge free dpph radicals was determined using the dpph assay. essential oils were dissolved in ethanol and a series of different concentrations (0.4-13.5 mg/ml and 0.3-10.3 mg/ml for eo isolated from red (rfeo) and orange flower petals (ofeo, respectively)) were prepared. ethanol solution of dpph radical (0.3 ml, 300 μmol solution (3×10-4 mol/l)) was added to 0.75 ml of the prepared essential oil solutions („sample”) and the absorbance was measured at 517 nm after 20 min, 40 min, 60 min, 90 min, and 120 minutes incubation with radical (as). absorbance at 517 nm was determined for ethanolic solution of dpph radical („control” ac), diluted in the aforementioned ratio (0.3 ml of the dpph radical of the given concentration with 0.75 ml ethanol added) as well as for the ethanolic solution of the essential oil which is not treated with dpph radical solution (“blank” ab). ethanol was used as a blank. free radical scavenging activity was calculated according to the formula: dpph radical scavenging capacity (%)=100-[(as-ab)x(100/ac)] all absorbances were measured on uv-vis varian-cary 100 conc. spectrophotometer. bht (dissolved in ethanol; concentration range 0.04-1.3 mg/ml) was used as a positive control. results and discussion qualitative and quantitative composition of essential oils the yields of pale yellow coloured essential oils were 0.03 g/100 g p.m and 0.01 g/100 g p.m for rfeo and ofeo, respectively. total ion chromatograms (tics) of eos studied are given in fig. 3, while their qualitative and quantitative composition is given in tab. 1. the chemical structures of the most abundant compounds are shown in fig. 4. 209 biologica nyssana ● 13 (2) december 2022: 205-216 stanojević et al. ● comparative analysis of chemical composition and antioxidant activity of essential oil isolated from orange and red marigold (tagetes patula l.) flower petals fig. 3. tic chromatogram of (a) ofeo, and (b) rfeo fig. 4. chemical structures of the most abundant compounds present in isolated eos 210 biologica nyssana ● 13 (2) december 2022: 205-216 stanojević et al. ● comparative analysis of chemical composition and antioxidant activity of essential oil isolated from orange and red marigold (tagetes patula l.) flower petals table 1. chemical composition of essential oils isolated from orange and red flower petals of t. patula l. no. tret, min compound riexp rilit method of identification content (%) c (mg/ml of eo) ofeo rfeo ofeo rfeo 1 7.92 sabinene 965 969a ri, ms tr tr 2 8.03 β-pinene 968 974a ri, ms, co-i tr tr 3 8.46 2-pentyl furan 983 984a ri, ms tr tr 4 10.11 (z)-β-ocimene 1,030 1,032a ri, ms 0.4 2.7 0.1 0.9 5 10.49 (e)-β-ocimene 1,040 1,044a ri, ms 0.2 0.1 6 10.92 γ-terpinene 1,051 1,054a ri, ms, co-i tr tr 7 12.06 terpinolene 1,081 1,086a ri, ms tr tr 8 12.25 p-cymenene 1,086 1,089a ri, ms tr tr 9 12.72 linalool 1,098 1,095a ri, ms, co-i 0.4 tr 0.1 tr 10 14.44 (e)-tagetone 1,132 1,139a ri, ms tr tr 11 15.98 terpinen-4-ol 1,169 1,174a ri, ms, co-i tr tr tr tr 12 16.58 p-cymen-8-ol 1,184 1,179a ri, ms tr tr 13 17.95 coahuilensol, methyl ether 1,216 1,219a ri, ms tr tr 14 18.19 (z)-ocimenone 1,222 1,226a ri, ms tr 0.3 tr 0.1 15 18.60 (e)-ocimenone 1,232 1,235a ri, ms tr tr 16 19.15 piperitone 1,245 1,249a ri, ms 0.3 0.1 17 20.30 isobornyl acetate 1,276 1,283a ri, ms tr tr 18 21.97 silphiperfol-5-ene 1,324 1,326a ri, ms tr tr 19 22.25 presilphiperfol-7-ene 1,330 1,334a ri, ms tr tr 20 22.89 piperitenone 1,337 1,340a ri, ms 0.5 1.5 0.1 0.5 21 23.04 α-terpinyl acetate 1,340 1,346a ri, ms tr tr 22 23.73 eugenol 1,357 1,356a ri, ms, co-i tr tr tr tr 23 23.93 piperitenone oxide 1,361 1,366a ri, ms tr tr 24 24.09 α-copaene 1,365 1,374a ri, ms tr tr tr tr 25 24.59 geranyl acetate 1,377 1,379a ri, ms 37.6 7.2 26 26.21 (e)-caryophyllene 1,416 1,417a ri, ms, co-i 31.6 69.4 6.0 22.8 27 26.51 α-trans-bergamotene 1,423 1,432a ri, ms tr tr 28 27.30 geranyl acetone 1,443 1,453a ri, ms tr tr tr tr 29 27.38 α-humulene 1,444 1,452a ri, ms 1.0 1.7 0.2 0.6 30 28.53 germacrene d 1,488 1,484a ri, ms 2.8 8.2 0.5 2.7 31 29.08 bicyclogermacrene 1,490 1,500a ri, ms 1.1 3.6 0.2 1.2 32 29.39 (e)-methyl isoeugenol 1,493 1,491a ri, ms tr tr 33 29.45 (e,e)-α-farnesene 1,495 1,505a ri, ms 0.6 0.2 34 29.56 β-bisabolene 1,498 1,505a ri, ms 12.5 2.4 35 30.10 γ-cadinene 1,511 1,513a ri, ms tr tr tr tr 36 30.78 (e)-γ-bisabolene 1,529 1,529a ri, ms 0.9 0.4 0.2 0.1 37 32.51 caryophyllene oxide 1,574 1,582a ri, ms 5.7 4.5 1.1 1.5 38 34.65 caryophylla4(12),8(13)-dien-5α-ol 1,631 1,639a ri, ms tr tr tr tr 211 biologica nyssana ● 13 (2) december 2022: 205-216 stanojević et al. ● comparative analysis of chemical composition and antioxidant activity of essential oil isolated from orange and red marigold (tagetes patula l.) flower petals 39 35.32 α-cadinol 1,648 1,652a ri, ms tr tr tr tr 40 36.63 shyobunol 1,684 1,688a ri, ms tr tr tr tr 41 36.81 eudesm-7(1l)-en-4-ol 1,688 1,698a ri, ms 0.9 tr 0.2 tr 42 37.18 (2e)-tridecenol acetate 1,698 1,703a ri, ms 0.8 tr 0.2 tr 43 41.65 hexahydrofarnesyl acetone 1,850 1,846b ri, ms 0.6 0.3 0.1 0.1 44 44.05 (5z,9e)-farnesyl acetone 1,897 1,889a ri, ms tr tr 45 44.27 5-(3-buten-1-ynyl)2,2'-bithiophene (bbt) 1,902 1,892c ri, ms 0.4 0.1 46 46.12 palmitic acid 1,960 1,959b ri, ms 1.2 1.9 0.2 0.6 47 49.29 5-(3-penten-1-ynyl)2,2-bithiophene (pbt) 2,053 2,043c ri, ms 0.7 1.5 0.1 0.5 48 53.28 2,2':5',2''-terthiophene (α-terthiophene) 2,181 2,171c ri, ms 0.9 0.3 49 55.35 tricosane 2,296 2,300a ri, ms, co-i 0.5 0.7 0.1 0.2 total identified 99.2 99.1 19.0 32.6 grouped components (%) (mg/ml of eo) monoterpene hydrocarbons (1, 2, 4-8) 0.4 2.9 0.1 1.0 oxygenated monoterpenes (9-21, 23, 25, 28) 38.5 2.1 7.4 0.7 sesquiterpene hydrocarbons (24, 26, 27, 29-31, 33-36) 49.9 83.9 9.5 27.6 oxygenated sesquiterpenes (37-41, 43, 44) 7.2 4.8 1.4 1.6 phenylpropanoids (22, 32) tr tr tr tr thiophenes (45, 47, 48) 0.7 2.8 0.1 0.9 others (3, 42, 46, 49) 2.5 2.6 0.5 0.8 tret.: retention time; ri lit retention indices from literature (aadams (2007); bbalogun et al. (2017); cszarka et al. (2007)); riexp: experimentally determined retention indices using a homologous series of n-alkanes (c8-c20 and c21-c40) on the hp-5ms column. ms: constituent identified by mass-spectra comparison; ri: constituent identified by retention index matching; co-i: constituent identity confirmed by gc co-injection of an authentic sample; tr = trace amount (<0.05%; <0.1 mg/ml); ofeo essential oil isolated from orange flower petals, rfeo essential oil isolated from red flower petals according to the results of gc/ms analysis, 30 and 42 compounds were identified in ofeo and rfeo, comprising 99.2%, and 99.1% of total eo composition, respectively. the most abundant groups of compounds in ofeo were sesquiterpene hydrocarbons (49.9%) and oxygenated monoterpenes (38.5%). on the other side, sesquiterpene hydrocarbons with 83.9% in the total essential oil composition were the dominant group in rfeo (tab. 1). among sesquiterpene hydrocarbons (e)-caryophyllene was the dominant compound in both eos, with 31.6% and 69.4%, in total ofeo and rfeo composition, respectively while β-bisabolene, with 12.5% was second most dominant compound in ofeo. β-bisabolene was not detected in rfeo. among oxygenated monoterpenes, the dominant one was geranyl acetate with 37.6% in total ofeo composition (structures given in fig. 3). it was not detected in rfeo. the results obtained are in agreement with the study by szarka et al. (2007). the authors hydrodistilled eo from t. patula flowers that had been air-dried, and the most abundant compound found there was (e)-caryophyllene, which accounted for 50.2% (compared to 69.4% in herein study). geographical origin plays an important role in the chemical diversity not just among species but even in the same species. for example, krishna et al. (2002) analysed eos of the capitula, leaves and shoots of tagetes patula l. raised in the cimap experimental biologica nyssana ● 13 (2) december 2022: 205-216 stanojević et al. ● comparative analysis of chemical composition and antioxidant activity of essential oil isolated from orange and red marigold (tagetes patula l.) flower petals 212 farm at lucknow (india). the main constituents of eo isolated from capitula were (z)-β-ocimene (19.9%), (z)-tagetenone (12.4%), (e)-tagetenone (10.4%), piperitenone (5.8%) and (e)-caryophyllene (15.1%) (krishna et al., 2002). on the other hand, the eo isolated from cultivated t. patula flowers grown in mandal (uttarakhand, india) contained β-ocimene (22.11%), α-terpinolene (14.59%), and (e)-caryophyllene (12.69%) as the most abundant compounds (negi et al., 2013). the most abundant components of the eo isolated from fresh flowers of t. patula grown in new delhi (india) were (e)caryophyllene (3.92-42.76%), germacrene-d (1.486.72%), (z)-tagetone (1.29-4.38), caryophyllene oxide (0.68-24.3%), and piperitone oxide (0.111.23) (tamut et al., 2019). in the eo isolated from t. patula flowers sampled during august in areas of erbil province (iraq), (e)-caryophyllene (20.59%), and (e)-ocimenone (12.08%) were the most abundant components while geranyl acetate was not identified (safar et al., 2020). zarate-escobedo et al. (2018) reported the presence of geranyl acetate for genus tagetes for the first time. they determined the chemical composition of the essential oil hydrodistilled from floral stems of 14 t. lucida populations from north and south of the state of mexico, where six types of soils and six climatic conditions were detected. the plant material was collected from september to october 2014 and in september 2015. in southern populations with a warm climate, the major compounds were monoterpenes: geranyl acetate (ranging from 12% to 40%) and β-ocimene (14% to 24%) depending on the location (zarate-escobedo et al., 2018). it is postulated that terpenoid compounds play a role in an ecological interaction of plants with biotic and abiotic factors of their environment by defending plants from herbivores and pathogens (toxins or repellents) or being the signals and rewards to pollinators. the constant evolution of new terpenoids structures is enabled by the evolution of new genes encoding new enzymes capable of making such new metabolites (pichersky & raguso, 2016). what is more, according to the available literature data single compounds such as bisabolene, (e)caryophyllene, camphor, (e)-β-farnesene, pinene, and linalol have been recognized as good repellents towards aphids and various pests (hori, 1998; isman, 2000; halbert et al., 2009; pascual-villalobos et al., 2017; dardouri et al., 2019). taking into account that (e)-caryophyllene and β-bisabolene were present in considerable amounts in the eos isolated in this study (tab. 1), they could be considered as a potential source of natural pesticides. it is well known that acyclic monoterpenes (ocimenones) including (z)-βand (e)-β-ocimene, (z)and (e)-tagetone and (z)and (e)-tagetenone are formed by chemical modification (such as hydrolysis, dehydration, oxidation and reduction which are catalyzed by specific enzymes) of either gpp or neryl pyrophosphate (npp). on the other hand, the biosynthesis of sesquiterpenes proceeds through the precursor farnesyl pyrophosphate (fpp) which is formed by condensation of gpp with one molecule of ipp (singh et al., 2015). given that terpenoids’ biosynthesis is genetically determined, finding the reason for the difference in chemical composition between ofeo and rfeo, regarding the most abundant components, goes beyond the scope of this paper. geranyl acetate is an acyclic monoterpenes ester with great economic value. it is widely used in cosmetic industry due to its „rose-like” odor. in cymbopogon spp. geranyl acetate is biosynthesed by acetylation of geraniol catalyzed by geraniol acetyl transferase (gat) (ganjewala & luthra, 2010). phosphatase (gppase) mediates the formation of geraniol while geranyl acetate esterase (gae) catalizes deacetylation of geranyl acetate into geraniol. thus, the content of geraniol depends on the relative activities of these three (gppase, gat, and gae) enzymes. geraniol was not identified in herein studied eo. the possible reason could be that the plant material was collected in the phase when gae (catalyzing transformation of geranyl acetate to geraniol) had little or no activity. having in mind that essential oils isolated from „geranylacetate rich” plants like palmarosa (cymbopogon martini (roxb.) wats. var. motia burk.) contained 4.3-14.8% of geranyl acetate (rajeswara rao et al., 2009); wild carrot (daucus carota l. ssp. carota) mature umbels 16.5% (staniszewska et al., 2005); lemongrass (cymbopogon flexuosus (steud) wats.) 25.9% (kulkarni et al., 1997); pastinocello fruits (daucus carota ssp. major) 34.2% (flamini et al., 2014); coriander (coriandrum sativum l.) fruits 46.27% (msaada et al., 2007); and the ofeo isolated in this paper (containing 37.6%) could be considered as a potential source of geranyl acetate. the thiophenes, with bbt, pbt, and α-terthienyl as representatives, were also identified, with 0.7% and 2.8% in ofeo and rfeo, respectively (tab. 1). although the thiophenes are the main secondary metabolites of tagetes roots in this paper they were identified in flower petals, which is in agreement with the study of szarka et al. (2007). namely, szarka et al. (2007) studied the composition of eos isolated from hairy roots, normal roots and flowers and the major thiophene of flower eo was pbt with 6.0% in the total eo composition (szarka et al., 2007). the identity of thiophenes identified in this paper was confirmed by comparing mass spectrum from the hit 213 biologica nyssana ● 13 (2) december 2022: 205-216 stanojević et al. ● comparative analysis of chemical composition and antioxidant activity of essential oil isolated from orange and red marigold (tagetes patula l.) flower petals list in mass spectra libraries but also with the mass spectra given in the study of szarka et al. (2006) as well as by comparing experimentally obtained retention indices with the retention indices given in the study of szarka et al. (2007). on the other side, in the study of arciniegas et al. (2020), the authors determined the antioxidant and photosensitizer activities of extracts and isolates of genus dyssodia (asteraceae, tageteae). the antioxidant activity was determined by the interactions with copper ion (cu2+) observed in epr, as well as by the dpph and the thiobarbituric reactive substances (tbars) methods. their photosensitizer activities were observed as the the abilities to produce 1o 2 by electron paramagnetic resonance (epr). they isolated seven thiophene derivatives, among others 2,2′:5′,2′′‐terthiophene, and 5‐(3‐buten‐1‐ynyl)‐2,2′‐bithiophene, which were identified in this study as well. both thiophenes had no antioxidant activity determined by the dpph assay, so they surely not contribute to the antioxidant activity of the eos studied in this paper. on the other side, α‐terthiophene and related compounds are known as photosensitizers by their activity to generate singlet oxygen (1o2) under uv irradiation regime (arciniegas et al., 2020). what is more, these compounds, and especially α-terthienyl show enhanced nematocidal activity in the presence of sunlight (uv-a), but also antibiotic, ovicidal, algicidal, larvicidal, and antifeedant activities (d’auria et al., 1987). antioxidant activity the antioxidant activity of isolated eos was determined by the dpph assay and compared both mutually, and with bht as a positive control. their dpph radical scavenging activity determined after 20 min, 40 min, 60 min, 90 min, and 120 min incubation with the dpph radical is shown in fig. 5. the ec50 values obtained from the graphs given in fig. 5 are presented in the tab. 2. according to the results obtained, antioxidant activity of both eos and bht was dependant on both concentration and incubation time with the dpph radical, as already stated (stanojević et al., 2016). generally, the duration of incubation time of dpph radical with samples depends on the type of sample, taking longer time for interaction with the weak antioxidants (bal et al., 2021). the effect of incubation time studied in this paper indicated that dpph radical scavenging activity of both eos and bht reached the maximum after 120 minutes of incubation (tab. 2). however bht, as a phenolic representative of synthetic antioxidants used in this paper, reduced the 50% of the initial dpph radical concentration (the ec50 value) after 20 minutes of incubation and showed ~12 and ~14 times stronger fig. 5. antioxidant activity of (a) ofeo, (b) rfeo, and (c) bht antioxidant activity in comparison to ofeo and rfeo after 120 minutes of incubation, respectively. therefore, the isolated eos had similar antioxidant biologica nyssana ● 13 (2) december 2022: 205-216 stanojević et al. ● comparative analysis of chemical composition and antioxidant activity of essential oil isolated from orange and red marigold (tagetes patula l.) flower petals 214 activity and both are weaker antioxidants than bht. comparing the chemical composition of isolated eos, the main groups were sesquiterpenes hydrocarbons (83.9% vs. 55.6%) and oxygenated monoterpenes (2.1% vs. 38.5%) in rfeo and ofeo, respectively. according to the study by ruberto & barrata (2000), oxygenated monoterpenes have better antioxidant activity in comparison to monoterpenes hydrocarbons, sesquiterpenes hydrocarbons, and oxygenated sesquiterpenes. the best antioxidant activity in the mentioned study showed phenolic compounds (such as thymol and carvacrol) due to their redox properties, and the ability to neutralize free radicals (ruberto & barrata, 2000). considering that ofeo contained considerable amount of oxygenated monoterpenes (38.5%) in comparison to rfeo (2.1%), its slightly better antioxidant activity could be ascribed to their presence, especially to the presence of geranyl acetate due to its capacity to reduce free radical stability via electron or hydrogen donating mechanisms (seema farhath et al., 2013). on the other side, phenolic compounds (particularly eugenol) were identified in traces in both isolated eos, so their weak antioxidant activity is not surprising. conclusions the essential oils hydrodistilled from dry red and orange tagetes patula l. flower petals cultivated in south-eastern serbia are rich sources of sesquiterpene hydrocarbons ((e)-caryophyllene in rfeo comprising 69.4% of total eo composition) and oxygenated monoterpenes (geranyl acetate in ofeo comprising 37.6% of total eo composition). they have also contained sulphurated tiophenes (bbt, pbt and ttp) with 2.8% and 0.7% in total rfeo and ofeo composition, respectively. having in mind that α-terthienyl, also called terthiophene (ttp) and 5-(3-penten-1-ynyl)-2,2-bithienyl (pbt) are phototoxic compounds, they should not be used as components in cosmetic products for applications on areas of skin exposed to sunshine. being weaker antioxidants in comparison to the synthetic antioxidant bht, they could not be used as its natural alternative. on the other side, the overuse of synthetic pesticides causes pest resistance and makes them one of the major pollutants in soil and water, as well as toxic substances for humans and animals. taking into account that (e)-caryophyllene and β-bisabolene present in considerable amounts in the eos isolated in this study are recognized as good repellents (besides thiophenes). the authors would like to draw attention to marigolds, not just as garden ornamental flowers but also as a potential source and appropriate basis for future development of naturally based herbicides. acknowledgements. this work was supported by the ministry of education, science and technological development of the republic of serbia under the program of financing scientific research work, number 451-0368/2022-14/ 200133. nataša simonović and aleksandra milenković are scholars of the ministry of education, science and technological development of the republic of serbia. references adams, r.p. 2007: identification of essential oil components by gas chromatography /mass spectrometry. carol stream, allured publishing corporation. illinois, usa. 804 p. arciniegas, a., gómez-vidales, v., pérezcastorena, a.l., nieto-camacho, a., villaseñor, j.l., romo de vivar, a. 2020: recognition of antioxidants and photosensitizers in dyssodia pinnata by epr spectroscopy. phytochemical analysis, 31(2): 252-261. bal, a., pati, s.g., panda, f., paital, b. 2021: modification of the time of incubation in colorimetric method for accurate determination of the total antioxidants capacity using 2,2-diphenyl-1picrylhydrazyl stable free radical. journal of applied biology & biotechnology, 9(4): 156-161. balogun, o.s., ajayi, o.s., adeleke, a.j. 2017: hexahydrofarnesyl acetone-rich extractives from hildegardia barteri. journal of herbs, spices & medicinal plants, 23(4): 393-400. dardouri, t., gautier, h., ben issa, r., costagliola, g., gomez, l. 2019: repellence of myzus persicae (sulzer) evidence of two modes of action of volatiles from selected living aromatic plants. pest management science, 75(6): 1571-1584. d’auria, m., de mico, a., d’onofrio, f., piancatelli, g. 1987. synthesis of naturally occurring bithiophenes: a photochemical approach. the journal of organic chemistry, 52(23): 5243table 2. the ec50 values of isolated eos and bht ec50, mg/ml incubation time ofeo rfeo bht 20 min 0.43±0.002 40 min 12.20±0.054 0.27±0.001 60 min 8.41±0.014 9.29±0.050 0.19±0.001 90 min 6.55±0.018 6.71±0.025 0.13±0.000 120 min 5.04±0.000 5.96±0.000 0.09±0.001 215 biologica nyssana ● 13 (2) december 2022: 205-216 stanojević et al. ● comparative analysis of chemical composition and antioxidant activity of essential oil isolated from orange and red marigold (tagetes patula l.) flower petals 5247. dos reis, v.m.s. 2010: dermatosis due to plants (phytodermatosis). anais brasileiros de dermatologia, 85(4): 479-489. flamini, g., cosimi, e., cioni, p.l., molfetta, i., braca, a. 2014: essential-oil composition of daucus carota ssp. major (pastinocello carrot) and nine different commercial varieties of daucus carota ssp. sativus fruits. chemistry & bidiversity, 11(7): 1022-1033. ganjewala, d., luthra, r. 2010: essential oil biosynthesis and regulation in the genus cymbopogon. natural product communications, 5(1): 163-172. halbert, s.e., corsini, d., wiebe, m., vaughn, s.f. 2009: plant-derived compounds and extracts with potential as aphid repellents. annals of applied biology, 154(2): 303-307. hori, m. 1998: repellency of rosemary oil against myzus persicae in a laboratory and in a screenhouse. journal of chemical ecology, 24: 1425-1432. isman, m.b. 2000: plant essential oils for pest and disease management. crop protection, 19(8-10): 603-608. krishna, a., mallavarapu, g.r., kumar, s., ramesh, s. 2002: volatile oil constituents of the capitula, leaves and shoots of tagetes patula l. journal of essential oil research, 14(6): 433-436. kulkarni, r.n., mallavarapu, g.r., baskaran k., ramesh s. 1997: essential oil composition of a citronella-like variant of lemongrass. journal of essential oil research, 9(4): 393-395. msaada, k., hosni, k., taarit, m.b., chahed, t., kchouk, m.e., marzouk, b. 2007: changes on essential oil composition of coriander (coriandrum sativum l.) fruits during three stages of maturity. food chemistry, 102: 1131-1134. negi, j.s., bisht, v.k., bhandari, a.k., sundriyal, r.c. 2013: essential oil contents and antioxidant activity of tagetes patula l. journal of essential oil bearing plants, 16(3): 364-367. parisi, a.v., schouten, p., downs, n.j., turner, j. 2010: solar uv exposures measured simultaneously to all arbitrarily oriented leaves on a plant. journal of photochemistry and photobiology b: biology, 99(2): 87-92. pascual-villalobos, m.j., cantó-tejero, m., vallejo, r., guirao, p., rodríguez-rojo, s., cocero, m.j. 2017: use of nanoemulsions of plant essential oils as aphid repellents. industiral crops and products, 110: 45-57. pichersky, e., raguso, r.a. 2018: why do plants produce so many terpenoid compounds?. new phytologist, 220(3): 692-702. priyanka, d., shalini, t., kumar navneet, v. 2013: a brief study on marigold (tagetes species): a review. international research journal of pharmacy, 4(1): 43-48. rajeswara rao, b.r., rajput, d.k., patel, r.p. 2009: essential oil profiles of different parts of palmarosa (cymbopogon martini (roxb.) wats. var. motia burk.). journal of essential oil research, 21(6): 519-521. ruberto, g., baratta, m.t. 2000: antioxidant activity of selected essential oil components in two lipid model systems. food chemistry, 69(2): 167174. safar, a.a., ghafoor, a.o., dastan, d. 2020: chemical composition, antibacterial and antioxidant activities of tagetes patula l. essential oil raised in erbil, iraq. journal of reports in pharmaceutical sciences, 9(1): 59-67. salehi, b., valussi, m., morais-braga, m.f.b., carneiro, j.n.p., leal, a.l.a.b., coutinho, h.d.m., vitalini, s., kręgiel, d., antolak, h., sharifi-rad, m., silva, n.c.c., yousaf, z., martorell, m., iriti, m., carradori, s., sharifirad, j. 2018: tagetes spp. essential oils and other extracts: chemical characterization and biological activity. molecules, 23(11): 2847. seema farhath, m.s., vijaya, p.p., vimal, m. 2013: antioxidant activity of geraniol, geranial acetate, gingerol and eugenol. research in pharmacy, 3(1): 1-6. singh, p., krishna, a., kumar, v., krishna, s. singh, k., gupta, m., singh s. 2015: chemistry and biology of industrial crop tagetes species: a review. journal of essential oil research, 28(1): 1-14. singh, y., gupta, a., kannojia, p. 2020: tagetes erecta (marigold) a review on its phytochemical and medicinal properties. current medical drug research, 4(1), article id 201. sparkman, d.o., penton, z.e., fulton, k.g. 2011: gas chromatography and mass spectrometry: a practical guide, 2nd ed. elsevier inc. oxford. usa, 611 p. staniszewska, m., kula, j., wieczorkiewicz, m., kusewicz, d. 2005: essential oils of wild and cultivated carrots the chemical composition and antimicrobial activity. journal of essential oil biologica nyssana ● 13 (2) december 2022: 205-216 stanojević et al. ● comparative analysis of chemical composition and antioxidant activity of essential oil isolated from orange and red marigold (tagetes patula l.) flower petals 216 research, 17(5): 579-583. stanojević, lj.p., stanojević, j.s., cvetković, d.j., ilić, d.p. 2016: antioxidant activity of oregano essential oil (origanum vulgare l.). biologica nyssana, 7(2): 131-139. szarka, s., héthelyi, é.b., lemberkovics, é., bálványos, i., szőke, é. farkas, e., kuzovkina, i.n. 2007: essential oil constituents of intact plants and in vitro cultures of tagetes patula l. journal of essential oil research, 19(1): 85-88. szarka, s., héthelyi, é., lemberkovics, é., kuzovkina, i.n., bányai, p., szőke, é. 2006: gc and gc-ms studies on the essential oil and thiophenes from tagetes patula l. chromatographia, 63(13): 67-73. tamut, o., singh, k.p., panwar s. 2019: variations in quantitative and qualitative composition of essential oils from leaves and flowers of french marigold (tagetes patula). international journal of current microbiology and applied sciences, 8(07): 1037-1042. tomova, b.s., waterhouse, j.s., doberski, j. 2005: the effect of fractionated tagetes oil volatiles on aphid reproduction, entomologia experimentalis et applicata, 115:153-159. zarate-escobedo, j., castañeda-gonzález, e.l., cuevas-sánchez, j.a., carrillo-fonseca c.l., ortiz-torres c., ibarra-estrada e., serrato-cruz m.a. 2018: aceite esencial de algunas poblaciones de tagetes lucida cav. de las regiones norte y sur del estado de méxico. revista fitotecnia mexicana, 41(2): 199-209. microsoft word 0401_stojanovic-radic_et_al biologica nyssana 1 (1-2) december 2010: 83-88 stojanović-radić, z. et al. antimicrobial activity and cytotoxicity of… 83 original article ! antimicrobial activity and cytotoxicity of commercial rosemary essential oil (rosmarinus officinalis l.) zorica stojanović-radić1*, marija nešić1, ljiljana čomić2, niko radulović3 1 university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, niš, serbia 2department of biology and ecology, faculty of sciences, university of kragujevac, radoja domanovića 12, kragujevac, serbia 3 university of niš, faculty of sciences and mathematics, department of chemistry, ćirila i metodija 2, niš, serbia * e-mail: lab3zmm@yahoo.com abstract: stojanović-radić, z., nešić, m., čomić, lj., radulović, n.: antimicrobial activity and cytotoxicity of commercial rosemary oil (rosmarinus officinalis l.). biologica nyssana, 1 (1-2), december 2010: 83-88. rosemary is well known as a spice and widely used plant in ethnomedicine worldwide. in this paper, commercial essential oil of rosemary was tested for antimicrobial and cytotoxic activity together with its effect on germination. antimicrobial activity testing showed moderate effect to both g-positive and gnegative bacteria. in order to determine its effect to the cell membrane, spectrophotometric analysis was performed. it was determined that rosemary affects the cell membrane of bacteria. cytotoxic activity of rosmarinus officinalis essential oil had been evaluated. as a plant object, germinative bulbs of allium cepa were used. cytotoxic activity that corresponded to the concentration of essential oil was determined. it had been noticed that rosemary essential oil affected mitotic phase i.e. it significantly slowed down the mitosis. also, investigation of rosemary essential oil's activity to germination was performed. it was determined that it had high effect to the germination. concentration of 5 mg/ml completely inhibited the germination of triticum vulgare. key words: essential oil, cytotoxicity, antimicrobial activity, effect on germination introduction ! essential oils are natural, concentrated, volatile aromatic compounds isolated from plants. these compounds posses a wide spectrum of pharmacological activities. the main advantage of natural agents is that they do not enhance the “antibiotic resistance”, a phenomenon encountered with the long term use of synthetic antibiotics. rosemary (rosmarinus officinalis l.) is plant belonging to the family labiateae. this plant is widely used for many purposes – it is well known as a culinary spice and frequently employed in the practice of aromatherapy (d a v i d s o n et al., 1989). also, it is used in ethnomedicine as general stimulant, for improvement of circulation, treatment of rheumatic pains, hyperglycemia and skin care (h a m e d o , 2009). rosemary is known to have antioxidant and antibacterial properties (s h a h i d i et al. 1992). p u t n a m et al. (2006) reported that rosemary essential oil inhibit osteoclast activity and increase bone density in vitro. also, cytotoxic activity of rosemary essential oil has been demonstrated by several authors (k h a f a g i et al. 2000; e l m e l e i g y et al. 2010). the essential oil of rosemary contains several compounds in high percentage and many others in traces. these major compounds determine the biological properties of the essential oil and can act 10th sfses • 17-20 june 2010, vlasina lake1 (1-2) • december 2010: 83-88 biologica nyssana 1 (1-2) december 2010: 83-88 stojanović-radić, z. et al. antimicrobial activity and cytotoxicity of… 84 in synergic manner or regulate one on another (f a i x o v a & f a i x , 2008). in the papers dealing with chemical composition of rosemary essential oil, α-pinene is reported as the major component, followed by 1,8 – cineole, camphene, β-myrcene, camphor and borneole (j a m s h i d i et al., 2009, m o g h t a d e r & a f z a l , 2009). some studies reported 1,8 – cineole as main component (f u et al., 2007, d e b e r s a c et al., 2001). rosemary is confirmed as antimicrobial agent against both gram-positive and gram-negative bacteria, as well as against fungi (v a l e r o & s a l m e r o n , 2003, p r a b u s e e n i v a s a n et al., 2006, f u et al. 2007a, m o g h t a d e r & a f z a l , 2009). in the present study, antimicrobial activity of commercially available rosemary essential oil was investigated against a panel of microorganisms for minimal inhibitory and bactericidal activity. mic and mbc were determined using broth microdilution method. the most sensitive determined bacterial strain was used to investigate the mode of action of this oil. in order to evaluate if the oil exhibits membrane damage effect, testing of the 260 nm absorbing material leakage was employed. also, cytotoxic activity of rosemary essential oil was determined. material and methods ! essential oil, bacterial strains and culture conditions essential oil of rosemary, produced by sinefarm, vršac, srbija, was purchased from the local pharmacy. bacterial strains used in this study were staphylococcus aureus atcc 25923, s. aureus (clinical isolate), escherichia coli (clinical isolate), bacillus subtilis atcc 6633 and klebsiella pneumoniae (clinical isolate). bacterial cultures were maintained on the nutrient agar (na) at the department of biology and ecology, microbiological laboratory, faculty of science and mathematics, niš. testing of antimicrobial activity the antimicrobial activity was evaluated using broth microdilution method (n c c l s , 2003). minimum inhibitory concentrations determination was performed by a serial dilution method in 96 well microtitre plates. bacterial strains were cultivated in mueller hinton agar at 37 ºc for 18 h. cultures were used for making bacterial suspensions, whose turbidity was adjusted to 0.5 mcfarland and confirmed using a spectrophotometer (uv-vis 1650 shimatzu, japan). final density of inoculum was 5 x 105. dilutions of stock rosemary essential oil solutions were made with mhb (mueller hinton broth) and inoculums were added to all wells. one inoculated well was included to allow control of the broth suitability for organism growth. one non-inoculated well, free of antimicrobial agents, was also included to ensure medium sterility. bacterial growth was determined by adding 20 µl of 0.5 % triphenyl tetrazolium chloride (ttc) aqueous solution (s a r t o r a t t o et al., 2004). minimal inhibitory concentration (mic) was defined as the lowest concentration of the oil inhibiting visible growth (red colored pellet on the bottom of the wells after the addition of ttc), while minimal bactericidal concentration (mbc) was defined as the lowest oil concentration killing 99.9 % of bacterial cells. to determine mbc, the broth was taken from each well without visible growth and inoculated in mha for 24 h at 37ºc. experiments were done in triplicate and the mean values are presented. testing the integrity of the cell membrane very important indication of disturbed membrane integrity is the release of intracellular components, such as large molecules dna and rna. since these nucleotides have strong uv absorption at 260 nm, they are described as 260 nm absorbing materials (l i u et al., 2009). leakage of 260 nm absorbing material was determined by measuring the absorbance of the culture supernatants according to the modified method of c a r s o n et al. (2002). briefly, the cultures of s. aureus were harvested and twice washed with pbs. after resuspension in pbs, cells were adjusted to absorbance of ~ 0.2. at time t = 0 min, essential oil was added to cell suspensions to give a final concentrations of mic and 2 x mic. aliquots of the treated cell suspensions were removed at regular time intervals, centrifuged at 10 000 x g for 5 min and absorbance of supernatant was measured at 260 nm and plotted against time. the experiments were done in triplicate and the mean values are presented. allium cepa test cytotoxicity was determined by calculating the mitotic index of allium cepa l. root cells after the 24 h treatment with rosemary essential oil. small onion bulbs were placed in 10 ml distilled water in petri dishes in an incubator at 25±1ºc for 24 h. after initiating growth, water had been replaced two more times (after every 24h). after 72 h, water had biologica nyssana 1 (1-2) december 2010: 83-88 stojanović-radić, z. et al. antimicrobial activity and cytotoxicity of… 85 been replaced with appropriate solution (appropriate concentration of essential oil of rosemary). distilled water and 5 % ethyl alcohol were used as controls. after the growth, root tips were collected and fixed. the fixative solution was glacial acetic acid / absolute alcohol (1/3 v/v). the root tips were kept in acetic-alcohol solution for 72 h. after fixation, the root tips were washed with water and then placed into a 70 % ethanol and stored in a refrigerator until use. for examination the root tips were hydrolyzed in 1n hcl, placed on microscopic slide in 1% acetoorcein and after a few minutes squashed. for each root tip the number of mitotic and total meristemic cells was counted in 9 fields using power (100 x) light microscope. cells manifesting different stages of mitosis i.e. interphase and prophase (p), metaphase (m), anaphase (a) and telophase (t) were recorded. the mitotic index (mi) was calculated using following formula: mitotic index = (p+m+a+t) / total cells germination test three different concentrations of essential oil were used to determine whether the oil has effect to germination of triticum vulgare seeds. twenty wheat grains were placed in small petri dishes and in an appropriate medium. distilled water and 5 % ethanol were used as controls. concentrations of rosemary essential oil were 5 mg/ml, 12.5 mg/ml, 25 mg/ml and 50 mg/ml. after 24 h of the treatment, seeds which germinated were counted and percentage of germination, compared to positive control was presented. results and disscusion the results of minimal inhibitory (mic) and minimal bactericidal concentrations (mbc) are presented in table 1. the testing showed significant antibacterial activity of rosemary with mic values ranging from 1.562 – 3.125 μl/ml, while mbc ranged from 1.562 to >25.00 μl/ml. the most sensitive strain was s. aureus with both mic and mbc values of 1.562 μl/ml, while the most resistant one was escherichia coli, whose bactericidal concentration exceeded the tested values. determined resistance of e. coli can be attributed to the outer cell envelope, the feature of all gramnegative bacteria, which makes them generally more resistant to all external agents. this is confirmed by higher mbc values of k. pneumoniae (gram negative strain). f u et al. (2007a) investigated antibacterial activity of rosemary and the results obtained in our study showed very good agreement with activity against e. coli, b. subtilis and s. aureus (1.250 μl/ml against all three bacteria) determined in that paper. m o g h t a d e r & a f z a l i (2009) reported activity of rosemary against both gram positive and negative strains. previous investigation of f u et al. (2007) reported activity of rosemary essential oil against propionibacterium acnes in the concentration of 0.56 mg/ml. in order to determine the mode of action of rosemary essential oil against the most sensitive of the tested bacteria, staphylococcus aureus atcc 25923, spectrophotometric method was employed. since damaged cell membrane increases its permeability, small molecular weight molecules such as phosphates and potassium ions first leach out from the cell, followed by larger molecules, such as dna and rna (c h e n et al, 2002). since these nucleotides have strong uv absorption at 260 nm, they are described as “260 nm absorbing materials”. the uv-vis study on the release of materials absorbing at 260 nm showed significant leakage proportional to the oil concentration (fig.1). on contrary, control did not showed any releasing of this material during 120 min period. this indicates membrane damage related to the addition of the rosemary essential oil. for testing of cytotoxicity, onion meristems (allium cepa l.) were used as the study material. they are considered as one of the best biological models for the study of cytotoxicity, genotoxicity and environmental pollutants (f i s k e s j o, 1985). allium cepa root meristems have been widely used table 1. minimal inhibitory concentrations (mic) and minimal bactericidal concentrations (mbc) of rosemary essential oil bacterial strain strain type mic (μl/ml) mbc (μl/ml) tetracycline (μg/ml) s. aureus atcc 25923 1.562 1.562 0.090 s. aureus clinical isolate 3.125 6.250 0.045 e. coli clinical isolate 3.125 >25.00 0.045 b. subtilis atcc 6633 1.562 6.250 0.090 k. pneumoniae clinical isolate 1.562 12.50 0.045 biologica nyssana 1 (1-2) december 2010: 83-88 stojanović-radić, z. et al. antimicrobial activity and cytotoxicity of… 86 for the evaluation of cytotoxicity and anti-mitotic activity of various compounds (s h e h a b , 1980; w i l l i a m s & o m o h , 1996; a l m e s h a l , 1987). in the present paper, cytotoxicity and antimitotic activity of essential oil of r. officinalis l. was tested using allium cepa test. the inhibitory effect of essential oil of rosemary on the mitotic activity of root meristems was evaluated and the effect was compared with distilled water and 5% ethyl alcohol. the results are presented in tab. 2. 0 20 40 60 80 100 120 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 ap so rb an ce a t 2 60 n m time (min) mic 2xmic control figure 1. release of 260 nm absorbing material from s. aureus atcc 25923 cells during the treatment with mic and 2 x mic concentrations of rosemary essential oil (od – optical density) table 2. mitotic index in allium cepa (l.) meristem following incubation with various concentrations of essential oil of rosmarinus officinalis (l.) concentration unit mi mi in % d h2o 0.424 42.40 etoh % 0.172 17.20 5.00 mg/ml 0.257 25.70 12.5 mg/ml 0.235 23.50 25.0 mg/ml 0.218 21.80 50.0 mg/ml 0.191 19.10 the results showed that different concentrations of essential oil have different percentage of activity, but all of them affect root growth, length and number. also, it was demonstrated that essential oil has inhibitory effect on proliferation of cells. higher concentrations of essential oil exhibit stronger inhibitory effect on mitosis (tab. 2). previous studies revealed that rosemary essential oil exhibits strong cytotoxicity against brine shrimp nauplii (k h a f a g i et al. 2000). the test which was taken is not only used for predicting cytotoxicity, but is also used as a predictor of antitumor and pesticide activity. data showed that rosemary essential oil except high cytotoxicity, might posses antitumor and pesticide potential. it is supposed that oxygenated monoterpenes of essential oil exhibit a variable degree of cytotoxicity. as a typical lipophilic substance, it passes through the cell wall and cytoplasm membranes and disrupts their structure (f a i x o v a & f a i x , 2008). k i v a n c & a k g ü l (1988) reported that the most active components of the essential oils were phenols, followed by aldehydes and ketons. one of the major compound of rosemary essential oil is 1, 8-cineole. a s a n o v a et al. (2003) demonstrated that this component has very strong cytotoxicity and pronounced antitumor action. it is possible that this component has the major role in cytotoxicity of this essential oil. 1, 8 cineole exhibits a strong toxic effect on eukaryotic cells (o b e n g o f o r i et al., 1997, s a n t o s et al., 2004). another potent compound is ß-caryophyllene. it was demonstrated that this substance may not have direct influence on cytotoxicity of essential oil, but may impact accumulation of some substances by increasing permeability of the plasma-membrane. in that way, it affects and increases cytotoxic effect of compounds with which it interacts (l e g a u l e & p i c h e t t e , 2007). according to all this facts, it is not surprising that concentration of only 5 mg/ml of rosemary essential oil totally inhibited germination of wheat. this was the lowest concentration tested. other concentrations also totally inhibited germination of triticum vulgare seeds. limonene, one of the components of rosemary essential oil, has ability to moderate proliferation of cells (m a n u e l e et al., 2008), and in that manner it affects cell mitosis. conclusion essential oil of rosemary was previously reported as an important antimicrobial agent with activity at very low concentrations. our study confirmed the previous results regarding mic and mbc values of this essential oil. for the first time, it was determined that rosemary essential oil exhibits activity by damaging the cell membrane of gram positive bacteria and further tests are necessary to explore the exact mechanism of this activity. the determined cytotoxic and antimicrobial activity of rosemary essential oil is of great importance in preservation of food products and also very important information for its pharmacological application. biologica nyssana 1 (1-2) december 2010: 83-88 stojanović-radić, z. et al. antimicrobial activity and cytotoxicity of… 87 references al-meshal i.a. 1987: mitodepressive effect of (-)cathinone, from caltha edulis (khat), on the meristematic region of allium cepa (l.) root tips. toxicon, 25 (4): 451-454. asanova, zh.k., suleimenov, e.m., atazhanova, g.a., dembitskii, a.d., pak, r.n., dar, a., adekenov, s.m. 2003: biological activity of 1, 8-cineole from levant wormwood. pharmaceutical chemistry journal, 37 (1): 2830. carson, f.c., mee, b.j., riley, t.v. (2002): mechanism of action of melaleuca alternifolia (tea tree) oil on staphylococcus aureus determined by time kill, lysis, leakage and salt tolerance assays and electron microscopy. antimicrobial agents and chemotherapy, 46 (6), 1914-1920. chen, c.z. & s.l. cooper 2002: interactions between dendrimer biocides and bacterial membranes. biomaterials 23 (16): 3359-3368. davidson, p.m., porish, m.e. 1989: methods for testing the efficacy of food antimicrobials. food technology 43 (1): 148-155. debersac, p., haydel, j.m., amiot, m.j., goudonnet, h., artur, y., suschetet, m., siess, m.h. 2001: introduction of cytochrome p450 and/or detoxication enzymes by various extracts of rosemary description of specific patterns. food and chemical technology, 39 (9): 907918. el-meleigy, m.a., ahmed, m.e., arafa, r.a., ebrahim, n.a., el-knolany, e.e. 2010: cytotoxicity of four essential oils on some human and bacterial cells. journal of applied sciences in environmental sanitation, 5(2): 143159. faixovà, z. & š. faix 2008: biological effects of rosemary (rosmarinus officinalis l.) essential oil. folia veterinaria, 52(3-4): 135-139. fiskejso, g. 1985: the allium test as a standard in environmental monitoring. hereditas, 102: 99112. fu, y.j., zu, y.g., chen, l.y., shil, x.g., wangl, z., sunl, s., efferth, t. 2007a: antimicrobial activity of clove and rosemary essential oils alone and in combination. phytotherapy research, 21 (10): 989–994. fu, y., zu, y., chen, l., efferth, t., liangh, h., liu, z., liu, w. 2007b: investigation of antibacterial activity of rosemary essential oil against propionibacterium acnes with atomic force microscopy. planta medica, 73 (12): 127580. hamedo h.a. & h.m. abdelmigid 2009: use of antimicrobial and genotoxicity potentiality for evaluation of essential oils as food preservatives. open biotechnology journal, 3 (50-56): 18740707. jamshidi r., afzali z., afzali, d. 2009: chemical composition of hydrodistillation essential oil of rosemary in different origins in iran and comparison with other countries. americaneurasian journal of agricultural & environmental sciences, 5 (1): 78-81. khafagi, i., dewedar a., farouk, s. 2000: in vitro cytotoxicity and antimicrobial activities of some common essential oils. egyptian journal of biology, 2: 20-27. kivanc, m. & a. akgül 1988: effect of some essential oil components on the growth of foodborne bacteria and synergism with some food ingredients. flavor and fragrance journal, 3: 95-98. legaule, j. & a. pichette 2007: potentiating effect of ß-caryophyllene on anticancer activity of αhumulene, isocaryophyllene and paclitaxel. journal of pharmacy and pharmacology, 59 (12): 1643-1647. liu, c., tao, y., gou, j., qin, d., liu, h., yan, s., feng, x. 2009: antimicrobial characteristic and mechanism of nano-fumed silica salt graftes n,n-dimethyl-n-tetradecylamine. life science journal, 6 (1): 52-54. manuele, m.g., ferraro, g., anesini, c. 2008: effect of tilia viridis flowers on the proliferation of a lymphoma cell line and on normal murine lymphocytes: participation of monoterpens specially limonene. phytotherapy research, 22 (11): 1520-1526. moghtader, m. & d. afzali 2009: study of the antimicrobial properties of essential oil of rosemary. american-eurasian journal of agricultural & environmental sciences, 5 (3): 393-397. nccls – national committee for clinical laboratory standards 2003: document m100s11. national committee for clinical laboratory standard, wayne, pa, usa. obeng-ofori, d., reichmuth, c.h., bekele, j., hassanali, a. 1997: biological activity of 1, 8cineole, a major component of essential oil of ocimum kenyese (ayobagira) against stored products beetles. journal of applied entomology 121 (2): 237-243. prabuseenivasan, s., jayakumar m. and s. ignacimuthu 2006. in vitro antibacterial activity of some plant essential oils. bmc complementary and alternative medicine, 6 (1): 39. biologica nyssana 1 (1-2) december 2010: 83-88 stojanović-radić, z. et al. antimicrobial activity and cytotoxicity of… 88 putnam, s.e., scutt, a.m., bicknell, k., priestley, c.m., williamson, e.m. 2006: natural products as alternative treatments for maintenance of bone health. phytotherapy research, 21 (2): 99-112. santos, f.a., silva, r.m., campos, a.r., de araújo, r.p., lima júnior, r.c.p., rao, v.s.n. 2004: 1, 8-cineole (eucalyptol), a monoterpene oxide attenuates the colonic damage in rats on acute tnbs-colitis. food and chemical toxicology, 42 (4): 579-584. sartoratto, a. machado, a.l.m., delarmelina, c., figueira, g.m., duarte, m.c.t., rehder, v.l.g. 2004. composition and antimicrobial activity of essential oils from aromatic plants used in brazil. brazilian journal of microbiology, 35, 275-280. shahidi, f., & p.d. wanasundara 1992: phenolic antioxidants. critical reviews in food science and nutrition, 32 (1): 67-103. shehab, a.s. 1980: cytological effects of medicinal plants in qatar ii. mitotic effect of water extract of teucrium pilosum on allium cepa (l.). cytologia (tokyo), 45 (1-2): 57-64. valero, m. & m.c. salmeron 2003: antibacterial activity of 11 essential oil against bacillus cereus in tyndallized carrot broth. international journal of food microbiology, 85 (1-2): 73–81. williams g.o. & l.e. omoh 1996: mitotic effects of the aqueous leaf extract of cymbopogon citratus in allium cepa (l.) root tips. cytobios, 87: 161-168. anticancer compounds from medicinal plants biologica nyssana 5 (2)  december 2014: 75-82 huseinović, s. et al.  morphological and ecological differentiation of the fruit … 75 original article received: 12 june 2014 revised: 10 october 2014 accepted: 20 december 2014 morphological and ecological differentiation of the fruit of fragaria vesca l. (rosaceae) from different habitats in bosnia and herzegovina samira huseinović1*, sanida osmanović1, zerina terzić1, marizela šabanović2 1university of tuzla, faculty of science, univerzitetska 4, 75000 tuzla, bosnia and herzegovina 2university of tuzla, faculty of pharmacy, univerzitetska 7, 75000 tuzla, bosnia and herzegovina * corresponding author: samira.huseinovic@untz.ba abstract: huseinović, s., osmanović, s., terzić, z., šabanović, m.: morphological and ecological differentiation of the fruit of fragaria vesca l. (rosaceae) from different habitats in bosnia and herzegovina. biologica nyssana, 5 (2), december 2014: 75-82. 30 individuals from each population of the fragaria vesca in the fruiting phenophase were collected in the ecologically different habitats. morphological variation was investigated using 10 populations and 300 individuals. all specimens were properly preserved in a formalin-acetic acid mixture and stored in herbarium. the fruits of fragaria vesca were investigated in order to determine the most variable and the most consistent morphological character, depending on the environmental factors under which they developed. each population and its belonging individuals were labeled with special numbers. the percentage of variability for the height of fruit is 24.338% and for the width of fruit is 25.747%. however, considering high resource values of strawberries, it is necessary to emphasize that the size of the fruit follows the overall size of an individual. key words: fragaria vesca l., fruits, morphological and ecological variability apstrakt: huseinović, s., osmanović, s., terzić, z., šabanović, m.: morfološka i ekološka diferencijacija plodova vrste fragaria vesca l. (rosaceae) na različitim staništima u bosni i hercegovini. biologica nyssana, 5 (2), decembar 2014: 75-82. na ekološki različitim staništima prikupljeno je po 30 individua iz svake populacije vrste fragaria vesca u fenofazi plodonošenja. morfološka varijabilnost je sagledana na osnovu 10 populacija i 300 jedinki. sve jedinke su konzervirane u fo-a smjesu ili pohranjene u herbarijumu. plodovi odabranih vrsta roda fragaria (fragaria vesca) su istraženi s ciljem da se odredi najvarijabilniji i najkonzistentniji morfološki karakter, u zavisnosti od ekoloških faktora pod kojima se razvijao. utvrđen je stepen varijabilnosti morfometrijskog karaktera za parametar: maksimalna širina ploda (p1), kretao se u rasponu 24,338%, a za širinu ploda (p2) iznosi 25,747%. na osnovu statističkih analiza utvrđeno je da plod jagode ne spada u grupu onih biljnih delova koji najviše variraju. ključne reči: fragaria vesca l., plodovi, morfološka i ekološka varijabilnost 5 (2) • december 2014: 75-82 biologica nyssana 5 (2)  december 2014: 75-82 huseinović, s. et al.  morphological and ecological differentiation of the fruit … 76 introduction strawberry, genus fragaria l., is a plant from the family rosaceae and subfamily rosoideae (p o t t e r et al., 2007). it has great economic importance and therefore it has been investigated from different aspects morphological (h a r r i s o n et al., 1997), systematic and biogeographic (s t a u d t , 1989, 2009), demographic (a n g e v i n e , 1983), genetic (g a l l e t t a & m a a s , 1990), molecular (d i m e g l i o et al., 2014) and other. the genus fragaria includes 20 wild species, three described naturally occurring hybrid species, and two cultivated hybrid species important to commerce (h u m m e r et al., 2011). the wild species are distributed in the north temperate and holarctic zones. the only species which is widespread in europe, asia and america is fragaria vesca l. (s t a u d t , 1989, 2009). in europe it is represented with 6 species (t u t i n et al., 1968; euro+med, 2006). the genus fragaria is represented by 3 species in bosnia and herzegovina f. vesca l., f. moschata weston and f. viridis weston (b e c k -m a n n a g e t t a , 1927; euro+med, 2006). fragaria vesca is the most widely distributed species. it is growing in a different natural habitats (forests, meadow, pasture and other) (b e c k m a n n a g e t t a , 1927) and therefore it is very variable. up to now, the morphological studies of this species included only vegetative parts of the plant (h u s e i n o v i ć & o s m a n o v i ć , 2010). the objective of this study was to investigate morphological and ecological differentiation of the fruits of fragaria vesca from different habitats in bosnia and herzegovina. material and methods plant material of the fragaria vesca was collected in the fruiting phenophase from ten sites around tuzla during 2013 (tab. 1). during the selection of the research sites, efforts were made to cover populations developed in different plant communities. field research the field research consisted of a determination of biotic habitat factors of each of the studied populations, establishing phenological data and collecting samples and herbarium material. during the fieldwork we also made an adequate photographic documentation. based on literature data (r e d ž i ć , 1997) we determined type of plant communities on each site. collecting samples of plant material on the selected ecologically diverse habitats, living material of the species fragaria vesca was collected. at least 30 individuals from the population in the fruiting phenophase were sampled from each locality. morphological variability was observed on the basis of 10 populations and 300 individuals. during the collection of plants in the field, keys for determination of plant species have been used (d o m a c , 2002; a i c h e l e , 2004). all specimens were properly preserved in a formalin acetic acid mixture, or stored in the herbarium. as the collection of plant material was run at the time of fruiting, each population and its dependent individuals were marked with special numbers. laboratory studies in addition to the morphometric and statistical processing of materials and data, the laboratory part of the study assumed also numerous activities: consulting the relevant literature, collecting and processing eco-climate data, creation of original illustrations, etc. table 1. list of plant collection sites site locality type of vegetation 1 kladanj (muška voda) abieti-fagetum fragarietosum vescae 2 gornja tuzla arrhenatherion elatioris 3 cerik-ljubače arrhenatherion elatioris 4 husino querco-pinetum nigrae 5 ilinčica ii agropyro-rumicion in zone tilio-quercetum petraeae 6 krojčica agrimonyo-fragarietum 7 simin han arrhenatherion elatioris 8 ilinčica i fragarion vescae in zone querco-carpinetum betuli 9 zlaća bridge fagetum montanum serpentinicum 10 zlaća road fagetum montanum serpentinicum biologica nyssana 5 (2)  december 2014: 75-82 huseinović, s. et al.  morphological and ecological differentiation of the fruit … 77 morphological studies of the species fragaria vesca on the localities of gornja tuzla, simin han, ilinčica i, ilinčica ii, krojčica, husino, cerik, zlaća (bridge), zlaća (road) and kladanj (muška voda), were performed in the laboratories of the faculty of science in tuzla. the following morphological characters were determined: maximum width of the fruit (p1) and maximum height of the fruit (p2). characters of the fruit besides all the above mentioned characters we studied the fruit as well, as an essential characteristic of the gene expression. according to its shape (p3), the fruit was placed into one of the following groups: rounded, globose, conical or elongated. statistical analysis evaluation and interpretation of individual character’s variability, the degree of correlation or relationship between individual characters and ecological parameters, as well as analysis of the significance of the obtained results were performed in accordance with the relevant statistical and biological and ecological patterns contained in the available software packages (anova, 2006; statistics spss). by methods of descriptive statistics we determined: the arithmetic mean, range, variance, standard deviation and coefficient of variation of individual characters in specific patterns. the significance of the identified differences between the arithmetic means was determined by the method of testing the difference between the arithmetic means of small independent samples (p e t z , 1983). results fragaria vesca has a pronounced ecological valence in relation to a complex of environmental factors. it inhabits a variety of volcanic and sedimentary rocks, as well as the different stages and types of automorphic soils. it was noted also at the serpentine peridotic substrate (locality m. voda, kladanj). its populations equally well developed on bare parent substrate, cleared land, fire sites, and at developed soil in hornbeam, pine and pine-oak forests. so far it has been recorded in all regions of the former yugoslavia, including bosnia and herzegovina, and the first varieties have been created in north america and chile, from where they were transferred to europe in the 17th and 18th century. variability of morphometric characters comparative morphological analysis of plant material collected from different habitats showed a significant degree of variability in a series of characters of the fragaria vesca. variability in character of the fruit observing these two characteristics (height and width of the fruit) on the total sample (whose number is 300 individuals for all ten sites) it can be seen that for the first property, the minimum and maximum values range from 4 to 18 mm, and for the second property they range from 4 to 14 mm. percentage of variability for the fruit height is 24.338% and for the width of the fruit is 25.747% (tab. 2). fig. 1: variations in height of the fragaria vesca fruits by locality (1-10) the height of the fruit (fig. 1) is the largest in populations from the localities ilinčica ii (5), krojčica (6), simin han (7) and zlaća road (10). populations from the sites (6) and (7) represent communities on cleared land. at the given localities, in forest conditions, the light was limited. after cutting down the forest, there was a rapid expansion of the strawberries population due to the optimization of conditions of its ecological valence. fruit width (fig. 2) is the largest in populations from the localities krojčica (6), simin han (7) and zlaća road (10). these sites represent communities of open habitats and forest edges suggesting that these are the communities which by their ecological characteristics provide optimum conditions for the growth of strawberries. observing present forms of fruit by localities, it can be seen that the globose ones were present in the largest proportion at the site krojčica (6), which is cleared land of a forest. the round fruits were biologica nyssana 5 (2)  december 2014: 75-82 huseinović, s. et al.  morphological and ecological differentiation of the fruit … 78 table 2. statistical data of the analyzed fragaria vesca characters in all localities location variable n x xs minx maxx range s v (%) total p1 386 8.486 0.105 4 18 14 2.065 24.338 p2 386 7.459 0.098 4 14 10 1.920 25.747 table 3. significance in variations of fruit characters significance site 1 p1 p2 p3 p1 0.066 0.009 p2 0.066 0.000 p3 0.009 0.000 site 2 p1 p2 p3 p1 0.000 0.068 p2 0.000 0.127 p3 0.068 0.127 site 3 p1 p2 p3 p1 0.098 0.130 p2 0.098 0.163 p3 0.130 0.163 site 4 p1 p2 p3 p1 0.218 0.026 p2 0.218 0.000 p3 0.026 0.000 site 5 p1 p2 p3 p1 0.000 0.284 p2 0.000 0.427 p3 0.284 0.427 site 6 p1 p2 p3 p1 0.000 p2 0.000 p3 site 7 p1 p2 p3 p1 0.049 0.989 p2 0.049 0.124 p3 0.989 0.124 site 8 p1 p2 p3 p1 0.002 0.528 p2 0.002 0.194 p3 0.528 0.194 site 9 p1 p2 p3 p1 0.020 0.461 p2 0.020 0.040 p3 0.461 0.040 site 10 p1 p2 p3 p1 0.043 0.005 p2 0.043 0.728 p3 0.005 0.728 fig. 2: variations in width of the fragaria vesca fruits by locality (1-10) fig. 3: variations in shape of the fragaria vesca fruits by locality (1-10) present at all sites, with the exception of the site (6) and they prefer forest edges and meadows. the conical were present most in localities kladanj (1), zlaća road (10) and simin han (7), which leads to the conclusion that they prefer cooler places. the strawberries with elongated fruits in the greatest proportion inhabit the site ilinčica ii (fig. 3, 4). correlation in variations of fruit characters we can conclude that in most cases there is a correlation between the width and shape of the fruit (tab. 3, fig. 5). ecological differentiation differences between morphological characteristics of populations originate from biologica nyssana 5 (2)  december 2014: 75-82 huseinović, s. et al.  morphological and ecological differentiation of the fruit … 79 differences in environmental factors prevailing in their habitats. determination of a high degree of variability of the analyzed characters indicates high interpopulation variability, which further points to the possible influence of the present environmental factors. the basis of the high degree of variability are genetic determinants of the species fragaria vesca. wide ecological valence in relation to the complex environmental factors resulted with spreading of this species in an environmentally very dynamic habitats, usually ecotones, where environmental factors of two different habitats collide (e.g. forests and meadows). as a specific answer to this kind of dynamic (changeable) value of environmental factors, there is expressed variability of morphological and meristic characters. these features have the function of population’s adaptation within the present environmental factors in order to achieve the best use of capacities and potentials of the habitats. given the very different types of habitats, and very different conditions in which the population can survive, the question is which habitat factors favor, and which limit the population to achieve its biological / ecological functions. in the aim of shedding light on the role of individual factors from the complex, a cluster analysis was performed on the basis of the observed morphometric characters. based on the character of the fruit fig. 6 shows high morphological similarity of populations from localities 8 and 9, i.e. from populations of the community habitats of fragarion vescae in the zone querco-carpinetum betuli and fagetum montanum serpentinicum. morphological characteristics of individuals of these populations are similar: individuals are of average height and have average values of the other monitored characters. fig 4. presence of the four basic groups of the fruit shape by locality (n – number of individuals) fig. 5: correlations for the height (p1), width (p2) and shape (p3) of the fruit fragaria vesca 0 5 10 15 20 25 30 35 40 n 45 kladanj muška voda gornja tuzla cerik ljubače husin o ilinčica ii krojčic a simin han ilincica i zlaća bridge zlaća road conical rounded globose elongate biologica nyssana 5 (2)  december 2014: 75-82 huseinović, s. et al.  morphological and ecological differentiation of the fruit … 80 fig. 6: cluster analysis of the populations of fragaria vesca in the investigated area based on the observed character of the fruit the next group of very similar populations is made up of the populations from sites 1 and 4, and the related population from locality 10. the complex of factors that govern the habitats of these populations is heterogeneous. at the localities 1 and 10, vegetation of the ecosystem of beech-fir forests on serpentine geological substrate has been developed. in contrast to that, the site 4 has developed vegetation of the ecosystem of pine and oak. although considering the temperature these are very different habitats, in ecological sense the sites are linked by having poor supply of water. in relation to the appearance of individuals in this group of populations (individuals are small, as well as most of individual characters), it can be assumed that the supply of water to habitats plays a major role in the possibilities of biomass production and dissemination of strawberries population. the group of populations of meadow ecosystems of the alliance arrhenatherion elatioris shows similarity with the populations developed in vegetation of fern colonies. individuals are high in growth (stems are elongated), the leaves are large and petioles are small. in the dense meadow complexes, individuals of strawberry, looking for sufficient quantities of light and heat, in a competitive fight, "invest" their energy in stem elongation and increase in all vegetative parts of the plant body (sites 3, 7 and 2). however, the greatest in growth are individuals that grow on the so-called cleared land (site 6, cleared land of the forest tilio-quercetum petraeae) and on the very edge of the forest (site 9, the edge of the forest fagetum montanum "serpentinicum.") this fact indicates that the strawberry as a species is very capable that through rapid expansion occupy new habitats before other species use nutrients that forest land offers. however, this also points to the fact that the lack of light and heat in a given period of development (conditions prevailing in assembled forest communities), stand out as the limiting factor for the expansion of its populations. a similar differentiation is shown also by the observed characters of the fruit. discussion variability in character of the fruit (i) the largest coefficient of variation in the height of the fruit was recorded at the site 10 and amounts 26.618%, the smallest on the site 7 and amounts 14.573%. (ii) in the variation of the width of the fruit, the largest coefficient of variation was recorded at the site 2 (23.863%) and the smallest amounts 14.667%, being recorded at the site 6. (iii) the coefficient of variation of the height of the fruit was 24.338%, and of the width of the fruit was 25.747%. thus, the fruit does not fall into the group of those plant parts that vary the most. however, due to the high resource values of strawberries, it is necessary to emphasize that the size of the fruit follows the overall size of individuals. as seen from the previous findings, the highest growth was achieved by individuals from the cleared land of the community tilio-quercetum petraeae, which indicates that strawberries in our conditions optimally thrive on mesophilic, well lit to somewhat shaded and relatively warm habitats of the mountain zone. biologica nyssana 5 (2)  december 2014: 75-82 huseinović, s. et al.  morphological and ecological differentiation of the fruit … 81 morphological differentiation the populations of species fragaria vesca in the study area were not morphologically uniform, but they contain more or less differentiated morphoeco-types (r e d ž i ć , 1990). the fact that in the populations of different geo-biocenosis the same morphological types of plants occured, and that these populations differ among themselves, however, shows that the quantitative participation of individual types in them is different (b a š i ć , 2004; k o v a č i ć , 2006). at the site of muška voda, kladanj, the size of specimens is relatively small compared to individuals from cleared land and open meadow habitats. the flowers are small, as well as the fruits, which are mostly elongated. individuals of the population at the locality of gornja tuzla developed within the community of arrhenatherion elatioris. flowers are also relatively small, as well as the fruits, which are mostly elongated. variations within the population are relatively large. individuals of the population at the site cerik ljubače (in the zone of the forest community querco-carpinetum) are characterized by the lowest average length and width of a rosette leaf midrib. individuals sampled at the site husino developed within the community sambucetum nigrae and are characterized by the fact that there have not been reported extremely high or low values for the observed characters. population individuals of the site ilinčica ii developed within the community fragarion vescae by the edge of querco-carpinetum betuli. the specimens are large, but still far less than those on the site krojčica, where maximum values for the most of the studied characters were recorded. individuals of the population at the site simin han developed within the community pteridiofragarietum vescae and are very variable among them. on the site ilinčica i, population has been developed within the community agropyrorumicion in anthropogenized conditions in the zone of mesophilic meadows. they do not stand out by the high growth, nor by the size of the fruit. individuals of the population zlaća (road) developed on the anthropogenized eutric cambisol within the community of dark deciduous forests, as well as their neighboring population. individuals of both populations are characterized by relatively low growth. ecological differentiation group of populations from meadow ecosystems of the alliance arrhenatherion elatioris showed similarity with the population developed in the vegetation of fern colonies and all other populations that are found in the open (nonforested) habitats (r e d ž i ć , 1987, 1988). individuals were high in growth (stems are elongated), the leaves were large, while petioles were small. in dense meadow complexes, individuals of strawberry, looking for sufficient quantities of light and heat, in a competitive fight, "invest" their energy in stem elongation and increase in all vegetative parts of the plant body (sites 2, 3 and 7). however, the greatest growth had the individuals that grew on the so-called cleared land (site 6, cleared land of the forest tilio-quercetum petraeae) and on the very edge of the forest (site 9, the edge of the forest fagetum montanum "serpentinicum"). this fact indicates that the strawberry as a species, is very capable to occupy new habitats by rapid expansion and before other species use nutrients that forest lands offer. (i) as a specific answer to this kind of dynamic (changeable) values of environmental factors there is an expressed variability of morphological and meristic characters, which has the function of adaptation of populations to present environmental factors, that is, the function of the best use of habitat's capacities and potentials (e r i k s s o n et al., 2003). (ii) the investigated morphologic characters are changing, and the emerged changes and differences among populations are not stable. further, stronger environmental isolation does not lead to a stronger morphological differentiation of the same character or group of characters. greater morphological differentiation is achieved by increasing the number of differentiating characters. in the second case, individual differential characters can lose that property and differentiation among populations is accomplished through other characters. (iii) since ecologically distant populations are simultaneously morphologically remote too, it could be assumed that those changes are still at the level of phenotypic variation. however, within the same population clearly separated groups of individuals (groups of clones) can be observed, among which there are no observed larger and clearer environmental differences. similar clones are located in ecologically different populations. therefore, a detailed knowledge of morphological variability and ecological differentiation of populations within the species is biologica nyssana 5 (2)  december 2014: 75-82 huseinović, s. et al.  morphological and ecological differentiation of the fruit … 82 essential condition for the full overview of biological potential and evolutionary status in which the analyzed group of plants is located. references aichele, d., 2004: was blűht denn da? kosmos naurfűhrer. stuttgart. angevine, m.w., 1983: variations in the demography of natural populations of the wild strawberries fragaria vesca and f. virginiana. journal of ecology, 71 (3): 959-974. bašić, n., 2004: morfološko – taksonomska istraživanja glogova (crategus l.) na području bosne i herzegovine. magistarki rad. sarajevo. beck-mannagetta, g., 1927: flora bosne, hercegovine i oblasti novog pazara. zemaljska štamparija; 6 – 8.sarajevo. dimeglio, l.m., staudt, g., yu, h., davis, t.m., 2014: a phylogenetic analysis of the genus fragaria (strawberry) using intron-containing sequence from the adh-1 gene. plos one, 9 (7), e102237. domac, r. 2002: flora hrvatske. školska knjiga. zagreb. 504p. eriksson, t., malin, s., hibbs., anne, d., yoder, s., charles, f., delwiche., michael, j. 2003: the phylogeny of rosoideae (rosaceae) based on sequences of theinternal transcribed spacers (its) of nuclear ribosomal dna and the trnl/f region of chloroplast dna. international journal of plant science, 164 (2): 197 – 211. galletta, g.j., maas, j.l. 1990: strawberry genetics. hortscience, 25 (8): 871-879. harrison, r., lubz, j., furnier, g., hancock, j., 1997: morphological and molecular variation among populations of octoploid fragaria virginiana and f. chiloensis (rosaceae) from north america. american journal of botany, 84 (5): 612-620. hummer, k.e., bassil, n, njuguna, w., 2011: fragaria, 17-44. in: c. kole (ed.), wild crop relatives: genomic and breeding resources, temperate fruits. springer-verlag berlin heidelberg. 247p. huseinović, s., osmanović, s. 2010: morphometric and meristic characteristics of the wild strawberry (fragaria vesca l.) on konjuh mountain. acta agriculturae serbica, 15 (30): 133-140. kovačić, s. 2006: srodstveni odnosi i korologija izofilnih i heterofilnih zvončića (campanula l., campanulaceae) primorskih dinarida. doktorska disertacija, zagreb. petz, b., 1983: osnovne statističke metode za nematematičare. sveučilišna naklada liber, zagreb. potter, d., eriksson, t., evans, r.c., oh, s., smedmark, j.e., morgan, d.r., kerr, m., robertson, k.r., arsenault, m., dickinson, t.a., campbell, c.s. 2007: phylogeny and classification of rosaceae. plant systematics and evolution, 266: 5-43. redžić, s., 1987: djelovanje faktora čistih sječa na spektar sistematske pripadnosti vrsta viših biljaka u nekim šumskim ekosistemima bosne. godišnjak biološkog instituta univerziteta u sarajevu, 40: 89-100. redžić, s., 1988: šumske fitocenoze i njihova staništa u uslovima totalnih sječa. godišnjak biološkog instituta univerziteta u sarajevu, 41: 1-260. redžić, s., 1990: morfološka diferencijacija populacija taksona potentilla malyana borbas. bilten društva ekologa bih, serija b, 5: 93-99. redžić s., 1997: karakteristike biljnog svijeta bosne i hercegovine. prirodno-matematički fakultet univerziteta u sarajevu. staudt, g., 1989: the species of fragaria, their taxonomy and geographical distribution. acta horticulturae, 265: 23–33. staudt, g., 2009: strawberry biogeography, genetics and systematics. acta horticulturae, 842: 71-84. tutin, t.g., heywood, v.h., burges, n.a., moore, d.m., valentine, d.h., walters, s.m., webb, d.a. (ed.), 1968: flora europaea, ii. cambridge, university press. london. microsoft word bn-sc-0201-11 crnobrnja-isailovic et al biologica nyssana 2 (1) september 2011: 81-84 crnobrnja-isailović, j. et al. occurrence of european adder… 81 short communication ! occurrence of european adder (vipera berus, viperidae, ophidia) on vlasina plateau (southeastern serbia) jelka crnobrnja-isailović*1, 2, jelena dinov3, & vladimir ranđelović1 1faculty of sciences and mathematics, university of niš, višegradska 33, 18000 niš, serbia; 2institute for biological research „siniša stanković“, despota stefana 142, 11000 beograd, serbia; 3biological society “dr sava petrović “, 18000 niš, serbia * e-mail: jelka@pmf.ni.ac.rs abstract: crnobrnja-isailović, j., dinov, j., ranđelović, v.: occurrence of european adder (vipera berus, viperidae, ophidia) on vlasina plateau (southeastern serbia). biologica nyssana, 2 (1), september 2011: 81-84. the european adder (vipera berus) is among the most widespread reptile species in europe, but it’s distribution at the balkan peninsula seems to be scarce and fragmented. as going toward the south, specimens were more frequently found in the vegetational zone of boreal forests and high alpine pastures. in the south-eastern serbia, recent occurrence of european adder was confirmed on the vlasina plateau, in june 2010, in a mosaic-complex of peat bogs and marsh vegetation. recent engagement of ecology students from university of niš in mapping actual local distribution of adder will help locating key spots for its conservation in this area. key words: distribution, european adder, vipera berus, vlasina plateau ! the european adder (vipera berus (l. 1758) viperidae) is among the most widespread reptile species in europe (g a s c et al., 1997). its distribution goes from the contact zone between pyrenean peninsula and central europe on the west; southern border follows the northern parts of the apennine peninsula and highlands of western, central, southern and eastern balkans. eastern part of species area spreads deeply into the central and western asia, over large part of former soviet republics, with southern border running through moldavia, central ukraine and southern russia (n i l s o n & a n d r e n , 1997). the european adder is concerned as boreal relict at the balkan peninsula. it was supposed to be widely distributed in lowlands during pleistocene glaciations, but only isolated refugial mountain populations persisted during interglacial periods, as it is in recent time (u r s e n b a c h e r et al., 2006). phylogeographic structure of this species undoubtedly points that the balkan population group was important source of the species genetic diversity, as one of three major mitochondrial lineages probably originated in the balkans (u r s e n b a c h e r et al., 2006). despite being evaluated globally as least concerned (lc) on the iucn red list of threatened species (c r n o b r n j a i s a i l o v i ć et al., 2008), european adder has been assigned as „scarce“ in serbia as far back as in r a d o v a n o v i ć (1951) book on amphibians and reptiles of yugoslavia. national conservation status of european adder in serbia is defined in anonymous (2010) as „strictly protected species“, what means prohibition of any collecting, killing or harming of specimens. on that way, detailed 2 (1) • september 2011: 81-84 biologica nyssana 2 (1) september 2011: 81-84 crnobrnja-isailović, j. et al. occurrence of european adder… 82 knowledge on distribution of this species in republic of serbia is necessary for appropriate design of further national conservation plans. historical records confirmed presence of european adder in vojvodina, but restricted on its hilly parts covered with mainly oak forests quercocarpinetum sensu lato (fruška gora mountain and the vršačke planine mountain – d ž u k i ć & p u r g e r , 1988) and relatively intact marshy areas (obedska bara – d ž u k i ć & p u r g e r , 1988). in the balkan part of serbia, published historical records of adder’s presence exist for the avala mountain (d ž u k i ć , 1972), but there is no confirmation about species recent occurrence there. as far as we know, european adder inhabits the kopaonik mountain (c r n o b r n j a & r o h a l j , 1988), the stara planina mountain (i v a n č e v i ć et al., 2007) and the zlatibor mountain (c r n o b r n j a i s a i l o v i ć , unpublished data), but many new records are still unpublished or deposited in manuscripts, as well. it is reliable to consider potential presence of this species on all the mountains in serbia where habitats typical for boreal woods and alpine pastures exist. specimens were more frequently found in the vegetational zone of boreal forests and high alpine pastures as going more toward the southern parts of the state. in southern serbia, communities potentially suitable for the european adder are distributed mostly on the altitudes higher than 1000 m. intensive degradation of natural potential vegetation communities stretch the area of potential occurrence of this species even more. on the vlasina plateau potential vegetation is represented by beech forests, but they were quite degraded. however, phytogeographical analysis shows that boreal has made a strong impact on the genesis of the flora and vegetation, which was particularly reflected in the significant presence of peat bogs (r a n đ e l o v i ć & z l a t k o v i ć, 2010). presence of european adder on the vlasina plateau was recently confirmed within utm square fn03 in the second decade of june 2010 (n42o43’ 9.9“, e22o19’ 59.1“, 1227 m altitude – figure 1.). one juvenile specimen was found dead on the magistral road, obviously killed by car while crossing the road. according to our knowledge, it was one year old juvenile female (number of supracaudal scales sc – less than 30), with 187 mm of total lenght (l) and 169 mm of snout-to-vent lenght (svl). natural habitat of european adder at the site near village vlasina rid is a mosaic-complex of peat bogs and marsh vegetation consisted of associations equisetoscirpetum silvaticae, deschampsietum caesoitosum, caricetum gracilis and caricetum goodenowii (r a n đ e l o v i ć & z l a t k o v i ć , 2010). sintopic amphibian species recorded there were triturus vulgaris, t. alpestris, bufo bufo and rana synklepton esculenta, while reptile species detected in the area were lacerta agilis and anguis fragilis. published records of occurrence of the european adder on the vlasina plateau, up to our knowledge, are restricted on information occurring in publication of institute for nature conservation of republic of serbia (a n o n y m o u s 2006). there was no detailed description of species distribution or about its preferable habitat types within the area of the vlasina plateau, that points to the lack of details important for future conservation strategies in situ. department of biology and ecology of faculty of sciences and mathematics in niš is already conducting conservation studies and monitoring in figure 1. new record of vipera berus on the vlasina plateau biologica nyssana 2 (1) september 2011: 81-84 crnobrnja-isailović, j. et al. occurrence of european adder… 83 the area, so recent engagement of ecology students there, under the scope of realisation of their master theses, will help facilitation of mapping actual local distribution of the european adder. the main objectives of their research will be evaluation of importance and suitability of different habitats on the vlasina plateau for the maintenance of viable populations of the european adder, as well as recording of most plausible threats that occur in the area. acknowledgements. we are grateful to our colleagues imre krizmanić and danko jović who kindly provided us information about literature data on herpetofauna of the vlasina plateau. this work was supported by faculty of sciences and mathematics of university of niš, touristic enterprise „vlasina“ and grant 173025 ministry of education and science of republic of serbia. references anonymous, 2006: predeo izuzetnih odlika vlasina, studija zaštite, beograd. anonymous, 2010: pravilnik o proglašenju i zaštiti strogo zaštićenih i zaštićenih biljnih vrsta biljaka, životinja i gljiva. prilog 2. strogo zaštićene vrste. službeni glasnik rs br 5/10. crnobrnja, j., rohalj, a., 1988: prilog poznavanju herpetofaune kopaonika. zbornik radova bid "josif pančić", beograd: 59 76. crnobrnja isailović, jelka, milan vogrin, claudia corti, valentin pérez mellado, paulo sa-sousa, marc cheylan, juan m. pleguezuelos, ljiljana tomović, bogoljub sterijovski, urlich joger, a. westerström, bartosz borczyk, benedikt schmidt, andreas meyer, roberto sindaco, dušan jelić 2008. vipera berus. in: iucn 2010. iucn red list of threatened species. version 2010.4. <www.iucnredlist.org>. džukić, g., 1972: herpetological collection of the belgrade museum of natural history. bull. mus.hist.natur. ser. b, liv. 27: 165-180. džukić, g., purger, j., 1988: significance of adder vipera berus (linnaeus, 1758) presence in vojvodina. arh.biol.sci, 40 (1-4): 13p-14p. gasc, j-p., cabela, a., crnobrnja-isailović, j., dolmen, d., grossenbacher, k., haffner, p., lescure, j., martens, h., martinez-rica, j.p., maurin, h., oliveira, m.l., sofianidou, t.s., veith, m., zuiderwijk, a., eds., 1997: atlas of amphibians and reptiles in europe. societas europaea herpetologica & museum nationall d' histoire naturelle (iegb/spn), paris, 496 p. ivančević, b., savić, s., sabovljević, m., niketić, m., tomović, g., zlatković, b., ranđelović, v., lakušić, d., ćetković, a., pavićević, d., krpoćetković, j., crnobrnja-isailović, j., puzović, s., paunović, m., 2007: pregled vrsta stare planine u srbiji. in: d. lakušić, a. ćetković, eds.: biodiverzitet stare planine u srbiji rezultati projekta: "p r e k o g r a n i č n a saradnja kroz upravljanje zajedničkim prirodnim resursima promocija umrežavanja i saradnje između zemalja jugoistočne evrope". regionalni centar za životnu sredinu za centralnu i istočnu evropu, kancelarija u srbiji, beograd: 159-219. nilson, g., andren, c. 1997: vipera berus. in: gasc, j-p., cabela, a., crnobrnja-isailović, j., dolmen, d., grossenbacher, k., haffner, p., lescure, j., martens, h., martinez-rica, j.p., maurin, h., oliveira, m.l., sofianidou, t.s., veith, m., zuiderwijk, a., eds.: atlas of amphibians and reptiles in europe. societas europaea herpetologica & museum nationall d' histoire naturelle (iegb/spn), paris: 388 389. radovanović, m., 1951: vodozemci i gmizavci naše zemlje. narodna knjiga, beograd. ranđelović, v. n., zlatković, b. k. 2010. flora i vegetacija vlasinske visoravni. prirodnomatematički fakultet, niš. ursenbacher, s., carlsson, m., helfer, v., tegelström, h., fumagalli, l., 2006: phylogeography and pleistocene refugia of the adder (vipera berus) as inferred from mitochondrial dna sequence data. molecular ecology, 15: 3425-3437. petrov & ocokoljić 2022, biologica nyssana 13(1) 13 (1) september 2022: 41-46 doi: 10.5281/zenodo.7117571 dendrometric analyses and determining biological characteristics of needles for the purpose of conservation and directed utilization of the spruce in southeastern serbia original article djurdja petrov faculty of forestry, university of belgrade, kneza višeslava 1, 11000 belgrade, serbia djurdja.stojicic@sfb.bg.ac.rs (corresponding author) mirjana ocokoljić faculty of forestry, university of belgrade, kneza višeslava 1, 11000 belgrade, serbia received: july 21, 2021 revised: september 16, 2022 accepted: september 21, 2022 abstract: this paper analyzes the morphological and dendrometric characteristics of three 97 years old european spruce trees, on čemernik mountain, in the municipality of crna trava, where european spruce is not autochthonous. the examined parameters were compared with 5 test european spruce trees, aged about 50 years, which are grown in the population, within a radius of 100 m, in identical environmental conditions. the results of this study confirm the excellent vitality of hundred-year-old trees at the researched location, which can be brought into direct correlation with the current climate changes. the selected trees have confirmed the best combinatorial ability, which is why they stand out as a good base for selection and production of planting material for establishing purpose culture, afforestation, application in landscape architecture, and also for timber production. the research confirmed that the analyzed hundred-year-old spruce trees are in excellent condition and high decorative value, with a crown formed from the ground, which is why they are proposed for protection as a natural monument of botanical character. key words: european spruce, crna trava, vitality, selection, genetic potential, conservation apstrakt: dendrometrijska naliza i utvrdjivanje bioloških svojstava četina smrče u cilju konzervacije i usmerenog korišćenja vrste u jugoistočnoj srbiji u radu su analizirane morfološke i dendrometrijske karakteristike tri stabla smrče starosti 97 godina, na čemerniku, na teritoriji opštine crna trava na kojoj smrča nije autohtona. istraživani parametri su poređeni sa pet test stabala smrče, starosti oko 50 godina, odgajenih u populaciji, u radijusu od 100 m, u identičnim uslovima. dobijeni rezultati potvrđuju odličnu vitalnost stogodišnjih stabala na istraživanoj lokaciji što se može dovesti u direktnu korelaciju sa aktuelnim klimatskim promenama. selektovana stabla potvrdila su kombinatornu sposobnost zbog čega se izdvajaju kao polazna za oplemenjivanje i proizodnju sadnog materijala, za podizanje namenskih kultura, pošumljavanje, pejzažnoarhitektonsku primenu, ali i dobijanje drvne mase. istraživanje je potvrdilo da su analizirana stogodišnja stabla smrče odličnog stanja i dekorativnosti, sa formiranom krošnjom od samog tla, zbog čega se predlažu za zaštitu kao spomenik prirode botaničkog karaktera. ključne reči: smrča, crna trava, vitalnost, selekcija, genetski potencijal, konzervacija introduction picea a. dietr. is a genus of evergreen coniferous trees in the family of pinaceae spreng. ex f. rudolphi. it comprises 37 spruce species (powo, 2022), one of which is european or norway spruce picea abies (l.) h. karst. besides european spruce, other autochthonous and allochthonous spruce species are also found in serbia. they include picea omorika (pancic) purk., picea orientalis (l.) peterm., picea pungens engelm., picea laxa (münchh.) sarg., picea engelmannii parry ex engelm. and picea sitchensis (bong.) carrière. (jovanović, 1970; schmidtvogt, 1974; matović & vujković, 1994; skrøppa, 2003; galović, 2015; cvjetićanin et al., 2016). the natural range of european spruce distribution © 2022 petrov & ocokoljić. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 41 14th symposium on the flora of southeastern serbia and neighboring regions stretches from siberia across the ural mountains and extends to northern europe and high mountain regions of central and southern europe (fukarek, 1970). fukarek (1970) stated that european spruces could not be found in the apennines and pyrenees. however, research conducted in 2016 (di pierro et al., 2016) confirmed the presence of european spruce in the far north of the apennines. in the south of europe, european spruce grows in two directions (cvjetković, 2018): eastwards (from the carpathian mountains, over the high mountains of western serbia and bulgaria to the rhodopes) and westwards (from the slopes of the eastern alps, across the dinaric alps, to the shar mountains and mount korab). however, it should be emphasized that throughout history spruce has undergone a significant shift in its range, as confirmed by pollen analyses according to which european spruce was limited to several refugia during the last glacial period, while the current range has resulted from its expansion from the refugia in the carpathians, dinaric alps and moscow region (huntley & birks, 1983). in serbia, european spruce is widely distributed in the mountains, such as tara, zlatibor, golija, zlatar, prokletije, and kopaonik, while it is not so common in the eastern and southern parts of the country: stara planina, suva planina, kučajske planine, beljanica and shar mountains (ivetić, 2004; banković et al., 2009; cvjetićanin et al., 2016). the range of distribution and genetic structure of european spruce are thought to have been shaped by historical events related to the last glaciation and postglacial colonisation processes (lagercrantz & ryman, 1990). while there was a decline in the natural distribution of european spruce in the past, there was a growing trend in the conversion of broadleaved forest habitats and abandoned agricultural land into coniferous forests. despite the high intrapopulation variability and adaptability of european spruce, abrupt distribution shifts are anticipated due to climate change. they will be reflected in the extinction of populations, primarily the edge populations growing along the distribution boundaries. according to some scenarios, numerous habitats in southern europe will be affected by climate change and european spruce will migrate north (falk & hempelmann, 2013). these predictions should cause some alarm since european spruce is one of the most common coniferous species in european forests; it is also a very important economic species; it has been widely naturalised outside its distribution range; in serbia, it covers 86,400 ha and accounts for about 5.2% of wood volume. bearing in mind the above and the fact that, due to its physical-geographical location, serbia is significantly affected by climate change, the research conducted in european spruce plantations on mount čemernik is of great importance. materials and methods according to morgenstern (1996), research on forest species can be divided into two categories: short-term and long-term tests. our research applied long-term tests of open-grown trees over 30 years of age. the suitability of the selected methods can be proved by kapeller et al. (2013) according to which the tests conducted at the age of 10 to 30 yield the most valuable research results related to species variability and adaptability. our research was conducted in a group of three solitary trees of picea abies (l.) h. karst. the trees were 97 years old (fig. 1). they were planted in the village of brod (42°84’88’’ north latitude and 22°28’39’’ east longitude) in 1925. the village is on mount čemernik, crna trava municipality, at an altitude of 856 m. the five control trees grew in a nearby spruce culture (within a 100 m radius) founded in the 1970s. 42 biologica nyssana ● 13 (1) september 2022: 41-46 petrov & ocokoljić ● dendrometric analyses and determining biological characteristics of needles for the purpose of conservation and directed utilization of the spruce in southeastern serbia fig. 1. three 100-year-old picea abies (l.) h. karst. trees 43 tree height, diameter at breast height (1.30 m), trunk girth, crown diameter, tree age, needle length, needle width, adaptability and vitality of the three analysed trees were taken as indicative values together with the differences between the group of three trees and five control trees that should facilitate the assessment of the species vitality. a total of 400 needles were collected for each tree (i.e. three 100-year-old trees and five control trees) which means that 3200 dimensions were measured for the length and another 3200 dimensions for the width of needles (fig. 2). biologica nyssana ● 13 (1) september 2022: 41-46 petrov & ocokoljić ● dendrometric analyses and determining biological characteristics of needles for the purpose of conservation and directed utilization of the spruce in southeastern serbia quantitative characteristics that included tree height, diameter at breast height, trunk girth, crown diameter and needle length and width (tab. 1, tab. 2) were biometrically processed using the statistica 10.0 software program and their limit of variability (minimum-maximum), medium values ( x ), standard deviations (s), coefficients of variation (v) and errors (s x , ss, sv) were determined. results and discussion field research in the municipality of crna trava in the village of brod recorded three trees of picea abies (l.) h. karst. of exceptional growth habit, excellent vitality and high decorative values. the three trees growing in a group stood out for their differential properties. tab. 1 shows the results of a comparative analysis of the growth elements of three investigated european spruce trees and five control trees. tree 1 reached a height of 52.9 m, a diameter at breast height of 118 cm and a crown diameter of 11.98 m. tree 2 reached a height of 53.1 m, a diameter at breast height of 103 cm and a crown diameter of 17.64 m. tree 3 reached a height of 50.94 m, a diameter at breast height of 84 cm and a crown diameter of 11.24 m. all three trees were around 100 years old and had abundant to moderately abundant yields and the highest ratings of vitality and decorativeness (5 and 5). they had broadly pyramidal crowns and shallow-cracked gray to gray-brown bark. the five control trees at the age of about 50 years old, which is the age suitable for the comparison of values, had significantly different dendrometric characteristics. the average parameters for the five control trees were as follows: trunk height – 24.16 m, diameter at breast height – 63.8 m, trunk girth – 2.01 m, and crown diameter – 8.2 m. fig. 2. analysed parameters of picea abies (l.) h. karst: length (l) and width (w) tree tree age (years) h (m) d1,30 (cm) trunk girth (m) crown diameter (m) fructification vitality (1-5) decorative value (1-5) 1 97 53.1 118 3.71 11.98 medium 5 5 2 97 52.9 103 3.24 17.64 abundant 5 5 3 97 50.94 84 2.66 11.24 abundant 5 5 4 ̴50 19.8 49 1.54 5.76 medium 3 2 5 ̴50 27.3 86 2.69 9.92 abundant 5 5 6 ̴50 26.6 74 2.33 8.74 abundant 5 4 7 ̴50 23.48 53 1.68 10.22 medium 4 4 8 ̴50 23.6 57 1.79 6.38 medium 3 3 table 1. comparative characteristics of three 100-year-old picea abies (l.) h. karst. trees and five trees planted in the 1970s 44 the analysis of statistical parameters (tab. 2) for the two characteristics of needles measured in trees at the age of 100 determined that the three investigated trees had the limit values of needle length ranging from 12 to 32 mm and from 0.8 to 2 mm for needle width. on the other hand, the five control trees aged 50 had the limit values ranging from 8 to 25 mm for needle length and from 0.5 to 1.5 mm for needle width. according to the literature (gajić & korać, 1972; ocokoljić & ninić-todorović, 2003; gebauer et al., 2011; caudullo et al., 2016; gebauer et al., 2019), spruce needles can be up to 25 mm long and more than 1 to 2 mm wide. as can be observed, the three investigated trees have significantly higher values. according to the results, the mean values of the needle length obtained for tree 1 were 22.49±0.05, tree 2 22.17±0.05, and tree 3 22.96±0, 06. the mean values of the needle width obtained for tree 1 were 1.26±0.00 for tree 2 1.11±0.00, and tree 3 1.22±0.00. on the other hand, the mean values of the needle length obtained for the five control trees (trees 4, 5, 6, 7 and 8) were 16.4±0.01 and the mean values of the needle width were 0.86±0.00. the obtained difference between the mean values was statistically significant. looking at the differential characteristics of the biologica nyssana ● 13 (1) september 2022: 41-46 petrov & ocokoljić ● dendrometric analyses and determining biological characteristics of needles for the purpose of conservation and directed utilization of the spruce in southeastern serbia table 2. statistical parameters of morphological characteristics of european spruce needles in the village of brod locality tree age (years) limit value ± s±ss v±sv (а) needle length (mm) tree 1 97 12-32 22.49 ± 0.05 3.25 ± 0.79 0.14 ± 0.01 tree 2 97 15-32 22.17 ± 0.05 3.21 ± 0.78 0.14 ± 0.01 tree 3 97 14-30.5 22.96 ± 0.06 3.25 ± 0.81 0.14 ± 0.00 tree 4 ̴50 9-21 15.19 ± 0.04 2.61 ± 0.54 0.17 ± 0.01 tree 5 ̴50 8-27 18.53 ± 0.05 3.68 ± 0.66 0.20 ± 0.01 tree 6 ̴50 10-25 18.04 ± 0.04 2.44 ± 0.64 0.13 ± 0.00 tree 7 ̴50 10-20 15.42 ± 0.04 2.20 ± 0.55 0.14 ± 0.00 tree 8 ̴50 8-20 14.83 ± 0.04 2.69 ± 0.52 0.18 ± 0.01 (b) needle width (mm) tree 1 97 0.8-2 1.26 ± 0.00 0.28 ± 0.04 0.22 ± 0.01 tree 2 97 0.9-2 1.11 ± 0.00 0.24 ± 0.04 0.22 ± 0.01 tree 3 97 0.8-15 1.22 ± 0.00 0.74 ± 0.04 0.60 ± 0.02 tree 4 ̴50 0.5-1.2 0.80 ± 0.00 0.19 ± 0.03 0.24 ± 0.01 tree 5 ̴50 0.5-2 1.02 ± 0.00 0.29 ± 0.04 0.28 ± 0.01 tree 6 ̴50 0.5-1.5 0.93 ± 0.00 0.18 ± 0.03 0.19 ± 0.01 tree 7 ̴50 0.5-1 0.77 ± 0.00 0.17 ± 0.03 0.22 ± 0.01 tree 8 ̴50 0.5-1 0.80 ± 0.00 0.21 ± 0.03 0.27 ± 0.01 group of three european spruce trees about 100 years old, it can be observed that these trees are more adaptive and ornamental than those planted 50 years later and growing in forest conditions. the data presented in tab. 1 and tab. 2 points to obvious differences in the analyzed properties between the three veteran trees and the five control trees. these differences can affect the cultivation of european spruce since the three selected veteran trees that stand out for their distinctive characteristics have considerable potential. as plus trees with a superior phenotype, they enable more successful and easier cultivation, due to better growth, higher level of adaptability, and remarkable decorativeness (compact crown formed from the ground level). further comparison of the phenotypic characteristics of the investigated 100-year-old spruce trees in the village of brod with literature data (caudullo et al., 2016; cvjetićanin et al., 2016) reveals that the analysed trees have reached maximum values, and thus can be used as starting material in the production of forest and ornamental seedlings. based on the conducted research, which included the age of trees, their vitality, decorativeness and position (sunny location for sciophilic european spruce), and their historical significance, all three trees are proposed to be protected as a botanical 45 biologica nyssana ● 13 (1) september 2022: 41-46 petrov & ocokoljić ● dendrometric analyses and determining biological characteristics of needles for the purpose of conservation and directed utilization of the spruce in southeastern serbia caudullo, g., tinner, w., de rigo, d. 2016: picea abies in europe: distribution, habitat, usage and threats. european atlas of forest tree species. publications office of the european union. luxembourg. 114-116 p. cvjetićanin, r., brujić, j., perović, m., stupar, v. 2016: dendrologija. univerzitet u beogradu, šumarski fakultet. 557 p. cvjetković, b. 2018: genetičko-fiziološka varijabilnost smrče (picea abies karst.) u testovima potomstva u bosni i hercegovini. doktorska disertacija. univerzitet u beogradu, šumarski fakultet. 351 p. di pierro, e.a., mosca1, e., rocchini, d., binelli, g., neale, d.b., la porta, n. 2016: climate-related adaptive genetic variation and population structure in natural stands of norway spruce in the southeastern alps. tree genetics & genomes, 12(2): 1-16. falk, w., hempelmann, n. 2013: species favourability shift in europe due to climate change: a case study for fagus sylvatica l. and picea abies (l.) karst. based on an ensemble of climate models. journal of climatology, 2013: 1-18. fukarek, p. 1970: areali rasprostranjenosti bukve, jele i smrče na području bosne i hercegovine. akademija nauka i umjetnosti bosne i hercegovine. knjiga 11: 235-256. gajić, m., korać, m. 1972: varijabilnost četina smrče (picea excelsa l.) u odnosu na različita staništa na planini goliji. simpozijum povodom proslave 50-godišnjice osnivanja i rada šumarskog fakulteta, beograd: 33-37. galović, v., šijačić-nikolić, m., šafhauzer, r., čortan, d., orlović, s. 2015: genetic differantion of norway spruce (picea abies (l) karst.) trees with differnet crown types from the montain golia. genetika, 47(3): 849–861. gebauer, r., volařík, d., urban, j., børja, i., nagy, n., drabløs, t., krokene, p. 2019: effects of mild drought on the morphology of sun and shade needles in 20-year-old norway spruce trees. iforest biogeosciences and forestry, 12: 27-34. gebauer, r., volarík, d., urban, j., børja, i., nagy, n., eldhuset, t., krokene, p. 2011: effect of thinning on anatomical adaptations of norway spruce needles. tree physiology, 31: 1103-1113. huntley, b., birks, h.j.b. 1983: an atlas of past and present pollen maps for europe, 0-13.000 years ago. cambridge university press. 650 p. ivetić, v. 2004: uticaj staništa i provenijencija na natural monument. conclusions in the village of brod in the municipality of crna trava, three european spruce trees were recorded at the age of 97. our research revealed great vitality and exceptional morphological characteristics of the three european spruce trees. this finding pointed out the necessity of preserving the trees so that their genetic potential can be fully exploited. the study of these 100-year-old trees also revealed their potential role in forestry. they can be of great significance in the production of planting material that would make a valuable forest resource with long-term adaptability to local environmental conditions. the analysed european spruce trees can also be greatly utilised in landscape architecture and horticulture where they can serve as starting material in breeding, pollen collection, controlled hybridisation and vegetative propagation, as well as in the identification of a new lower taxon of compacta type. the analyzed 100-year-old trees should also be protected in order to preserve the national biodiversity of species for which indigenous species are extremely important. they are also important for genetic variation, i.e. as well-adapted genotypes of specific phenotype, they greatly contribute to genetic diversity. their significance is supported by research studies that stress the issue of european spruce extinction due to climate change, and the issue of reproductive material transfer which is usually unknown, and in europe, it is mostly from geographically and ecologically undefined sources. therefore, effort must be put in to find new resistant and productive genotypes and through plant breeding, preserve the species and increase its productivity. the exquisite habit, outstanding features, high condition ratings, age, and historical significance these three 100-year-old spruce trees have for čemernik and serbia make them worthy candidates for a botanical natural monument designation. acknowledgements. the ministry of education, science and technological development is financing the scientific research work of the university of belgrade faculty of forestry in 2022 on the basis of the agreement on the realization number: 451-03-9 / 2022-14 / 200169. references banković, s., medarević, m., pantić, d., petrović, n. 2009: nacionalna inventura šuma republike srbije šumski fond republike srbije. ministarstvo poljoprivrede šumarstva i vodoprivrede republike srbje, uprava za šume. beograd. 244 p. razvoj juvenilnih kultura smrče (picea albies /l./ karst) na goliji. magistarski rad. univerzitet u beogradu, šumarski fakultet. beograd. jovanović, b. 1970: rod picea a. dietr. in: josifović m. (ed.), flora sr srbije, 1. srpska akademija nauka i umetnosti, beograd. kapeller, s., schüler, s., huber, g., božič, g., wohlgemuth, t., klumpp, r. 2013: provenance trials in alpine range – review and perspectives for applications in climate change. in: cerbu, g.a., hanewinkel, m., gerosa g., jandl, r. (eds.), management strategies to adapt alpine space forests to climate change risks: 233-256, in tech. lagercrantz, u., ryman, n. 1990: genetic structure of norway spruce (picea abies), concordance of morphological and allozymic variation. evolution, 44: 38-53. matović, m., vujković, lj. 1994: nova forma smrče (picea abies /l./ karst.) i njene ekološko-morfološke i dekorativne karakteristike. savetovanje “zelenilo u urbanističkom razvoju grada beograda”, zbornik radova, udruženje inženjera beograda: 277 284. 46 biologica nyssana ● 13 (1) september 2022: 41-46 petrov & ocokoljić ● dendrometric analyses and determining biological characteristics of needles for the purpose of conservation and directed utilization of the spruce in southeastern serbia morgenstern, e. 1996: geographic variation in forest trees, genetic basis and application of knowledge in silviculture. ubc press. vancouver. 214 p. ocokoljić, m., ninić-todorović, j. 2003: priručnik iz dekorativne dendrologije. univerzitet u beogradu, šumarski fakultet. beograd. 169 p. powo the international plant names index and world checklist of selected plant families 2022. published on the internet at http://www.ipni.org and http://apps.kew.org/wcsp/ schmidt-vogt, h. 1974: das natürliche verbreitungsgebiet der fichte (picea abies (l.) karst.) in eurasien. allgemeine forstund jagdzeitung, 145: 185–197. skrøppa, t. 2003: euforgen technical guidelines for genetic conservation and use for norway spruce (picea abies). international plant genetic resources institute. rome. 6 p. microsoft word 0603_jaksic_momirovic biologica nyssana 1 (1-2) december 2010: 123-130 jakšić, p., momirović, m. contribution to understanding the origin… 123 original article ! contribution to understanding the origin and the genesis of the nišava riverside valley fauna predrag jakšić*, milan momirović university of niš, faculty of science and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia *e-mail: jaksic@pmf.ni.ac.rs abstract: jakšić, p., momirović, m.: contribution to understanding the origin and the genesis of the nišava riverside valley fauna. biologica nyssana, 1 (1-2), december 2010: 123-130. the fact that very early biologists registered an extraordinary faunistic richnes in the greater area of niš (nišava’s riverside) is pointed out. the conditions that contributed to this variety are analyzed. the importance of geological history of this region is explained, from the ancient lake phase trough the glacial epochs including the present state. the dilemma about lake terraces and the water level of the neogene lake system is discussed. a list of gorges, speleological objects, springs, mountain tops as habitats of endemic and relict species is given. all the endemic and relict species in these habitats are listed. key words: fauna origin, fauna genesis, nišava riverside, serbia introduction ! the area of the nišava river valley attracted the attention of geographers and biologists very early. as early as 1891 đ. j o v a n o v i ć wrote about the caves in the gorge and in 1909 p. j a n k o v i ć devoted a comprehensive monograph to the development nišavska valley. among botanists s. p e t r o v i ć researched the flora of this region on two occasions (in 1882 and 1885, respectively). at the very early stages of research of the ponišavlje fauna, both botanists and zoologists noticed the diversity and large numbers of endemic and relic species. thus, for example, p a v l o v i ć (1912) in his monograph on the land snails in serbia pointed out the presence of numerous endemic species, and based on this data he produced one of the first zoogeographic maps in serbia, where the nišava valley was tagged as an area in which tertiary relics existed. subsequent studies of many botanists and zoologists had this view only updated and confirmed. the reason for this abundance is explained using geological and tectonic history of the area, its central position in the balkan peninsula and the topographic disposition of the terrain. from the valley of niš, through the surrounding hills and cliffs of numerous rivers, the suva planina mt., the vidlič mt. and other mountains of the wider area and geological complexity orographical areas offer good macroclimatic and microclimate conditions, and the significant complexity of habitat necessary for the existence of various elements of the flora and the fauna. besides the gorges, which serve as an excellent refuge, there are also wells and springs, numerous caves, forest litter (bedding) and xerothermal habitats on the tops of the surrounding mountains. the central position of this area in the balkan peninsula, in turn, allowed the presence of various elements of the fauna: central-european, pannonian, dacian, pontic, east asian, etc. 10th sfses • 17-20 june 2010, vlasina lake1 (1-2) • december 2010: 123-130 biologica nyssana 1 (1-2) december 2010: 123-130 jakšić, p., momirović, m. contribution to understanding the origin… 124 material and methods the area considered includes the headwaters of the svrljig timok, the reka river valley, the nišava valley from bela palanka to niš (gorge), the corresponding slopes of the svrljiška and suva planina mt., the jelašnica and prvokutinska gorges, caves and springs in the area. in the basin of svrljig timok, taken into consideration are okapina 1, okapina 2, tor, bezimena okapina, great okapina, small okapina, okapina at ostenjak, the church ruins okapina 1, the church ruins okapina 2, the church ruins okapina 3, the ripaljka cave, tunnel cave; in nišava basin, the objects considered are the okapina on the jecava, the eagle gap, mečija duvka, celenkina duvka, golema duvka, ladarova duvka, goveda duvka, great okapina 1, great okapina 2, okapina near the tunnel , small okapina, popova duvka (savnjak) paramanska okapina, ogorelička cave, the cave at zavrte, jovina duvka, great balanica, small balanica, popova duvka (brljevski stone), svinjarska duvka, radonjino konopljište, tunnel cave. wells and springs taken into consideration include bojanine vode and vrelo on the suva planina mt. finally, mountain peaks of xerothermic character taken into consideration are ramnište 951m, crni vrh 1164 m, mečji vrh 1184 m and golemi vrh 1535 m, all on the suva planina mt. on this geographic terrain the presence of tertiary relict species and postglacial xerothermic steppe species is analyzed. in this work the method of causal induction method was used (js mill) in which the conclusion is based on the search for causes. this method has many variations, and we applied the mode of agreement and difference, a method of reasoning where cause and effect correlate. geological-tectonic history and the history of climate change in the area are observed as a cause and the abundance of the species as a result. results representatives of nišava valley flora and fauna of preglacial age and xerothermic relics a) representatives of the flora. among tertiary relics we can cite the following species: juglans regia, syringa vulgaris, corylus colurna, acer intermedium, staphylea pinnata, daphne laureola, dictamnus albus, edraianthus serbicus and, especially, endemorelict species micromeria cristata, parietaria serbica, ramonda serbica and ramonda nathaliae. from the period of the relatively warm and dry interglacial (xerothermal) remained the steppe relics: adonis vernalis, prunus fruticosa, prunus tenela, asparagus tenuifolius, sternbergia colchiciflora, hyacinthella leucophea, ranunculus illyricus, cachrys alpina et al. (l a z a r e v i ć et al., 2007). b) representatives of the fauna. a survey of the fauna is limited to two groups of invertebrates: mollusca and arthropoda. significant contribution to the knowledge of mollusca in this area was given by p a v l o v i c (1912). the author cites the following relict and endemic species in the gorge: cabinet annularis stud, hyaline nitens michaud., was also found near the village prekonoge, zonitoides nitidus müll., patula solaria mke., eulota fruticum müll., vallonia pulchella müll., fruticicola ( monache) incarnata müll., campylaea trizon zgl., carthusian carthusian müll., buliminus (zebra) detritus müll., buliminus (ena) obscura müll., chondrula tridens müll., chondrula (mastus) venerabilis ziege., caecilianella acicula müll., orcula doliolum brug, pupa frumentum drp., modicella avenacea brug., pupilla muscorum l., isthmia minutissima hartm., isthmia salurensis reinhardt, clausiliastra laminate mont., alinda plicata drp., alinda biplicata mont., herilla frauenfeldi zelebor, carinigera eximia mollendo . the last one listed is an endemic species growing on the bare and sun-exposed limestone and found in bela palanka at vrelo in the gorge on the road village mokro, above the modro spring, near the village of krupac and also in pleš on nišavska side. finally, there are also types of carychium minimum müll., in both sićevo and jelašnička gorge acme serbica clessin was found, and ericia costulata (zgl.) rossmore. species uncinaria petrovici pavlović was found on vidlič above visočke ržane. species kuzmićia pygmaea mollendo. is distributed only in southern and southeastern serbia, in the analyzed area it was found on a suva planina mt., in the jelašnička and sićevo gorges, on pleš mt. and the village of prekonoge. different groups of arthropod fauna have also been the subject of intensive research by many authors. thus p l j a k i ć (1977) in this area identified the presence of many primitive species of lower crustaceans from oniscoidea: hyloniscus riparius (cl koch, 1838) in the sićevo gorge, hyloniscus transsylvanicus verhoeff, 1901 in the spring at ostrovica, hyloniscus kopaonicensis buturović, 1960 in the upper and lower dušnik, trichoniscus naissensis pljakić, 1977 in the three biologica nyssana 1 (1-2) december 2010: 123-130 jakšić, p., momirović, m. contribution to understanding the origin… 125 caves (donja cave in refuge, great belanica and kojina cave) and trichoniscus bononensis sotirova pljakić, 1977 in the cave kravljansko pester. the summation of known diplopoda in this area was given by makarov et al. (2004). the authors cite several cavernlike species: dorypetalum degenerans (latzel, 1884) and megaphyllum austriacum (latzel, 1884) from a cave in the barski reed, svarogosoma bozidarcurcici makarov, 2003 from a cave in sava’s fall (trem), nopoiulius kochii (gervais, 1847) from the great belanica and typhloiulus (typhloiulus) nevo makarov, mitić & curcio, 2002 from the great cave. the opilionida identified are paranemastoma (buresiolla) bureschi (roewer, 1926) and cyphophthalmus serbicus (hadži, 1973) in cerjanska caves. in the pseudoscorpiones group ć u r č i ć et al. (2004) found the species roncus remesianensis ćurčić, 1981, roncus timacensis ćurčić, 1981 and roncus jelasnicae ćurčić and dimitrijevic, 2009. the recognized species of spiders (araneae) include pholcomma gibbum (westring, 1851), steatoda triangulus (walckenaer, 1802) and lepthyphantes leprosus (ohlert, 1865) in košuća cave (kravlje), lepthyphantes trnovensis (drensky, 1931) in the cave near niš and mettelina merianae (scopoli, 1763) in jelašnička cave. among ticks (ixodidae), the species found include eschatocephalus vespertilionis cl koch, 1844 at ogorelačkoj cave. the presence of numerous representatives of insecta has been determined. the representatives of the species staphylinidae are bisnius cephalotes (gravenhorst, 1802) and aleochara (xenochara) funebris wollaston, 1864 from the ogorelačka cave, and the species quedius (microsaurus) mesomelinus skoraszewskyi korge, 1961 and atheta (alaobia) caving erichson, 1839 from both belanica and the ogorelačka cave. the representatives of the beetles (carabidae) are the species duvalius (paraduvalius) winkler (jeannel, 1923) from the ogorelačka cave, and straight prekonoga oven and laemostenus (pristonychus) terricola punctatus (dejean, 1828) from the ogorelačka cave, and the species trechus (trechus) irenis csik, 1912 from the cerjanska cave. finally, the orthoptera (raphidophoridae) species observed are troglophilus neglectus vlasinensis maran, 1958 in the belanica and ogorelačka cave. the ogorelačka cave has long been known for existence of the cave bear (d j o r d j e v i ć , 1891). j o v a n o v i ć (1891) established its presence in the prekonoška cave. herak (1947) proved that the cave bear in the balkan peninsula is not indigenous but has in fact come from the region of the alps at a later date diluvium. discussion from the description it is evident that the presence of the glacial elements in the analyzed region is reduced to a minimum. in association with the pine (sorbet-mughetum jn.) on the steep, cold northern slopes of the highest part of the suva planina mt. at about 1400 m above sea level, all the way to the highest peaks, some of the glacial relics can be found, such as eg. dryas octopetala. this means that the glaciation in this region has significantly disrupted the starting wildlife. if the climatic factor was not crucial, then it can be assumed that the proper interpretation of the geological history is the key to understanding the origin and genesis of the recent flora and fauna in the nišava riverside valley. in the geological-tectonic history of the area the most important event was the lake stage. from the point of view of modern science, it is surprising that as early as 1909, 100 years ago, j a n k o v i ć published an extensive study of the history of the development of the nišavska valley. the author of this work claims: "the niška valley is a drained pool of a large neogen lake, which existed probably until the end of the pliocene." in the same paragraph, the author presents another observation: "the niška valley with its configuration and its main plastic lines independent of the nišava valley and its actions, because the river formed only after the runoff neogen lake"; also extremely important is that we note that the author of this work gives the original drawings of the land and sea during the three phases of the neogen. the suva planina mountain, the pleš, the vidlič mt. and other ranges are presented in these maps as islands. j a n k o v i ć in summarizing his results highlighted the existence of two systems of terraces: the high abrasion (lake) and a system of low (fluvial, river) terraces and that by them we can see that the great lakes declined successively and in breaks. the assessed absolute levels and relative levels of lake shores in stages are also determined. c v i j i ć (1913) in his study of the rtanj mountain pays attention to rtanj lake plasticity. cvijić found the presence of lake terraces at 940-830 m, then at 680-670 m, and at the level of 600 m, he also links these terraces to pont. writing about the prvokutinska gorge, which was cut by kutina, a left tributary of nišava, m a r t i n o v i ć (1976) linked its genesis with the lake stage, the end of the miocene and stated that the area of today's gorge was the lake narrow of the niš lake basin. by analyzing the formation of dolomite in neogen lake sediments in the niš area of the morava riverside, n i k o l i ć & j a n j i ć (1986) came to the conclusion that in certain phases of the existence of biologica nyssana 1 (1-2) december 2010: 123-130 jakšić, p., momirović, m. contribution to understanding the origin… 126 the lake there occurred the salinisation of the water environment, an increased salinity, higher temperatures and evaporation, causing a deposit of dolomite. by analyzing the neogen series of the litostratigraphic column, the authors established the first cycle of sedimentation in the lower miocene, the second cycle of sedimentation taking place during the middle miocene, the third cycle of sedimentation proceeding during the mio-pliocene, and after that the last phase was implemented in the genesis of neogen sedimentation. a similar view was proposed by a n d j e l k o v i ć et al. (1991), who pointed out that during the pontian the marine environment had been reduced to remnants of paratethys in the pannonian and dacian areas, while in other parts of the former sea came to a loss of salinity and the formation of wetlands covered by dense vegetation taxodium forests. during the dacian era, the complete withdrawal of paratethys from serbia occurred, this territory, therefore, existing as a land mass from 12 (12.5) million years ago until the present day. m a r t i n o v i ć (1968) compiled a very detailed survey of the genesis of the sićevo gorge. he singled out six lake levels, i.e. six lake terrace: the highest abrasive track at the level of 1020 1000 m; the second terrace is at 860 m, the third terrace at 830-820 m; the next level of 760 750 m has been developed in the sićevo gorge and on the outskirts of the niš valley; the fifth level is a distinct riparian terraces in the gorge at a height of 720-700; the last level was at an altitude of 680-660 m; this level is also present along the perimeter of the niš valley (where it is represented at two levels due to intense tectonics of the terrain). martinović says that up to a height of 720 m terrace is of pannonian age (upper miocene, about 12-13 million years), lower than 460 m of pontus (pliocene, about 11 to 11.5 million years) and below this level are the diluvial ones; the last lowest terrace is recent. the pontic era climate in serbia is characterized by relatively high temperature and high humidity, as evidenced by the sediments and deposits of tacsodium. during the dacian era, the climate did not significantly change, which is proven by the process of paleocrustification (a n d j e l k o v i ć et al., 1991). in the debate about the level of neogen lakes in the south of serbia, m i l i ć (1982) showed that there was no communication between pannonian lake and aegean lake through grdelica gorge or other possible locations. but the author also pointed out that such communication was possible in the upper miocene and that the link extended west of the mount kukavica to the preševo-kumanovo basin. the author specifieds that this connection had existed during the pannonian. however, the views put forward by j a n k o v i ć (1909), c v i j i ć (1913) and others are opposed by the contemporary authors. thus, p e t r o v i ć d. (1988) critically examined cvijić’s understanding that during the upper and lower pliocene there existed a unique aegean lake that cut high terraces of 760-740 and 670 m. the shallow character of pont sediments, as encountered in the nišava riverside valley, and their low height exclude the possibility that the pontic water in general could reach the height of the coastal line of 940 m and above, and cut the surface at a height of 850 m. the existence of epigenetic gorges excludes the possibility that under the height of their upper edges surface abrasions could be carved, because the central lake level had to be above them. if we take the contour line of 857 m as a reference point selected as the middle of the three phases that janković presented on his maps [which is approximately equal to the highest correlated level terrace of 873 meters of the current sea levels, according to n i k o l i ć (1991)], then we come to the conclusion that the islands could not be used to maintain tertiary elements of flora and fauna (fig. 1). fig. 1. a reconstruction of the land and sea on the contour line of 857 m, as understood by janković (1909). the legend: 1 sea, 2 land, 3 river network, 4 caves analyzed in this paper, svrp the svrljiške planine mt., sp the suva planina mt.. such an image completely excludes the existence of gorges, caves and springs, all modern caves (represented by circles in the drawing) would in that time have been submerged. the abrasion and fluvial terraces seen in the field are real; their biologica nyssana 1 (1-2) december 2010: 123-130 jakšić, p., momirović, m. contribution to understanding the origin… 127 existence is not doubted by anyone. on the given maps of land and sea during the sarmat, pannonian and pontian, a n d j e l k o v i ć et al. (1991) specified the lines of the lake in that period, showing its successive reduction. these maps match well with the maps of jovanović for the period of pannon. however, substantial differences appear in the level andjelkovic et al. emphasize that this is a shallow lake, unlike jovanović’s view. finally, andjelkovic et al. pointed out that during the pontian there occurred a positive epirogenesis, an upheaval of the land that led to the withdrawal of the sea and the transition of plain lands to hill lands. now, we can set a theory that in this way the terraces were successively raised to the level seen today or that the level of the sea was more or less constant, and that the marking of terraces went successively, and that, due to positive epirogenesis after forming, they would be raised to this level. n i k o l i ć (2007), in a study of abrasive terraces, definitely confirmed this view. the author provided a precise series of successive levels of abrasion terrace for serbia (especially in the catchment area of south morava); according to the data, the altitude of the abrasion terrace showed these values: 60, 75, 100, 120, 140, 175, 200, 220, 240, 260 and 280 m. in the same article the author presented the data on a curve of eustatic fluctuations of the mediterranean sea in the pleistocene, based on which it is shown that the sea level used to be between + 280 in the gűnz 1 period gűnz 2 (560 thousand years ago), over 120 in wűrm 3 (about 30 thousand years), to the present level of 0 m. caves and species found in them can be the key to solving the dilemma presented here. according to the hypothesis of j a n k o v i ć (1909), as shown in figure 1, all existing caves during the neogen and until the end of the pliocene were submerged; it is unlikely that they existed at all. therefore, it follows that they were developed only after the runoff of the neogen lake, their maximum age being 11.5 million years. in speleology it is a common knowledge that in the dinarides and carpatho-balkanides there are old cave systems of preglacial age. most caves of the alpine arc are of glacial age, some of them are only 40,000 to 50,000 years old. there is little data about caves in eastern serbia. g a v r i l o v i ć (1967) described the genesis of the vlaška cave in the resava spring. the cave was formed by water, its formation probably began in early pleistocene and the formation of main channels in late pleistocene. a similar view was expressed by p e t r o v i ć (1977) in discussion about the genesis of the bogovinska cave. the author concluded that the formation of the bogovinska cave is related to the first half of the pleistocene, it being the oldest stage in the formation of this cave. we also have reliable data for the zlotska cave. the underground stream had built the channels of the zlotska cave during the late pleistocene and the formation was completed in holocene (brown, 1957/58). in the relative proximity of the area that was analyzed in this paper is the seselačka pećura in soko banja. p e t r o v i ć (1984) concluded that it was a younger object, formed at the turn of pleistocene to holocene. bearing in mind the relative closeness of these caves with other speleological objects in the nišava valley, as well as an exact geological-tectonic history and identical climatic conditions, we can conclude that the caves in the nišava valley are also of pleistocene age. this viewpoint can serve as an explanation for significant differences in cavern-like fauna of cave systems in eastern serbia (which are relatively poor in the fauna) and cave systems of dinarides (who hold one of the richest fauna in the world). cave systems in eastern serbia were formed at the end of the glaciation, when most termophile species of the tertiary age had already extinct. outside the context of this paper we present an interesting observation. specifically, the caves of eastern serbia have developed as part of two carbonate platforms: “kučajsko-tupižnička carbonate platform” and “miročka carbonate platform”. both platforms were developed in the mesozoic, in the upper jurassic and lower cretaceous (about 150 million years ago). "overlaying" the map of distribution of carbonate rocks in serbia with the map of neogen basins in serbia shows that limestone areas were not flooded by neogen waters they existed as dry land (figure 2). so the question is: why are there no old cave systems to them? one possible explanation is that at the time the position of these masses was much closer to the equator, and in such circumstances, in the absence of cold waters the forming of caverns was difficult. r i b e r a et al. (2010) analyzed the age of 57 species of cave-dwelling beetles from leptodirini (leiodidae, cholevinae). the authors concluded that the colonization of cavern like habitat in the pyrenees had begun shortly after their formation in the early oligocene. so, today's abundance of species from this group is understandable having in mind that their evolution took 35 million years. the authors also concluded autochthony of this group; it has evolved in the same area where its ancestors used to be. the pyrenees are represented by another tertiary relic ramonda myconi (fam. biologica nyssana 1 (1-2) december 2010: 123-130 jakšić, p., momirović, m. contribution to understanding the origin … 128 fig. 2. mutual spatial relationship – right, between the neogene basins of serbia (j a n k o v i c , 1990) left, and the range of karst (g a v r i l o v i c , 1976) in the middle. gesneriaeceae), this species makes it possible to make a complete parallel with the area that we have analyzed in this paper. as the species mentioned in the pyrenees, our ramonda serbica and ramonda nathaliae are tertiary relicts. but while in the caves of the pyrenees there are 57 analyzed species from leptodirini, to this time there are no species of this group in analyzed caves of nišava valley. by this means, the opinions of g a v r i l o v i ć (1967) and p e t r o v i ć (1977) are confirmed about the pleistocenic age of caves in eastern serbia. the known leptodirini are leiodidae from eastern serbia, the representatives of the endogean and not of the cavern-like fauna. (c u r č i ć et al., 2009). why did this group fail at adaptive radiation of cavern-like habitats? probably because the continuous temperature stability of the region and the presence of optimum moisture content in litter and endogean conditions allowed a comfortable existence. the area of deciduous forests, which dominate the carpatho-balkan mountains of eastern serbia, is characterized by the presence of a powerful layer of organo-mineral complexes; that is why the representatives of the endogean fauna did not have an ecophysiological need to retreat to cavern-like habitats. the areas affected by the influence of the pleistocene climate, but which were not covered in ice, have a well-preserved terrestrial fauna. we conclude that the process of formation of cavern-like fauna in the carpatho-balkanides started, but has not yet been completed. but, on the other hand, cavern-like leptodirini of the pyrenees have their analogues in eastern serbia. an analogy is found in the group pseudoscorpiones according to c u r č i ć et al. (2004) out of 25 species from this group distributed in eastern serbia, 20 of them live in caves, while only 5 species have been found outside the cave. conclusion the fauna of the nišava riverside valley is represented by species from different geochronologycal epochs: the species of tertiary age as the oldest (represented by thermophile species), the species of the pleistocene age (represented by glacial species) and species of the holocene age, of postglacial character, as the youngest (represented by xerothermic relicts). the opinion of j a n k o v i ć (1909) on the relationship between the land and the sea during the neogen must be taken with caution. the opinion of p e t r o v i ć (1988) is more likely, that in this area a shallow sea existed and the surrounding terrain upheaved successively. the current lake terraces explain the stages of ground upheaval, rather than the stages of the sea outflow. based on cavern-like fauna composition, and based on analogy of genesis of some caves in eastern serbia, it is certain that the cavern-like objects in the nišava riverside valley are of pleistocene age. the forming process of the cavern-like fauna has begun; in some groups it has advanced far (pseudoscorpiones), in some groups it is still in its infancy (leptodirini, leiodidae), but it certainly has not yet reached the scale of what we encounter in the dinarides. on the other hand, the species of tertiary age are abundantly represented in the gorges, springs and wells, especially in forest litter, in damp and shady habitats. also, they can be found in high mountain habitats of oromediteranic thermophilic character. biologica nyssana 1 (1-2) december 2010: 123-130 jakšić, p., momirović, m. contribution to understanding the origin… 129 references cvijić, j., 1913. rtanj. glasnik geografskog društva, 2: 279-298, beograd. ćurčić, b.p.m. dimitrijević, n.r., legakis, a., 2004. the pseudoscorpions of serbia, montenegro and the republic of macedonia. belgrade-athens. ćurčić, s.b., brajković, m.m., ćurčić, b.p.m., 2007. the carabids of serbia. belgrade – vienna. ćurčić, s.b., makarov, s.e. and ćurčić, b.p.m., 2009. a revision of magdelainella jeannel, with a description of m. studenicae sp. n. (coleoptera, leiodidae, leptodirini), a new endogean beetle from serbia. archives of biological sciences, 61(4): 757-765, belgrade. deltshev, c. c., 1996. a faunistic and zoogeographical review of the spiders (araneae) of the balkan peninsula. the journal of arachnology, washington, hors series (1): 141151. deltshev, c. c., ćurčić, b. p. m., blagoev, g. a., 2003. the spiders of serbia. belgrade – sofia. drensky, p., 1936. katalog der echten spinnen (araneae) der balkanhalbinsel. sbornik na bulgarskata akademiya na naukite, sofia, 32(15): 3p-4p. đorđević, z. i vasiljević, b., 1993-94. zaštita sićevačke klisure – vrednosti, problemi i koncept zaštite. [protection of sićevačka klisura (the sicevo gorge), values, problems and protection approach]. zaštita prirode, 46-47: 221-244, beograd. đurović p., 1998. spelološki atlas srbije, geografski institut jovan cvijić sanu, zavod za zaštitu prirode srbije, geografski fakultet univerziteta u beogradu, posebna izdanja geografskog instituta jovan cvijić, knjiga 52, beograd. gavrilović, d., 1967. spuštanje podzemnog toka u krasu na primeru vlaške pećine. (über die senkung der unteriridischen wasserläufe im karst am beispiel der höhle vlaška pećina) zbornik radova pmf, geografski zavod, xiv: 17-27, beograd. gavrilović d., gavrilović lj., 1998. kras stare planine, zbornik radova geografskog fakulteta univerziteta u beogradu, 48, str. 5-25, beograd. grebenščikov, o., 1950. o vegetaciji sićevačke klisure. (la végétation de la gorge de sićevo.) glasnik prirodnjačkog muzeja srpske zemlje, b 3-4: 175-191, beograd. grimm, u., 1985. die gnaphosidae mitteleuropas (arachnida, araneae). abbhandlungen des naturwissenschaftlichen vereines in hamburg, hamburg, 26: 1-318. herak, m., 1947. starost i sistematske značajke spiljskog medvjeda hrvatske. (geological age and taxonomical characters of the cave-bear of croatia) geološki vjesnik geološko-rudarskog instituta ministarstva industrije i rudarstva u zagrebu, i: 12-47+tab. i-v, zagreb. janković, m.m., stevanović, v., 1981. prilog poznavanju fitocenoza sa srpskom ramondijom (ramonda serbica panč.) u klisurama severnih ogranaka šar-planine. ekologija, 16(1): 1-34, beograd. janković, p., 1909. istorijski razvitak nišavske doline. ska, beograd. jovanović, đ., 1891. fauna prekonoške pećine. geološki anali balkanskog poluostrva, iii: 300317, beograd. jovanović, đ., 1891. sićevačka klisura, pećine, dupke i potkapine. otadžbina, 10(29): 401-419. karaman, i., 2008. cyphophthalmi of serbia (arachnida, opiliones). in: pavićević, d. & perreau, m. (eds.): advances in the studies of the fauna of the balkan peninsula. institute for nature conservation of serbia. pp.: 97118.beograd. kolosváry, g., 1938. sulla fauna arachnologica della jugoslavia. rassegna faunistica, roma, 16(3-4): 3-23. kostić, m., 1955. o ulozi i značaju sićevačke klisure za saobraćaj, naseobine i ljudska kretanja. geografski zavod prirodno-matematičkog fakulteta univerziteta u beogradu, sv. ii. beograd. kostić, m. i martinović, ž., 1967. ostrovičke terme – prilog proučavanju termalnih izvora srbije. zbornik radova geografskog instituta «jovan cvijić», knj. 21. beograd. lazarević, p., mitrović, v. i zlatković, b., 2007. flora and vegetation of the sićevo and jelašnica gorge. in: trajković. s. and branković, s. (ed.) sićevo and jelašnica gorge. environment status monitoring. zavod za zaštitu prirode srbije i građevinsko-arhitektonski fakultet u nišu. beograd. makarov, e. s., ćurčić, p.m. b., tomić, t.v., legakis, a., 2004. the diplopods of serbia, montenegro, and the republic of macedonia. belgrade – athens. martinović ž., 1968. sićevačka klisura, zbornik filozofskog fakulteta u prištini, priština. martinović, ž., 1973. hidrogeološke odlike severnog dela niške kotline [hydrogeologic characteristics of the northern part of the niš ravine (depresion, basin)]. zbornik radova biologica nyssana 1 (1-2) december 2010: 123-130 jakšić, p., momirović, m. contribution to understanding the origin… 130 prirodno-matematičkog fakulteta u prištini, 1: 133-155, prtiština. martinović, ž., 1976. prvokutinska klisura (defile de prva kutina). zbornik radova prirodnomatematičkog fakulteta u prištini, c 4: 113-138, priština. mihailović, d., 2004. istraživanja pećinskih arheoloških nalazišta u slivu timoka i nišave. zbornik radova odbora za kras i speleologiju sanu, viii: 135-144, beograd. mihailović, d., 2008. istraživanja paleolita na području niške kotline između 2004. i 2006.godine. zbornik radova odbora za kras i speleologiju sanu, ix: 105-118, beograd. milić, č., 1959. prilog poznavanju geneze crvenice na suvoj planini. zbornik radova san, lxvii, geografski institut, knj. 16: 89-104. beograd. milić, č., 1982. cvijićeve hipoteze o postanku i evoluciji moreuza na balkanskom poluostrvu. (hypotheses de cvijić sur l'origine et l'evolution des detroits dans la peninsule balkanique). in: naučno delo jovana cvijića, sanu, pp.: 95-109, beograd. mišić, v., 1970. reliktna šumska vegetacija sićevačke klisure. arhiv bioloških nauka, 22(14): 1p-2p, beograd. mišić, v., 1981. šumska vegetacija klisura i kanjona istočne srbije. institut za biološka istraživanja “siniša stanković”, beograd. mitić, d. i stanojević, m., 2008. šume srednjeg ponišavlja. centar za naučna istraživanja univerziteta u nišu i sanu. niš. mladenović, t., 1973. neka izdašnija kraška vrela u slivu nišave. glasnik srpskog geografskog društva. liii(2): 51-59. beograd. nešić, d., pavićević, d., belij, s., 2005. results of the complex speleological research of northwest part of the svrljiške mountains. in: stevanović z. and milanović p. (eds.): water resources and environmental problems in karst. pp.: 795-800. belgrade. nešić, d., 2007. results of speleological investigations of the sićevo and jelašnica gorges. in: trajković s. and branković s. (eds.) sićevo and jelašnica gorges environment status monitoring. institute for nature conservation of serbia. belgrade. nikolić, p., janjić, s., 1986. dolomiti u jezerskim neogenim naslagama niškog dela pomoravlja. xi kongres geologa jugoslavije, mineralne sirovine, knjiga 4: 341-355, tara. nikolić, s., 1991. terase i neotektonika u nekim oblastima srbije i mediterana. geološki anali balkanskog poluostrva, 55(2): 75-90, beograd. nikolić, v. i diklić, n., 1966. zajednica žalfije i rudinskog pelina – artemisio-salvietum officinalis (salvia officinalis – artemisia lobelii grebenschikov 1950) u sićevačkoj klisuri. glasnik prirodnjačkog muzeja u beogradu, b 21: 5-21, beograd. pavićević, d., zatezalo, a., popović, m., 2007. fauna of sićevo gorge caves arthropods. in: trajković s. and branković s. (eds.) sićevo and jelašnica gorges environment status monitoring. institute for nature conservation of serbia. belgrade. pećinar, m., 1961. hidrologija termalnih vrela niške banje i njihova zaštita od rashlađivanja i mućenja. glas ccxvii sanu, vol. 5: 35-66, beograd. petković, k., 1930. geološki sastav i tektonski sklop suve planine. ska, posebna izdanja, knjiga lxxvi: 1-136, beograd. petrović, d., 1977. morfologija i geneza bogovinske pećine. zbornik radova geografskog instituta „jovan cvijić“, 29: 92-102, beograd. petrović, d., 1988. problem jezerskih površi na obodu panonskog i vlaško-pontiskog basena u srbiji. zbornik radova geografskog instituta „jovan cvijić“, 40: 69-78, beograd. petrović, j., 1951. o epigenetskoj sutesci ostrovičke reke. glasnik srpskog geografskog društva, xxxi(1), beograd. petrović j., 1976. jame i pećine srbije, vojnoizdavački zavod, beograd. pljakić, m., 1977. taksonomsko-biogeografski odnosi primitivnih evolutivnih serija nižih oniscoidea jugoslavije posebno elemenata kavernikole faune srbije. sanu, posebna izdanja, knjiga div. beograd. ribera, j., fresneda, j., bucur, r., izquierdo, a., vogler, a., salgado j. and cieslak, a., 2010. ancient origin of a western mediterranean radiation of subterranean beetles. evolutionary biology, 10: 29-42. stojićević, d., 1929. pravi pauci u srbiji. araneae sund. muzej srpske zemlje, posebno izdanje, 19, beograd. pp. 1-65. zlatković, b., 1999. flora sićevačke klisure. (the flora of sićevo gorge). diplomski rad, pp. 1127. univerzitet u novom sadu, pmf, institut za biologiju. novi sad. (manuscript). zlatković, b. i ranđelović, v., 1993-94. ugroženost i zaštita flore sićevačke klisure. [endangerment and protection of flora of sićevačka klisura (the sicevo gorge)]. zaštita prirode, 46-47: 191-199, beograd. microsoft word bn020205 djurdjevic biologica nyssana 2 (2) december 2011: 129-130 đrđević a., đurić m. blepisanis vittipennis (reiche 1877) (coleoptera:… 129 short communication ! blepisanis vittipennis (reiche 1877) (coleoptera: cerambycidae), a new longhorn beetle for serbia aca đurđević1, milan đurić2 1university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia 2habiprot, belgrade, serbia * e-mail: djukiamphibia@gmail.com abstract: đurđević, a., đurić, m.: blepisanis vittipennis (reiche 1877) (coleoptera: cerambycidae), a new longhorn beetle for serbia, biologica nyssana, 2 (2), december 2011: 129-130. a longhorn beetle species, blepisanis vittipennis, known so far in europe but only from greece and bulgaria, was recorded for the first time in the fauna of serbia, increasing number of recorded longhorn beetle species to 238. key words: blepisanis vittipennis, cerambycidae, preševo, serbia. blepisanis vittipennis (r e i c h e , 1877) is one of two representatives of the genus blepisanis in europe. the other species, b. melanocephala (f a b r i c i u s , 1787) inhabits only island of sicily, while b. vittipennis is distributed in se balkan peninsula, caucasus, turkey and near east (h o s k o v e c & r e j z e k , 1991-2011). in the balkan peninsula, the species is reported as common in continental greece and bulgaria (s a m a , 2002), as well as in the near east turkey (ö z d i k m e n & t u r g u t , 2008). as a part of first systematic entomological research in serbia, on june 4 2011, a group of researchers visited poorly investigated region in southeast of serbia, miratovac village in vicinity of preševo. the most interesting habitats were found to the east of the village, at an altitude of 551 m, situated at 42°16΄20˝ n, 21°39΄1.6˝ e. the location is an area with a strong submediterranean influence, probably felt more than anywhere else in serbia. the biotope comprises mostly of fragmented quercus pubescens forest and dry, rocky pastures, despite the fact that wider area is predominantly used for crop planting. during that survey, one specimen of blepisanis vittipennis was found on dianthus sp. the series of photographs was taken, but since authors did not recognize species and significance of the finding, specimen was not collected for further analysis (fig. 1). good photographs enabled dr. nastas ilić to determine species and emphasize importance of this finding. first systematic review of longhorn beetles in serbia (i l i ć , 2005) listed a total of 245 taxa of this family, but some were marked with a question mark due to unreliable findings, some listed as species at the time were given status of subspecies. therefore, having in mind the most widely used taxonomy (fauna europaea, 2011), list of longhorn beetles recorded in serbia comprised 237 species (habiprot, 2005-2011), and with this finding comes up to 238. the finding of blepisanis vittipennis in serbia is significant because it increases the species areal towards the west, and vicinity of the location to macedonia implies that presence of the species in that country is probable. 2 (2) • december 2011: 129-130 biologica nyssana 2 (2) december 2011: 129-130 đrđević a., đurić m. blepisanis vittipennis (reiche 1877) (coleoptera:… 130 figure 1. blepisanis vittipennis near miratovac village. photo: milan đurić acknowledgements. the authors want to express their gratitude to dr. nastas ilić for unselfish support and sharing of his immense knowledge on longhorn beetles, and to bojan zlatković for plant determination, and also, to miloš popović and ivan simonović for their relentless support during field surveys. references fauna europaea, 2011. group coordinator audisio, p. fauna europaea version 2.4, web service available online at http://www.faunaeur.org habiprot, 2005-2011: alciphron – a database on insects of serbia. habiprot. belgrade. hoskovec, m. & rejzek, m. 1999-2011. cerambycidae, web service available online at http://www.cerambyx.uochb.cz/ ilić, n. 2005. strižibube srbije (coleoptera; cerambycidae) faunistički pregled, autorsko izdanje, beograd, 180pp. özdikmen, h. and turgut, s. 2008. new species and a new subspecies of the subgenus phytoecia (blepisanis) pascoe, 1866 from turkey (coleoptera: cerambycidae: lamiinae). mun. ent. zool. (3) 2: 568-581. sama, g.., 2002. atlas of the cerambycidae of europe and the mediterranean area vol. 1, nakladatelství kabourek, zlín (czech republic), 173 pp. djebbouri & terras 2022, biologica nyssana 13(1) 13 (1) september 2022: 1-9 doi: 10.5281/zenodo.7117398 community structure with particular reference to the effect of grazing in forest formations of saïda (algeria) original article mohammed djebbouri laboratory of water resources and environment, department of biology, faculty of science, university of saïda dr. tahar moulay, saida, 20000, algeria conservation of forests in the wilaya of saïda general directorate of forestry (gdf), algeria mohammeddjebbouri@yahoo.com (corresponding author) mohamed terras laboratory of water resources and environment, department of biology, faculty of science, university of saïda dr. tahar moulay, saida, 20000, algeria received: august 16, 2021 revised: may 04, 2022 accepted: may 24, 2022 abstract: to better understand and manage the forest vegetation of the saïda district (types of vegetation present, floristic compositions, possible types of degradation), a total of 215 phytoecological surveys were carried out in the study area. in each survey, the intensity of the grazing and the presence or absence of regeneration of the tree layer were noted. the plant groups were determined using a modified twinspan classification. for each vegetation group, the diagnostic species were selected based on their fidelity index (phi coefficient). we identified 9 plant groups and selected 147 diagnostic species. the results of this study showed that in the surveys carried out, there were more than 80% of moderately to heavily grazed, while in more than 70% of the studied plots, there was no regeneration of the main tree species. this alarming situation implies the rapid and effective implementation of protection and conservation measures. key words: structure, forest formations, grazing, saïda, algeria apstrakt: struktura zajednica sa posebnim osvrtom na efekat ispaše u šumskim formacijama saïda (alžir) kako bi se bolje razumelo i upravljalo šumskom vegetacijom okruga saïda (prisutni tipovi vegetacije, floristički sastav, mogući tipovi degradacije), u oblasti istraživanja prikupljeno je ukupno 215 fitocenoloških snimaka. za svaku sastojinu utvrđen je intenzitet ispaše i prisustvo ili odsustvo regeneracije sprata drveća. biljne grupe su utvrđene korišćenjem modifikovane twinspan klasifikacije. za svaku vegetacijsku grupu, dijagnostičke vrste su odabrane na osnovu vrednosti indeksa “vernosti” (phi koeficijenta). identifikovali smo 9 biljnih grupa i selektovali 147 dijagnostičkih vrsta. rezultati ove studije su pokazali da u više od 80% izvršenih istraživanja postoji prekomerna ili umerena ispaša, dok u više od 70% ispitivanih lokaliteta nema regeneracije glavnih vrsta drveća. ova alarmantna situacija zahteva brzu i efikasnu implementaciju mera zaštite i konzervacije. ključne reči: struktura, šumske formacije, ispaša, saïda, alžir introduction algeria is part of the mediterranean basin, which has been described as one of the 34 “hotspots” on the planet (myers et al., 2000). indeed, myers et al. (2000) consider that the mediterranean countries hold almost 4.5% of the world’s flora. in recent decades, a number of stakeholders have threatened important habitats and their associated plants. an estimate of the extinction rate of higher mediterranean plants is 0.15 percent of the total, representing 37 species presumed extinct, while 4,251 plant taxa are threatened (greuter, 1994). it is therefore urgent to improve vegetation management. the identification and description of plant groups, as well as their relationship to the environment, is a vital step in understanding arid environments (al harthy & grenyer, 2019). first, different plant communities are considered as substitutes for ecosystem delineation, and community definitions therefore help to understand the macro-ecological differences between biosphere components (al harthy & grenyer, 2019). indeed, using ecological species groups and the presence-absence or coverage of their indicator species can help with the development of management plans and the selection of preferred practices for the long-term management of ecosystems, such as monitoring environmental © 2022 djebbouri & terras. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 1 changes, assessing management effectiveness, and providing warning signals for impending ecological changes (barnes et al., 1982; siddig et al., 2016). effective conservation planning also includes identifying and describing plant communities (kent, 2011; brown et al., 2013). in algeria, knowledge on the structure of forest and preforest vegetation is still incomplete (meddour, 2012). in this context, the present study aims to improve our knowledge on the structure of forest and preforest groupings in the saïda region and to obtain an accurate inventory and a detailed diagnosis of existing plant groupings, possibly for mapping plant groups (elaboration of the typology). the final objective was always to better understand and manage natural areas (types of vegetation present, floristic compositions, conservation issues, possible types of degradation...). the results are intended for biodiversity specialists, managers of natural areas and/or naturalists. materials and methods study area the study area (forest formations and preforests), located in the district of saïda in the northwest of algeria with an area of about 174,361 hectares, between 35°6’23.68” and 34°35’9.14” latitudes north and 0°25’10.89” west at 0°47’28.58” longitudes east (fig. 1). vegetation sampling the inventory of flora is based on the 215 floristic surveys carried out through the forests area of the saïda region during the years 2017-2018-2019 2 biologica nyssana ● 13 (1) september 2022: 1-9 djebbouri & terras ● community structure with particular reference to the effect of grazing inforest formations of saïda (algeria) fig. 1. location of study area. projection system is utm zone 30-n, wgs84 3 between the periods of march to mid-june. the sampling plan used to analyze the vegetation is subjective. this is the simplest and most intuitive form of choosing as samples areas that appear particularly homogeneous and representative (gounot, 1969; terras, 2011). the identification of the inventoried plant species was carried out using the new flora of algeria and the southern desert regions (quézel & santa, 19611962). in this study, the synonymic index of north africa was used for the botanical nomenclature of species inventoried (dobignard & chatelain, 20102013). a total of 215 records were collected in the turboveg database (hennekens & schaminée, 2001). floristic classification and statistical analysis the phytosociological data were analyzed using the modified twinspan (two-way indicator species analysis) method (hill, 1979; roleček et al., 2009), integrated into the program juice 7.0 (tichý, 2002). the minimum group size has been set at 3 and the coverage thresholds have been set at 0%, 5% and 25%. the whittaker beta diversity index (whittaker, 1972) was used for the analysis of the heterogeneity of groups, because it provides balanced classifications, respecting the size and heterogeneity of the group, but also because of its robustness (roleček et al., 2009). to find an optimal cluster depth in the dendrogram produced by the modified twinspan algorithm, we used the optimclass procedure (tichý et al., 2010), which is also implemented in juice. optimclass was applied in mode 1. in naming plant groupings, we followed the informal approach of menz et al. (2012) and ik-gürsoy et al. (2016) meaning that the nomination of plant groups always includes the name of the most diagnostic species and the species with the highest coverage. grazing intensity to obtain an estimate of the grazing pressure in the study area, we used the following scale (medjahdi, 2001; medjahdi, 2010): 0 absent; 1 present but without causing damage other than on the herbaceous layer (very low); 2 fairly significant damage to the herbaceous layer and to the foliage of the bushy layer (low); 3 significant damage to the bushy layer (moderately); 4 the heavily degraded forest undergrowth (height); 5 almost total disappearance of the bushy layer (heavily). biologica nyssana ● 13 (1) september 2022: 1-9 djebbouri & terras ● community structure with particular reference to the effect of grazing inforest formations of saïda (algeria) calculation of diversity indices for each floristic survey the following indices were calculated using the juice 7.0 software (tichý, 2002): richness-index, shannon-index, eveness index and simpson-index. species richness (s), shannon-wiener diversity index (h′) and eveness index were calculated to describe the pattern of species diversity for each floristic record (i.e. alpha diversity α), as well as for each plant group identified by twinspan (that is to say the beta diversity β). the differences between the mean values of the diversity indices between the plant groups were evaluated using a nonparametric analysis of variance with the kruskal-wallis test and the multiple comparison of the mean ranks. the differences were considered significant if p<0.05. this analysis was performed using spss 24.0 (statistical package for the social sciences). to study species diversity and its relationship to the variables measured in this study, the data collected was examined using the pearson correlation coefficient, which aims to draw a brief conclusion about the factors likely to affect species diversity. stratification and recouvrement to bring out the main lines of the vertical structure of the vegetation, only three strata were retained: arborescent, shrub and herbaceous. these three strata are identified according to the scale: tree stratum: (3 meters and more), shrub stratum (between 0.50 m and 3 m), herbaceous stratum (includes all nonwoody species, these species do not exceed 0.50 m in general). in each floristic survey we noted: the percentage of the overall recovery and the recoveries of the three identified strata, the presence or absence of the natural regeneration of the tree layer. results and discussion plant groups and diagnostic species free based on the twinspan analysis, and after the optimclass1 analysis, nine plant groups were identified. considering a cut-off value of 0.30 for the fidelity coefficient f, 58 diagnostic species were selected. these groups each have a particular floristic composition (tab. 1). in its study in the same area terras (2011) identified seven plant groups. this indicates the wide variation in the results of this study and the study conducted by terras (2011). there are several reasons why a gap exists. this could be the difference in scale of the surveys, the difference in methods and style of assessment as well as the variation in anthropozoic factors such as fires and grazing (fig. 2). the overall recovery rate and the contribution of 4 biologica nyssana ● 13 (1) september 2022: 1-9 djebbouri & terras ● community structure with particular reference to the effect of grazing inforest formations of saïda (algeria) table 1. synoptic table of vegetation sampling and species fidelity values in the nine groups group n 1 2 3 4 5 6 7 8 9 number of floristic surveys 6 47 5 16 34 4 39 11 53 total number of species 15 83 35 54 136 23 99 62 144 group 1: asparagus albuspistacia atlantica asparagus albus l. 87.5 -- -- -- -- -- -- -- --pistacia atlantica desf. 75 -- -- -- -- -- -- -- --ballota hirsuta benth. 69.9 -- -- -- -- -- -- -- --psychine stylosa desf. 55.5 -- -- -- -- -- -- -- --capsella bursa-pastoris (l.) medik. subsp. ábursa-p 55.5 -- -- -- -- -- -- -- --calendula arvensis (vaill.) l. 54.8 -- -- -- -- -- -- -- --group 2: olea europaea l. subsp. europaea tetraclinis articulata olea europaea l. subsp. europaea --64 -- -- -- -- -- -- --drimia fugax (moris) stearn --41.4 -- -- -- -- -- -- --group 3: papaver rhoeas hordeum murinum papaver rhoeas l. -- --94.2 -- -- -- -- -- --clypeola jonthlaspi subsp. microcarpa (moris) arca -- --73.5 -- -- -- -- -- --avena sterilis l. -- --69 -- -- -- -- -- --carduus getulus pomel -- --67.9 -- -- -- -- -- --hordeum murinum subsp. leporinum (link) arcang -- --61.4 --18.1 -- -- -- --galium viscosum vahl -- --61 -- -- -- -- -- --hohenackeria exscapa (steven) koso-pol. -- --61 -- -- -- -- -- --coronilla scorpioides (l.) w.d.j. koch -- --61 -- -- -- -- -- --dasypyrum hordeaceum (coss. & durieu) p. -- --61 -- -- -- -- -- --lolium perenne l. -- --61 -- -- -- -- -- --valerianella coronata (l.) dc -- --54 -- -- -- -- -- --group 4: juniperus oxycedrus subsp. oxycedrus quercus ilex juniperus oxycedrus l. subsp. oxycedrus -- -- --46.6 28.7 -- -- -- --ferula communis l. --26.4 --34.6 28.8 -- -- -- --group 5: neatostema apulum macrochloa tenacissima neatostema apulum (l.) i.m. johnst. -- -- -- --50 -- -- -- --carthamus pinnatus desf -- -- -- --46.3 -- -- -- --paronychia arabica subsp. cossoniana j. gay ex ba -- -- -- --44.6 -- -- -- --plantago albicans l. -- -- -- --40 -- -- -- --atractylis cancellata l. -- -- -- --36.4 -- -- -- --anacyclus pyrethrum (l.) link -- -- -- --34.2 -- -- -- --echinaria capitata (l.) desf. -- -- -- --34.2 -- -- -- --scandix australis l -- -- -- --32.6 -- -- -- --group 6: quercus faginea quercus faginea lam. -- -- -- -- --100 -- -- --each stratum to the overall recovery are summarized in fig. 3. the overall recovery rate of the identified groups is high. this importance is mainly due to the strong contribution of the shrub layer. these values confirm once the openness of the environment and the role played by the shrub layer in the fight against erosion, the stability of the plant cover and the protection of the phytocenosis despite the attacks to which it is subjected (medjahdi, 2010; terras, 2011). if we consider only the floristic richness (tab. 2), we notice that the obtained results are logical, that is to say that the richness is decreased when the intensity of grazing is increased. in a recent study (herrero‐jáuregui & oesterheld, 2018), it was found that the effect of grazing intensity on species 5 biologica nyssana ● 13 (1) september 2022: 1-9 djebbouri & terras ● community structure with particular reference to the effect of grazing inforest formations of saïda (algeria) richness and diversity was negative for arid systems. according to our results, the correlation between grazing intensity and tree cover was absent, which also showed the high grazing intensity for all the plant groups identified in this study. a negative correlation was recorded between richness and tree cover rate (tab. 2). the average wealth index was higher in groups 3, 5, 8 characterized by a low tree cover which is between 0% and 14% (fig. 3, fig. 4). according to quézel (2000), the matorrals constitute the vegetation structures undoubtedly the most remarkable of the maghreb, because of their floristic richness. similarly, medjahdi (2010) indicates for the mountains of traras that the floristic richness of smilax aspera l. -- -- -- -- --84.1 -- -- --nerium oleander l. -- -- -- -- --79 -- -- --dittrichia viscosa (l.) greuter -- -- -- -- --68.6 -- -- --mentha suaveolens ehrh. -- -- -- -- --68.6 -- -- --geranium molle l. -- -- -- -- --68.6 -- -- --oxalis pes-caprae l. -- -- -- -- --68.6 -- -- --group 7: pinus halepensis pinus halepensis mill. -- -- -- -- -- --53.5 --23 helianthemum cinereum subsp. rotundifolium -- -- -- -- -- --33.9 -- --group 8: ziziphora hispanica macrochloa tenacissim ziziphora hispanica l. -- -- -- -- -- -- --58.6 --alyssum simplex rudolphi -- -- -- -- -- -- --58 --globularia alypum l. subsp. alypum -- -- -- -- -- -- --55.2 29.4 reseda collina müll. arg. -- -- -- -- -- -- --52.6 --teucrium polium l. -- -- -- -- -- -- --51.4 18.7 erysimum grandiflorum desf. -- -- -- -- -- -- --50 --achillea santolinoides lag. -- -- -- -- -- -- --50 --ebenus pinnata aiton -- -- -- -- -- -- --48 --erodium botrys (cav.) bertol. -- -- -- -- -- -- --45 --group 9: rosmarinus eriocalyx tetraclinis articulata rosmarinus eriocalyx jord. & fourr. -- -- -- -- -- -- -- --56.7 cistus creticus l. -- -- -- -- -- -- -- --43.9 quercus coccifera l. -- -- -- -- -- -- -- --37.6 cistus clusii dunal -- -- -- -- -- -- -- --34.5 macrochloa tenacissima (l.) kunth -- -- -- -- -- -- -- --34 lavandula stoechas l. -- -- -- -- -- -- -- --31.9 genista quadriflora munby -- -- -- -- -- -- -- --30.3 thymus munbyanus subsp. ciliatus (desf.) g -- -- -- -- -- -- -- --30.2 tetraclinis articulata (vahl) mast. --56.3 -- -- -- -- -- --35.5 quercus ilex l. -- -- --55.7 43.8 -- -- -- --asphodelus ramosus l. -- -- --32.5 38.6 -- -- -- --the habitats is maximum for the matorrals more or less open, it decreases in the case of closure of the medium. the values of the shannon index are less strong overall (2.8 bits) and the equitability varies between 0.4 and 0.9, but they are higher in wooded matorrals than in closed formations (fig. 3, fig. 4). these results are confirmed by the negative correlation that has been recorded between the values of the shannon index and the coverage rate (p<0.05) (tab. 2). according to orth & girard (1996), the shannon index has high values for species with similar recoveries and takes low values when a few species have high recoveries, whereas equitability tends towards 0 when a species has a very strong overlap and tends towards 1 when all species have the same importance. this is due to the presence of rare and infrequent taxa. no quantitative studies on pastures have already been carried out in the saïda region, so it is difficult to measure changes in vegetation composition and diversity. nevertheless, based on the results of this study, it was evident that most of the groups identified in this study are severely disturbed by grazing. this is one of the main threats, indicated as more than 80% of the sites surveyed were moderately to heavily grazed and the rest showing signs of disturbance. during the period of study and continuous observation of the vegetation, in more than 70% of the sites studied there was no regeneration of the main tree species such as pistacia atlantica, 6 biologica nyssana ● 13 (1) september 2022: 1-9 djebbouri & terras ● community structure with particular reference to the effect of grazing inforest formations of saïda (algeria) fig. 2. some of the anthropogenic actions in the pre-forest regions of saïda (photos: djebbouri. m). a: burnt stand of aleppo pine (djebel aoun); b: pasture (hssasna); c: misdemeanors and illegal cuts; d: land clearing. (tendfelt) massif; e: aleppo pine groups spared by the fires 7 biologica nyssana ● 13 (1) september 2022: 1-9 djebbouri & terras ● community structure with particular reference to the effect of grazing inforest formations of saïda (algeria) fig. 3. the overall recovery rate and the contribution of each stratum to the overall recovery in each group of plants richness index shannon index simpson index eveness index grazing intensity tree cover (%) richness index 1 0.830** 0.634** 0.499** -0.269** -0.367** shannon index 1 0.940** 0.824** -0.176* -0.657** simpson index 1 0.833** -0.129 -0.733** eveness index 1 0.032 -0.697** grazing intensity 1 -0.025 tree cover (%) 1 table 2. pearson correlation between measured variables fig. 4. mean values of diversity indices (means ± standard error) in the identified groups of plant species. different letters in each column indicate a significant difference at the 0.05 level according to the kruskal wallis test biologica nyssana ● 13 (1) september 2022: 1-9 8 djebbouri & terras ● community structure with particular reference to the effect of grazing inforest formations of saïda (algeria) tetraclinis articulata and olea europaea, quercus ilex subsp. ballota (desf.) samp., pinus halepensis, quercus faginea, which is indisputably due to grazing (χ2 = 18.129, p = 0.0001). this has already been underlined by benabedli (1996) who specifies that: “the course in forest formation constitutes a very degrading factor by its aggressiveness and the damage it causes to the vegetation and to the soil. source of partial or total removal of plant cover, the unregulated route imposes the following degradation process: total exploitation of the herbaceous layer, consumption of young shoots, seedlings and suckers, grazing of the palatable bushy layer, pruning of the shrub layer”. louni (1994) also indicates that overgrazing thus has the consequence of eliminating by browsing young regenerations, low branches and suckers and the effects of trampling on the ground are also serious. indeed, this phenomenon of overgrazing has never ceased to endanger the entire forest heritage of the saïda region. the excess livestock destroys the protective plant cover, while by trampling the surface of the soil powdery and by compacting it, it reduces the permeability of the soil, and therefore its water reserves (ayoub et al., 2019). conclusions the forest vegetation of the saïda region has not been described in detail before. the need for classification and detailed description has become necessary. the present study allowed a better knowledge of the floristic composition and the structural characteristics through the 09 identified plant groups. the vegetation of the study area is quite rich and consists of 406 plant species. nine (9) groups were discriminated against and the g9 group (rosmarinus eriocalyx tetraclinis articulata) is the richest in terms of number of species with 136 species. the diversity and equity indices show that there is a high diversity of species within the groupings and that many species participate in the recovery. the structure of the stands shows the dominance of the shrub character of the woody stratum in the groups. this testifies to the state of degradation and disturbance of the vegetation in this area subject to overgrazing and arid climatic conditions and further reveals the regressive trends in the overall dynamics of the vegetation. this alarming situation requires the rapid and effective implementation of protection and conservation measures. in order to maintain this biological wealth and safeguard this diversity of natural habitats, which remain strongly threatened due to the accentuation of the constraints of anthropogenic origins in these regions. references al harthy, l., grenyer, r. 2019: classification and ordination of the main plant communities of the eastern hajar mountains, oman. journal of arid environments, 169:1-18. ayoub, a., habib, b.a., abelkrim, k., mohamed, z., raphael, g. 2019: effects of overgrazing on the physico-chemical and biological properties of semiarid forest soils in western algeria. indian journal of ecology, 46(4): 745-750. barnes, b.v., pregitzer, k.s., spies, t.a., spooner, v.h. 1982: ecological forest site classification. journal of forestry, 80(8): 493-498. benabdeli, k. 1996: mise en évidence de l’importance des formations basses dans la sauvegarde des écosystèmes forestiers: cas des monts de dhaya (algérie occidentale). ecologia mediterranea, 22(3): 101-112. brown, l.r., du preez, p.j., bezuidenhout, h., bredenkamp, g.j., mostert, t.h., collins, n.b. 2013: guidelines for phytosociological classifications and descriptions of vegetation in southern africa. koedoe, 55(1): 1-10. djebbouri, m., terras, m. 2020: effects of aleppo pine (pinus halepensis mill) plantation on plant biodiversity in the high plains of saïda (algeria). asian journal of research in biosciences, 29-36. dobignard, a., chatelain, c. 2010-2013: index synonymique de la flore d’afrique du nord. conservatoire et jardin botanique de genève. gounot, m. 1969: méthodes d’étude quantitative de la végétation. masson et cie, paris. 314 p. greuter, w. 1994: extinctions in mediterranean areas. philosophical transactions of the royal society b: biological sciences, 344(1307): 41-46. hennekens, s. m., schaminée, j. h. 2001: turboveg, a comprehensive data base management system for vegetation data. journal of vegetation science, 12(4): 589-591. herrero‐jáuregui, c., oesterheld, m. 2018: effects of grazing intensity on plant richness and diversity: a meta‐analysis. oikos, 127(6): 757-766. hill, m.o. 1979: a fortran program for arranging multivariate data in an ordered twoway table by classification of the individuals and attributes. twinspan. cornel university press, ithaca, ny, usa. 48 p. ik-gürsoy¸ i.d., uğurlu, e., oldeland, j. 2016: plant communities, diversity and endemism of the kula volcano, manisa, turkey. plant biosystems-an international journal dealing with all aspects of plant biology, 150(5): 1046-1055. kent, m. 2011: vegetation description and data analysis: a practical approach. john wiley & sons, new jersey, états-unis. 448 p. louni, d. 1994: les forêts algériennes. forêt méditerranéenne.t: xv, 7: 59–63. meddour, r. 2012: bioclimatologie, phytogeographie et phytosociologie en algerie: exemple des groupements forestiers et preforestiers de kabylie djurdjureenne. phd thesis. universite mouloud mammeri. algerie. medjahdi, b. 2001: réponse de la végétation du littoral des monts des trara (ouest algérien) aux différents facteurs de dégradation. mém. magistère: université de tlemcen. algerie. medjahdi, b. 2010: reponse de la vegetation du littoral oranais aux perturbations: cas des monts des trara (nord-ouest de l’algerie). phd thesis. université de tlemcen. algerie. menz, g., alvarez, m., möseler, b. m., mogha, n.g., handa, c., oyieke, h.a., misana, s.b., boehme, b., mwita, e.j., kamiri, h.w. 2012: floristic classification of the vegetation in small wetlands of kenya and tanzania. biodiversity & ecology, 4: 63–76. myers, n., mittermeier, r.a., mittermeier, c.g., da fonseca, g.a., kent, j. 2000: biodiversity hotspots for conservation priorities. nature, 403(6772): 853. orth, d., girard, c.m. 1996: espèces dominantes et biodiversité: relation avec les conditions édaphiques et les pratiques agricoles pour des prairies des marais du cotentin. ecologie, 27(3): 171. quézel, p. 2000: réflexions sur l’évolution de la flore et de la végétation au maghreb méditerranéen. paris edition, ibis.117 p. quézel, p., santa, s. 1961-1962: nouvelle flore de l’algérie et des régions désertiques. centre nat edition, paris. 1170 p. roleček, j., tichý, l., zelený, d., chytrý, m. 2009: modified twinspan classification in which the hierarchy respects cluster heterogeneity. journal of vegetation science, 20(4): 596-602. siddig, a.a., ellison, a.m., ochs, a., villarleeman, c., lau, m.k. 2016: how do ecologists select and use indicator species to monitor ecological change? insights from 14 years of publication in ecological indicators. ecological indicators, 60: 223-230. terras, m. 2011: typology, mapping of forest stations and modeling of forest stands. for forests in the province of saida. phd thesis. université of tlemcen. algeria. tichý, l. 2002: juice, software for vegetation classification. journal of vegetation science, 13(3): 451-453. tichý, l., chytrý, m., hájek, m., talbot, s.s., botta‐dukát, z. 2010: optimclass: using species‐ to‐cluster fidelity to determine the optimal partition in classification of ecological communities. journal of vegetation science, 21(2): 287-299. whittaker, r.h. 1972: evolution and measurement of species diversity. taxon: 213-251. djebbouri & terras ● community structure with particular reference to the effect of grazing inforest formations of saïda (algeria) biologica nyssana ● 13 (1) september 2022: 1-9 9 microsoft word bn-oa-0201-05 cavlovic et al biologica nyssana 2 (1) september 2011: 39-44 čavlović d. et al. allochthonous woody taxa in zasavica ecosystem 39 original article ! allochthonous woody taxa in zasavica ecosystem dragana čavlović, mirjana ocokoljić*, dragica obratov-petković faculty of forestry, university of belgrade, serbia * e-mail: mirjana.ocokoljic@sfb.rs abstract: čavlović, d., ocokoljić, m., obratov-petković, d.: allochtonous woody taxa in zasavica ecosystem. biologica nyssana, 2 (1), september 2011: 39-44. special nature reserve zasavica is an important wetland in serbia. therefore, it was designated as “special nature reserve of the first category“ in 1997. moreover, it has been included in the national network of ramsar sites (the ramsar convention on wetlands of international importance), in 2006. considering importance of special nature reserve zasavica and the fact that stability of ecosystems can be disturbed by introducing alien species, we analyzed extent and coverage of allochthonous woody taxa within the reserve region. researching area comprised 1821 hectares. we found 21 allochthonous woody taxa that belong to magnoliophyta subdivision. the most individuals are located within the first zone of protection. key words: alburnoides bipunctatus, cyprinidae, mesohabitat, phenotype plasticity introduction ! the ecological impact of fast spreading introduced plant species (invasive species) on natural communities has been analysed in numerous articles (e l t o n 1958; d r a k e et al. 1989; d i c a s t r i et al. 1990). some alien tree species used in commercial forestry and agroforestry cause major problems as invaders of natural and seminatural ecosystems. exotic plant species are considered as one of major threats to biodiversity (r i c h a r d s o n , 1998, r i c h a r d s o n et al., 2000). in this article we analyzed the coverage of allochthonous woody taxa within the zasavica reserve region. special nature reserve zasavica is a marshy lowland, located in north-west mačva, within the municipalities sremska mitrovica and bogatić. it is named after the watercourse zasavica which is 33.1 km long, 80 m wide. during normal water levels, the river is 2.5 metres deep on average. zasavica forms six large meanders. the terrain surrounding the meanders is marshland (p a v i ć , 2001). in 1997 zasavica was designated as “special nature reserve of the first category“ by the government of the republic of serbia and as a protected area of exceptional importance for the republic (official gazette rs, 19/97). in 2006 the reserve was included in the ramsar list of wetlands of international importance. the reserve is covered by a two-degree regime of protection: 1150 ha are the first degree, and 671 ha are the second category of protection. taking into account that, by intentional or unintentional introduction of allochthonous woody species to wet ecosystems, their stability is disturbed, this study analyses the state and the influence of allochthonous woody species to the area of the reserve (daisie, 2010). materials and methods during investigation period that covered two complete vegetation seasons (march 2009 to october 2010), we recorded each individual of 2 (1) • september 2011: 39-44 biologica nyssana 2 (1) september 2011: 39-44 čavlović d. et al. allochthonous woody taxa in zasavica ecosystem 40 allochthonous woody taxa that were determined according to š i l i ć , 1998. for each individual we assessed the age, phytopathological and entomological damage. we used separate scales to assess the vitality and ornamental value of individuals (o c o k o l j i ć , et al. 2009). we used the combined abundance-coverage scale (b r a u n -b l a n q u e t , 1965) to assess the importance of each species within investigated area. results and discussion during the research, we recorded 1850 individuals of 21 allochthonous woody taxa. ten species originate from the north american continent and ten from asia. populus x euramericana is a hybrid species. its presence in zasavica was expected because it grows in plantations (46,600 individuals) of the fe sremska mitrovica. all species belong to magnoliophyta subdivision. acer negundo l. (fam. aceraceae) this species is represented throughout the reserve with 95 individuals. their age varies from 10 to 60 years. combined abundance-coverage value of the species is 3. average mark of vitality is 4, and depending on individuals, vitality ranges from 2 to 5. average mark of ornamental value is 3, and it varies from 2 to 4. there are no phytopathological and entomological injuries on the trees. ailanthus altissima (mill.) sw. (fam. simarubaceae) this species is represented throughout the reserve with 120 individuals. tree age ranges from 10 to 20 years. coverage is evaluated by mark 3. average mark of vitality is 4, and depending on individuals, vitality ranges from 2 to 5. average mark of ornamental value is 5. there are no phytopathological and entomological injuries on any trees. amorpha fruticosa l. (fam. leguminosae) this species is represented throughout the reserve with 550 individuals. average age is 20 years. coverage is evaluated by mark 4, and at some places the abundance attains mark 5. average mark of vitality is 4, and depending on individuals, vitality ranges from 3 to 5. average mark of ornamental value is 4, and it varies from 4 to 5. no phytopathological and entomological damage is observed in any of the individuals. according to snr zasavica data, the removal of false indigo was performed in 2008. caragana arborescens lam. (fam. leguminosae) c. arborescens is recorded at the site ribnjača bara as a single individual. its age is 10 years. coverage is evaluated by mark +. vitality mark is 3, and mark of ornamental value is also 3. this specimen does not show any phytopathological and entomological damage. catalpa bignonioides walt. (fam. bignoniaceae) c. bignonioides is recorded in this area only at the locality cerik as one tree aged about 20 years. its coverage is marked +. its vitality is low (mark 1) and its ornamental value is also evaluated by the same mark. gleditsia triacanthos l. (fam. cesalpiniaceae) this species is recorded at two localities in the reserve: jovača and široka bara. there are 25 individuals on these two sites. tree age ranges from 10 to 20 years. coverage is evaluated by mark 1. average mark of vitality is 4, and depending on individuals, vitality ranges from 3 to 5. average mark of ornamental value is also 4, and it varies from 2 to 5. there are no phytopathological and entomological injuries on any trees. based on the analysis at these localities, it is concluded that gleditsia triacanthos l. occurs mainly along the roads in minor line groups. maclura aurantiaca nutt. (fam. moraceae) m. aurantiaca is recorded at two localities in the reserve: jovača and prekopac with 20 individuals. tree age ranges from 10 to 20 years. coverage is evaluated by mark 1. average mark of vitality is 3, and depending on individuals, vitality ranges from 2 to 5. average mark of ornamental value is 5. there are no phytopathological and entomological injuries on any trees. mahonia aquifolium (pursh) nutt. (fam. berberidaceae) m. aquifolium is recorded as one individual at the site jovača. the shrub age is about 10 years. its coverage is therefore marked with +. vitality and ornamental value are evaluated as 4. morus alba l. (fam. moraceae) m. alba is represented throughout the reserve with 65 individuals. tree age is from 20 to 30 years. coverage is evaluated by mark 3. all the analysed specimens show exceptional vitality and ornamental value (average mark 5). there are no biologica nyssana 2 (1) september 2011: 39-44 čavlović d. et al. allochthonous woody taxa in zasavica ecosystem 41 phytopathological and entomological injuries on any individuals. morus nigra l. (fam. moraceae) m. nigra is represented throughout the reserve with 46 individuals. tree age is 30 to 50 years. coverage is evaluated by mark 3. vitality is evaluated with 5. its average mark of ornamental value is also the highest (5). there are no phytopathological and entomological injuries on any trees. parthenocissus quinquefolia (l.) planch. (fam. vitaceae) p. quinquefolia is a climber, which is recorded at the site široka bara (1 individual). its age is about 10 years. coverage is evaluated by mark +. vitality mark is 3, and the mark of ornamental value is 4. there are no phytopathological and entomological injuries on this tree. populus alba 'pyramidalis' l. (fam. salicaceae) p. alba 'pyramidalis' is a taxon which is represented throughout the reserve with 60 individuals. tree age ranges from 20 to 30 years. coverage is evaluated by mark 1, because the trees at the study locality are solitary. average mark of vitality is 4, and depending on individuals, vitality ranges from 3 to 5. average mark of ornamental value is 4, and it varies from 3 to 5. there are no phytopathological and entomological injuries on any trees. only male individuals are represented. populus deltoides marshall (fam. salicaceae) p. deltoides is recorded at two locality in reserve: cerik and vrbovac with 11 individual. tree age ranges from 20 to 30 years. coverage is evaluated by mark 2. average mark vitality is 4, and depending on individuals, vitality ranges from 2 to 5. average mark of ornamental value is 4, and it varies from 3 to 5. there are no phytopathological and entomological injuries on any trees. prunus cerasifera ehrh. (fam. rosaceae) p. cerasifera is represented throughout the reserve with 450 individuals. this taxon grows both as a shrub and as a tree; the age is up to 20 years. coverage is evaluated by mark 4, and at the site valjevac, its coverage is 5. its average mark of vitality is 4, and depending on individuals, vitality ranges from 3 to 5. average mark of ornamental value is 5. there are no phytopathological and entomological injuries on any individuals. reduction of individuals was performed during the previous period. aerial photographs from 2008, 2009 and 2010 show the aggressive spreading and invasion of the territory of the pasture valjevac. quercus borealis michx. (fam. fagaceae) q, borealis is recorded only at the localty jovača as one individual, its age is about 20 years. coverage is evaluated by mark +. this exceptionally vital tree, with regular crown, is evaluated with mark 5, and the mark of ornamental value is also the highest (5). there are no phytopathological and entomological injuries on this tree. robinia pseudoacacia l. (fam. leguminosae) this species is represented throughout the reserve with 400 individuals. tree age varies from 10 to 30 years. coverage is evaluated by mark 4. average mark of vitality is 5. average mark of ornamental value is also 5. there are no phytopathological and entomological injuries on any trees. salix babylonica l. (fam. salicaceae) this is recorded in the reserve at the following localities: modran, gaj, cerik and zovik with 4 individuals. tree age ranges from 10 to 30 years. coverage is evaluated by mark 1. as for tree vitality, all 4 trees are evaluated with mark 5, and also their ornamental value is excellent (5). there are no phytopathological and entomological injuries on any trees. salix matsudana koidz. (fam. salicaceae) s. matsudana is recorded in the reserve at the localities: modran, cerik and zovik with three individuals. tree age ranges from 10 to 30 years. coverage is evaluated by mark +. average mark of vitality is 4, and the marks range from 3 to 5. ornamental value is evaluated 4 (all three trees). there are no phytopathological and entomological injuries on any trees. sophora japonica l. (fam. leguminosae) this is recorded at the localty modran as one individual. the observed tree age is about 20 years. coverage is evaluated +. its vitality is excellent (5), and also its ornamental value (5). ulmus pumila l.(fam. ulmaceae) this is recorded in the reserve at the localty ribnjača bara with three individuals. tree age is from 10 to 20 years. coverage is evaluated by mark +. all trees are evaluated with mark 4 both for vitality and for ornamentalness. there are no phytopathological and entomological injuries on any trees. biologica nyssana 2 (1) september 2011: 39-44 čavlović d. et al. allochthonous woody taxa in zasavica ecosystem 42 conclusion analysing trees and shrubs in snr zasavica, we recorded 1850 individuals of alien species. half of the analysed taxa occupy the second zone of protection, and 50 % grow both in the first and in the second zones of protection. total number of recorded taxa is 20 and all of them belong to the subdivision magnoliophyta. the most abundant taxon is amorpha fruticosa (550 individuals). on the other side, we recorded only one individual of caragana arborescens, catalpa bignonioides, quercus borealis and sophora japonica. the comparative analysis shows an exceptionally high vitality (4) of the alien species. this indicates on their good adaptation to environmental conditions within the study area. the species with the highest vitality (mark 5) are amorpha fruticosa, morus alba, morus nigra, quercus borealis, robinia pseudoacacia and salix babylonica. the lowest vitality (mark 1) was detected for catalpa bignonioides. high marks support the thesis of good adaptability of allochthonous taxa. the comparative analysis of the marks of ornamental value of all individuals shows that the average mark is 4. at the taxon level, the highest average mark (mark 5) is attained by: ailanthus altissima, maclura aurantiaca, morus alba, morus nigra, prunus cerasifera, quercus borealis, robinia pseudoacacia, salix babylonica and sophora japonica, and the lowest by catalpa bignonioides (mark 2). the research confirms the occurrence of spontaneous spreading in 30 % of taxa: acer negundo, ailanthus altissima, amorpha fruticosa, gleditsia triacanthos, prunus cerasifera and robinia pseudoacacia. in 20 %: acer negundo, ailanthus altissima, amorpha fruticosa and prunus cerasifera, spreading can be characterised as invasiveness. all invasive taxa are characterised by extremely good coppicing power. their spreding potential is high since their seeds are readily dispersed by wind to very distant places or zoochoriously. this study indicates that the long-term monitoring is necessary to detect the effect of allochthonous woody taxa on the stability of ecosystems in the zasavica nature reserve. acknowledgements. this paper was realized as a part of the project "studying climate change and its influence on the environment: impacts, adaptation and mitigation" (43007) financed by the ministry of education and science of the republic of serbia within the framework of integrated and interdisciplinary research for the period 2011-2014. references antić, m., jovanović, b., jović, n., avdalović, v. 1973: projekat klasifikacije šumskih zemljišta u jugoslaviji, simpozijum iz šumarske pedologije, tjentište, 1973. izdanje an b i h, knjiga 5, sarajevo, 1975. braun-blanquet,j. 1965. phlanzensoziologie. grundzuge der vegetatinskunde. 3rd ed. springer, wien, n. y. daisie 2010: handbook of alien species in europe http:// www.europe-aliens.org. di castri, f., hansen, a.j. & debussche, m. (eds). 1990. biological invasions in europe and the mediterranean basin. kluwer academic publishers, dordrecht. drake, j., mooney, h.a., di castri, f., groves, r., kruger, f.j., rejmanek, m. & williamson, m. (eds) (1989) biological invasions. a global perspective. wiley, chichester. elton, c.s. 1958. the ecology of invasions by animals and plants. methuen, london. erdeši j. 1971: fitocenoze šuma jugozapadnog srema, srem, sremska mitrovica. erdeši j., janjatović g. 2001: šumski ekosistemi rezervata „zasavica“, „zasavica 2001“, monografija, prirodno-matematički fakultet, institut za biologiju, novi sad i goranskoekološki pokret, sremska mitrovica. nejgebauer, v. et al. 1971: pedološka karta sr srbije i sap vojvodine, 1:50000. novi sad. sfr jugoslavija. obratov-petković d., popović i., stanković m. 2007: diverzitet lekovitih biljaka specijalnog rezervata prirode zasavica, zasavica 2007, naučno-stručni skup, pokret gorana sremska mitrovica. ocokoljić, m., ninić-todorović, j. 2003: priručnik iz dekorativne dendrologije, šumarski fakultet, beograd. pavić, d., kovačević, t. 2001: hidrološke karakteristike sliva zasavice, „zasavica 2001“, monografija, prirodno-matematički fakultet, institut za biologiju, novi sad i goranskoekološki pokret, sremska mitrovica. petković, b., obratov-petković, d. 2003: botanika sa praktikumom, beograd. richardson, d. m. , pyšek , p., rejmanek, m., barbour , m., g., danepanetta , f., carol j. 2000. naturalization and invasion of alien plants: concepts and definitions. diversity and distributions, 6, 93-107. biologica nyssana 2 (1) september 2011: 39-44 čavlović d. et al. allochthonous woody taxa in zasavica ecosystem 43 richardson, d. m. 1998. forestry trees as invasive aliens. conservation biology, 12, 18-26 službeni glasnik republike srbija br 51/95: rešenje prethodnog zaštite prirodnog dobra zasavica. stilinović, s. 1987: proizvodnja sadnog materijala šumskog i ukrasnog drveća i grmlja, institut za šumarstvo šumarskog fakulteta, beograd. šilić, č. 1988: atlas drveća i grmlja, svjetlost, sarajevo, zavod za udžbenike i nastavna sredstva, beograd. šilić, č. 1990: ukrasno drveće i grmlje, svjetlost, sarajevo, zavod za udžbenike i nastavna sredstva, beograd. tomić, z. 2004: šumarska fitocenologija, šumarski fakultet, beograd. vasin, j., sekulić, p., hadžić, v., bogdanović, d., pucarević, m. 2004: stepen zagađenja nepoljoprivrednog zemljišta u vojvodini, zbornik radova, sveska 40, naučni institut za ratarstvo i povrtarstvo, novi sad. vukićević, e. 1996: dekorativna dendrologija, šumarski fakultet, beograd. biologica nyssana 2 (1) september 2011: 39-44 čavlović d. et al. allochthonous woody taxa in zasavica ecosystem 44 microsoft word 0502_lazarevic_lazarevic novo biologica nyssana 1 (1-2) december 2010: 105-109 lazarević, j., lazarević, s. possibilities for production and application of… 105 original article ! possibilities for production and application of native cyclamen neapolitanum in landscape architecture and horticulture jelena lazarević1*, slobodan lazarević2 1 biotechnical faculty department of forestry, mihaila lalića 1, 81000 podgorica, montenegro 2 kopernikova 18, 11 000 belgrade, serbia *e-mail: ena.lazarevic@gmail.com abstract: lazarević, j., lazarević, s.: possibilities for production and application of native cyclamen neapolitanum in landscape architecture and horticulture. biologica nyssana, 1 (1-2), december 2010: 105-109. cyclamen neapolitanum ten is autochthonous in montenegro and serbia in wide range of habitats, from mediterranean-seaside, to sub mediterranean and mountain ones, where it grows on fresh, porous soils, rich in humus and carbonates. it is perennial hardy herbaceous plant with underground tuber, which goes in dormancy trough the period of physical drought. c. neapolitanum flourishes during the autumn, forming after near ground rosette of leaves, which lasts to the next summer, so it is extraordinary suitable for perennials, especially alpinums, individual green areas and also for vertical greening of buildings (balconies, terraces). reproduction is generative. generative reproduction of c. neapolitanum was examined with seed collected in gorica (podgorica, mne), petrovac (mne), and palojce (grdelicka george, serbia). two different artificial substrates were used, as well as classical technologies of plant production with single seed sowing in tresset pots, which were later used to transplantation. seed germination was high (more then 85%), for the seeds originated from all three localities, and also in both of substrates. further loses in plant production were not evident, and development of seedlings as well as transplanted plants, was good. based on conducted experiments, here are given the proposals for economical commercial production, and also for cultivation of plants on green areas. key words: cyclamen neapolitanum, serbia, montenegro, seed germination, commercial production. introduction ! cyclamen l. is a genus of 23 species of flowering plants, native in mediterranean region, along with krimea, caucasus, mountains of middle europe and cappadocia. the most popular among cyclamen is c. persicum miller, originated from ciprus, syria and israel, with their cultivars, which are one of leading flower pot cultures for more then 100 years. during the long period of cultivation, numerous cultivars have been created, differing among each other according to size of entire plant, flower color and size, and petals shape. natural size of c. persicum is about 30 cm in height, and about 18-20 cm in diameter (above ground). according to size of plant, there is a whole group of selected cultivars with “small sizes”not more then 10 cm in height, and 915 cm in diameter, called mini cyclamen, which became very popular pot culture. but those mini cyclamen, as almost all c. persicum cultivars do not tolerate low temperatures, so they are appropriate only as a pot culture in interior and exterior. cyclamen neapolitanum ten is autochthonous in montenegro and serbia in wide range of habitats, from mediterranean seaside, to submediterranean and mountain ones, where it grows on fresh, porous soils, rich in humus and carbonates. it is perennial hardy herbaceous plant 10th sfses • 17-20 june 2010, vlasina lake1 (1-2) • december 2010: 105-109 biologica nyssana 1 (1-2) december 2010: 105-109 lazarević, j., lazarević, s. possibilities for production and application of… 106 with underground tuber, which, as the other cyclamen species, goes in dormancy trough the period of physical drought. c. neapolitanum flourishes during the autumn, forming after near ground rosette of leaves, which lasts to the next summer. the flowers appear in late summer and autumn before leaves, and are distinguished by the base of the petals flaring outwards into a pentagonal throat with 10 lobe-like teeth (auricles). tubers are very large, up to 10 cm in diameter, with roots growing from the upper surface only. leaves are robust and tough, very variable, usually with 5-9 shallow lobes but often rounded or lance shaped, usually toothed but not cartilaginous, blade green with silvery mottling of great variety. leaf stalks are long, creeping and then upstanding. flowers are 11.5 cm long, with varying shade of rose pink with a dark crimson blotch at the throat, with faint scent. the fruit is five-chambered capsule, containing numerous sticky seeds, about 2 mm in diameter in the base. the capsule is surrounded by sepals until fruit ripening. during the ripening, fruiting stalks are curling up in a clock-spring-like spiral and settle (condescend) to ground level, and 1-2 cm underground, taking the capsule in peat layer in depth optimal for seed germination. that’s happened in the end of plant vegetation, when the leaves start drying, i.e. when the more then half leaves are already dry. when is ripe, capsule is about 1, 5 (1-2) cm in diameter. (p a p a z o v a a n t o n o v a , 1964; g r u n e r t , 1968; p o l u n i n & h u x l e y , 1978). c. napolitanum in serbia was recorded and described by p a n č i ć (1974). it belongs to mediterranean floral element, and its native areal is widespread to middle and eastern parts of south europe, south italy and greece. in montenegro it grows in maquia, with erica arborea, spartum junceum near the adriatic coast, but also in sub mediterranean and mountain region in primarily oak forests or pine cultures, in deciduous and evergreen shrubberies and woods on fresh, porous soils, rich in humus and carbonates. in serbia is widespread in mountain regions around južna morava, in shrubberies and deciduous forests around bujanovac, predejane, lebane, vučje (leskovac), stalać, kruševac, niška banja, jelašnica (niš) and koritnik (prizren), on warm, but shaded places, on fresh, porous soils, rich in humus and carbonates (n i k o l i ć , 1972). c. neapolitanum is according to size of underground spring very close to mini cyclamen, but it withstand low temperatures, so there is a possibility for its application in exterior. it is extraordinary suitable for perennials, especially alpinums, individual green areas and also for vertical greening of buildings (balconies, terraces). during the october and november, in phase of flowering, in flower markets in vranje, leskovac, niš and podgorica, one can find c. neapolitanum planted in pots. for that purpose, tubers have been being taken directly from the native habitats. this way of popularization and cultivation of cyclamen can not be accepted from the standpoint of biodiversity protection. besides, their cultivation and plant production by generative reproduction is possible, easy and economical. we investigated the possibility of production of c. neapolitanum for commercial purposes, and first of all we were interesting in production of cyclamen as perennial plant, and for tuber production. we would like to underline that fact, because the mostly produced c. persicum cultivars are produced because of flowers, and their decorative values: as pot plants, or even as cut flowers. in that case, in regime of forced plant production, production cycles last about 12-13 months, or even 8-9 months in greenhouses, with strong fertilization and also phytohormone application. (b a c h a m , 1985; b o o d l e y , 1981). period of production of plant with aim to get tuber, which could support plant development on open, in gardens or green areas, and during the number of years, lasts much longer. material and methods seed collection generative reproduction of c. neapolitanum was examined with seed collected from 3 localities. collecting of seeds was done during the last decade of may. about 25 fruits of c. neapolitanum were collected per locality. the fruits were in ripening (5 days). the seeds are cleaned, counted and measured on 0.0001g accuracy. table 1. the main characteristics of chosen localities, according to geological base and climate locality n e altitude geological base climate petrovac (mne) 42˚15’ 18˚56’ 0 m limestone mediterranean podgorica(mne) 42˚26’ 19˚17’ 54 m limestone submediterranean predejane (serbia) 42˚50’ 22˚08’ 276 m limestone and metamorphic rocks moderate continental biologica nyssana 1 (1-2) december 2010: 105-109 lazarević, j., lazarević, s. possibilities for production and application of… 107 experiment design two different artificial substrates were used in the experiment: floradur b and floradur b: vermiculite 7:3. floradur b fine is substrate made of peat (70% of white peat and 30 % of black peat) with addition of sand and perlite, with super fine structure and with ph 5.2-6.0. it contain 70-150 mg/l n, 80-180 mg/l p2o5, 140-220 mg/l k2o and 0.5-1,1 mg/l salts. floradur b with addition of vermiculite (7:3) was also used, and vermiculite added in order to perform substrate porosity. 120 seeds per locality were separated and treated by benlate wp 50 (0.05%) and used for sowing. seed sowing and entire testing were carried out in open, with environmental conditions modified by shading: 80% during the plant dormancy (summer), and 20% during the period of plant development (autumn to spring). moderation of environmental condition was influenced by sand layer 0.03 m3/m2, placed on the bottom of bad. this sand layer contributes to fine regulation of air humidity, and consequently moderate temperature. besides, it assures good drain off substrate, and also protection against ground insects, worms and snails. classical technologies of plant production with single seed sowing in peat strips were used. strips were filled with substrate and marked. sowed strips were placed on bed previously filled with sand in 3 cm layer. seed sowing was done on jiffi strips 4x5, (product no. 30051590 ) (750 plants per m2), first transplanting in jiffy strips 6x6 was done in the september of next (second) growing year, and the second transplantation in jiffy pots 10x 8, in next september (third year of cultivation) soil moisture, and consequently air humidity, was maintained manually. phases of plant development under the examination were: from sowing to germination and plant development during the vegetation period. cultivation from sowing to germination cultivation of sowed seed have taken place in modified climate conditions, with 80-100% shading, with even watering and on temperature of 20-22˚ c. cultivation of seedlings and young plants seedlings were watered, and preventive phytopathological and entomological protection was made. in first half of september shading was reduced to 20%, and in those conditions plants have stayed until the end of vegetation period. in second half of may next year, when the 50% of assimilation leaf mass were dry, shading of 80% was established, and the watering was stopped. from the beginning of june, plants were, practically, in dormancy. first transplantation has been done during the september, second year from seed sowing. transplantation was done on jiffi strips 6x6 cm (300 plants per m2) (product no. 30052200 ). second transplantation was done in next september, third year from seed sowing. jiffi pots 10x8 (single-100 plants per m2) (product no. 30033500) were used. as a measure of plant development, the total number of developed leaves, also as general appearance and condition of plant was estimated. statistical analysis the data were analyzed by oneway analysis of variance (anova) and significant differences among treatments were separated by duncan’s test (p<0.05) for diameters of collected fruits, number of seeds per fruit and seed mass. number of days from sowing to germinations, and also all other later comparing of numbers of leaves formed per plant, also as tuber diameters, were compared on the base of mean value. we consider that those parameters, though varying in some manner, are not needed to be analyzed statistically, because they do not contribute to technological process of c. neapolitanum commercial production in frame postulated with this investigation. results and discussion average number of seeds per fruit, mass of seed and characteristics of seed germination are shown in the table 2. statistical analyses anova with duncan test (sig 0.05) showed that there were no significant differences among material collected at different localities according to diameter of fruits, number of seed per fruit, and mass of seed. seeds were sown on 10th of june. germination started on 5th of july and lasted to july 12th, i.e. germination lasted 25-32 days. seed germination was high (more then 80%), for the seeds originated from all three localities, and also in both of substrates. there were no differences according to germination in substrate 1 and substrate 2, but germination lasts shorter in substrate 2, which was more porous. as important characteristic, we would like to emphasize that the percent of technical germination was quite high according to c. persicum (p a p a z o v a -a n t o n o v a , 1964). biologica nyssana 1 (1-2) december 2010: 105-109 lazarević, j., lazarević, s. possibilities for production and application of… 108 table 2. number per fruit and mass of c. neapolitanum seed divided from different localities, and characteristics of seed germination seed origin f diam nmb of seeds mass of 10 seeds s gs gd % g petrovac (mne) 1.170 a 19.6 a 0.1701 a 1 25 7 82 2 27 4 84 podgorica (mne) 1.289 a 20.8 a 0.1803 a 1 26 4 84 2 26 3 86 predejane (serbia) 1.298 a 20.4 a 0.1753 a 1 25 6 80 2 27 5 85 f diam –diameter of collected fruits , s n number of seed per fruit, maverage mass of 10 seeds (g), s sowing substrate, gsstarting of germination (nmb. of days after sowing), dgduration of germination (nmb. of days), %-percent of germinated seeds table 3. number of leaves per plant, developed during the four year of investigation seed origin substrate number of leaves per plant sept ii may ii t r a n s p l a n t. may iii t r a n s p l a n t. may iv petrovac (mne) 1 2-3 (2.5) 4-5 (4.7) 8-9 (8.2) 13-18 (15.5) 2 2-3 (2.8) 5 (5.0) 8-9 (8.9) 15-18 (16.5) podgorica (mne) 1 2-3 (2.8) 4.5 (4.5) 8-9 (8.9) 12-17 (14.5) 2 3 (3.0) 5 (5.0) 10 (10.0) 14-18 (16,0) predejane (serbia) 1 2-3 (2.9) 4-5 (4.8) 9-10 (9.6) 14-18 (16) 2 3 (3.0) 5 (5.0) 10 (10.0) 16-17 (16.5) average number of leaves per plant developed during the four years of investigation was shown in table 3. differences in number of leaves formed in september of first year of cultivation and may of second year of cultivation were not evident. number of leaves is mostly equal independently of locality, with lightly decreased number of leaves on substrate 2. in september of second year of cultivation, there were no differences in number of formed leaves. mostly because of needs for developing above-ground spring, transplanting in single pots was done in september third year of cultivation. in that moment, the tubers were about 2 cm in diameter, independently on treatments. during the september and october the flowers were formed, 13 per plant. in regard to small number of flowers per plant, plants have not proved commercial yet. at the end of that vegetation period, in may in fourth year of cultivation, number of leaves per plant was 12-18. the diameter of tubers was about 4 cm in that moment. for following growing season (in fourth year of cultivation) the plants were fit for market: as tubers, as plantlet in peat pots, or as pot plants, transplanted in earthenware. the experiments show that the flowering starts at the same time, independently of seed origin or substrate used, in third year of cultivation, and also without differences according to developed leaf mass. average number of leaves was bigger on substrate 2. the tubers were equal independently of seed origin and substrate also in the end of vegetation season in fourth year of plant cultivation. on the manner previously described, c. neapolitanum could be generatively reproduced economically. cultivation was done under the natural environmental conditions with shading. sterile conditions of substratum and pots deplete the possibility of pests and diseases appearances. in whole cycle of plant production only 2 transplantings exist, without damaging of root system. humidity, attained by watering is applied only in first 4 months in continuum, and after that only when necessary. plants obtained this way could be used on individual and public green areas, especially in perennials because of late flowering of c. neapolitanum and retaining of leaves during the whole winter, until the end of may. cultivation measures which could be applied are the same as for the other perennes with tubers or bulbs resistant on low temperatures. by supplying market with plants produced this way, one could not only actualize it in application, but also protect their natural genofund and habitats, meaning stoppage of taking tubers from native habitats, and without harm to the wild plants and populations. biologica nyssana 1 (1-2) december 2010: 105-109 lazarević, j., lazarević, s. possibilities for production and application of… 109 conclusion c. hederifolium has a great value as a herbaceous perennial plant for use on individual or public green areas because of its late flowering and retaining of leaves during the whole winter, especially because of its withstanding on low winter temperatures. characteristics of seeds, and consequently developed plants, collecting from native populations of c. neapolitanum in montenegro (petrovac and podgorica) and serbia (predejane) were investigated on two artificial substrates (floradur b and floradur b: vermiculite 7:3). seeds collected from all tree localities have practically the same value, and can be used in generative plant reproduction. seed germination was high (more then 80%), for the seeds originated from all three localities, and also in both of substrates. further loses in plant production were not evident, and development of seedlings as well as transplanted plants, was good. plant development is better on substrate 2 in phase of plant sowing and first transplanting, so we suggest it (floradur b: vermiculite 7:3) for sowing and first transplanting, and substrate 1 (floradur b) for second transplanting. usage of jiffy strips and jiffy pots during cultivation assures good aeration of soil, which, as we believe, contributes to good results. references bacham, t. 1985: cyclamen-faster crops are here, in ball g.j. (ed)the ball red book a reston book, prentice-hall, new jersey: 440-448 boodley, j.w. 1981: the commercial greenhouse, delmar publishers inc, new york: 296-300. grunert, c. 1968: bunte blumen im garten, neumann verlag, melsungen, bez. kassel, 176177. kolić, b. 1983: šumarska ekoklimatologija sa osnovama fizike atmosfere, naučna knjiga, beograd 365-377 nikolić, v. 1972: rod cyclamen, in josifović, m. (ed), flora sr srbije iii: 500-502, srpska akademija nauke i umetnosti, beograd. pančić, j. 1976: flora kneževine srbije i dodatak flori kneževine srbije. ponovljeno izdanje, sanu, beograd papazova-antonova, g. 1964: cvetarstvo, zemzidat, sofija schacht, w. 1976: blumen europas, p.13-22, 142149. paul parey, berlin und hamburg polunin, o., huxley, a. 1978: flowers of the mediterranean, p. 140-142, chato and windus, london anticancer compounds from medicinal plants biologica nyssana 5 (1)  september 2014: 53-61 vulić, i. et al.  taxonomic composition and community structure... 53 original article received: 14 august 2014 revised: 3 september 2014 accepted: 10 september 2014 taxonomic composition and community structure of trichoptera (insecta) on the territory of the city of niš (serbia) ivana vulić, ivana vasov, ana savić university of niš, faculty of science and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia e-mail: anka@pmf.ni.ac.rs abstract: vulić, i., vasov, i., savić, a.: taxonomic composition and community structure of trichoptera (insecta) on the territory of the city of niš (serbia). biologica nyssana, 5 (1), septemeber 2014: 53-61. the research of trichoptera community was performed within the territory of the city of niš on 10 localities. these localities have different physical and chemical characteristics ranging from hot springs to cold karst streams. some of them are with a measurable human influence (in the city of niš), while others are practically without it (dušničko vrelo). a total number of 16 genera trichoptera within 11 families were noted. number of identified species was 20. it was determined that the highest species diversity is in localities with the least human influence. during this study, the presence of the species that has the status of a strictly protected species in republic of serbia was recorded. the largest number of localities belongs to β – mesosaprobic waters. it has been found that group trichoptera is suitable for independent application in bioindicator purposes. key words: trichoptera, nišava river, saprobiological analysis, diversity, taxonomic composition, community structure apstract: vulić, i., vasov, i., savić, a.: taksonomski sastav i struktura zajednice trichoptera (insecta) na teritoriji grada niša. biologica nyssana, 5 (1), septemeber 2014: 53-61. istraživanje zajednice trichoptera sprovedeno je na teritoriji grada niša na 10 istraživanih lokaliteta. lokaliteti su različitih fizičko-hemijskih karakteristika u rasponu od termalnih izvora do hladnih kraških tekućica. neki od njih su pod značajnim antropogenim uticajem (u samom gradu nišu) dok su drugi gotovo bez njega (dušničko vrelo). konstatovano je ukupno 16 rodova trichoptera u okviru 11 familija. identifikovano je ukupno 20 vrsta. utvrđeno je da je specijski diverzitet najveći na lokalitetima sa najmanjim antropogenim uticajem. tokom istraživanja konstatovana je vrsta koja ima status strogo zaštićene vrste u republici srbiji. najveći broj lokaliteta pripada beta mezosaprobnim vodama. ustanovljeno je da je ova grupa pogodna za samostalnu primenu u bioindikatorske svrhe. ključne reči: trichoptera, diverzitet, reka nišava, saprobiološka analiza, taksonomski sastav, struktura zajednice 5 (1) • september 2014: 53-61 biologica nyssana 5 (1)  september 2014: 53-61 vulić, i. et al.  taxonomic composition and community structure... 54 introduction order trichoptera is the largest among the orders of primarily aquatic insects, which include: ephemeroptera, plecoptera, odonata and megaloptera. its diversity is similar to that of the order diptera (h o l z e n t h a l et al., 2007). trichoptera are considered to be the most common group of macro-invertebrates in the freshwater ecosystems. they are widespread and inhabit a variety of habitat types, within the multiple trophic levels (s a v i ć 2013). trichoptera larvae are a very important component of trophic dynamics and energy flows in the aquatic ecosystems they inhabit. many species of trichoptera are sensitive to pollution, so their presence and relative abundance are used in biological assessment and monitoring of water quality (h o l z e n t h a l et al., 2007). there are 23 families of trichoptera recorded in europe (including arctopsychidae and ptilocolepidae, but excluding apataniidae, because it is considered a subfamily of limnephilidae) and a total of 133 genera, including the genus alpopsyche (b o t o s a n e a n u & g i u d i c e l l i , 2004, according to w a r i n g e r & g r a f , 2013) with about 1100 recent species (i b r a h i m i et al., 2012). according to the available data, the trichoptera fauna of the balkan peninsula includes 21 families and a high number of 90 genera. within the territory of serbia, trichoptera larvae were investigated as part of macrozoobenthos studies in mountain streams and rivers. the adult forms were studied by a small number of authors, including m a r i n k o v i ć -g o s p o d n e t i ć (1975, 1980) and r a d o v a n o v i ć (1931, 1935, 1953), as cited by ž i v i ć et al. (2000). the first checklist of the trichoptera fauna in serbia included 173 species (ž i v i c et al., 2002). the trichoptera fauna of serbia currently includes 186 species, 49 genera and 18 families (ž i v i c et al., 2006). figure 1: map of the studied area with localities biologica nyssana 5 (1)  september 2014: 53-61 vulić, i. et al.  taxonomic composition and community structure... 55 table 1. database of localities and gps coordinates locality name of locality n е altitude (m) 1 niška banja spring 43° 17.468' 22° 00.749' 266 2 niška banja near jelašnica road 43° 17.903' 22° 00.498' 212 3 nišava r. near sićevo 43° 19.893' 22° 04.429' 233 4 dušničko vrelo spring 43 o 10.433’ 22° 07.033' 501 5 dušnička reka r. 43 o 10.297’ 22° 07.056’ 466 6 kutinska reka r. 43° 09.820' 22° 05.972' 395 7 banja topilo upstream from warm water discharge 43° 26.889' 21° 52.726' 256 8 banja topilo downstream from warm water discharge 43° 26.894' 21° 52.692' 253 9 nišava r. at bulevar (near the mouth of gabrovačka reka r.) 43° 19.517' 21° 55.266' 195 10 nišava r. near medoševac 43° 19.330' 21° 52.375' 189 material and methods material was collected in november and december 2012 and february and may 2013 at 10 representative sites at the nišava river and its tributaries (fig. 1). selected sites were located at points above and below the pollutant discharge. choice of sampling sites was heavily influenced by need for variety in following factors: altitude, bedrock, slope, flow rate, bottom substrate, microclimate conditions etc. localities 1 and 2 are thermal water bodies at niška banja. in contrast, localities 4 and 5 are a typical cool karst spring (locality 4) and upper course of a karst river (locality 5). locality 6 is the tributary of the nišava river-kutinska reka river. the following two localities are at the miljkovačka reka river: locality 7 is above the thermal springs of banja topilo, while locality 8 is located below the same thermal springs. localities 9 and 10 are at the nišava river, in the urban area (tab. 1). explorations were conducted once a month, during a single day in order to accurately compare chemical parameters, which change a lot faster than the biotic parameters. physical parameters were measured directly at the sampling sites. the following physical characteristics were determined: temperature, turbidity and conductivity. temperature and conductivity were measured using a device photometer system pc multidirect (lovibond ®) while turbidity of water was determined using the lovibond pc checkit device. chemical parameters were mostly measured directly, using the photometer system pc multidirect (lovibond ®) device (the saturation and oxygen concentration, ph). biochemical oxygen demand (bod5) was obtained using the standard methodology, recommended by apha (1999). sampling was conducted by using a square frame kick net (35 × 35 cm, mesh size 300 μm). the treated area of bottom surface was 35 x 35 cm. the bottom substrate was disturbed and macroinvertebrate individuals were separated from the substrate and carried by a stream as water was entering the sampling net. samples were then fixed in 4% formaldehyde, placed in labeled plastic bags and transported to the laboratory. in the laboratory, the samples were rinsed under the tap, using two screens with larger and smaller diameter of buds. a total of 40 samples were analyzed. separated materials have been sorted in transparent bottles containing 70% ethanol and later determined by use of identification keys. the determination of each individual was performed by using a binocular magnifying glass and microscopes in specially equipped laboratories at the center for biological and environmental monitoring of surface waters in the city of niš (bioekocen), at the faculty of science and mathematics, university of niš. the observation and measurements of very small body parts were done by using binocular leica mz 16a stereomicroscope with leica dfc320 digital camera and leica dm2500 microscope system with leica dfc490 digital camera. the individuals of the order trichoptera were identified by using the following keys: individuals from families glossosomatidae, beraeidae, biologica nyssana 5 (1)  september 2014: 53-61 vulić, i. et al.  taxonomic composition and community structure... 56 table 2. average values of physical-chemical parameters at the studied localities locality turbidity temperature ph conductivity oxygen mg/l oxygen % bod5 shading % 1 0.35 30.6 7.5 477 3.59 48.5 5.14 40 2 3.63 23.5 7.7 466 4.74 58 5.57 15 3 1.91 12.4 7.9 421 5.81 64.25 5.33 15 4 0.12 10.4 7.6 246 7.29 61.54 4.02 0 5 2.24 10.1 7.9 290 7.82 91.25 9.68 40 6 4.88 9.2 7.9 364 8.77 113.75 10.72 2 7 1.82 11.8 8.1 404 7.87 71 9.12 20 8 2.6 8.2 8 435 8.3 101.25 8.82 5 9 1.93 11.3 8.1 459 6.83 84.25 5.91 6 10 2.29 11.9 8.1 518 5.77 84.75 5.73 5 table 3. average abundance of trichoptera species (individuals/ m 2 ) localities 1 2 3 4 5 6 7 8 9 10 glossosomatidae agapetus ohripes curtis 1834 2.72 beraeidae beraea pullata (curtis 1834) 277.55 46.26 8.16 5.44 beraeodes minutus (linnaeus 1761) 2.72 2.72 hydropsychidae cheumatopsyche lepida (pictet 1834) 165,9 8.16 111.56 2.72 hydropsyche angustipennis (curtis 1834) 2.72 hydropsyche contubernalis mclachlan 1865 78.9 2.72 2.72 hydropsyche fulvipes curtis 1834 5.44 2.72 hydropsyche instabilis (curtis 1834) 95.25 2.72 76.2 8.16 hydropsyche pellucidula (curtis 1834) 2.72 117.01 2.72 5.44 8.16 hydropsyche sp. 2.72 limnephilidae halesus radiatus (curtis 1834) 51.7 43.54 stenophylax permistus mclachlan 1895 24.49 21.77 2.72 16.33 lepidostomatidae lasiocephala basalis (kolenati 1848) 43.54 leptoceridae setodes sp. 21.77 27.21 8.16 psychomyiidae lype reducta (hagen 1868) 2.72 tinodes unicolor (pictet 1834) 8.16 psychomyia pusilla ( fabricius 1781) 38.09 5.44 32.65 163.26 phryganeidae phryganea bipunctata retzius 1783 8.16 polycentropodidae neureclipsis bimaculata (linnaeus 1758) 2.72 rhyacophilidae rhyacophila dorsalis (curtis 1843) 2.72 2.72 2.72 rhyacophila fasciata hagen 1859 8.16 2.72 8.16 sericostomatidae sericostoma personatum (kirby & spence 1826) 2.72 5.44 biologica nyssana 5 (1)  september 2014: 53-61 vulić, i. et al.  taxonomic composition and community structure... 57 limnephilidae, lepidostomatidae, phryganeidae, psychomyiidae and leptoceridae were identified according to the w a l l a c e et al. (1990) identification key, while individuals belonging to families rhyacophilidae, polycentropodidae and hydropsychidae were identified by using the e d i n g t o m and h i l d r e w (1995) identification key. saprobity is expressed according to the saprobity index s, in accordance with the method devised by pantle buck (1955). s stands for saprobic index of the communities, h for the relative abundance and s for the saprobic value, which is characteristic for each species. for needs of saprobiological analysis, lists of indicator species by sladaček were used (s l a d a č e k 1973), while for the evaluation of the frequency of species, a modified scale of 6 degrees to rusev-in was used (r u s e v 1993). results during the analysis of the physical-chemical parameters, it was determined that locality 1 stood out from the rest due to low values of turbidity, high temperatures and minimal values of average concentration of oxygen. the lower value of turbidity was observed only at the locality 4. the highest values of turbidity were recorded at the locality 6. ph values at all localities were close to the values characteristic for neutral water (about 7). lower alkalinity values were noticed at localities 7, 8, 9 and 10 (tab. 2). the greatest number of species was recorded at the locality 4 (11 species). localities with high species diversity were locality 8 (9 species) and locality 3 (8 species). most individuals were sampled at locality 3 a total of 187. the lowest number of species was recorded at locality 2 only two species. no trichoptera individuals were recorded at localities 1 and 10 (tab. 3). the family psychomyiidae was represented by three genera, families beraeidae, hydropsychidae and rhyacophilidae by two genera, while other families were represented by one genus each. five species belonging to the genus hydropsyche were recorded, while other genera were represented by single species only. the species hydropsyche pellucidula (curtis 1834) was observed at five of the investigated localities while species cheumatopsyche lepida (pictet 1834), stenophylax permistus mclachlan 1895, hydropsyche instabilis (curtis 1834), beraea pullata (curtis 1834) and psychomyia pusilla (fabricius 1781) were recorded at four investigated localities each. the following species were recorded at only a single locality: agapetus ohripes curtis 1834 at locality 4, hydropsyche angustipennis (curtis 1834) at locality 3, lype reducta (hagen 1868) at locality 5, tinodes unicolor (pictet 1834) at locality 8, while phryganea bipunctata retzius 1783 and neureclipsis bimaculata (linnaeus 1758) were both recorded only at locality 3. the saprobity index is presented as part of autumn, winter or spring aspect. the autumn aspect included samples collected in november 2012, the winter aspect included samples collected in december 2012 and february 2013, while the spring aspect included samples collected in may 2013. in the autumn aspect, indicator species were present at localities 3, 5 and 8 (tab. 4). the saprobity index has shown that all three sites had β mesosaprobic water (β), which belongs to the class ii water. in the winter aspect, the lowest value of saprobity index was recorded at the locality 4. during this period, saprobity index values at all sites were within the limits characteristic for β mesosaprobic water, except at locality 4 where the value of saprobity index indicated oligosaprobic water. in the spring aspect, the saprobity index values at localities 5, 6 and 7 were within the range typical for oligosaprobic water, while the values recorded at localities 3 and 9 indicated β mesosaprobic water (fig. 2). following indicators of saprobity were used in this research: genus hydropsyche and species sericostoma personatum, rhyacophila septentrionis (i.e. rhyacophila fasciata), neureclipsis bimaculata and stenophylax permistus. all species recorded during this study were typical for the european continent and known for many european countries (fig. 3). all of them were also included in the checklist of the trichoptera fauna in serbia from 2006. localities where trichoptera species were recorded are also the new sites for these species in republic of serbia. biologica nyssana 5 (1)  september 2014: 53-61 vulić, i. et al.  taxonomic composition and community structure... 58 table 4. saprobity index (s) values for all aspects locality aspect 1 2 3 4 5 6 7 8 9 10 autumn 1.95 1.55 1.95 winter 1.95 1.82 1.4 1.75 1.62 1.95 spring 1.95 1.25 1.25 1.25 1.95 figure 2. saprobity index values in autumn, winter and spring aspect figure 3. comparison of the european genera and families with genera and families found within the territory of city of niš discussion the maximum value of temperature was observed at locality 1, which is a thermal spring at niška banja. at this locality, thermal pollution was dominant. the lowest values of turbidity were recorded at the locality dušničko vrelo, which may be explained by the fact that this site is located in a place still under minimal anthropogenic influence. in the period from november 2012 to may 2013, a total of 16 genera within 11 families were recorded. 20 species were identified, while two taxa were identified to genus level. the trichoptera fauna in niš contained a high percentage (47.83%) of european families and 11.63% of european genus biologica nyssana 5 (1)  september 2014: 53-61 vulić, i. et al.  taxonomic composition and community structure... 59 genera, which is a significant percentage considering that this order is insufficiently explored in our country. the greatest number of species was recorded at the locality dušničko vrelo (11 species), while the largest number of individuals was recorded at locality nišava river near sićevo. no species of trichoptera were recorded at localities niška banja spring and nišava river near medoševac. in comparison to the number of species of trichoptera previously recorded in other rivers in serbia where the trichoptera or other macrozoobenthos groups were studied, the diversity of the trichoptera assemblage in niš is greater than the diversity values observed in the following rivers: vlasina (s c h m i d t -k l o i b e r et al., 2008), ribnica and lepenica (j o v i ć et al. 2006), jablanica (s t e f a n o v i ć 2009), banja river (ž i v i ć et al. 2000), vrelska padina i ivanštica (đ u k n i ć et al. 2010), zapadna morava (n o v a k o v i ć 2013), pčinja (s i m i ć & s i m i ć 2003), južna morava (n o v a k o v i ć 2012), velika morava (m a r k o v i ć et al. 2011), pusta reka (ž i v i ć et al. 2001). the greater diversity of this order was noticed at rivers temska and visočica (ž i v i ć et al. 2005) and golijska moravica (đ i k a n o v i ć et al. 2008), with respect to the diversity of the trichoptera community in area of city of niš. the diversity of the trichoptera community in the study area is equally high as the diversity recorded along the entire flow of the nišava river (s a v i ć 2012). of all of the recorded species, beraeodes minutus (linnaeus 1761) stands out as one of the strictly protected river invertebrates in the republic of serbia (r a d u l o v i ć et al. 2012). this species was recorded in december 2012 at the locality dušničko vrelo and in february 2013 at the locality banja topilo downstream, at the place where hot water discharge enters the river. in autumn and winter, the saprobity index was within the limits characteristic of β-mesosaprobic waters. the saprobity was lower in spring due to the prevalence of indicators of oligosaprobic waters (or the species stenophylax permistus). low values of the saprobic index may be associated with variations of water level in nišava river. this river is characterized by highest water levels in spring months when rainfall and water from thawed snow are at the peak. periods of high water level lead to dilution of pollutants that continue to influence the decrease in the saprobity index. most of the studied localities at βmesosaprobic water bodies belong to class ii waters. the results obtained during this study correspond to the results collected during the hydro-biological research of the nišava river, according to the water framework directive (2000/60/ec), when it was concluded that at the studied sites the saprobic index was within the limits of β-mesosaprobic waters (b r a n k o v i ć et al. 2007). conclusion during this study it was concluded that hydropsychidae was the most common family in the study area. out of the recorded total of 16 genera, the most common genus was hydropsyche. recorded species belonging to this genus inhabited both the polluted and the minimally contaminated locations. therefore it was concluded that within the order trichoptera the taxonomic level of the genus should not be considered reliable for bio-indicator purposes. there are no literature data indicating that the sampling sites chosen for this study were previously used for studying the trichoptera species in the republic of serbia. the presence of a species with the status of a strictly protected species in the republic of serbia in studied localities provides additional significance for this area as an area of hydrobiological importance. as the sampled sites belonged to the oligosaprobic and β-mesosaprobic zone, this area may be considered to have great potential for production of high-quality drinking water. references anonymous, 2000: okvirna direktiva o vodama. water framework directive, 2000/60/ec, official journal of the european communities l327, 1-72. brajković, m., 2004: zoologija invertebrata. zavod za udžbenike i nastavna sredstva, beograd. branković, s., simić, v. & trajković, s., 2007: hidrobiološka istraživanja reke nišave prema okvirnoj direktivi o vodama. zbornik radova građevinsko-arhitektonskog fakulteta, 22: 143148. đikanović, v., jakovčev-todorović, d., nikolić, v., paunović, m. & cakić, p., 2008: qualitative composition of communities of aquatic macroinvertebrates along the course of the golijska moravica river (west-central serbia). archives of biological sciences, 60(1): 133-144. đuknić, j., bjelanović, k., durutović, a. & jovanović, v., 2010: ecological survey of macroinvertebrate communities in the vrelska padina and the ivanštica rivers (eastern serbia). balwois 2010: 1-12. biologica nyssana 5 (1)  september 2014: 53-61 vulić, i. et al.  taxonomic composition and community structure... 60 edington, j. m. & hildrew, a. g., 1995: a revisited key to the caseless caddis larvae of the british isles with notes on their ecology. scientific publications freshwater biological association, 53: 6 -133. grginčević, m., pujin, v., 1998: hidrobiologija: priručnik za studente i poslediplomce 3. dopunjeno i prerađeno izdanje. ekološki pokret grada novog sada, novi sad. holzenthal, r. w., blahnik, r. j., prather, a. l & kjer, k. m., 2007: order trichoptera kirby,1813 (insecta), caddisflies. in: zhang, z.-q. & shear, w.a. (eds): linnaeus tercentenary: progress in invertebrate taxonomy. zootaxa, 1668: 639 698. ibrahimi, h., kučinić, m., gashi, a. & grapcikotori, l., 2012: the caddisfly fauna (insecta, trichoptera) of the rivers of the black sea basin in kosovo with distributional data for some rare species. zookeys 182: 71-86. jović, a., paunović, m., stojanović, b., milošević, s. & nikolić, v., 2006: aquatic invertebrates of the ribnica and lepenica rivers: composition of the community and water quality. archives of biological sciences, 58(2): 115-119. kerovec, m., 1986: priručnik za upoznavanje beskralješnjaka naših potoka i reka. snl, zagreb. kumanski, k., 1997: contribution to the caddisfly fauna (trichoptera) of the central-western part of the balkan peninsula. lauterbornia 31: 73-82. marković, v., atanacković, a., tubić, b., vasiljević, b., simić, v., tomović, j., nikolić, v. & paunović, m., 2011: indicative status assessment of the velika morava river based on aquatic macroinvertebrates. water research and management, 1(3): 47-53. novaković, b., 2012: indicative ecological status assessment of južna morava river based on aquatic macroinvertebrates. water research and management, 2 (4): 45-50. novaković, b., 2013: indicative ecological status assessment of the zapadna morava river basin on aquatic macroinvertebrate community. water research and management, 3 (2): 3742. olah, j., 2011: new species and records of balkan trichoptera. folia historico naturalia musei matraensis, 35: 111-121. paunović, m., tomović, j., kovačević, s., zorić, k., žganec, k., simić, v., atanacković, a., marković, v., kračun, m., hudina, s., lajtner, j., gottstein, s. & lucič, a., 2012: macroinvertebrates of the natural substrate of the sava river-preliminary results. water research and management, 2(4): 33-39. radulović, s., boon, p. j., laketić, d., simonović, p., puzović, s., živković, m., jurca, t., ovuka, m., malaguti, s. & teodorović, i., 2012: preliminary checklist for applying sercon (system for evaluating rivers for conservation) to rivers in serbia. archives of biological sciences, 64(3): 1037-1056. savić, a., ranđelović, v., đorđević, m., karadžić, b., đokić, m. & krpo-ćetković, j., 2013: the influence of environmental factors on the structure of caddisfly (trichoptera) assemblage in the nišava river (central balkan peninsula). knowlege and management of aquatic ecosystems, 409: 26-43. savić, a., 2012: ekološka analiza zajednice makrozoobentosa reke nišave, doktorska disertacija, biološki fakultet, univerzitet u beogradu. simić, v. & simić, s. 2003: macroalgae and macrozoobenthos of the pčinja river. archives of biological sciences, 55(3-4):121-132 sladeček, v., 1973: system of water quality from the biological point of view. ergebnisse der limnologie 7: 280 stefanović, k. s., nikolić, p. v., tubić, p. b., tomović, m. j., atanacković, d. a., simić, m. v. & paunović. m. m., 2009: aquatic macroinvertebrates of the jablanica river, serbia. archives of biological sciences, 61(4): 787-794. schmidt-kloiber, a., graf, w., moog, o., lorenz, a. & hering, d., 2008: the indicator database for european freshwater invertebrates. proceedings of the first conference on faunistics and zoogeography of european trichoptera, 55: 85 -88. uherkovich, a. & nogradi, s., 2002: trichoptera from the balkans and asia minor in hungarian and a dutch collection. a janus pannonius muzeum évkönyve, 44-45(1999-2000): 33-42. vasov, i., 2014: ekološke karakteristike zajednice trichoptera na teritoriji grada niša. master rad, prirodno-matematički fakultet, univerzitet u nišu. vulić, i., 2014: taksonomski sastav i struktura zajednice trichoptera na teritoriji grada niša. master rad, prirodno-matematički fakultet, univerzitet u nišu. wallace, i.d., wallace, b. & philipson, g. n., 1990: a key to the case-bearing caddis larvae of britain and ireland. scientific publication freshwater biological association, 51: 1-237. waringer, j. & graf, w., 2013: key and bibliography of the genera of european trichoptera larvae. zootaxa, 2: 101-151. biologica nyssana 5 (1)  september 2014: 53-61 vulić, i. et al.  taxonomic composition and community structure... 61 živić, i., marković, z. & brajković, b., 2001: macrozoobenthos in the pusta reka river, left tributary of the south morava river. archives of biological sciences, 53(3-4): 109-122. živić, i., marković, z. & brajković, b., 2002: dynamics and distribution of macrozoobenthos in the toplica river, a tributary of the kolubara. archives of biological sciences, 54(1-2): 19-27. živić, i., marković, z. & ilić, j., 2005: composition, structure and seasonal dynamics of macrozoobenthos in the temska and visočica rivers (serbia). archives of biological sciences, 57(2): 107-118. živić, i., marković, z. & brajkovič, m., 2000: a contribution to the study of the trichoptera (insecta) fauna in the toplica river. acta entomologica serbica, 5(1/2): 35-46. živić, i., marković, z. & brajković, m., 2003: the diversity of trichoptera larvae in the južna morava river basin. archives of biological sciences, 55(3-4): 33-34. živić, i., marković, z. & brajković, m., 2002: a contribution to the study of the trichoptera fauna in serbia over the period 1980-2001. archives of biological sciences, 52 (1-2): 15-16. živić, i., marković, z. & brajković, m., 2006: new trichoptera for the fauna of serbia. acta entomologica serbica, 11(1/2): 51-60. živić, i. & marković, z., 2006: saprobiological analysis of water of the southern morava river (a second order tributary of the danube in serbia) on the basis of macrozoobenthos as a bioindicator. proceedings 36th international conference of iad: 301-306. biologica nyssana 5 (1)  september 2014: 53-61 vulić, i. et al.  taxonomic composition and community structure... 62 anticancer compounds from medicinal plants biologica nyssana 5 (2)  december 2014: 113-121 ilić milošević, i. et al.  tritrophic accociations of lysiphlebus fabarum… 113 original article received: 5 december 2014 revised: 18 december 2014 accepted: 20 december 2014 tritrophic associations of lysiphlebus fabarum (marshall) (hymenoptera, braconidae, aphidiinae) in serbia marijana ilić milošević1*, maja lazarević1, olivera petrović-obradović2, vladimir žikić1 1university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia 2faculty of agriculture, nemanjina 6, 11081 belgrade – zemun, university of belgrade, serbia * corresponding author: marijanailic83@yahoo.com abstract: ilić milošević, m., lazarević, m., petrović-obradović, o., žikić, v.: tritrophic associations of lysiphlebus fabarum (marshall) (hymenoptera, braconidae, aphidiinae) in serbia. biologica nyssana, 5 (2), december 2014: 113-121. one hundred and eighty-six tritrophic parasitoid-aphid plant associations were established between lysiphlebus fabarum and 64 aphids with 148 different plant species from the territory of serbia. we identified 18 aphid genera of which the genus aphis was represented by 38 species. the most frequent interactions between the parasitoid and the hosts were the ones of aphis fabae, a. craccivora and a. ruborum on various plants comprising one third of the total trophic interactions listed here. key words: parasitoids, trophic chains, distribution, aphids, host plant apstrakt: ilić milošević, m., lazarević, m., petrović-obradović, o., žikić, v.: tritrofičke asocijacije vrste lysiphlebus fabarum (marshall) (hymenoptera, braconidae, aphidiinae) u srbiji. biologica nyssana, 5 (2), decembar 2014: 113-121. na teritoriji srbije je sada ustanovljeno 186 tritrofičkih asocijacija između parazitoida, biljnih vaši i biljaka. vrsta lysiphlebus fabarum je registrovana na 64 vrste biljnih vaši koje napadaju 148 biljnih vrsta. utvrđeno je da se biljne vaši grupišu u 18 različitih rodova, od kojih je najzastupljeniji bio rod aphis sa 38 vrsta. najčešće parazitirane vrste biljnih vaši od strane l. fabarum bile su aphis fabae, a. craccivora i a. ruborum prikupljene sa mnogih biljaka, formirajući oko jednu trećinu svih zabeleženih trofičkih interakcija. key words: parazitoidi, trofički lanci, distribucija, biljne vaši, biljke introduction the subfamily aphidiinae (hymenoptera: aphidiidae) represents a small group of solitary endoparasitoid wasps of aphids including many important species acting as biological control agents (b r e w e r & e l l i o t t , 2004; l e v i e et al., 2005). they are very useful in crop fields, as well as in greenhouses and non-arable fields such as meadows and forests reducing the number of pest aphids 5 (2) • december 2014: 113-121 biologica nyssana 5 (2)  december 2014: 113-121 ilić milošević, i. et al.  tritrophic accociations of lysiphlebus fabarum… 114 (s t a r ý , 1970, 1974; h a g v a r & h o f s v a n g , 1991; k a v a l l i e r a t o s et al., 2004). one genus in subfamily aphidiinae which has been studied in detail is lysiphlebus foerster, which comprises about 25 species worldwide, 16 occuring in the european fauna (y u et al, 2013; van achterberg, 2013). on the territory of serbia, ten species were recorded including the two that have already been introduced here: l. orientalis starý & rakhshani, 2006 and l. testaceipes (cresson, 1880) (p e t r o v i ć et al., 2013; ž i k i ć et al., 2015). according to the wing venation, this genus could fall into two species groups. the “fabarum” group includes the species with a long metacarpal vein while in “testaceipes” group the species have a short metacarpal vein (p e t r o v i ć et al., 2015). lysiphlebus fabarum (marshall, 1896) is a polyphagous aphid parasitoid that attacks more than a hundred of aphid species in various habitats (y u et al., 2013). they often exploit small aphids, usually from the genus aphis l., including some serious pests, e.g. the black bean aphid, a. fabae scopoli, the cowpea aphid, a. craccivora koch and the cotton aphid, a. gossypii (glover) (k a v a l l i e r a t o s et al., 2004; s t a r ý , 2006) but also the target aphid genus for l. fabarum is brachycaudus van der goot, e.g. the plum-thistle aphid, b. cardui (l.) and the leaf-curling plum aphid, b. helichrysi (kaltenbach). likewise, the species l. fabarum has a tendency to attacks the antprotected aphid colonies. its distribution is very wide, including the whole palaearctic, as well as australia and oceania. in europe, l. fabarum is found in the fauna of andorra, bulgaria, the canary islands, corsica, croatia, czech republic, france, germany, great britain, greece, hungary, italy, latvia, lithuania, madeira, montenegro, poland, russia, sicily, slovakia, slovenia, spain, and the netherlands. there are few studies regarding tritrophic associations of l. fabarum, and its diversity in agroecosystems in serbia (t o m a n o v i ć & b r a j k o v i ć , 2001; k a v a l l i e r a t o s et al., 2004; ž i k i ć et al., 2012). in this study we present the parasitoid-aphidplant trophic associations of l. fabarum registered on the territory of serbia. the knowledge about parasitoid-aphid-plant associations appears to be necessary for a better implementation of programs of biological control of aphids as an economically important group of pests. also, host-plant associations are important for better knowledge of biodiversity of species l. fabarum. material and methods we used the data from the collections of lysiphlebus fabarum sampled during the period 1989-2014 from numerous localities throughout serbia. our samples represent cut branches infested by aphids, sometimes with visible signs of parasitization. this plant material., with or without mummified aphids, was put in small plastic meshcovered boxes to enable ventilation, and then placed in a growth cabinet (22.5°c, 65% relative air humidity, 16:8 h, light:dark photoperiod) until the parasitoid emergence. before leaving the samples in the growth cabinet, some live adult aphids were preserved in 90% ethyl-alcohol for identification. parasitoid material and plants were deposited at the faculty of sciences and mathematics, department of biology and ecology, university of niš, serbia and the faculty of biology, university of belgrade, serbia. in order to obtain some data relevant literature was used (ž i k i ć et al., 2012). the aphids used in this study were from the collection of dr olivera petrović-obradović (faculty of agriculture, university of belgrade, serbia). the nomenclature of aphids is given according to r e m a u d i è r e and r e m a u d i è r e (1997), whereas the nomenclature of plants is based on flora europaea (1964, 1968, 1972, 1976, 1980) and g r e u t e r et al. (1984, 1986, 1989). results and discussion the results shown in appendix 1 refer to the long time period of investigation of lysiphlebus fabarum and all the interactions with aphids infesting analysed plants on the territory of serbia. analysing established trophic chains, we found 186 tritrophic parasitoid-aphid-plant associations including 64 hosts on 148 plants. the aphids belong to 18 genera. it is evident that the species belonging to the genus aphis, were most frequently recorded in this investigation, about 38 taxa. the species a. fabae and a. craccivora made a great number of associations with different plants. we registered 57 trophic chains for a. fabae together with two subspecies, a. fabae cirsiiacanthoidis scopoli and a. fabae solanella theobald, species a. fabae forming 75 interactions with different plant species. as far as the species aphis craccivora is concerned, we counted 30 associations with plants. the third most commonly encountered host species was a. ruborum, forming 11 associations. biologica nyssana 5 (2)  december 2014: 113-121 ilić milošević, i. et al.  tritrophic accociations of lysiphlebus fabarum… 115 conclusion based on such a large number of recorded associations of the parasitoid lysiphlebus fabarum and the previous studies regarding this species, l. fabarum is a suitable candidate for the biological control programs of various aphid species. future investigations should be focused on interactions between many aphid species and l. fabarum, especially the very close relation between l. fabarum and polyphagous aphid, aphis fabae as its host. through different methods, such as the level of parasitism success, field campaign, and the introduction or detection of bacterial symbionts using suitable molecular markers have already been applied in the pioneering work by v o r b u g e r et al. (2009). acknowledgements. we wish to thank dr. željko tomanović and dr. andjeljko petrović (faculty of biology, university of belgrade) for the loan of specimens; saša stanković (faculty of sciences and mathematics, university of niš) for his help in sampling material and dr. nickolas kavallieratos (benaki phytopathological institute, athens, greece). this study was supported by the ministry of education, science, and technological development of the republic of serbia (iii43001). references brewer, m. j., elliott, n. c. 2004: biological control of cereal aphids in north america and mediating effects of host plant and habitat manipulations. annual review of entomology, 49: 219–242. greuter, w., burdet, h. m., long, g. 1984: medchecklist 1. conservatoire et jardin botaniques de la ville de gèneve, med-checklist. trust of optima, gèneve, 330 pp. greuter, w., burdet, h. m., long, g. 1986: medchecklist 3. conservatoire et jardin botaniques de la ville de gèneve, med-checklist. trust of optima, gèneve, 395 pp. greuter, w., burdet, h. m., long, g. 1989: medchecklist 4. conservatoire et jardin botaniques de la ville de gèneve, med-checklist. trust of optima, gèneve, 458 pp. hågvar, e. b., hofsvang, t. 1991: aphid parasitoids (hymenoptera: aphidiidae): biology, host selection and use in biological control. biocontrol news and information, 12 (1): 13-42. kavallieratos, n. g., tomanović, ž., starý, p., athanassiou, c. g., sarlis, g. p., petrović, o., niketić, m. a., anagnou-veroniki, m. 2004: a survey of aphid parasitoids (hymenoptera: braconidae: aphidiinae) of southeastern europe and their aphid-plant associations. applied entomology and zoology, 39 (3): 527–563. levie, a., legrand, m. a., dogot, p., pels, c., baret, p., hance, t. 2005: mass releases of aphidius rhopalosiphi (hymenoptera: aphidinae), and strip management to control of wheat aphids. agriculture, ecosystems and environment, 105:17–21. petrović, a., mitrović, m., ivanović, a., žikić, v, kavallieratos, n. g., starý, p., mitrovskibogdanović, a., tomanović, ž., vorburger, c. 2015: genetic and morphological variation in sexual and asexual parasitoids of the genus lysiphlebus – an apparent link between wing shape and reproductive mode. bmc evolutionary biology (in press). petrović, a., mitrović, m., starý, p., petrovićobradović, o., žikić, v., tomanović, ž., vorburger, c. 2013: lysiphlebus orientalis (hymenoptera: braconidae), a new invasive aphid parasitoid in europe – evidence from molecular markers. bulletin of entomological research, 103 (4): 451-457. remaudière, g., remaudière, m. 1997: catalogue des aphididae du monde – of the world’s aphididae. homoptera aphidoidea. inra editions, paris, 473p. starý, p. 1970: biology of aphid parasites (hymenoptera: aphidiidae) with respect to integrated control. dr. w. junk, the hague. 643 pp. starý, p. 1974: taxonomy, origin, distributionand host range of aphidius species(hymenoptera, aphidiidae) in relation to biological control of the pea aphid ineurope and north america. zeitschrift furangewandte entomologie, 77 (2): 141-171. starý, p. 2006: aphid parasitiods of the czech republic. academia, prague, czech republic. tomanović, ž., brajković, m. 2001: aphid parasitoids (hymenoptera, aphidiidae) of agroecosystems of the south part of the pannonian area. archives of biological sciences, 53 (1-2): 57-64. tutin, t. g., heywood, v. h., burges, n. a., moore, d. m., valentine, d. h., walters, s. m., webb, d. a. (eds.), 1964-1980: flora europaea, i-v. cambridge university press. london. van achterberg, c., 2013. fauna europaea: braconidae. in: fauna europaea: hymenoptera: symphyta + ichneumonoidea, fauna europaea, version 2.6.2. available through: http://www.faunaeur.org vorburger, c., sandrock, c., gouskov, a., castañeda, l. e., ferrari, j. 2009: genotypic variation and the role of defensive biologica nyssana 5 (2)  december 2014: 113-121 ilić milošević, i. et al.  tritrophic accociations of lysiphlebus fabarum… 116 endosymbionts in an all-parthenogenetic host– parasitoid interaction. evolution, 63 (6):1439-50. yu, d. s., van achterberg, c., horstmann, k. 2013. world ichneumonoidea 2011. taxonomy, biology, morphology and distribution. taxapad (scientific names for information management), interactive catalogue, ottawa available on: www.taxapad.com. žikić, v., ilić-milošević, m., stanković, s., petrović, a., petrović-obradović, o., kavallieratos, n., starý, p., tomanović, ž. 2012: aphidiinae (hymenoptera: braconidae) of serbia and montenegro – tritrophic interactions. acta entomologica serbica, 17 (1/2): 83-105. žikić, v., stanković, s. s., ilić milošević, m., petrović-obradović, o., petrović, a., starý, p., tomanović, ž. 2015: first detection of lysiphlebus testaceipes (cresson) (hymenoptera: aphidiinae) in serbia: an introduced species invading europe?. northwestern journal of zoology, 11 (1): 97-101. appendix 1. list of registered tritrophic associations plant aphid finding althaea officinalis l. aphis fabae novi sad, 10.06.1997, žt aphis davletshinae hille ris lambers surčin, 20.05.1989, op aphis fabae glover zemun, galenika, 07.06.2011, ap amaranthus retroflexus l. aphis fabae belgrade, crveni krst, 05.07.1996, žt; lebane, konjino, 06.07.2010, ss. arctium lappa l. aphis fabae suva planina mt., vetanska reka river, 20.07.1997, žt; sićevačka klisura gorge, ostrovica, 23.06.2012, ss; 28.05.2013, mđ; tara mt., mitrovac, 25.06.2013, vž atriplex patula l. aphis craccivora palićko jezero lake, 06.09.1995; žt zemun, 09.07.1996, op; ballota nigra l. aphis balloticola szelegiewicz belgrade, avala mt.,01.06.1996, žt; obedska bara swamp, 07.06.1997, žt; stara pazova, 10.06.1997, žt; belgrade, radmilovac, 18.06.1999, žt; niš, popovac, 25.06.2010, vž; niš, bubanj, 05.06.2013, mđ aphis spp. zemun, izbis, 30.05.2011, ap; beta vulgaris l. aphis fabae zemun, 27.09.1990, žt; capsella bursa pastoris (l.) medik. aphis fabae čemernik, 04.08.2011, ss; capsicum annuum l. myzus persicae kikinda, 23.06.1997, žt; carduus acanthoides l. aphis fabae stara planina mt., balta berilovac, 22.07.1997, žt; slankamen, 24.06.2011, žt; brachycaudus cardui (l.) zrenjanin, 20.06.1996, žt; užice, trešnjica, 23.07.1996, žt; beljanica mt., 03.08.1996, žt; bečej, 03.08.1996, žt; mol, 03.08.1996, žt; rusko selo, 03.08.1996, žt; sefkerin, 03.08.1996, žt; perlez, 23.06.1997, žt; stara planina mt., glogovac, 16.07.1997, žt; stara planina mt., balta berilovac, 20.07.1997, žt; stara planina mt., ćuštica, 20.07.1997, žt; beljanica mt., 12.07.1998, op; pčinja canyon, prohor pčinjski, 10.06.2014, vž; uroleucon sp. mordvilko surduk, 21.06.2011, žt; carduus crispus l. brachycaudus carduii (l.) vlasina plateau, čemernik, 04.08.2011, ss; carduus nutans l. brachicaudus carduii (l.) niš, popovac, 23.06.2012, ss; carduus sp. aphis fabae kragujevac, ilićevo, 06.06.2011, amb;valjevo, petnica, 12.06.2011, ap; brachycaudus carduii (l.) vlasinsko jezero lake, 06.08.2010, ss;đerdap, veliki kazan, 28.05.2011, žt; valjevo, petnica, 12.06.2011, ap; dukat mt., 06.08.2011, ap; carlina acaulis l. aphis carlinae (borner 1940) šara mt., ošljak, 04.07.1995, op; centaurea cyanus l. aphis celastrii matsumura divčibare, 08.08.2000, op; lebane, konjino, 31.05.2014, ss; aphis fabae lebane, konjino, 31.05.2014, ss; centaurea sp. brachycaudus cardui (l.) kopaonik mt., brzeće, 29.07.2010, ss; cephalaria transsylvanica (l.) schrad. aphis craccivora niš, duvanište, 13.06.2013, pm; chamaecytisus sp. aphis craccivora dukat mt., 29.06.2012, ss; biologica nyssana 5 (2)  december 2014: 113-121 ilić milošević, i. et al.  tritrophic accociations of lysiphlebus fabarum… 117 plant aphid finding chaenopodium album l. aphis fabae zemun, 30.10.1990, op; belgrade, botanical garden, 29.06.1996, žt; niš, kunovica, 16.10.1998, vž; niš, popovac, 22.05.2010, vž; niš, popovac, 22.05.2010, ss; niš, popovac, 10.07.2010, vž; novi belgrade, block 62, 28.05.2011, žt; sićevačka klisura gorge, sićevo, 04.06.2011, ss; zemun, galenika, 07.06.2011,ap; slankamen, 24.06.2011, žt; lebane, konjino, 01.06.2013, ss; sićevačka klisura gorge, sićevo, 06.06.2013, vž; tara mt., derventa canyon, 25.06.2013, vž; sićevačka klisura gorge, sićevo, 17.07.2013, ss; aphis spp. majur, 21.06.2011, žt; chrysanthemum sp. aphis fabae glover kanjiža, 08.06.1995, žt; cichorium intibus l. aphis intybi koch bela crkva, 13.06.1996, žt; kragujevac, 05.07.2011, amb; niš, matejevac, 23.06.2012, vž; lebane, konjino, 31.05.2014, ss; aphis spp. negotin, 01.07.2011, žt; aphis craccivora tara, derventa canyon, 03.07.2012, vž; cirsium acanthoides l. aphis fabae lebane, konjino, 31.05.2014, ss; brachycaudus cardui (l.) lebane, konjino, 31.05.2014, ss; cirsium arvense (l.) scop. aphis fabae vlasinsko jezero lake, 11.08.2006, vž; vlasinsko jezero lake, 06.08.2010, vž; belgrade, ostružnica, 06.07.2011, žt; vlasinsko jezero lake, 03.08.2011, vž; vlasinsko jezero lake, 28.06.2012, vž; vlasinsko jezero lake, 15.06.2013, ss; vlasinsko jezero lake, 21.07.2013, ss; merošina, oblačinsko jezero lake, 15.06.2014, ss; aphis fabae cirsiiacanthoidis scopoli surčin, 29.05.1993, žt; vlasinsko jezero lake, 01.08.1996, žt; vlasina plateau, čemernik, 24.07.1997, žt; belgrade, kovilovo, 11.06.1999, žt; belgrade, radmilovac, 18.06.1999, žt; suva planina mt., glogovac, 16.07.1999, žt; zrenjanin, ečka, 31.05.2001, žt; kopaonik mt., brzeće, 29.07.2010, vž; vlasinsko jezero lake, 06.08.2010, vž; vlasina plateau, čemernik, 07.08.2010, ss; vlasinsko jezero lake, 21.07.2013, ss; aphis fabae solanella theobald valjevo, petnica, 12.06.2011, ap; uroleucon aeneum (hille ris lambers) kopaonik, 27.07.2010, vž; zemun, 02.06.2011, žt; vlasina plateau, čemernik, 1400 m, 04.08.2011, vž; bajina bašta, 06.07.2012, vž; merošina, oblačinsko jezero lake, 15.06.2014, ss; cirsium eriophorum (l.) scop. aphis fabae cirsiiacanthoidis scopoli vlasinsko jezero lake, 1250 m, 21.07.2013, ss; brachycaudus cardui (l.) suva planina mt., korube, 17.07.1997, žt; vlasinsko jezero lake, 1250 m, 21.07.2013, ss; cirsium lanceolatum (l.) scop. brachycaudus cardui (l.) beljanica mt., 12.07.1997, žt; aphis fabae cirsiiacanthoidis scopoli stara planina mt., balta berilovac, 20.07.1997, žt; cirsium palustre (l.) scop. aphis fabae cirsiiacanthoidis scopoli goč mt., 13.07.2011, žt; brachycaudus cardui (l.) vlasinsko jezero lake, 21.07.2013, ss; cirsium sp. aphis fabae cirsiiacanthoidis scopoli kopaonik mt., 17.07.2013, ss; cirsium vulgare (savi) ten. brachycaudus cardui (l.) bosilegrad, jarešnik, 22.07.2013, ss; clematis sp. aphis spp. užice, djetinja canyon, 26.06.1999, žt; coreopsis verticillata l. aphis fabae zemun, 17.10.1991, op; coronilla varia l. aphis craccivora niš, pantelej, 28.06.2012, vž; niš, trošarina, 26.04.2014, vž; corydalis sempervirens (l.) pers. aphis fabaei glover niš, pantelej, 01.06.2013, vž; crepis biennis l. aphis crepidis (börner) mol, 23.06.1997, žt; aphis spp. žitište, 22.07.1997, žt; crepis foetida l. uroleucon cichorii (koch) niš, jasenovik, 12.06.2011, mđ; vlasinsko jezero lake, 1250 m, 21.07.2013, ss; aphis gr craccivora niš, pantelej, 01.06.2013, vž; crepis sp. aphis spp. niš, bubanj, 31.05.2013, mđ; aphis craccivora niš, donji matejevac, 05.06.2013, vž; niš, tvrdjava, 05.06.2013, mđ; aphis crepidis (börner) vlasinsko jezero lake, 1250 m, 28.06.2012, ss; cucumis sativus l. aphis fabaei glover užice, carina, 05.08.1996, žt; biologica nyssana 5 (2)  december 2014: 113-121 ilić milošević, i. et al.  tritrophic accociations of lysiphlebus fabarum… 118 plant aphid finding daucus carota l. semiaphis dauci (fabricius) carska bara, 02.07.1996, op; daucus sp. semiaphis dauci (fabricius) kruševac, slobodište, 13.07.2012, zk; digitalis ambigua murray aphis fabae tara, derventa canyon, 03.07.2012, vž; dorycnium herbaceum vill. aphis craccivora zlatarsko jezero lake, 20.06.1998, žt; epilobium angustifolium l. aphis spp. kopaonik mt., 27.07.2010, vž; epilobium sp. aphis spp. sombor, 16.07.1996, op; euphorbia cyparissias l. aphis spp. niš, bubanj, 11.05.2013, mđ; euonymus japonicus l.f. aphis fabae lebane, konjino, 31.05.2014, ss; aphis fabae solanella theobald lebane, konjino, 31.05.2014, ss; euonymus europaeus l. aphis fabae belgrade, botanical garden, 17.06.1997, žt; filipendula ulmaria (l.) maxim. macrosiphum cholodkovskyi (mordvilko) dukat mt., 29.06.2012, vž; foeniculum vulgare mill. cavariella sp. del guercio sićevačka klisura gorge, sićevo, 28.05.2013, zk; galium aparine l. aphis craccivora vlasinsko jezero lake, 06.06.2014, ss; aphis fabae belgrade, dušanovac, 20.05.1993, žt; niš, tvrdjava, 05.06.2013, mđ; vlasinsko jezero lake, 06.06.2014, ss; genista tinctoria l. aphis genistae (börner) belgrade, ostružnica, 17.06.2010, žt; gentiana asclepiadea l. aphis gentianae (börner) stara planina mt., babin zub, 19.07.1994, op; geranium sp aphis spp. zemun, galenika, 07.06.2011, ap; hedera helix l. aphis hederae kaltenbach zemun, 13.06.2011, op; zemun, 14.06.2011, op; niš, trošarina, 18.05.2013, mđ; niš, pantelej, 01.06.2013, vž; heracleum sphondylium l. aphis fabae belgrade, botanical garden, 29.06.1996, žt; suva planina mt., toponički put, 19.07.1997, žt; hypochaeris radicata l. uroleucon sp. mordvilko adžine livade, žeželj, 01.06.2011, žt; nasonovia sp. mordvilko adžine livade, žeželj, 01.06.2011, žt; iris germanica l. aphis newtoni theobald belgrade, 13.10.1997, žt; knautia drymeia heuff. macrosiphum knautiae holman sićevačka klisura gorge, ostrovica, 28.05.2013, mđ; kochia scoparia (l.) schrad. aphis sp. lebane, konjino, 06.07.2010, ss; lactuca sp. aphis craccivora lebane, konjino, 01.06.2013, ss; lamium sp. aphis spp. zemun, 23.06.1995, op; smederevo, lugavčina, 06.06.1999, op; laserpitium halleri crantz aphis spp. zemun, altina, 09.06.2011, ap; dobanovci, 17.06.2011, ap; levisticum officinale w. d. j. koch aphis fabae užice, 14.08.1999, žt; lilium sp. aphis spp. belgrade, zvezdara, 10.05.1997, žt; lolium italicum a. braun sitobion avenae (fabricius) obrenovac, 15.06.1989, op; lolium perene l. aphis fabae lebane, konjino, 06.06.2010, ss; lotus corniculatus l. aphis craccivora kruševac, slobodište, 22.06.2013, zk; lycopus europaeus l. aphis spp. valjevo, petnica, 17.05.1998, žt; lythrum salicaria l. aphis sp. dobanovci, 17.06.2011, žt; sićevačka klisura gorge, sićevo, 28.05.2013, zk; malus domestica borkh. aphis pomi de geer novi belgrade, 15.11.1992, op; niš, donji matejevac, 05.06.2013, vž; dysaphis plantaginea (passerini) niš, pasi poljana, 27.05.2013, vž; malus sp. aphis pomi de geer zemun, galenika, 18.06.2011, ap; malva sylvestris l. aphis umbrella (börner) belgrade, kalemegdan, 29.09.1990, op; novi belgrade, 23.11.1991, op; zemun, 07.12.1991, op; belgrade, radmilovac, 22.06.1995, op; višnjica, 15.06.2011, op; niš, mediana, 21.05.2013, ss; niš, quay, 02.07.2013, mđ; niš, trošarina, 28.09.2013, vž; niš, mediana, 25.10.2013, vž; matricaria chamomilla l. pro parte aphis fabae lebane, konjino, 01.06.2013, ss; matricaria inodora l. aphis fabae surčin, 12.05.1989, op; matricaria perforata mérat aphis fabae vlasinsko jezero lake, 05.08.2011, ss; medicago lupulina l. aphis craccivora užice, djetinja canyon, 26.06.1999, op; medicago minima (l.) bartal. aphis craccivora vlasinsko jezero lake, 05.08.2011, ss; medicago sativa l. aphis craccivora kosmaj mt., 24.05.1995, žt; palićko jezero lake, 06.09.1995, žt; bečej, 20.06.1996, žt; zrenjanin, 20.06.1996, žt; žabalj, biologica nyssana 5 (2)  december 2014: 113-121 ilić milošević, i. et al.  tritrophic accociations of lysiphlebus fabarum… 119 plant aphid finding 20.06.1996, žt; belgrade, besni fok, 10.06.1997, žt; kač, 10.06.1997, žt; perlez, 23.06.1997, žt; senta, 23.06.1997, žt; valjevo, petnica, 28.06.1997, ap; zemun, 20.05.2011, ap; novi belgrade, block 45, 06.06.2011, zk; bešnjaja mt., 14.06.2011, žt; zemun, galenika, 17.06.2011, ap; slankamen, 24.06.2011, žt; kragujevac, ilićevo, 06.07.2011, amb; melilotus album medik. aphis craccivora vlasinsko jezero lake, 28.06.2012, vž; melilotus officinalis (l.) pall. aphis craccivora žitište, 22.06.1997, žt; negotin, 01.07.2011, op; mentha aquatica l. aphis affinis del geurcio surčin, 20.05.1989, op; belgrade, radmilovac, 12.06.1995, op; užice, duboko, 18.06.1995, žt; belgrade, radmilovac, 09.06.2011, op; mentha longifolia (l.) huds. aphis afinis del geurcio vlasinsko jezero lake, 15.06.2013, ss; mentha piperita l. aphis affinis del geurcio kruševac, 18.06.2013, zk; musa sapientia cavendish aphis spiraecola patch kruševac, 21.06.2013, zk; aphis fabae kruševac, 21.06.2012, zk; nepeta nuda l. aphis nepetae kaltenbach bosilegrad, jarešnik, 22.07.2013, vž; aphis frangulae kaltenbach dukat mt., 06.08.2011, ap; aphis origani passerini beljanica mt., 12.07.1998, žt; oenanthe stenoloba schur aphis fabae vlasinsko jezero lake, 05.08.2011, mim; oenothera biennis l. aphis oenotherae oestlund novi belgrade, block 45, 05.06.2002, žt; onopordum acanthium l. brachicaudus carduii (l.) preševo, slavujevac, 10.06.2014, ss; papaver somniferum l. aphis fabae kanjiža, 08.06.1995, žt; pastinaca sativa l. aphis fabae belgrade, radmilovac, 22.08.1996, op; aphis spp. vlasinsko jezero lake, 05.08.2011, mim; phaseolus vulgaris l. aphis fabae kovačica, 22.06.1996, žt; aphis craccivora užice, duboko, 18.06.1995, žt; phragmites australis (cav.) trin. hyalopterus pruni (geoffroy) belgrade, radmilovac, 10.05.1995, op; picris hieracioides l. hyperomyzus picridis (börner & blunck) palićko jezero lake, 06.09.1995, žt; plantago lanceolata l. aphis fabae vlasinsko jezero lake, 05.08.2011, mim; polygonum aviculare l. myzus persicae suva planina mt., toponički put, 19.07.1997, žt; aphis polygonata (nevsky) zemun, 05.06.2011, ap; zemun, galenika, 09.06.2011, ap; polygonum lapathifolium l. capitophorus sp. van der goot niš, pantelej, 13.10.3013, vž; populus tremula l. capitophorus sp. van der goot niš, popovac, 22.05.2010, vž; prunus cerasifera ehrh. hyalopterus pruni (geoffroy) belgrade, karađorđev park, 08.06.1996, žt; phorodon humuli novi belgrade, 17.06.1993, op; prunus cerasifera pisardi (carrière) l. h. bailey phorodon humuli niš, mediana, 21.05.2013, vž; prunus cerasus l. myzus cerasi niš, pmf, 10.05.2013, vž; brachycaudus helichrysi belgrade, radmilovac, 10.05.1995, op; prunus persica (l.) batsch brachycaudus schwartzi belgrade, 02.05.1995, op; belgrade, radmilovac, 10.05.1995, op; smederevo, lugavčina, 24.05.1995, op; niš, pasi poljana, 27.05.2013, vž; punica granatum l. aphis nasturtii kaltenbach belgrade, crveni krst, 17.07.1996, žt; ranunculus sp. aphis fabae sićevačka klisura gorge, ostrovica, 28.05.2013, mđ; ribes sp. aphis schilderi (börner) belgrade, konjarnik, 06.06.2011, žt; robinia pseudacacia l. aphis craccivora deliblatska peščara sands, devojački bunar, 29.06.1992, op; niš, pmf, 02.06.2012, vž; slankamen, 24.06.2011, žt; rosa canina l. aphis spp. brestovik, 27.05.2011, žt; aphis spp. smederevo, 27.05.2011, žt; rosa sp. aphis spp. koštunići, 17.06.2011, žt; rubus sp. aphis ruborum bešnjaja mt., 14.06.2011, žt; sićevačka klisura gorge, ostrovica, 29.05.2010, vž; zemun, 06.06.2011, op; koštunići, 17.06.2011, žt; sićevačka klisura gorge, sićevo, 01.06.2012, ss; sićevačka klisura gorge, ostrovica, 12.05.2013, vž; tara mt., derventa canyon, 26.06.2014, ss; rubus caesius l. aphis ruborum kragujevac, čumić, 14.05.2011, amb; sićevačka klisura gorge, sićevo, 04.06.2011, vž; ivanovići, 05.06.2011, žt; kragujevac, biologica nyssana 5 (2)  december 2014: 113-121 ilić milošević, i. et al.  tritrophic accociations of lysiphlebus fabarum… 120 plant aphid finding grošničko jezero lake, 05.06.2011, amb; ljig, 05.06.2011, žt; kragujevac, ilićevo, 06.06.2011, amb; kragujevac, košutnjačko brdo, 13.06.2011, amb; aphis idaei sićevačka klisura gorge, sićevo, 23.06.2012, vž; rubus discolor boiss. aphis ruborum preševo, slavujevac, 10.06.2014, ss; aphis idaei tara mt., derventa canyon, 03.07.2012, ss; rubus fruticosus l. aphis idaei niš, matejevac, 23.06.2012, vž; rubus idaeus l. aphis ruborum čačak, zdravljak, 24.05.2000, žt; aphis idaei valjevo, petnica, 18.05.1998, žt; banja vrujci spa, berkovac, 30.04.2000, žt; rumex acetosella l. aphis fabae kač, 10.06.1997, žt; rumex obtusifolius l. aphis fabae belgrade, dušanovac, 29.05.1993, žt; rumex sp. aphis fabae tara mt., derventa canyon, 03.07.2012, vž; niš, trošarina, 22.05.2013, mđ; sićevačka klisura gorge, sićevo, 28.05.2013, zk; salix cinerea l. aphis farinosa j. f. gmelin šara mt., 21.07.1995, op; aphis farinosa j. f. gmelin zlatar mt., aljinovići, 12.07.1991, op; salvia nemorosa l. aphis salviae walker uzdin, 22.06.1997, žt; salvia officinalis l. aphis salviae walker sićevačka klisura canyon, ostrovica, 28.05.2013, mđ; salvia sclarea l. aphis craccivora niš, pantelej, 23.06.2012, vž; salvia sp. aphis craccivora niš, bubanj, 05.06.2013, mđ; salvia verticillata l. aphis spp. l. beljanica mt., resava, 11.07.1998, žt; crna trava, 22.07.2001, op; dukat mt., 29.06.2012, ss; sanguisorba minor scop. aphis sp. l. preševo, 05.06.2011, op; kalna, 11.06.2011, op; aphis sanguisorbae (schrank) niš, donji matejevac, 05.06.2013, vž; pčinja canyon, gornji starac, 10.06.2014, ss; scabiosa argentea l. aphis sp. l. pčinja canyon, prohor pčinjski, 10.06.2014, vž; macrosiphum sp. passerini pčinja canyon, prohor pčinjski, 10.06.2014, vž; solanum nigrum l. aphis fabae solanella theobald obedska bara swamp, 15.06.1996, žt; apatin, prigrevica, 27.09.1997, žt; srbobran, 27.09.1997, žt; vršac, 02.10.1997, žt; sonchus oleraceus l. uroleucon sonchi (l.) brestovik, 27.05.2011, op; symphytum officinale l. aphis fabaei glover obedska bara swamp, 02.06.2011, op; sićevačka klisura gorge, 28.05.2013, mđ; aphis spp. ečka, 31.05.2001, žt; valjevo, petnica, 20.05.1996, ap; tamarix sp. brachyunguis tamaricis (lichtenstein) niš, niška banja spa, 06.06.2013, vž; tanacetum parthenium (l.) sch. bip. aphis fabae šara mt., muržička reka river, 21.07.1995, op; beljanica mt., suvaja, 11.07.1997, op; tanacetum vulgare l. metopeurum fuscoviride stroyan vlasinsko jezero lake, 06.08.2010, ss; vlasinsko jezero lake, 15.06.2013, ss; jerma canyon, 19.07.2013, ss; vlasinsko jezero lake, 21.07.2013, ss; taraxacum officinale weber aphis taraxicicola blackman belgrade, dušanovac 08.05.1993, žt; belgrade, dušanovac, 29.05.1993, žt; belgrade, crveni krst, 14.06.1997, žt; tecoma radicans (l.) juss. aphis spiraecola patch niš, pmf, 21.05.2013, ss; thymus sp aphis serpylli koch zlatar mt., 13.07.1991, op; tordylium maximum l. aphis spp. suva planina mt., vetanska reka river, 16.07.1997, žt; tragopogon sp. aphis spp. belgrade, botanical garden, 24.06.1999, žt; trifolium sp. aphis craccivora surčin, 15.06.2011, op; therioaphis trifolii monell surčin, 15.06.2011, op; trifolium lagopus pourr. aphis craccivora pčinja canyon, prohor pčinjski, 10.06.2014, bz; trifolium pratense l. aphis craccivora beljanica mt., 31.07.1996, op; kruševac, slobodište, 13.07.2012, zk; trigonella procumbens (besser.) rchb. aphis craccivora uzdin, 22.06.1997, žt; kostolac, 02.06.2001, žt; triticum aestivum l. anoecia corni (fabricius) suva planina mt., vetanska reka river, 17.06.1997, žt; triticum vulgare vill. sitobion avenae (fabricius) leskovac, 01.06.2013, vž; typha latifolia l. rhopalosiphum nymphaeae (l.) niš, niška banja spa, 23.07.2013, vž; urtica dioica l. aphis fabae brestovik, 27.05.2011, op; smederevo, 27.05.2011, op; aphis sp. niš, popovac, 22.05.2010, vž; sićevačka klisura gorge, sićevo, 29.05.2010, ss; biologica nyssana 5 (2)  december 2014: 113-121 ilić milošević, i. et al.  tritrophic accociations of lysiphlebus fabarum… 121 plant aphid finding aphis urticata j. f. gmelin smederevo, lugavčina, 15.04.1990, op; kokin brod, 12.07.1991, žt; belgrade, dušanovac, 08.05.1993, žt; surčin, 23.05.1993, žt; surčin, 02.05.1995, žt; kanjiža, 08.06.1995, žt; belgrade, radmilovac, 20.05.1996, žt; omoljica, 13.06.1996, žt; čenta, 20.06.1996, žt; užice, trešnjica, 23.07.1996, žt; belgrade, radmilovac, 10.06.1997, žt; novi sad, 10.06.1997, žt; belgrade, radmilovac, 13.05.1998, žt; niš, popovac, 10.05.2010, vž; lebane, konjino, 06.06.2010, ss; stara planina mt., bojanine vode, 11.07.2010, ss; sićevačka klisura gorge, sićevo, 04.06.2011, vž; rogojevac, 16.06.2011, op; sićevačka klisura gorge, sićevo, 23.06.2012, ss; kruševac, pakašnica, 11.05.2013, zk; sićevačka klisura gorge, sićevo, 12.05.2013, vž; sićevačka klisura gorge, sićevo, 28.05.2013, zk; niš, bubanj, 31.05.2013, mđ; lebane, konjino, 01.06.2013, ss; sićevačka klisura gorge, sićevo, 06.06.2013, vž; sićevačka klisura gorge, sićevo, 22.04.2014, vž; urtica urens l. aphis fabae kumane, 20.06.1996, žt; valeriana officinalis l. aphis fabae vlasinsko jezero lake, 21.07.2013, ss; verbascum nigrum l. aphis verbasci schrank vlasinsko jezero lake, 05.08.2011, vž; verbascum sp. aphis verbasci schrank gornjane, 06.07.1990, op; belgrade, 12.10.1990, op; kopaonik mt., brzeće, 25.08.1998, op; lebane, konjino, 01.07.2012, ss; niš, popovac, 08.06.2013, vž; viburnum opulus l. aphis viburni scopoli belgrade, žarkovo, 19.04.1990, op; vicia cracca l. aphis craccivora vlasinsko jezero lake, 05.08.2011, mim; lebane, konjino, 31.05.2014, ss; prokuplje, bresničić, 15.06.2014, vž; vicia cordata wulfen aphis craccivora niš, mediana, 21.05.2013, ss; zea mays l. aphis fabae beljanica mt., 30.07.1996, op; microsoft word 0602_stankovic_et_al biologica nyssana 1 (1-2) december 2010: 117-122 stanković s. et al. betula species as host plants for various insects… 117 original article ! betula species as host plants for various insects parasitized by braconids (hymenoptera: braconidae) in serbia saša stanković*, vladimir žikić, marijana ilić university of niš, faculty of science and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia e-mail: sasasta@gmail.com abstract: stanković, s., žikić, v., ilić, m.: betula species as host plants for various insects parasitized by braconids (hymenoptera: braconidae) in serbia. biologica nyssana, 1 (1-2), december 2010: 117-122. this work presents braconid wasps which can be found on insects attacking birches, especially betula alba and b. pubescens (betulaceae) which are autochthonous in serbia. we have found 49 braconid species from 26 genera on 40 phytophagous insects; one from the hemimetabolous order: homoptera: homoptera and three from the holometabolous orders: coleoptera, hymenoptera and lepidoptera. registered braconid species belong to the subfamilies: aphidiinae, braconinae, doryctinae, euphorinae, exothecinae, microgastrinae, orgilinae, rhysipolinae and rogadinae. although most of the registered phytophagous insects pose a significant threat to betula species, the two species: epirrita autumnata (geometridae) and lymantria dispar (noctuidae) are the most important, because they can defoliate entire forests when their populations are in gradation. also, there are two buprestid pests agrilus anxius native to north america and a. planipennis (buprestidae) from central asia which are considered as potentially invasive species. key words: betula, braconidae, hosts, tritrophic associations, serbia introduction ! braconidae wasps comprise at least 50.000 species of solitary or gregarious ectoand endoparasitoids that parasitize larvae of their hosts, as well as eggs and very rarely recorded, the adult stage. the body size of braconid wasps is in range from 0.5 mm to over 20 cm including the “fake ovipositor” (s t a r ý , 1970, 1988; s h a w & h u d d l e s t o n , 1991). there are two strategies among braconidae wasps: idiobionts that permanently parasitize their host before laying eggs and konobionts that allow the host to continue developing while the larvae feed within the host’s body. the great majority of braconidae are cosmopolitan and polyphagous insects. some species show different degrees of host specificity. there are programs of utilization of braconid wasps and other insects as biological control agents starting from b a i r d (1958), m a t h e w s (1974), l e w i s et al. (1977, 1990), over the fieldworks such as biocontrol of olive fruit flies (s i m e et al., 2006; y o k o y a m a et al., 2008), to brand-new, k u l a et al. (2010). not much data exists about the insect pests feeding on birch and their parasitoids from the family braconidae in europe, especially in serbia. some sporadically records exist in the following journals: (t o m a n o v i ć & k a v a l l i e r a t o s , 2002; k a v a l l i e r a t o s et al., 2002, 2004). world fauna of braconidae which attack pests in the birch communities is poorly investigated as well. birches are autochthones for the greater part of europe, as well as for the territory of serbia. we have chosen to present this work in this journal because the first issue is dedicated to vlasina lake, which is the place full of native birches: betula alba and b. pubescens (betulaceae). most important parasitoids 10th sfses • 17-20 june 2010, vlasina lake1 (1-2) • december 2010: 117-122 biologica nyssana 1 (1-2) december 2010: 117-122 stanković s. et al. betula species as host plants for various insects… 118 in biological control of the species from the genus agrilus (coleoptera: buprestidae), could be spathius agrili yang 2005, (braconidae) found in the usa on european birch betula alba (yang et al. 2008). the host range of s. agrili is presented by 18 wood boring insects belonging to the genus agrilus, who are present on the other host plants such as fraxinus, prunus and alianthus. material and methods the examined parasitoid wasps for this study were collected from the hosts feeding on betula alba and b. pubescens all over serbia. in the tritrophic associations: parasitoid-host-plant include all hosts found in europe. one part of specimens is deposited at the faculty of sciences and marthematics, department of biology and ecology, university of niš, serbia while the rest of specimens are at the faculty of biology, university of belgrade, serbia. identification of birch species was done according to v u k i ć e v i ć (1970). results and discussion the parasitoid subfamilies, genera and species and their hosts are arranged in four tables as trophic associations connected by (x). the total number of 49 braconids species from 26 genera belong to the following subfamilies: aphidiinae, braconinae, doryctinae, euphorinae, exothecinae, microgastrinae, orgilinae, rhysipolinae and rogadinae. we have found 42 host species belonging to four insect orders: coleoptera, homoptera, hymenoptera and lepidoptera (tab. 1). all registered host species are phytophagous insects feeding on leaves or they are xylophagous on birches. the trophic relations between aphidiinae and aphid hosts (homoptera: aphididae) are presented in the table 2. the species callaphis flava (aphididae) is registered as the host of four aphidiinae wasps: aphidius aquilus mackauer, 1961, praon flavinode (haliday, 1833), trioxys betulae (marshall, 1896) and t. cirsii (curtis, 1831). parasitoid wasp aphidius aquilus attack three of four recorded aphids. the ectoparasitoid groups of braconidae witch parasitize coleopteran and hymenopteran host larvae are shown in the table 3. the subfamily braconinae is presented by five parasitoids of coleoptra species coming from curculionidae and cerambycidae. bracon obscurator nees 1811 was found as parasitoid of the genus trypodendron: t. domesticum and t. signatum (curculionidae). for buprestid chrysobothris affinis (buprestidae) we noted four natural enemies: glyptomorpha pectoralis (brullé 1832), pseudovipio castrator (fabricius 1798), vipio appellator (nees 1834) from the subfamily braconinae and doryctes leucogaster (nees 1834) from the subfamily doryctinae. the species iphiaulax impostor (scopoli 1763) parasitizes aegomorphus clavipes and leiopus nebulosus from the family of lonhorned beetles (cerambycidae). braconidae attacking the caterpillars of various lepidopteran families found on birches belong to the genus bracon are b. abbreviator nees 1834, b. discoideus wesmael 1838, b. hebetor say 1836, b. intercessor nees 1834, b. romani fahringer 1927 and b. variegator spinola 1808 (tab. 4). among bracon species the most important is polyphagous b. intercessor which attacks various insects from coleopteran families, especially curculionidae and lepidoptera, sessidae synanthedon culiciformis, paranthrene tabaniformis. another important species is bracon hebetor recorded from many larvae of crambidae, gelechiidae, noctuidae and tortricidae (milonas, 2005); an important natural enemy of indian meal moth plodia interpunctella, (pyralidae), common as pest of stored products. subfamily doryctinae is presented by six species from four genera attacking different hosts form the order coleoptera: dendrosoter protuberans (nees 1834), doryctes leucogaster (nees 1834), ontsira ignea (ratzeburg 1852), o. imperator (haliday 1836), spathius curvicaudis ratzeburg 1844 and s. rubidus (rossi 1794), tab. 3, and from lepidoptera, only one but the most important and extremely polyphagous species, lymantiria dispar from the family noctuidae. it is interesting that the presence of any spathius parasitoid of agrylus cynaescens –species group (buprestidae), native in europe, but an invasive alien pest in north america is not reported in europe (j e n d e k & g r e b e n n i k o v , 2009). one of the candidates for the biological control of a. cynaescens is spathius rubidus known as polyphagous on various coleopteran families: buprestidae, cerambycidae, curculionidae, and rhizophagidae as well as xiphydriidae from the order of hymenoptera. in addition to this species, we report it as parasitoid of two species of agrylus: a. angustulus and a. hastulifer (buprestidae) and leiopus nebulosus and rhagium mordax from the family of cerambycidae. diversity and some parasitoid-host data are the best known for this subfamily among ectoparasitic (b e l o k o b y l s k i j & ž i k i ć , 2009). from the ectoparasitoid subfamily exothecinae we registered only two species: colastes braconius haliday 1833 parasitizes on heterarthrus vagans sawfly (tenthredinidae) and on another extremely polyphagous species epirrita autumnata geometridae), biologica nyssana 1 (1-2) december 2010: 117-122 stanković s. et al. betula species as host plants for various insects… 119 table 1. the list of pests on betula alba and b. pubescens order family species homoptera aphididae callaphis flava mordvilko, 1928 euceraphis betulae (koch 1855). euceraphis punctipennis (zetterstedt 1828) symydobius oblongus (von heyden, 1837). coleoptera buprestidae agrilus angustulus (illiger 1803) agrilus cyanescens ratzeburg 1837 agrilus hastulifer ratzeburg 1837 chrysobothris affinis (fabricius 1794) dicerca berolinensis (herbst 1779) curculionidae scolytus ratzeburgi janson 1856 trypodendron domesticum (linnaeus 1758) trypodendron signatum (fabricius, 1787) cerambycidae aegomorphus clavipes (schrank 1781) leiopus nebulosus (linnaeus 1758) rhagium mordax (de geer 1775) hymenoptera tenthredinidae fenusa pumila leach 1817 fenusella nana (klug 1816) heterarthrus vagans (fallen 1808) scolioneura betuleti (klug 1816) xiphydriidae xiphydria prolongata (geoffroy 1785) lepidoptera geometridae cyclophora pendularia (clerck 1759) epirrita autumnata (borkhausen 1794) hypagyrtis unipunctata (haworth 1809) gracillariidae caloptilia betulicola (hering 1928) heliozelidae heliozela betulae stainton 1890 noctuidae euproctis chrysorrhoea (linnaeus 1758) hyphantria cunea (drury 1773) lymantria dispar (linnaeus 1758) orthosia gothica (linnaeus 1758) orthosia incerta (hufnagel 1766) lasiocampidae malacosoma neustria (linnaeus 1758) psychidae megalophanes viciella (denis & schiffermuller 1775) sesiidae synanthedon culiciformis (linnaeus 1758) tortricidae acleris hastiana (linnaeus 1758) adoxophyes orana fischer von rösslerstamm 1834) apotomis sororculana (zetterstedt 1839) archips rosana (linnaeus 1758) pandemis cerasana (hubner 1786) pandemis heparana (denis & schiffermuller 1775) geometridae cyclophora pendularia (clerck 1759) hypagyrtis unipunctata (haworth 1809) coleophoridae coleophora serratella (linnaeus 1761) and shawiana catenator (haliday 1836) on caterpillars of caloptilia betulicola (gracillariidae). subfamily rogadinae assembles eight species of aleiodes: a. circumscriptus (nees 1834), a. gastritor (thunberg 1822), a. nigricornis wesmael 1838, a. pallidator (thunberg 1822), a. rossicus (kokujev 1898) and a. sanctihyacinthi (provancher 1880), distributed mostly in whole europe (aydogdu & beyarslan, 2006) and petalodes compressor (herrich-schäffer 1838). the species a. pallidator has been recorded as parasitoid of four lepidopteran larvae: cyclophora pendularia (geo table 2. subfamily aphidiinae and the aphid hosts c al la ph is fla va e uc er ap hi s be tu la e e uc er ap hi s pu nc tip en ni s s ym yd ob iu s ob lo ng us aphidius aquilus x x x betuloxys compressicornis x x praon flavinode x trioxys betulae x x trioxys cirsii x biologica nyssana 1 (1-2) december 2010: 117-122 stanković s. et al. betula species as host plants for various insects… 120 metridae), lymantria dispar, megalophanes viciella (psychidae) and apotomis sororculana (tortricidae) (tab. 4). the last registreted ectoparasitic braconidae subfamily is rhysipolinae, with only one species, rhysipolis meditator (haliday 1836) on caloptilia betulicola (gracillariidae). table 3. subfamilies of ectoparasitoid braconidae on coleopteran and hymenopteran hosts. t ry po de nd ro n do m es tic um t ry po de nd ro n si gn at um s co ly tu s ra tz eb ur gi a gr ilu s cy an es ce ns a gr ilu s an gu st ul us a gr ilu s ha st ul ife r c hr ys ob ot hr is a ffi ni s d ic er ca b er ol in en si s a eg om or ph us c la vi pe s le io pu s ne bu lo su s r ha gi um m or da x f en us a pu m ila f en us el la n an a h et er ar th ru s va ga ns s co lio ne ur a be tu le ti x ip hy dr ia p ro lo ng at a braconinae bracon obscurator x x glyptomorpha pectoralis x iphiaulax impostor x x pseudovipio castrator x vipio appellator x doryctinae dendrosoter protuberans x x doryctes leucogaster x ontsira ignea x ontsira imperator x x spathius curvicaudis x spathius rubidus x x x x x exothecinae colastes braconius x shawiana catenator x x x x table 4. subfamilies of ectoparasitoid braconidae on lepidopteran hosts c yc lo ph or a pe nd ul ar ia h yp ag yr tis u ni pu nc ta ta c al io ph ili a be tu lic ol a h el io ze la b et ul ae e pi rr ita a ut um na ta ly m an tr ia d is pa r e up ro ct is s im ili s h yp ha nt ria c un ea o rt ho si a go th ic a o rt ho si a in ce rt a m al ac os om a ne us tr ia m eg al op ha ne s vi ci el la s yn an th ed on c ul ic ifo rm is a cl er is h as tia na a do xo ph ye s or an a a po to m is s or or cu la na a rc hi ps r os an a p an de m is c er as an a p an de m is h ep ar an a braconinae bracon abbreviator x bracon discoideus x bracon hebetor x bracon intercessor x bracon romani x bracon variegator x x doryctinae doryctes leucogaster x exothecinae colastes braconius x rogadinae aleiodes circumscriptus x aleiodes gastritor x x aleiodes nigricornis x x aleiodes pallidator x x x x aleiodes rossicus x x x aleiodes sanctihyacinthi x aleiodes signatus x aleiodes testaceus, x petalodes compressor x rhysipolinae rhysipolis meditator x biologica nyssana 1 (1-2) december 2010: 117-122 stanković s. et al. betula species as host plants for various insects… 121 in this study we have found the data of three endoparasitic braconid subfamilies: euphorinae, microgastrinae and orgilinae (tab. 5). as bracon obscurator, cosmophorus regius niezabitowski 1910 (euphorinae) shows the same affinity to the species of trypodendron (tab. 2). two species of meteorus, m. pulchricornis (wesmael 1835) and m. meteorus, m. pulchricornis (wesmael 1835) and m. versicolor (wesmael 1835) attack the most common pest lymantria dispar. polyphagous epirrita autumnata is the host of solitary endoparasitoid zele deceptor (wesmael 1835). mostly gregarious, microgastrinae are presented by nine members on betula spesies that eight of them attack lymantria dispar: three species of apanteles: a. lacteicolor viereck 1911, a. melanoscelus (ratzeburg 1844) and a. xanthostigma (haliday 1834), one species of cotesia: c. lomerata (linnaeus 1758), microgaster hospes marshall 1885, and three species of protapanteles: p. fulvipes (haliday 1834), p. liparidis (bouche 1834) and p. immunis (haliday 1834). larvae of epirrita autumnata is parasitized by cotesia jucunda (marshall 1885). there is only one species of orgilinae, orgilus punctulator (nees 1811) has been found as pest of coleophora serratella (coleophoridae). table 5. subfamilies of euphorinae, microgastrinae and orgilinae and their hosts. tr yp od en dr on d om es tic um tr yp od en dr on s ig na tu m e pi rri ta a ut um na ta ly m an tri a di sp ar c ol eo ph or a se rra te lla euphorinae cosmophorus regius x x meteorus pulchricornis x meteorus versicolor x zele deceptor x microgastrinae apanteles lacteicolor x apanteles melanoscelus x apanteles xanthostigma x cotesia glomerata x cotesia jucunda x microgaster hospes x protapanteles fulvipes x protapanteles liparidis x protapanteles immunis x orgilinae orgilus punctulator x conclusion we noted that pest insects make no difference on which betula species they feed. the most important pests on betula as well as other trees are polyphagous species lymantria dispar and epirrita autumnata, because they can defoliate entire forests when their populations are in gradation. we have found that larvae of lymantria dispar on birches are attacked by 11 parasitoid species from the subfamilies braconinae, rogadinae, euphorinae and microgastrinae. the moth epirrita autumnata can be very dangerous in birch forests. the example from northern sweden shows that this pest’s caterpillars can defoliate birch forests, causing death of stems of the polycormic trees as a result (t e n o w , et al., 2004). there are three buprestid pests of the genus agrilus found on betula alba and b. pubescens: a. angustulus, a. cyanescens, a. hastulifer. they are extremely polyphagous wood borers. for now there is no data of the natural enemies of a. cyanescens. agrilus anxius, which is native to north america and a. planipennis from central asia are considered as potentially invasive species. acknowledgements. the authors wish to express their sincere thankfulness to dr. ž. tomanović (faculty of biology, belgrade, serbia). for the present work the authors were supported by the ministry of science and environment protection of the republic of serbia (grant no. 143006b). references aydogdu, m., beyarslan, a. 2006: first records of aleiodes wesmael, 1838 species in east marmara region of turkey (hymenoptera: braconidae: rogadinae). acta entomologica slovenica, 14(1): 81–88 baird, a. b. 1958: biological control of insects and plant pests in canada. prc 10th int. cong. ent. 4: 483-485. belokobylskij, s. a., žikić, v., 2009: new data on cyclostome braconid subfamilies doryctinae, exothecinae, rogadinae and braconinae (braconidae: hymenoptera) of serbia and neighbouring territories. acta entomologica serbica. 14(1): 65-71 georgiev, g. 2005: bioecological characteristics of bracon intercessor nees (hymenoptera: braconidae) as a parasitoid of the poplar clearwing moth, paranthrene tabaniformis (rott.) (lepidoptera: sesiidae) in bulgaria. journal of pest science, 78: 161-165 biologica nyssana 1 (1-2) december 2010: 117-122 stanković s. et al. betula species as host plants for various insects… 122 jendek, j., grebennikov, v. v. 2009: revision of the agrilus cyanescens species-group (coleoptera: buprestidae) with description of three new species from the east palaearctic region. zootaxa, 2139: 43–60. kavallieratos, n.g., athanassiou, c., stathas, g., tomanović ž. 2002: aphid parasitoids (hymenoptera, braconidae) on citrus: seasonal abundance, association with the species of host plant and sampling indices. phytoparasitica, 30: 365-377. kavallieratos, n. g., tomanović, ž., starý, p., athanassiou, c. g., sarlis, g. p., petrović, o., niketić, m. a., anagnou-veroniki, m., 2004: a survey of aphid parasitoids (hymenoptera: braconidae: aphidiinae) of southeastern europe and their aphid-plant associations. appl. entomol. zool. 39 (3): 527–563. kula, r. r., boughton, a.j., pemberton, r.w., 2010: stantonia pallida (ashmead) (hymenoptera: braconidae) reared from neomusotima conspurcatalis warren (lepidoptera: crambidae), a classical biological control agent of lygodium microphyllum (cav.) r. br. (polypodiales: lygodiaceae). proceedings of the entomological society of washington, 112(1): 61-68. lewis, w.j., nordlund, d.a., gross, h.r., jr., jones, r.l., jones, s.l., 1977: kairomones and their use for management of entomophagous insects: v. moth scales as a stimulus for predation of heliothis zea (boddie) eggs by chrysopa carnea stephens larvae. j. chem. ecol. 3: 483-487. lewis, w.j., vet, l.e.m., tumlinson, j.h., van lenteren, j.c., papaj, d.r., 1990: variations in parasitoid foraging behavior: essential element of a sound biological control theory. environmental entomology 19: 1183-1193. mathews, r.w., 1974: biology of braconidae. annual review of entomology 19: 15-32. milonas, p.g., 2005: influence of initial egg density and host size on the development of the gregarious parasitoid bracon hebetor on three different host species. biocontrol, 50: 415-428. shaw, m.r., huddleston, t., 1991: classification and biology of braconid wasps (hymenoptera: braconidae). royal entomological society of london 7, 11: 1-126. sime, k.r., daane, k.m., nadel, h., funk, c.s., messing, r.h., andrews jr, j.w., johnson, m.w., pickett, c.h., 2006: diachasmimorpha longicaudata and d. kraussii (hymenoptera: braconidae), potential parasitoids of the olive fruit fly. biocontrol science and technology, 16(2): 169-179. starý, p., 1988: aphidiidae. aphids, their biology, natural enemies and control. vol. 2b (a. k. minks and p. harrewijn eds.). elsevier, amsterdam, 171–184 pp. starý, p., 1970: biology of aphid parasites (hymenoptera:aphidiidae). dr w. junk, the hague, 643 p. tomanović, ž., kavallieratos, n.g., 2002: trioxys haliday (hymenoptera: braconidae: aphidiinae) in serbia and montenegro. acta entomologica serbica, 7(1/2): 67-81 vukićević, e., 1970: rod betula l. in: josifović, m., ed.: flora sr srbije, 2. sanu, beograd. yang. z-q., wang, x-y., gould, j.r., wu, h., 2008: host specificity of spathius agrili yang (hymenoptera: braconidae), an important parasitoid of the emerald ash borer. biological control, 47: 216–221. yokoyama, v.y., rendo, p.a., sivinski, j., 2008: psyttalia cf. concolor (hymenoptera: braconidae) for biological control of olive fruit fly (diptera: tephritidae) in california. environ. entomol. 37(3): 764-773. tenow, o., bylund, h., karlsson, p. s., hoogesteger, j., 2004: rejuvenation of a mountain birch forest by an epirrita autumnata (lepidoptera: geometridae) outbreak. acta oecologica, 25(1/2): 43-52. radulović et al. 2022, biologica nyssana 13(2) 13 (2) december 2022: 179-189 doi: 10.5281/zenodo.7437345 the essential oil composition of different parts of artemisia absinthium and its antibacterial activity against phytopathogenic bacteria original article maja radulović faculty of biology, university of belgrade, studentski trg 16, 11000 belgrade, serbia maja.radulovic@bio.bg.ac.rs (corresponding author) milan gavrilović faculty of biology, university of belgrade, studentski trg 16, 11000 belgrade, serbia nemanja rajčević faculty of biology, university of belgrade, studentski trg 16, 11000 belgrade, serbia tamara janakiev faculty of biology, university of belgrade, studentski trg 16, 11000 belgrade, serbia ivica dimkić faculty of biology, university of belgrade, studentski trg 16, 11000 belgrade, serbia pedja janaćković faculty of biology, university of belgrade, studentski trg 16, 11000 belgrade, serbia received: october 04, 2022 revised: december 01, 2022 accepted: december 02, 2022 abstract: the composition of essential oil (eo) from different aerial parts of artemisia absinthium l. (asteraceae), from serbia, was analyzed. shoots without leaves and inflorescences (swli), leaves (l), and inflorescences (i) were subjected to hydrodistillation using the clevenger-type apparatus. analysis of eo was done by gas chromatography with ms of flame ionization detection (gc/ms and gc/fid). microdilution method was used to determine the minimum inhibitory concentration (mic) and minimum bactericidal concentration (mbc). organoleptic characteristics and quantitative and qualitative differences were documented between examined eos. in total, 17 compounds were identified in swli, 11 in l and 18 in i. the principal constituent in all eos was trans-thujone (swli 23.56%, l 60.41%, and i 46.16%). the most susceptible strains were xanthomonas campestris pv. campestris, erwinia amylovora and rathayibacter tritici. the strongest activity was shown for swli eo against e. amylovora (0.03 mg/ml). the same mic values were observed for l eo against e. amylovora and r. tritici (0.09 mg/ ml). the equal activity for all eos tested was detected against x. campestris (0.13 mg/ml). key words: wormwood, asteraceae, hydrodistillation, volatile terpenes, antibacterial activity apstrakt: sastav etarskog ulja iz različitih delova vrste artemisia absinthium i njegova antibakterijska aktivnost protiv fitopatogenih bakterija u ovom radu je analiziran sastav etarskog ulja (eu) iz različitih nadzemnih delova vrste artemisia absinthium l. (asteraceae) iz srbije. izdanci bez listova i cvasti (iblc), listovi (l) i cvasti (c) podvrgnuti su hidrodestilaciji pomoću klevendžerove aparature. analiza eu je izvršena upotrebom gh/ ms (gasne hromatografije i masene spektrometrije). mikrodilucionom metodom određena je minimalna inhibitorna koncentracija (mik) i minimalna baktericidna koncentracija (mbk). utvrđeni su prinos (%, w/w), organoleptičke karakteristike (boja i miris), kao i kvantitativne i kvalitativne razlike između ispitivanih etarskih ulja. ukupno je identifikovano 17 komponenti u eu iblc, 11 u eu l i 18 u eu i. dominantno jedinjenje u svim etarskim uljima je trans-tujon (iblc 23,56%, l 60,41%, i c 46,16%). najosjetljiviji sojevi bili su xanthomonas campestris pv. campestris, erwinia amylovora i rathayibacter tritici. najveću aktivnost pokazalo je eu iblc protiv e. amylovora (0,03 mg/ml). iste mik vrednosti dobijene su za eu l protiv e. amylovora i r. tritici (0,09 mg/ml). sva testirana etarska ulja pokazala su istu aktivnost protiv x. campestris (0,13 mg/ml). ključne reči: pelin, asteraceae, hidrodestilacija, isparljivi terpeni, antibakterijska aktivnost introduction artemisia absinthium l. (anthemideae, asteraceae), commonly known as wormwood, is a perennial semi-shrubby plant up to 120 cm high (gajić, 1975; bora & sharma, 2011) which grows in dry habitats in eurasia, north and south america (deans & kennedy, 2001). this herb is the main ingredient of one of the most popular alcoholic beverages – absinthe (padosch et al., 2006). wormwood is known for its medical properties both in traditional medicine and in modern pharmacology (nguyen et al., 2018; © 2022 radulović et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 179 ahamad, 2019). as a remedy, wormwood is used for stomachache, kidney and gallbladder diseases, insomnia, and for the treatment of many other diseases and disorders (judzentiene et al., 2012). in serbia, a few ethnobotanical studies showed that it has been traditionally used as a diuretic and against respiratory, urinary, and digestive system disorders (jarić et al., 2015; živković et al., 2020). the most significant active constituents of the wormwood are volatile compounds and bitter substances which have attracted the interest of many researchers and producers throughout the world. numerous studies have shown that wormwood displays significant intraspecific variation in the terpene constituents of the eo (basta et al. 2007; mohammadi et al., 2015). many previous researchers have examined eos from herba of a. absinthium and have shown that the main component was bicyclic monoterpene – thujone (juteau et al., 2003; blagojević et al., 2006). both isomeric forms, cisand trans-thujone were present, but the eo was characterized by a higher percentage of trans-thujone (benkhaled et al., 2020). besides thujone, which is convulsant and has toxic activity (olsen, 2000), some other natural products were found in the oil: (z)-epoxyocimene, chrysanthenyl acetate (juteau et al., 2003; nguyen et al., 2017), caryophyllene oxide, p-cymene, 1,8-cineole (basta et al., 2007), sabinene, sabinyl acetate, α-phellandrene (mihajilov-krstev et al., 2014), camphor, and chamazulene (benkhaled et al., 2020). the chemical composition of wormwood eo varies significantly not only depending on the individual genetic variability, but also according to phenological stage and the plant part used (nguyen & németh 2016; nguyen et al., 2018). however, data regarding eo composition of separated plant parts are scarce. there are few papers dealing with variability in volatile composition regarding plant organ in wormwood, mainly aerial parts and roots (blagojević et al., 2006), or leaves and flowers (judzentiene & budiene, 2010; riahi et al., 2013), while shoots without leaves and inflorescences have not been analyzed to date. the wormwood eo has a broad spectrum of biological properties: antioxidant, antimicrobial (mihajilov-krstev et al., 2014; riahi et al., 2015), antiparasitic (yildiz et al., 2011), cytotoxic activity (taherkhani, 2014), and insecticidal and repellent effect (mihajilov-krstev et al., 2014). many previous studies investigated the antibacterial activity of wormwood eo, but they have been mainly focused on human pathogens (blagojević et al., 2006; mihajilov-krstev et al., 2014), while only a few studies were focused on phytopathogens (kordali et al., 2005). unfortunately, crop loss represents a problem due to plant diseases caused by insects and plant pathogens. since widely used synthetic chemicals in plant disease control are associated with undesirable effects and some toxic residues in the products (isman, 2000), there has been a growing interest in research concerning alternative pesticides and active antimicrobial compounds, including the plant extracts and eos. to date, there are no data regarding the antimicrobial activity of separated parts of wormwood against phytopathogens, but there are only few studies against human pathogens (joshi, 2013; vieira et al., 2016). investigation of the eo composition of different plant parts as well as their antimicrobial activity deserves special attention, bearing in mind that a specific chemical composition could significantly affect biological activities. therefore, the objectives of the present work were to (1) isolate and analyze the composition of eo of separated parts of a. absinthium; (2) investigate their antibacterial properties against selected phytopathogenic bacterial strains; and (3) highlight their potential future importance in chemophenetics and agriculture. materials and methods plant material plant material of artemisia absinthium was collected during the flowering period in village reka, near kladovo (eastern serbia, 44°29’35’’n, 22°24’55’’ e) in august 2021. the plants were identified using appropriate professional literature (gajić, 1975). a voucher specimen was deposited at the herbarium of the university of belgrade – faculty of biology, institute of botany and botanical garden „jevremovac” (beou 17804). the collected material was dried at room temperature and then divided into three groups: (1) shoots without leaves and inflorescences (swli), (2) leaves (l), and (3) inflorescences (i). isolation of essential oil separated plant material (swli – 172 g, l – 72 g and i – 124 g) was chopped and subjected to hydrodistillation using a clevenger type apparatus for 3 h, according to the procedure described in ph. eur. 6. (european directorate for the quality of medicines, 2007). the obtained eos were stored at 4 °c before the analysis by gas chromatography with ms of flame ionization detection (gc/ms and gc/ fid). gc-fid and gc/ms analysis the gc-fid and gc/ms analyses were carried out with an agilent 7890 a apparatus equipped with 180 biologica nyssana ● 13 (2) december 2022: 179-189 radulović et al. ● he essential oil composition of different parts of artemisia absinthium and its antibacterial activity against phytopathogenic bacteria biologica nyssana ● 13 (2) december 2022: 179-189 radulović et al. ● he essential oil composition of different parts of artemisia absinthium and its antibacterial activity against phytopathogenic bacteria 181 a 5975 c mass-selective detector (msd), a flame ionization detector (fid), and an hp-5 msi fusedsilica cap (column length 30 m, diameter 0.25 mm, film thickness 0.25 mm). the oven temperature was programmed linearly, rising from 60 °c to 240 °c at 3 °c/min; the injector temperature was 220 °c; the detector temperature was 300 °c, and the transferline temperature was 240 °c. the carrier gas was he (flow rate: 1.0 ml/min at 210 °c, constant pressure mode) at an injection volume of 1 μl and a split ratio of 10:1. electron impact mass spectra (ei-ms; 70 ev) were acquired over the m/z range 40–550. library search and mass spectral deconvolution and extraction were performed using the nist amdis (automated mass spectral deconvolution and identification system) software, version 2.64.113.71, with the retention index (ri) calibration data analysis parameters set to the strong level and a 10% penalty for compounds without a ri. the ris were experimentally determined using the standard method involving retention times (rt) of n-alkanes, which were injected after the essential oil under the same chromatographic conditions. the search was performed against our home-made library, containing 4972 spectra. the relative contents of identified compounds were computed from the gc peak areas. tested bacterial strains and growth conditions antibacterial activity was tested using one gram-positive (rathayibacter tritici), and five gram-negative phytopathogenic bacterial strains previously identified: agrobacterium tumefaciens, erwinia amylovora, pantoea alli, pseudomonas oryzihabitans, xanthomonas campestris pv. campestris. the bacterial strains were cultured in lb medium (composition g/l: tryptone 10, yeast extract 5, nacl 5) for 24 h at 30 °c. suspensions were prepared in phosphate saline buffer (1×pbs, sigma aldrich, usa) in the final concentration of 106 cfu/ml. mic assay microdillution method (mic assay) described in a previous study (ristivojević et al., 2016) was used to determine the minimum inhibitory concentrations (mic) and minimal bactericidal concentrations (mbc) of the a. absinthium essential oils. twofold serial dilutions with lb medium in 96-well microtiter plates were performed. except for the sterility control, each well was inoculated with 20 μl of bacterial suspensions (1×106 cfu/ml), reaching a final volume of 200 μl. besides a negative control, a sterility control, and control for the solvent (meoh), the antibiotics gentamicin and kanamycin were tested as positive controls in the final concentration of 0.025 mg/ml. the final concentration of methanol (meoh) as a solvent was 10%. all dilutions were done in duplicate, and the results were expressed in mg/ml. after reaching the final volume, 22 μl of resazurin as an indicator was added, and the 96well microtiter plates were incubated for 24 h at 30 °c. according to the resazurin reaction, the lowest concentration, which showed no change in color was defined as the mic. the lowest concentration that after sub-culturing did not show bacterial growth overnight was defined as the mbc value. results and discussion organoleptic characteristics, yield and composition of investigated eos the yield and organoleptic characteristics of the eos of separated parts of a. absinthium are given in tab. 1. the lowest yield (w/w) was recorded for swli eo (0.05%), while the highest was recorded for l eo (0.29%). this may be in relation with the abundance of glandular trichomes and the presence of secretory canals in the plant parts. although secretory canals are found only in the stem, while glandular trichomes were found both on the stem and leaves of a. absinthium (janaćković et al., 2019), it can be considered that certain mass of leaves, used for distillation, contain more glandular trichomes which contribute to higher eo yield. riahi et al. (2013) recorded variation between yields of the flowers and leaves eos. the highest yield was recorded for flowers eo (2.98%) whereas leaves eo exhibited a lower yield (1.87%) (riahi et al. 2013). on the contrary, we have shown that leaves produced more eo than inflorescences (tab. 1). it was noted that the color of obtained eos had changed over time. the l and i eo samples were light orange at the beginning of distillation but became dark brown at the end of distillation. the swli eo was orange-brown at the beginning and during distillation, but then changed color and became brown at the end of distillation. after 24 h in the refrigerator, the color of this oil was greenish, and after a few days, it was light yellow (tab. 1). wormwood eos, obtained from aerial parts of the plant, has a dark green to orange or dark brown color (sensitive to ultraviolet/visible light) (judzentiene, 2016). dark blue eo was reported by riahi et al. (2013) and msaada et al. (2015). on the other hand, according to pino et al. (1997), the eo obtained from the dried leaves and flowering tops of wormwood varied from dark green to brown or dark brownish green. different colors of eo can be a consequence of the presence/absence of different constituents. besides the color, the smell of eos was also specific. all of the obtained eos had a specific and 182 biologica nyssana ● 13 (2) december 2022: 179-189 radulović et al. ● he essential oil composition of different parts of artemisia absinthium and its antibacterial activity against phytopathogenic bacteria unsavory odor. swli eo had an odor very similar to the smell of tobacco. l eo showed the strongest and most unpleasant fragrance in comparison with the other two samples. this smell was described as „typically wormwood”, which reminds of bitter constituents. i eo was also recognized as fragrance with bitter characteristics. because of high concentrations of volatile terpenes, especially in leaves and inflorescences, the eo of this species has strong aromatic smell (nguyen & németh, 2016). phytochemical analyses of investigated eos showed some qualitative and quantitative differences. the conducted gc-fid and gc/ ms analyses detected and identified a total of 27 compounds (17 in the swli eo, 11 in l eo, and 18 in i eo). all compounds are listed in tab. 2. more compounds were identified in i eo, than in l eo, which is in agreement with the investigation done by riahi et al. (2013). the dominant group of compounds in the l eo and i eo were oxygenated monoterpenes (81.80% and 62.63%, respectively). on the other hand, swli eo was not dominated by terpenes (other compounds including fatty acid derivates, esters, were found in higher abundance, 62.15%). this finding is important as there are no studies dealing with eo from shoots without leaves and inflorescences. the l eo was also rich in monoterpene hydrocarbons (15.26%), while i eo and swli eo contain these compounds in smaller quantities (8.94% and 7.85%, respectively). sesquiterpenes were present only in two analyzed eos, but in low amounts (2.94% in l eo and 1.19 in i eo). thus, monoterpenes were found in higher abundance than sesquiterpenes both in l eo and i eo, which also showed nguyen et al. (2018). on the other hand, swli eo lacks sesquiterpenes, while l eo lacks other compounds. blagojević et al. (2006) documented differences between aerial parts eo and root eo, where monoterpenes (84.6%) dominated in the aerial parts eo, while aliphatic esters (64.5%) were major compounds of the root eo. in addition, a predominance of oxygenated monoterpenes (81.489.1%) was documented in aerial parts eo, while the root eo showed high ratios of hydrocarbon monoterpenes (43.8–55.1%) and monoterpene esters (36.6–41.5%) (llorens-molina & vacas 2015). the dominant compound in all three eos was trans-thujone (in swli eo 23.6%, in l eo 60.4%, in i eo 46.2%). wormwood is usually known and reported to be rich in thujone (meschler & howlett, 1999; juteau et al., 2003). the concentration of trans-thujone was the highest in the l eo (60.4%), which is in agreement with riahi et al. (2013). however, judzentiene & budiene (2010) determined higher ratios of thujone in flowers eo (5.3-10.4%) than in leaves eo (0.0-8.9%). it should be noted that for the praxis the ratio of plant parts may play an important role in reaching lower thujone levels of the drug. in our study, less thujone content was detected in inflorescences eo, thus a. absinthium drug should contain more flowers than leaves which was also concluded by nguyen et al. (2019). on the other hand, it was shown that thujone is not necessarily the main compound of the essential oil of wormwood (nguyen et al., 2017). in addition, nguyen et al. (2017) showed that trans-thujone is the major compound, however, in some cases, cisthujone reached comparable percentages, while no sample was found where this latter one would have been the only isomer. in this study, we documented that swli eo and i eo lack cis-thujone. other dominant compounds were as follows: sample dry plant material (g) obtained oil (g) yield (%, w/w) smell color start of distillation end of distillation after 24 h after few days swli1 172 0.09 0.05 strong; unpleasant; like tobacco orangebrown dark brown green light yellow l 72 0.21 0.29 strong; very unpleasant; bitter; typically wormwood light orange dark brown dark brown dark brown i 124 0.26 0.21 less unpleasant; bitter light orange dark brown dark brown dark brown table 1. yield and organoleptic characteristics of eos of separated parts of a. absinthium 1swli shoots without leaves and inflorescences; l leaves; i inflorescences 183 biologica nyssana ● 13 (2) december 2022: 179-189 radulović et al. ● he essential oil composition of different parts of artemisia absinthium and its antibacterial activity against phytopathogenic bacteria table 2. composition of eos of different parts of a. absinthium no ri* compounds % swli1 l i 1 923 α-thujene 1.12 1.01 2 943 α-fenchene 1.65 3 971 sabinene 1.51 6.23 4.17 4 1024 p-cymene 4.68 7.08 2.62 5 1031 1,8-cineole 2.67 4.28 6 1059 γ-terpinene 0.83 1.14 7 1104 linalool 4.54 8 1110 cis-thujone 1.12 9 1122 trans-thujone 23.56 60.41 46.16 10 1134 (z)-epoxyocimene 2.93 15.23 5.22 11 1137 iso-3-thujanol 0.99 12 1178 terpinen-4-ol 1.38 2.42 13 1228 nerol 3.51 14 1291 lavandulyl acetate 3.49 15 1425 lavandulyl isobutanoate 9.81 1.27 16 1488 β-selinene 1.19 17 1491 neryl isobutanoate 5.73 2.44 18 1511 geranyl isobutanoate 11.65 3.15 19 1513 lavandulyl 2-methylbutanoate 4.03 1.52 20 1578 geranyl butanoate 8.94 7.97 21 1584 caryophyllene oxide 2.94 22 1585 neryl isovalerate 5.29 6.40 23 1603 fatty acid ester 4.25 24 1609 geranyl isovalerate 2.35 25 2008 fatty acid ester 2.47 1.78 26 2014 fatty acid ester 2.72 27 2082 fatty acid ester 4.16 total monoterpenes 37.85 97.06 71.57 monoterpene hydrocarbons 7.85 15.26 8.94 oxygenated monoterpenes 30.00 81.80 62.63 total sesquiterepenes 0.00 2.94 1.19 sesquiterpene hydrocarbons 0.00 0.00 1.19 oxygenated sesquiterpenes 0.00 2.94 0.00 other 62.15 0.00 27.24 total 100.00 100.00 100.00 no. of compounds 17 11 18 contents of the total essential oil composition are given as percentages; -: not detected; *linear retention index was calculated for all compounds using the following formula: lri=100•(trs-trn)/((trn+1-trn)+100•n; other non terpenoid compounds, i.e. alyphatic and aromatic hydrocarbons; ms spectra of unidentified fatty acids are given in the appendix 1. 1swli shoots without leaves and inflorescences; l leaves; i inflorescences 184 biologica nyssana ● 13 (2) december 2022: 179-189 radulović et al. ● he essential oil composition of different parts of artemisia absinthium and its antibacterial activity against phytopathogenic bacteria geranyl isobutanoate and lavandulyl isobutanoate (11.65%, 9.81%, respectively) in swli eo; (z)epoxyocimene and p-cymene (15.23%, 7.08%, respectively) in l eo; and geranyl butanoate and neryl isovalerate (7.97%, 6.40% respectively) in i eo. some constituents were specific for certain examined eo. for example, six compounds were found only in swli eo: α-fenchene, nerol, lavandulyl acetate, geranyl isovalerate, and two fatty acid esters; three were found only in l eo: cis-thujone, iso-3-thujanol and caryophyllene oxide; while three were found only in i eo: linalool, β-selinene and one fatty acid ester. nguyen et al. (2018) also showed qualitative differences in eo profiles in a. absinthium thujone chemotype, e.g. geranyl-p-cymene, nuciferol esters and two unknown compounds were present only in the flower oil. also, neryl-isobutanoate, nerylisovalerate and caryophyllene oxide were much more present in flower oil than in the leaves oil (nguyen et al., 2018). however, we have shown herein that neryl-isobutanoate was in a higher amount in swli eo (5.73%) than in i eo (2.44%), while l eo lacks this compound. on the other hand, the amount of neryl-isovalerate was slightly lower in swli than in i eo (5.29% and 6.40, respectively). caryophyllene oxide was found only in the l eo. the relative amount of sabinene was higher in the l eo compared to i eo (6.23% and 4.17%, respectively), which is in accordance with the results obtained by riahi et al. (2013). blagojević et al. (2006) showed that the main compound in the aerial parts eo of wormwood was trans-thujone (β-thujone), while α-fenchene was the main compound found in root oil. α-fenchene was recorded only in swli eo. in addition, llorens-molina & vacas (2015) documented (z)epoxyocimene, (z)-chrysanthenyl acetate and linalool as the main compounds in aerial parts eo, while β-myrcene and α-fenchene were the main compounds in the root oil. (z)-epoxyocimene was recorded in all three eos, (z)-chrysanthenyl acetate and β-myrcene were not recorded, while linalool was found only in i eo. significant differences in eo composition were observed also between leaves and flowers. ariño et al. (1999) reported some quantitative differences in eo profile from leaves and flowers, e.g., cis-epoxyocimene was found in higher ratios in leaf oil, which is in accordance with our study. also, ariño et al. (1999) showed that cischrysanthnyl acetate, which we have not detected, was the dominant compound in the flower oil. in addition, riahi et al. (2013) showed that camphor was present only in flower oil but not in the leaves oil. on the other hand, bornan-2-one, was found in leaf oil, whereas flower oils were lacking in this compound. neither camphor nor bornan-2-one have been detected in our examined eos. differences in the composition of the essential oils of each part of the plant, especially in the flower heads, may have chemophenetic significance in future research, given that their components are very significant and genetically stable due to their repellent role in flower protection. antibacterial activity of a. absinthium eo the results of mic and mbc are given in tab. 3. it was shown that examined eos exhibited various degrees of antibacterial activities depending on tested bacterial strains (tab. 3). in general, tested oils had moderate antibacterial activity. mic assay indicates that the most sensitive bacterial strains were erwinia amylovora, rathayibacter tritici, and xanthomonas campestris pv. campestris, while pseudomonas oryzihabitans and pantoea alli were the most resistant. the strongest effect was shown for swli eo against e. amylovora (mic 0.03 mg/ ml). also, eo from l demonstrated high activity against e. amylovora and r. tritici (mic 0.09 mg/ ml). equal activity against x. campestris (mic 0.13 mg/ml) was detected for all three tested eos. antibacterial activity of wormwood eo against human pathogens was documented in several studies. it was shown that a. absinthium eo had a significant antimicrobial effect against human pathogenic bacteria isolated from wounds and stools of patients (mihajilov-krstev et al., 2014) with minimal inhibitory/bactericidal concentrations ranging from <0.08 to 2.43 mg/ml and from 0.08 to 38.80 mg/ml, respectively. riahi et al. (2015) reported that majorly gram-positive bacterial strains showed more sensitivity against the wormwood eos. in another study, the wormwood eo has shown very pronounced antibacterial activity, with inhibitory and bactericidal concentrations ranging from 4.72 against gram-negative to 37.80 mg/ml against grampositive bacterial strains (stanković et al., 2016). the study by kordali et al. (2005) demonstrated by disk diffusion method that a. absinthium eo at 600-1200 μ/disk concentrations was active against a limited number of bacterial strains, including phytopathogens: curtobacterium flaccumfaciens, pseudomonas syringae pv. maculicola, p.syringae pv. syringae (rk-470), xanthomonas axanopodas pv. vesicatoria, and x. pelargonii. different inhibitory effects of tested eos may be attributed to the differences in the biological properties of the main compounds, differences in composition in the oil and their synergistic effects. this result is in agreement with delaquis et al. (2002), who reported that the biological activity of different eos was the result of the interaction between their total chemical compounds, mainly with additive and 185 biologica nyssana ● 13 (2) december 2022: 179-189 radulović et al. ● he essential oil composition of different parts of artemisia absinthium and its antibacterial activity against phytopathogenic bacteria synergistic effects. the action mode of antimicrobial agents essentially depends on the type of the treated microorganism in relation to the structure of their cell wall and the outer membrane (shan et al., 2007). considering that there are a large number of different compounds in eo, it is most likely that their antibacterial activity is a result of targeting different cell components and consequently causing several types of damage (cha et al., 2005; sadaka et al., 2013). that could explain the broad antibacterial activity of tested a. absinthium eo against both gram-negative e. amylovora and x. campestris pv. campestris and gram-positive r. tritici were observed in the present study. it was confirmed that monoterpene ketones, α –and β-thujone, could be responsible for antibacterial potential against gram-positive and gram-negative bacteria (juteau et al., 2003; blagojević et al., 2006; tsiri et al., 2009; mihajilov-krstev et al., 2014). according to these, high amounts of thujone in all tested eos could be responsible for antibacterial activity. also, these substances can be important in agriculture as potential agents in pest control. conclusions herein, we presented a detailed analysis of the eo composition of the a. absinthium different parts (shoots without leaves and inflorescences, leaves, and inflorescences) and their antimicrobial activity. trans-thujone was the dominant compound in all three eos. on the other hand, the organoleptic characteristics, yield, number of identified components, the dominant group of compounds, the amount of the dominant compound, as well as the presence and amount of other compounds differed among the tested eos. these differences indicate the different biological functions of different components in different parts of the plant. moreover, the strongest effect was shown for swli eo, which may be in relation to the dominance of non-terpene compounds present in the eo. although the biochemical and physiological processes behind these differences in eo profiles still await explanation, other artemisia species should be included in future similar studies in order to get better insight into eo variability and antimicrobial activity of different plant parts. acknowledgements. financial support was provided by the ministry of education, science and technological development of the republic of serbia (451-03-68/202214/200178). references ahamad, j. 2019: a pharmacognostic review on artemisia absinthium. international research journal of pharmacy, 10(1): 25-31. ariño, a., arberas, i., renobales, g., arriaga, s., domínguez, j.b. 1999: seasonal variation in wormwood (artemisia absinthium l.) essential oil composition. journal of essential oil research, 11: 619–622. basta, a., tzakou, o., couladis, m., pavlović, m. 2007: chemical composition of artemisia absinthium l. from greece. journal of essential oil research, 19: 316-318. benkhaled, a., boudjelal, a., napoli, e., baali, f., ruberto, g. 2020: phytochemical profile, table 3. antibacterial activity of different parts of a. absinthium eos phytopathogenic strains artemisia eos (mg/ml) gentamicin (mg/ml) kanamycin (mg/ ml)swli l i mic mbc mic mbc mic mbc mic mbc mic mbc xanthomonas campestris pv. campestris 0.125 0.5 0.125 0.25 0.125 0.25 0.008 0.006 0.008 0.006 pseudomonas oryzihabitans 1 >1 0.75 1 1 >1 0.016 0.013 0.016 0.0125 erwinia amylovora 0.031 0.5 0.094 0.0625 0.25 0.5 0.004 0.003 0.008 0.006 rathayibacter tritici 0.125 0.25 0.094 0.0625 0.031 0.008 0.006 0.003 0.025 pantoea alli >1 0.75 1 >1 0.006 0.025 0.006 0.025 agrobacterium tumefaciens 0.5 1 0.75 1 0.5 1 >0.400 >0.400 not detected in the range of tested concentrations biologica nyssana ● 13 (2) december 2022: 179-189 radulović et al. ● he essential oil composition of different parts of artemisia absinthium and its antibacterial activity against phytopathogenic bacteria 186 antioxidant activity and wound healing properties of artemisia absinthium essential oil. asian pacific journal of tropical biomedicine, 10(11): 496. blagojević, p., radulović, n., palić, r., stojanović, g. 2006: chemical composition of the essential oils of serbian wild-growing artemisia absinthium and artemisia vulgaris. journal of agricultural and food chemistry, 54(13): 4780-4789. bora, k.s., sharma, a. 2011: the genus artemisia: a comprehensive review. pharmaceutical biology, 49: 101-109. cha, j.d., jeong, m.r., jeong, s.i., moon, s.e., kim, j.y., kil, b.s., song, y.h. 2005: chemical composition and antimicrobial activity of the essential oils of artemisia scoparia and a. capillaris. planta medica, 71(2): 186–190. deans, s.g., kennedy, a.i. 2001: artemisia absinthium. in: wright c.w. (ed.), artemisia. 1: 7990, crc press, london, uk. delaquis, p.j., stanich, k., girard, b., mazza, g. 2002: antimicrobial activity of individual and mixed fractions of dill, cilantro, coriander and eucalyptus essential oils. international journal of food microbiology, 74: 101–109. european directorate for the quality of medicines, european pharmacopoeia, sixth ed., council of europe, 2007 (isbn: 9789287160546) gajić, m. 1973: artemisia. in: josifović m. (ed.), flora of serbia vii: 122 p., sanu, belgrade. isman, m.b. 2000: plant essential oils for pest and disease management. crop protection, 19: 603-608. janaćković, p., gavrilović, m., rančić, d., dajić-stevanović, z., giweli, a.a., marin, p.d. 2019: comparative anatomical investigation of five artemisia l. (anthemideae, asteraceae) species in view of taxonomy. brazilian journal of botany, 42(1): 135-147. jarić, s., mačukanović-jocić, m., djurdjević, l., mitrović, m., kostić, o., karadžić, b., pavlović, p. 2015: an ethnobotanical survey of traditionally used plants on suva planina mountain (south-eastern serbia). journal of ethnopharmacology, 175: 93108. joshi, r.k. 2013: volatile composition and antimicrobial activity of the essential oil of artemisia absinthium growing in western ghats region of north west karnataka, india. pharmaceutical biology, 51(7): 888-892. judzentiene, a. 2016: wormwood (artemisia absinthium l.) oils. in: essential oils in food preservation, flavor and safety: 849-856, academic press. judzentiene, a., budiene, j. 2010: compositional variation in essential oils of wild artemisia absinthium from lithuania. journal of essential oil bearing plants, 13(3): 275-285. judzentiene, a., budiene, j., gircyte, r., masotti, v., laffont-schwob, i. 2012: toxic activity and chemical composition of lithuanian wormwood (artemisia absinthium l.) essential oils. records of natural products, 6(2). juteau, f., jerkovic, i., masotti, v., milos, m., mastelic, j., bessière, j.m., viano, j. 2003: composition and antimicrobial activity of the essential oil of artemisia absinthium from croatia and france. planta medica, 69(02): 158-161. kordali, s., kotan, r, mavi, a., cakir, a., ala, a., yildirim, a. 2005: determination of the chemical composition and antioxidant activity of the essential oil of artemisia dracunculus and of antifungal and antibacterial activities of turkish artemisia absinthium, a.dracunculus, artemisia santonicum, and artemisia spicigera essential oils. journal of agricultural and food chemistry, 53(24): 9452-9458. llorens-molina, j.a., vacas, s. 2015: seasonal variations in essential oil of aerial parts and roots of an artemisia absinthium l. population from a spanish area with supramediterranean climate (teruel, spain). journal of essential oil research, 27: 395–405. meschler, j. p., howlett, a.c. 1999: thujone exhibits low affinity for cannabinoid receptors but fails to evoke cannabimimetic responses. pharmacology biochemistry and behavior, 62(3): 473-480. mihajilov-krstev, t., jovanović, b., jović, j., ilić, b., miladinović, d., matejić, j., rajković, j., đorđević, lj., cvetković, v., zlatković, b. 2014: antimicrobial, antioxidative, and insect repellent effects of artemisia absinthium essential oil. planta medica, 80(18): 1698-1705. mohammadi, a., sani, t., ameri, a., imani, m., golmakani, e., kamali, h. 2015: seasonal variation in the chemical composition, antioxidant activity, and total phenolic content of artemisia absinthium essential oils. pharmacognosy research, 7: 329. msaada, k., salem, n., bachrouch, o., bousselmi, s., tammar, s., alfaify, a., marzouk, b. 2015: chemical composition and antioxidant and antimicrobial activities of wormwood (artemisia absinthium l.) essential oils and phenolics. journal of chemistry, 1–12. 187 biologica nyssana ● 13 (2) december 2022: 179-189 radulović et al. ● he essential oil composition of different parts of artemisia absinthium and its antibacterial activity against phytopathogenic bacteria nguyen, h.t., inotai, k., radácsi, p., tavaszisárosi, s., ladányi, m., zámboriné-németh, é. 2017: morphological, phytochemical and molecular characterization of intraspecific variability of wormwood (artemisia absinthium l.). journal of applied botany and food quality, 90: 238-245. nguyen, h.t., németh, z.é. 2016: sources of variability of wormwood (artemisia absinthium l.) essential oil. journal of applied research on medicinal and aromatic plants, 3(4): 143-150. nguyen, h.t., radácsi, p., gosztola, b., rajhárt, p., németh, é.z. 2018: accumulation and composition of essential oil due to plant development and organs in wormwood (artemisia absinthium l.). industrial crops and products, 123: 232-237. nguyen, h.t., radácsi, p., rajhárt, p., németh, é. 2019: variability of thujone content in essential oil due to plant development and organs from artemisia absinthium l. and salvia officinalis l. l. journal of applied botany and food quality, 92: 100-105. olsen, r.w. 2000: absinthe and γ-aminobutyric acid receptors. proceedings of the national academy of scieces. usa 97, (pp. 4417–4418). padosch, s.a., lachenmeier, d.w., kröner, l.u. 2006: absinthism: a fictitious 19th century syndrome with present impact. substance abuse treatment, prevention, and policy 1(14). pino, j.a., rosado, a., fuentes, v. 1997: chemical composition of the essential oil of artemisia absinthium l. from cuba. journal of essential oil research, 9(1): 87–89. riahi, l., chograni, h., elferchichi, m., zaouali, y., zoghlami, n., mliki, a. 2013: variations in tunisian wormwood essential oil profiles and phenolic contents between leaves and flowers and their effects on antioxidant activities. industrial crops and products, 46: 290-296. riahi, l., ghazghazi, h., ayari, b., aouadhi, c., klay, i., chograni, h., cherif, a., zoghlami, n. 2015: effect of environmental conditions on chemical polymorphism and biological activities among artemisia absinthium l. essential oil provenances grown in tunisia. industrial crops and products, 66: 96-102. ristivojević, p., dimkić, i., trifković, j., berić, t., vovk, i., milojković-opsenica, d., stanković, s. 2016: antimicrobial activity of serbian propolis evaluated by means of mic, hptlc, bioautography and chemometrics. public library of science, 11(6): 157-097. sadaka, f., nguimjeu, c., brachais, c.h., vroman, i., tighzert, l., couvercelle, j.p. 2013: review on antimicrobial packaging containing essential oils and their active biomolecules. innovative food science and emerging technologies, 20: 350. shan, b., cai, y.z., brooks, j.d., corke, h. 2007: the in vitro antibacterial activity of dietary spice and medicinal herb extracts. international journal of food microbiology, 117(1): 112–119. stanković, n., mihajilov-krstev, t., zlatković, b., matejić, j., jovanović, v.s., kocić, b., čomić, l. 2016: comparative study of composition, antioxidant, and antimicrobial activities of essential oils of selected aromatic plants from balkan peninsula. planta medica, 82(07): 650-661. taherkhani, m. 2014: in vitro cytotoxic activity of the essential oil extracted from artemisia absinthium. iranian journal of toxicology, 8(26): 1152-1156. tsiri, d., graikou, k., pobłocka-olech, l., krauzebaranowska, m., spyropoulos, c., chinou, i. 2009: chemosystematic value of the essential oil composition of thuja species cultivated in poland — antimicrobial activity. molecules, 14(11): 47074715. vieira, t.m., dias, h.j., medeiros, t.c., grundmann, c.o., groppo, m., heleno, v.c., martins, c.h.g., cunha, w.r., crotti, a.e.m., silva, e.o. 2017: chemical composition and antimicrobial activity of the essential oil of artemisia absinthium asteraceae leaves. journal of essential oil bearing plants, 20(1): 123-131. yildiz, k., basalan, m., duru, o., gokpinar, s. 2011: antiparasitic efficiency of artemisia absinthium on toxocara cati in naturally infected cats. turkish journal of parasitology, 35(1): 10–14. živković, j., ilić, m., šavikin, k., zdunić, g., ilić, a., stojković, d. 2020: traditional use of medicinal plants in south-eastern serbia (pčinja district): ethnopharmacological investigation on the current status and comparison with half a century old data. frontiers in pharmacology, 11: 1020. biologica nyssana ● 13 (2) december 2022: 179-189 radulović et al. ● he essential oil composition of different parts of artemisia absinthium and its antibacterial activity against phytopathogenic bacteria 188 appendix appendix 1. ms spectra of unidentifies compounds from artemisia absinthium essential oil lri1603 lri2008 189 biologica nyssana ● 13 (2) december 2022: 179-189 radulović et al. ● he essential oil composition of different parts of artemisia absinthium and its antibacterial activity against phytopathogenic bacteria lri2014 lri2082 microsoft word bn-oa-0201-07 jovanovic_et_al biologica nyssana 2 (1) september 2011: 51-58 jovanović, b. et al. assessment of heavy metal load in chub liver… 51 original article ! assessment of heavy metal load in chub liver (cyprinidae – leuciscus cephalus) from the nišava river (serbia) boris jovanović*1, željko mihaljev2, stevan maletin3, dušan palić4 1interdepartmental toxicology program and fisheries biology program, departments of biomedical sciences and natural resource ecology and management, iowa state university, ia, usa 2scientific insitute „novi sad“ for veterinary medicine, novi sad, serbia 3faculty of agriculture, university of novi sad, serbia 4department of biomedical sciences, the college of veterinary medicine, iowa state university, ia, usa * e-mail: prcko@iastate.edu abstract: jovanović, b., mihaljev, ž., maletin, s., palić, d.: assessment of heavy metal load in chub liver (cyprinidae leuciscus cephalus) from the nišava river (serbia). biologica nyssana, 2 (1), september 2011: 51-58. the nišava river plays an important role as the source for both drinking water and agricultural irrigation due to its hydrological and geomorphological characteristics as the largest river in the region of southeast serbia. in this study we used the liver of the chub (leiciscus cephalus) as a tool for biomonitoring heavy metal accumulation along the river. chub specimens were sampled from two localities (one at the border with bulgaria and a second in the city of niš). concentrations were estimated for six heavy metals (iron, cadmium, copper, zinc, lead and manganese) in chub liver. low bioconcentration level was observed for most of the metals and the concentrations corresponded to the nominal concentration in livers of fish inhabiting metal unpolluted streams and rivers. however, cadmium concentration in the chub liver exceeded 0.5 mg kg-1, a several hundred folds increase from nominal concentration indicating a potential toxic exposure of the fish and of the stream ecosystem to this heavy metal. hepatosomatic indices were calculated and tested for the impact of metal concentrations on liver size. a decrease of the hepatic index was observed in fish with higher cadmium concentration, suggesting a possible impact on the health of the chub population in the nišava river. key words: biomonitoring, heavy metals, hepatosomatic index, leuciscus cephalus, nišava river introduction ! rivers represent the most complex aquatic systems in terms of transport and interactions of heavy metals with geochemical and biological processes. large surface to volume ratios typical of flowing waters support extensive interactions with the atmosphere, constant exchange of gases, and the introduction of material suspended in atmospheric precipitations. oxygen saturation in the surface layer of the water allows for sustained high redox potential, causing quick degradation of organic matter and maintenance of the oxidative state of iron and manganese as suspended colloid hydroxides (a l l a r d et al., 2004; w a r r e n & h a a c k 2001). chemical assays of water and sediments have traditionally been used as tools in ecotoxicological analysis, but the variable chemistry of freshwater streams limits their application in observing cumulative effects of contaminants on the stream biota. biomonitoring of hazardous substances in tissues of aquatic organisms have been successfully 2 (1) • september 2011: 51-58 biologica nyssana 2 (1) september 2011: 51-58 jovanović, b. et al. assessment of heavy metal load in chub liver… 52 applied in recent years as indicators of heavy metal pollution (e v a n s et al., 2000; l a m a s et al, 2007; t e o d o r o v i ć , 2001; v a n d e r o o s t et al., 2003; w a g n e r & b o m a n 2003). furthermore, the use of fish as bioindicators appears more timeand cost-effective when compared with the effort required for repetitive water chemistry sampling and the cost of assessing pollutants with standardized chemistry and toxicity assays (b a r b o u r , 1999). water use and consumption of fish contaminated with heavy metals have deleterious effects on human health, which was widely acknowledged after a series of events in the period from 1953 to 1960 when several thousand people died in minamata bay in japan as a result of poisoning caused by the consumption of mercurycontaminated fish (s m i t h & s m i t h 1975). observed toxic effects raised public safety and human health concerns repeatedly since minamata, prompting legislators to set limits on the lead, cadmium and mercury levels detected in the fish muscle, but other heavy metals and fish tissues were not included in the european union (eu) regulations (e u r o p e a n c o m m i s s i o n , 2001; e u r o p e a n c o m m i s s i o n , 2002). fish can absorb heavy metals through epithelial or mucosal surfaces of the skin, gills and gastrointestinal tract. metal ions can exert toxicity effects either directly at the gill surface (fast binding reactions), or passing through the gills and causing adverse effects in the internal organs (s i m k i s s & t a y l o r , 1989). however, the majority of heavy metal load is commonly ingested via water or feedstuff and transported to the bloodstream and liver via mucosal membranes and hepatic blood supply. therefore, heavy metals are accumulated in the liver and subjected to a detoxication process. being the primary target of metal deposition, the liver can be effectively used as a biomonitoring organ for heavy metal pollution of water ecosystems (i c e s , 1991; d e n n y et al., 1995; j ø r g e n s e n & p e d e r s e n , 1994; s w a i b u h l w a n g a et al.,, 2003; t e o d o r o v i ć , 2001). the presented study describes heavy metals loads in leuciscus cephalus l. 1758 cyprinidae livers in the nišava river, which is the major supply point for irrigation and drinking water to south-east serbia. research aims were to: a) determine the concentrations of six heavy metals (fe, cd, cu, zn, mn and pb) in l. cephalus liver; and b) assess the impact of the heavy metals on the size of l. cephalus liver as the major detoxification organ. using fish livers as bioindicators of water quality in streams appears to be a rapid and economical method for screening waterways for the presence of heavy metals. furthermore, collected information on water quality can be used to establish a broad categorization of the examined stream and has the potential to be used by environmental managers and policy makers to improve the management of the river, following the example of the nišava river canyon section, “sićevačka gorge nature park”, which is currently under strict state protection because of its biodiversity and for providing a habitat for a number of endemic and relict species. material and methods stream and sampling site description the nišava river with its tributary ginska is a 202 km long with a watershed area of 4068 km2. it is situated in south-east serbia and bulgaria (upper one fourth of the river) and it is the largest tributary of the južna figure 1. the geographic region of the nišava river, the two sampling stations (l1 and l2) are indicated with arrows biologica nyssana 2 (1) september 2011: 51-58 jovanović, b. et al. assessment of heavy metal load in chub liver… 53 morava river, part of the southern danube river watershed (g a v r i l o v i ć & d u k i ć , 2002). the nišava has two distinct parts: upstream of the town of bela palanka it is a narrow, rapid mountain stream ranging from 6 to 76 m in width and 0.6 to 2 m in depth with velocity from 1 to 2.5 m s-1 and flow of 0.3-270 m3 s-1 of water. after bela palanka and all the way to its mouth near the village of lalinac, the nišava becomes a meandering stream with increased width (15-100 m), depth (1-3 m), and flow that carries 3-700 m3 s-1 of water (b r a n k o v i ć , 1997). l. cephalus sampling was performed on two sites in may of 2005 (fig. 1). the nišava river at ivko’s watermill, 1 km upstream from the city of dimitrovgrad was selected as site l1. this is the first section of the river as it enters serbia and there are no larger settlements upstream to this sampling site. in this section the river has a width of 6 m, and a depth of no more than 1 m. the bottom is composed of gravel, and the banks are covered with thick vegetation. the majority of the benthofauna is composed of ephemeroptera and trichoptera larvae, while in the ichthyofauna the most dominant species are: alburnoides bipunctatus bloch, 1782 cyprinidae, barbus peloponnesius valenciennes, 1842 cyprinidae and l. cephalus. as site l2, the nišava river access point at the greek consulate in the city of niš was selected. the selected site is located on the last section of river, about 10 km before its mouth. the greek consulate site is located downstream of the niš city limits and other heavily populated areas (cities of dimitrovgrad, pirot, and bela palanka). the site was selected due to the high potential for heavy metal contamination from several upstream industrial areas. at this site, the nišava river is about 80 m wide, and between 0.2 and 1.2 m deep. the bottom is composed of gravel, and both sides of the river are paved with no vegetation. the icthyofauna is rich, with the predominant species being a. bipunctatus, barbus barbus l. 1758 cyprinidae, b. peloponnesius, l. cephalus, alburnus alburnus l. 1758 cyprinidae, chondrostoma nasus l. 1758 cyprinidae and rhodeus sericeus pallas, 1776 cyprinidae. fish sampling and tissue preparation l. cephalus is one of the few species that inhabit the nišava through the whole serbian section of the river, can be collected with low catch per unit effort using only basic sport fishing gear, and its trophic level (omnivore – insectivore) potentially allows for bioaccumulation of heavy metals in the organs due to both water and nutritional intake. chub sampling was performed by an angling technique using hook and line. upon capture, specimens were immediately anesthetized in chloroform, euthanized, and stored in a field fridge at 0-4 °c. wet weight and total length of each specimen was measured in the laboratory using digital scales (kern 440-33, d=0.01g). the age of specimens was determined using standard scale method (apha, 1995). to avoid contamination of samples, dissection was carried out using stainless steel instruments and latex gloves without talcum, and collected livers were weighed. analyses of heavy metals in l. cephalus livers l. cephalus liver samples were treated using standard nitric acid-perchloric acid digestion (apha, 1995). briefly, 10 ml of hno3 and hclo4 has been added to the sample and heated until the samples dissolved. dissolved sample was diluted to 100 ml with deionized water in volumetric flask. all digests were incubated for a minimum of five hours at room temperature, before being transferred to a conical flask, and then refrigerated for 48 h at 4 °c to allow complete settling before aspiration and analysis with a flame atomic absorption spectrometer (varian, spektr aa-10). a total of 10 samples were prepared for each locality. concentrations of fe, cd, cu, zn, mn, and pb were expressed as mg kg-1 of wet weight, as well as cumulative concentrations (the summary of fe, cd, cu, zn and mn). data analysis prior to any further analysis, all data were subjected to tests of normality (kolmogorovsmirnov) and homogeneity of variance (levene’s), and no deviations were detected. analysis of variance (anova; (s t a t s o f t , 1996)) was used to determine differences in metal loads between localities. to determine if the size of specimens was associated with the concentrations of metals in the liver, spearman’s rank correlation tests (s t a t s o f t , 1996) were conducted between metal concentrations from chub liver and total length of fish specimen (s w a i b u h l w a n g a , 2003). the hepatosomatic index was calculated according to the formula: hsi=m/m*100, where m is the mass of the liver; and m is the mass of the fish specimen. spearman’s rank correlation test (s t a t s o f t , 1996) was also used to explore the correlation level between the hepatosomatic index and concentrations of metals in the liver, as well as to test hypotheses as to the possible negative impacts to fish due to high metal concentrations. biologica nyssana 2 (1) september 2011: 51-58 jovanović, b. et al. assessment of heavy metal load in chub liver… 54 results total length of fish specimens ranged from 13.6 to 20.5 cm, with the age ranging from 2 years to 3 years. the smallest specimen weighed 24.6 g, while the largest one weighed 86.1 g. no correlations between fish total length and metal concentration in the liver were observed, neither with single metal concentrations nor with cumulative concentrations (spearman’s rank correlation, p > 0.05). mean concentrations of heavy metals in chub liver at the l1 site for fe, cd, cu, zn and mn are 76.73, 0.98, 2.98, 38.83 and 3.47 mg kg-1 wet weight; while at the l2 site, concentrations were of the same order of magnitude: 93.95, 0.57, 6.94, 31.81 and 2.82 mg kg-1 wet weight, respectively (fig. 2). in both sites, pb was below the detection limit of the instrument. the comparison of data from the two sites revealed a significantly increased concentration of cadmium and zinc (fig. 3) at site l1 (anova: f = 5.2, p < 0.05; f = 4.7, p < 0.05 respectively). there were no differences in fe, cu and mn concentrations. l1 fe cd cu zn mn -20 0 20 40 60 80 100 120 l2 fe cd cu zn mn -20 0 20 40 60 80 100 120 figure 2. heavy metal concentration in chub liver at site l1 and site l2. values on the y-axis represent mg kg-1 wet weight. boxes refer to standard errors and whiskers refer to standard deviations correlation tests between hepatosomatic index and metal concentration in liver can be used to infer potential deleterious effects of heavy metal bioaccumulation. the concentration of cadmium in fish collected at site l1 is strongly and negatively correlated with liver size in chub (spearman’s rank correlation, r = -0.87, p < 0.001), which suggests potential influence of a metal on fish metabolism (fig. 4). all other concentrations of metals, singular and cumulative, from either site did not show any significant correlation. hepatosomatic index was 1.36 + 0.11 (standard error of the mean) for site l1 and 1.57 + 0.12 for site l2. ±std. dev. ±std. err. mean 22 26 30 34 38 42 46 50 l1 zn l2 zn b* ±std. dev. ±std. err. mean 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 l1 cd l2 cd a* figure 3. differences in concentration of cadmium and zinc at localities l1 (3a) and l2 (3b). values on the y-axis represent mg kg-1 wet weight. boxes refer to standard errors and whiskers refer to standard deviations. * indicates that the effect is statistically significant (anova p<0.05) discussion progressive accumulation of metal residues during the aging process in fish has been discussed recently, and results presented in this study are in accordance with published data (s w a i b u h biologica nyssana 2 (1) september 2011: 51-58 jovanović, b. et al. assessment of heavy metal load in chub liver… 55 l w a n g a et al., 2003; vinagre et al., 2004). it has been demonstrated that in relatively young fish progressive accumulation is not significant (except for mercury), and it was proposed that only fish from 1+ to 4+ years old should be used for such analysis (t e o d o r o v i ć , 2001). most of the concentrations of heavy metals in l. cephalus liver were close to the nominal concentrations (t e o d o r o v i ć , 2001) that are present in livers of fish inhabiting metal unpolluted streams and rivers. however, concentrations of cd were several hundreds folds greater than the nominal concentrations (t e o d o r o v i ć , 2001). cd ls i 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 0.2 0.6 1.0 1.4 1.8 2.2 l1 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 cd 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 ls i l2 figure 4. linear regression (spearman’s rank correlation, r = 0.87, p < 0.001) of hepatosomatic index (y-axis) and concentration of cadmium in chub liver mg kg-1 wet weight from locality l1 (xaxis) and r= 0.07, p >> 0.05 from locality l2 a recent comparable study was conducted on the nišava river using b. peloponnesius as a model on four sites, including site l2 of the present study (b r a n k o v i ć , 2003), and a significantly lower concentration of lead (nil vs. 0.76 mg kg-1) and cadmium (0.57 vs 1.82 mg kg-1) were detected in the l. cephalus liver compared to b. peloponnesius. lower concentrations of pb and cd detected after two years could suggest that the water quality had improved, but also can reflect different ecotoxicological effects of heavy metals on two analyzed species. b. peloponnesius is a strict benthivore species and could be more likely to accumulate heavy metals when compared to l. cephalus, which also feed in the upper water column. cadmium concentration in chub liver was higher in locality l1, which was expected to be the less polluted site. it is unclear why this phenomenon occurred, as there are no known pollution sources upstream of the site on the serbian side of the border, unless cd is found in the waters flowing in from nearby bulgaria. cadmium at observed concentrations has the potential to affect the metabolism of the fish (fig. 4) and could account for the increase of zinc concentration in the livers on this locality (fig. 3). zinc is an antagonist to cadmium and adaptation has been observed that in the presence of toxic concentrations of cd, zinc can be more rapidly absorbed by receptor spots in the gills in order to lower cd toxicity (w i c k l u n d et al., 1988). although the concentration of the heavy metals in the liver can be used to estimate the overall heavy metal burden in the stream that the aquatic biota can uptake, it is unrelated to food safety of the fish used in human consumption. therefore, it is strongly advisable to compare all suspicious metal concentrations with the proposed maximal allowed concentration cited in the regulations before fish is used for this purpose. the cd concentration in the liver was converted to muscle concentration using the bioconcentration index for carp, in order to check if there are any possible threats for human health, (c i n i e r d e c o n t o et al., 1999). the maximal possible cd concentration was used as output corresponding to 53 µg l-1 exposure of cd for 127 days as described (c i n i e r d e c o n t o et al., 1999). a calculated estimate placed cd concentration in fish muscle at 0.07 mg kg-1 wet weight at site l1 and 0.04 mg kg-1 wet weight at l2. according to current eu legislation (european c o m m i s s i o n , 2002), the l1 values could represent potential threats to human health as the maximum allowed concentrations (mac) for cadmium in freshwater fish musculature are 0.05 mg kg-1 wet weight. however, serbian laws are much looser and the mac for cadmium is 0.1 mg kg-1 wet weight (a n o n , 1992). nevertheless, the observed concentrations of cd exceed european standards on locality l1 and suggest a pressing need for frequent monitoring and a stronger biologica nyssana 2 (1) september 2011: 51-58 jovanović, b. et al. assessment of heavy metal load in chub liver… 56 environmental policy in order to maintain the water quality in the nišava river. in conclusion, the concentrations of cadmium and zinc are increased in l. cephalus livers from the upper part of the river, and cd concentration is high enough to suggest a certain degree of effects on the metabolism of l. cephalus, also involving a possible buffer-like interaction with zinc. historical information on the presence of cadmium in the upper parts of the river system is not available, and we therefore propose that further investigations should also include the bulgarian river section. a potential risk to human health is observed due to increased cd concentration in fish tissues collected from the upper nišava river system. acknowledgments. we are very thankful to stefano mariani for the critical reading of the manuscript, and adolfo carrillo cabello for the help with english grammar. references allard, t., menguy, n., salomon, j., calligaro, t., weber, t., calas, g., benedetti, m.f., 2004: revealing forms of iron in river-borne material from major tropical rivers of the amazon basin (brazil). geochimica et cosmochimica acta, 68(14): 3079-3094. anon, 1992: pravilnik o količinama pesticida, metala i metaloida i drugih otrovnih supstancija, hemioterapeutika, anabolika i drugih supstancija koje se mogu nalaziti u namirnicama. službeni list sfrj. broj 5, član 8. 67-85. apha, 1995: standard methods for examination of water and wastewater, american public health association, american water work association and water pollution control federation. washington, dc, american public health association. barbour, m.t., gerritsen, j., snyder, b.d., stribling, j.b., 1999: rapid bioassessment protocols for use in streams and wodeable rivers: periphyton, bentic macroinvertebrates and fish, epa 841-b-99-002. u.s. enviromental protection agency; office of water; washington, d.c. branković, s., 1997: biosistematske i hematološke karakteristike riba iz gornjeg i donjeg toka reke nišave. diplomski rad. prirodno-matematički fakultet. priština, univerzitet u prištini. 42. branković, s., 2003> risk assessment of ichthyofauna using biochemical monitoring of heavy metals in river systems. faculty of occupational safety. magistarska teza, university of niš. cinier de conto, c., petit-ramel, m., faure, r., garin, d., bouvet, y. 1999. kinetics of cadmium accumulation and elimination in carp cyprinus carpio tissues. comparative biochemistry and physiology part c, 122(3): 345-352. denny, p., bailey, r., tukahirwa, e., mafabi, p., 1995: heavy metal contamination of lake george (uganda) and its wetlands. hydrobiologia, 297(3): 229-239. european commission. 2001: decree 466/2001. 08.3.2001. european commission. official journal of the european communities. l 77/1. european commission. 2002: decree 221/2002, 06.2.2002. european commission. official journal of the european communities. l 37:4-6. evans, c.w., hills, j.m., dickson, j.m.j., 2000: heavy metal pollution in antarctica: a molecular ecotoxicological approach to exposure assessment. journal of fish biology, 57: 8-19. gavrilović, lj., dukić, d., 2002: reke srbije. zavod za izdavanje udžbenika, beograd. ices, 1991: statistical analysis of the ices cooperative monitoring programme data on contaminants in fish liver tissue and mytilus edulis (1978-1988) for the determination of temporal trends. ices. 176. jørgensen, l.a., pedersen, b., 1994. trace metals in fish used for time trend analysis and as environmental indicators. marine pollution bulletin, 28(4): 235-243. lamas, s., fernández, j.a., aboal, j.r., carballeira, a., 2007: testing the use of juvenile salmo trutta l. as biomonitors of heavy metal pollution in freshwater. chemosphere, 67(2): 221-228. simkiss, k., taylor, m.g., 1989: metal fluxes across the membranes of aquatic organisms. reviews in aquatic sciences, 1: 173-188. smith, w.e., smith, a.m., 1975: minamata. new york, holt, rinehart and winston. first edition. 192 p. statsoft, inc. 1996: statistica for windows (computer program manual). tulsa, ok. http://www.statsoft.com. swaibuh lwanga, m., kansiime, f., denny, p., scullion, j., 2003: heavy metals in lake george, uganda, with relation to metal concentrations in tissues of common fish species. hydrobiologia, 499(1): 83-93. teodorović, i., 2001: metal pollution index – contribution to freshwater monitoring. belgrade zadužbina andrejević. 102 p. van der oost, r., beyer, j., vermeulen, n.p.e., 2003: fish bioaccumulation and biomarkers in environmental risk assessment: a review. environmental toxicology and pharmacology, 13(2): 57-149. biologica nyssana 2 (1) september 2011: 51-58 jovanović, b. et al. assessment of heavy metal load in chub liver… 57 vinagre, c., frança, s. , costa, m.j., cabral, h.n., 2004: accumulation of heavy metals by flounder, platichthys flesus (l i n n a e u s 1758), in a heterogeneously contaminated nursery area. marine pollution bulletin, 49(11-12): 11091113. wagner, a., boman, j., 2003: biomonitoring of trace elements in muscle and liver tissue of freshwater fish. spectrochimica acta part b: atomic spectroscopy, 58(12): 2215-2226. warren, l.a., haack, e.a., 2001: biogeochemical controls on metal behaviour in freshwater environments. earth-science reviews, 54(4): 261-320. wicklund, a., runn, p., norrgren, l., 1988: cadmium and zinc interactions in fish: effects of zinc on the uptake, organ distribution, and elimination of 109 cd in the zebrafish, brachydanio rerio. archives of environmental contamination and toxicology, 17(3): 345-354. biologica nyssana 2 (1) september 2011: 51-58 jovanović, b. et al. assessment of heavy metal load in chub liver… 58 microsoft word bn020202 nahirnic_lepidoptera biologica nyssana 2 (2) december 2011: 00-00 nahirnić a. supplements of butterfly fauna… 107 original article ! supplements to butterfly fauna (hesperioidea and papilionoidea) of grza river gorge (eastern serbia) ana nahirnić faculty of biology, university of belgrade, studentski trg 16, 11 000 belgrade, serbia * e-mail: ananahirnic@gmail.com abstract: nahirnić, a.: supplements to butterfly fauna (hesperioidea and papilionoidea) of grza river gorge (eastern serbia), biologica nyssana, 2 (2), december 2011: 107-117. the aim of this article is report of butterfly fauna of grza river gorge (eastern serbia). during 2008-2009. nineteen field visits have been made, 11 localities and 12 types of habitats were investigated. a faunistic account of totally 84 recorded butterfly species is given compiling the data: 30 butterfy species previously published by other authors and 81 species from our research. we recorded 54 species new to the investigated territory. zoogeographical composition of all species is done. comparison of the data concerning two others valleys, lazarev canyon and the ibar river gorge studied by jakšić (2008), was made in order to show the specific fauna elements. key words: butterflies, grza river gorge introduction ! the butterfly fauna of grza river gorge has not been truly studied up to now. only two sources provide data on butterfly species present in the area (v a n s w a a y et al., 2007; j a k š i ć 2011). these references give us the list of only 29 species recorded at a single locality, mountain home "đorđe živković" and one more species for grza territory, respectively . grza river gorge is located 20 km eastern from paraćin, at the southern side of kučaj mountains as a part of carpatho-balkanides. examined territory includes the grza river gorge from vrelo to the mouth of čestobrodica river extending over javorački vrh mt. to the north-east, veliki kozji rog mt. to the east and pištolje to the west. grza river gorge is eight km long spreading north south and two km from east to west. this whole region is intersected by numerous brooks. the most important springs are malo vrelo and vrelo. grza river gorge has been carved during glacial and postglacial phase. the supstrate is predominantly calcareous combine with limestone, including neogene lake sediments, tertiary volcanic stones, karst and partly andesite and freshwater middle miocene sediments. the climate of the investigated region is temperate continental. climate and vegetation inversion is characteristic for the grza river gorge. in gorges air is cooler at lower altitudes. as gorge sides shelter valley from wind, atmosphere is calm and moist. this inversion provides suitable climate conditions for mesophile plants such as fagus moesiaca (k. maly) czecz which is dominant edificator of forests in this gorge. oak forests are distributed at higher altitudes above colline beech forests. the preliminary number of vascular plants comprises 346 species (z a j i ć & l a k u š i ć , 2004). according to m i š i ć (1981) relict and polydominant plant communities are typical for the grza river gorge. what makes this territory interesting for biological studies is a significant 2 (2) • december 2011: 107-117 biologica nyssana 2 (2) december 2011: 107-117 nahirnić a. supplements of butterfly fauna… 108 presence of the large number of relict and endemorelict species from the tertiary age and the elements of the sub-mediterranean flora materials and methods sampling of butterflies was done by using an entomological net, and after that the specimens were mounted and conserved. the material is in author’s collection. taxonomical order and the nomenclature were given according to k a r s h o l t & r a z o w s k y (1996) and van s w a a y et al. (2010), respectively. nineteen field examinations were done during 2008-2009, from april to september. localities on the map marked by numbers, the altitude, utm 10 x 10 km code and the habitat codes are listed on fig. 1. determination of habitats was done on the basis of eunis habitat classification l a k u š i ć (2005a) and l a k u š i ć et al. (2005), and partly h i l l et al. (2004). corresponding habitat types to d a v i e s & m o s s (2002) were identified following the l a k u š i ć (2005b). habitat types f3.242c, g1.691, g1.696 and g1.761 don’t have corresponding habitat types in international eunis habitat classification. as an alternative, these four habitat types were related to serbian national classification of habitats (l a k u š i ć & m e d a r e v i ć , 2010). serbian eunis habitat classification codes of these habitats were given together with codes and their names in english from serbian national classification. next habitat types were investigated: e grasslands and lands dominated by forbs, mosses or lichens: e1 dry grasslands: e1.2 perennial calcareous grassland and basic steppes (different than all e1.22 investigated in this study), e1.22 arid subcontinental steppic grassland (festucion valesiacae), e1.74 calamagrostis epigejos stands, e2.2 low and medium altitude hay meadows, e3.4 moist or wet eutrophic and mesotrophic grassland, e5 woodland fringes and clearings and tall forb stands: e5.21 xero-thermophile fringes, e5.22 mesophile fringes; f heathland, scrub and tundra: f3.242c b2.511 oriental hornbeam [carpinus orientalis] thicket; g woodland, forest and other wooded land: g1.691 a3.22 colline beech [fagus moesiaca] forests, g1.696 a3.22c polydominant relict colline beech [fagus moesiaca] forest, g1.761 a2.111 moesian hungarian oak [quercus frainetto] and turkey oak [quercus cerris] woods, g1.a46 southeastern european ravine forests. list of investigated localities. 1. mouth of čestobrodica to grza, 290 m a.s.l., ep 55, е2.2, e3.4, e5.21, e5.22, g1.a46. 2. weekend estate "grza", 305-337 m a.s.l., ep 56, e2.2, g1.691. 3. veliki kozji rog mt., 570 m a.s.l., ep 56, e1.74. 4. mountain home "đorđe živković", 423 m a.s.l., ep 56, e1.22, e2.2, e5.22 g1.691, g1.761. 5. pištolje, 547 m a.s.l., ep 56, e1.22, e5.22. 6. vrelski krš, 650 m a.s.l., ep 56, f3.242c. 7. vrelo, 415 m a.s.l., ep 56, g1.691. 8. vrelska padina, 679 m a.s.l., ep 56, e1.22, g1.761. 9. barski vrh мt., 785 m a.s.l., ep 56, e1.2, g1.696. 10. javoračke padine, 778 m a.s.l., ep 56, e2.2, e5.22. 11. javorački vrh mt., 930 m a.s.l., ep 56, f3.242c. figure 1. map of investigated localities results the following list includes all data of butterflies recorded in grza river gorge. all new data were made by the author. biologica nyssana 2 (2) december 2011: 107-117 nahirnić a. supplements of butterfly fauna… 109 superfamily hesperioidea family hesperiidae 1. erynnis tages (linnaeus, 1758) new data: weekend estate "grza" (e2.2), 20.04.2009; mouth of čestobrodica to grza (e5.21), 12.07.2009. 2. carcharodus alceae (esper, 1780) new data: weekend estate (e2.2), 13.06.2009. 3. pyrgus sidae (esper, 1784) new data: vrelska padina (e1.22), 14.06.2009. 4. pyrgus malvae (linnaeus, 1758) new data: weekend estate "grza" (e2.2), 20.04.2009; mouth of čestobrodica to grza (e5.21), 10.5.2009. 5. thymelicus lineola (ochsenheimer, 1808) published data: grza (380-420 m, utm: ep 46), 15.07.2005. van swaay et al. (2007). new data: weekend estate "grza" (e2.2), 13.06.2009; mountain home "đorđe živković" (e1.22), 07.07.2009; mouth of čestobrodica to grza (e5.21), 12.07.2009. 6. thymelicus sylvestris (poda, 1761) published data: grza (380-420 m, utm: ep 46), 15.07.2005. van swaay et al. (2007). new data: mouth of čestobrodica to grza (e2.2), 13.06.2009; pištolje (e1.22), 09.07.2009. 7. thymelicus acteon (rottemburg, 1775) new data: mouth of čestobrodica to grza (e2.2), 13.06.2009; mountain home "đorđe živković" (e2.2), 14.06.2009; vrelska padina (e1.22), 14.06.2009. 8. ochlodes sylvanus (esper, 1777) published data: grza (380-420 m, utm: ep 46), 15.07.2005. van swaay et al. (2007). new data: mountain home "đorđe živković" (e2.2), 28.07.2008; mountain home "đorđe živković" (e1.22), 01.08.2008; mouth of čestobrodica to grza (e2.2), 13.06.2009; vrelska padina (e1.22), 14.06.2009; mountain home "đorđe živković" (e2.2), 07.07.2009; vrelo (g1.691), 08.07.2009; pištolje (e1.22, e5.22), 09.07.2009; mouth of čestobrodica to grza (e5.21), 12.07.2009; mouth of čestobrodica to grza (e2.2), 12.07.2009; weekend estate "grza" (e2.2), 13.07.2009; vrelska padina (e1.22), 15.07.2009; barski vrh (e1.2), 15.07.2009; javoračke padine (e2.2), 15.07.2009; javorački vrh (f3.242c), 15.07.2009; mouth of čestobrodica to grza (e5.21), 02.09.2009. superfamily papilionoidea family papilionidae 9. zerynthia polyxena (denis & schiffermüller, 1775) new data: mouth of čestobrodica to grza (e2.2), 10.05.2009. 10. zerynthia cerisy (godart, 1824) new data: mouth of čestobrodica to grza (e5.21), 10.05.2010; mouth of čestobrodica to grza (e2.2) 13.06.2009, larvae. 11. parnassius mnemosyne (linnaeus, 1758) new data: vrelska padina (e1.22), 14.06.2009. 12. iphiclides podalirius (linnaeus, 1758) published data: grza (380-420 m, utm: ep 46), 15.07.2005. van swaay et al. (2007). new data: mountain home "đorđe živković" (e2.2), 28.07.2008; mountain home "đorđe živković" (e1.22), 29.07.2008; vrelska padina (e1.22), 31.07.2008; javoračke padine (e2.2), 31.07.2008; mountain home "đorđe živković" (e1.22), 01.08.2008; vrelo (g1.691), 01.08.2008; veliki kozji rog (e1.74), 02.08.2008; mouth of čestobrodica to grza (e2.2), 04.08.2008; weekend estate "grza" (e2.2), 20.04.2009; mouth of čestobrodica to grza (e5.21), 10.05.2009; mouth of čestobrodica to grza (e3.4, e5.21), 13.06.2009; weekend estate (e2.2), 13.06.2009; mountain home "đorđe živković" (e2.2), 14.06.2009; mountain home "đorđe živković" (e2.2), 07.07.2009; vrelo (g1.691), 08.07.2009; pištolje (e1.22, e5.22), 09.07.2009; mouth of čestobrodica to grza (e2.2, e5.21), 12.07.2009; javoračke padine (e2.2), 15.07.2009. 13. papilio machaon (linnaeus, 1758) new data: javoračke padine (e2.2), 31.07.2008; mouth of čestobrodica to grza (e2.2), 10.05.2009; mouth of čestobrodica to grza (e2.2), 02.09.2009. family pieridae 14. leptidea sinapis (linnaeus, 1758) published data: grza (380-420 m, utm: ep 46), 15.07.2005. van swaay et al. (2007). new data: javoračke padine (e2.2), 31.07.2008; weekend estate "grza" (e2.2), 20.04.2009; mouth of čestobrodica to grza (e3.4), 10.05.2009, 2$m; mouth of čestobrodica to grza (e5.21), 13.06.2009; biologica nyssana 2 (2) december 2011: 107-117 nahirnić a. supplements of butterfly fauna… 110 mountain home "đorđe živković" (e2.2), 14.06.2009; vrelska padina (e1.22), 14.06.2009; mountain home "đorđe živković" (e1.22, e.22), 07.07.2009; vrelo (g1.691), 08.07.2009; pištolje (e1.22, e5.22), 09.07.2009; mouth of čestobrodica to grza (e5.21), 12.07.2009; weekend estate "grza" (e2.2), 13.07.2009; mouth of čestobrodica to grza (e2.2, e3.4, e5.21), 12.07.2009; vrelska padina (e1.22), 15.07.2009; barski vrh (e1.2), 15.07.2009; javoračke padine (e2.2), 15.07.2009; mouth of čestobrodica to grza (e2.2, e3.4, e5.21), 02.09.2009. 15. antocharis cardamines (linnaeus, 1758) new data: weekend estate "grza" (e2.2), 20.04.2009; mouth of čestobrodica to grza (e3.4), 10.05.2009. 16. aporia crataegi (linnaeus, 1758) new data: mouth of čestobrodica to grza (e2.2, e5.21), 13.06.2009; weekend estate (e2.2), 13.06.2009; mountain home "đorđe živković" (e2.2), 14.06.2009; vrelska padina (e1.22), 14.06.2009. 17. pieris rapae (linnaeus, 1758) published data: grza (380-420 m, utm: ep 46), 15.07.2005. van swaay et al. (2007). new data: mountain home "đorđe živković" (e2.2), 28.07.2008; javoračke padine (e2.2), 31.07.2008; vrelo (g1.691), 01.08.2008; mountain home "đorđe živković" (e2.2), 03.08.2008; weekend estate "grza" (e2.2), 20.04.2009; mouth of čestobrodica to grza (e2.2, e5.21), 10.05.2009; mouth of čestobrodica to grza (e2.2, e5.21), 13.06.2009; weekend estate (e2.2), 13.06.2009; mountain home "đorđe živković" (e2.2), 14.06.2009; vrelska padina (e1.22), 14.06.2009; mountain home "đorđe živković" (e1.22, e2.2), 07.07.2009; vrelo (g1.691), 08.07.2009; pištolje (e1.22, e5.22), 09.07.2009; mouth of čestobrodica to grza (e5.21), 12.07.2009; mouth of čestobrodica to grza (e2.2, e3.4, e5.21), 12.07.2009; weekend estate (e2.2), 13.07.2009; vrelska padina (e1.22), 15.07.2009; barski vrh (e1.2), 15.07.2009; javoračke padine (e2.2), 15.07.2009; javorački vrh (f3.242c), 15.07.2009; mouth of čestobrodica to grza (e2.2, e3.4, e5.21), 02.09.2009. 18. pieris napi (linnaeus, 1758) published data: grza (380-420 m, utm: ep 46), 15.07.2005. van swaay et al. (2007). new data: weekend estate "grza" (e2.2), 20.04.2009; mouth of čestobrodica to grza (e2.2, e3.4, e5.21) 10.05.2009; mouth of čestobrodica to grza (e5.21, g1.a46), 13.06.2009; mouth of čestobrodica to grza (e5.21), 12.07.2009; mouth of čestobrodica to grza (e5.21), 02.09.2009. 19. colias erate (esper, 1805) new data: mountain home "đorđe živković" (e2.2), 28.07.2008; mountain home "đorđe živković" (e1.22), 01.08.2008. 20. colias croceus (fourcroy, 1785) published data: grza (380-420 m, utm: ep 46), 15.07.2005. van swaay et al. (2007). new data: mountain home "đorđe živković" (e2.2), 28.07.2008; mountain home "đorđe živković" (e1.22), 29.07.2008; mouth of čestobrodica to grza (e2.2), 04.08.2008; mountain home "đorđe živković" (e2.2), 08.07.2009; mouth of čestobrodica to grza (e2.2), 13.06.2009; mouth of čestobrodica to grza (e2.2), 12.07.2009; mouth of čestobrodica to grza (e2.2, e5.21), 02.09.2009. 21. colias hyale (linnaeus, 1758) new data: mouth of čestobrodica to grza (e5.21), 12.07.2009; mouth of čestobrodica to grza (e2.2, e5.21), 02.09.2009. 22. gonepteryx rhamni (linnaeus, 1758) published data: grza (380-420 m, utm: ep 46), 15.07.2005. van swaay et al. (2007). new data: javoračke padine (e5.22), 31.07.2008; weekend estate "grza" (e2.2), 20.04.2009, 2$m; mouth of čestobrodica to grza (e5.21, e5.22), 10.05.2009; mouth of čestobrodica to grza (e5.21), 13.06.2009; mountain home "đorđe živković" (e5.22), 14.06.2009; vrelska padina (e1.22), 14.06.2009; mountain home "đorđe živković" (e2.2), 07.07.2009; weekend estate "grza" (e2.2), 13.07.2009; mouth of čestobrodica to grza (e5.21, e5.22), 12.07.2009; javorački vrh (f3.242c), 15.07.2009. family riodinidae 23. hamearis lucina (linnaeus, 1758) new data: mountain home "đorđe živković" (e2.2), 28.07.2008; mouth of čestobrodica to grza (e2.2), 10.05.2009. family lycaenidae 24. lycaena phlaeas (linnaeus, 1761) new data: mouth of čestobrodica to grza (e2.2), 13.06.2009; mountain home "đorđe živković" (e2.2), 14.06.2009; vrelska padina (e1.22), biologica nyssana 2 (2) december 2011: 107-117 nahirnić a. supplements of butterfly fauna… 111 14.06.2009; pištolje (e1.22), 09.07.2009; mouth of čestobrodica to grza (e2.2), 02.09.2009. 25. lycaena dispar (haworth, 1802) new data: mouth of čestobrodica to grza (e2.2), 13.06.2009; vrelska padina (e1.22), 14.06.2009; mouth of čestobrodica to grza (e2.2), 02.09.2009. 26. lycaena virgaureae (linnaeus, 1758) published data: grza (380-420 m, utm: ep 46), 15.07.2005. van swaay et al. (2007). new data: vrelska padina (e1.22), 14.06.2009; weekend estate (e2.2), 13.07.2009. 27. lycaena tityrus (poda, 1761) new data: mouth of čestobrodica to grza (e3.4), 10.05.2009; mouth of čestobrodica to grza (e2.2), 02.09.2009. 28. lycaena alciphron (rottemburg, 1775) published data: grza (380-420 m, utm: ep 46), 15.07.2005. van swaay et al. (2007). new data: vrelska padina (e1.22), 14.06.2009. 29. lycaena thersamon (esper, 1784) new data: vrelska padina (e1.22), 14.06.2009. 30. favonius quercus (linnaeus, 1758) new data: vrelska padina (g1.761), 02.08.2008. 31. callophrys rubi (linnaeus, 1758) new data: vrelska padina (e1.22), 14.06.2009. 32. satyrium ilicis (esper, 1779) new data: mouth of čestobrodica to grza (e5.21, e5.22, g1.a46), 13.06.2009; vrelska padina (g1.761), 14.06.2009; mouth of čestobrodica to grza (g1.a46),12.07.2009. 33. cupido argiades (pallas, 1771) new data: mouth of čestobrodica to grza (e2.2, e3.4, e5.21), 02.09.2009. 34. celastrina argiolus (linnaeus,1758) published data: grza (380-420 m, utm: ep 46), 15.07.2005. van swaay et al. (2007). new data: mouth of čestobrodica to grza (e5.21), 13.06.2009; vrelska padina (e1.22), 14.06.2009; mountain home "đorđe živković" (e1.22, e2.2), 07.07.2009; vrelo (g1.691), 08.07.2009; mouth of čestobrodica to grza (e1.22), 13.07.2009; weekend estate "grza" (e2.2), 13.07.2009. 35. scolitantides orion (pallas, 1771) new data mouth of čestobrodica to grza (e5.21), 10.05.2009; mouth of čestobrodica to grza (e2.2, e5.21, g1.a46), 12.07.2009. 36. iolana iolas (ochsenheimer, 1816) new data: mountain home "đorđe živković" (e1.22), 39.07.2008. 37. phengaris alcon (doherty, 1891) new data: javoračke padine (e2.2), 15.07.2009. 38. plebejus argus (linnaeus, 1758) new data: mountain home "đorđe živković" (e1.22), 01.08.2008; mountain home "đorđe živković" (e2.2), 03.08.2008. 39. plebejus idas (linnaeus, 1761) new data: vrelska padina (e1.22), 14.06.2009. 40. aricia agestis (denis & schiffermüller, 1775) new data: mouth of čestobrodica to grza (e2.2, e5.21), 02.09.2009. 41. aricia artaxerxes (fabricius, 1793) new data: pištolje (e1.22), 09.07.2009. 42. polyommatus semiargus (rottemburg, 1775) new data: mouth of čestobrodica to grza (e2.2), 13.06.2009; mountain home "đorđe živković" (e2.2), 14.06.2009; mountain home "đorđe živković" (e1.22), 07.07.2009. 43. polyommatus amandus (schneider, 1792) new data: mouth of čestobrodica to grza (e2.2), 13.06.2009; mountain home "đorđe živković" (e2.2), 14.06.2009. 44. polyommatus thersites (cantener, 1835) new data: mouth of čestobrodica to grza (e2.2, e5.21), 13.06.2009; vrelska padina (e1.22), 14.06.2009. 45. polyommatus icarus (rottemburg, 1775) new data: mouth of čestobrodica to grza (e3.4), 10.05.2009; mouth of čestobrodica to grza (e2.2, e3.4, e5.21), 13.06.2009; weekend estate "grza" (e2.2), 13.06.2009; mountain home "đorđe živković" (e2.2), 14.06.2009; mountain home "đorđe živković" (e1.22), 07.07.2009; mouth of čestobrodica to grza (e2.2, e5.21), 12.07.2009; mouth of čestobrodica to grza (e2.2, e5.21), 02.09.2009. biologica nyssana 2 (2) december 2011: 107-117 nahirnić a. supplements of butterfly fauna… 112 46. polyommatus daphnis (denis & schiffermüller, 1775) new data: mountain home "đorđe živković" (e2.2), 28.07.2008; vrelska padina (e1.22), 31.07.2008; veliki kozji rog (e1.74), 02.08.2008; pištolje (e5.22), 09.07.2009; mouth of čestobrodica to grza (e5.21, g1.a46), 12.07.2009; weekend estate (e2.2), 13.07.2009; vrelska padina (e1.22), 15.07.2009; javoračke padine (e2.2), 15.07.2009. family nymphalidae 47. argynnis paphia (linnaeus, 1758) published data: grza (380-420 m, utm: ep 46), 15.07.2005. van swaay et al. (2007). new data: mountain home "đorđe živković" (e2.2), 28.07.2008; mountain home "đorđe živković" (e1.22), 29.07.2008; vrelska padina (e1.22), 31.07.2008; javoračke padine (e2.2), 31.07.2008; mountain home "đorđe živković" (e1.22), 01.08.2008; vrelo (g1.691), 01.08.2008; mountain home "đorđe živković" (e2.2), 03.08.2008; mouth of čestobrodica to grza (e2.2), 04.08.2008; mouth of čestobrodica to grza (e5.21), 13.06.2009; weekend estate "grza" (e2.2), 13.06.2009; mountain home "đorđe živković" (e2.2), 14.06.2009; mountain home "đorđe živković" (e2.2, e5.22), 07.07.2009; vrelo (g1.691), 08.07.2009; pištolje (e1.22, e5.22), 09.07.2009; mouth of čestobrodica to grza (e2.2, e5.21), 12.07.2009; barski vrh (e1.2), 15.07.2009; vrelska padina (e1.22), 15.07.2009; javoračke padine (e2.2), 15.07.2009; javorački vrh (f3.242c), 15.07.2009; mouth of čestobrodica to grza (e2.2, e5.21), 02.09.2009. 48. argynnis aglaja (linnaeus, 1758) new data: mountain home "đorđe živković" (e2.2), 07.07.2009; javoračke padine (e2.2), 15.07.2009. 49. argynnis adippe (denis & schiffermüller, 1775) published data: grza (380-420 m, utm: ep 46), 15.07.2005. van swaay et al. (2007). 50. argynnis niobe (linnaeus, 1758) new data: mountain home "đorđe živković" (e2.2), 14.06.2009. 51. issoria lathonia (linnaeus, 1758) new data: mountain home "đorđe živković" (e2.2), 28.07.2008; javoračke padine (e2.2), 31.07.2008; mountain home "đorđe živković" (e2.2), 03.08.2008; mouth of čestobrodica to grza (e2.2), 04.08.2008; mouth of čestobrodica to grza (e5.21), 10.05.2009; mouth of čestobrodica to grza (e5.21), 13.06.2009; weekend estate "grza" (e2.2), 13.06.2009; mountain home "đorđe živković" (e2.2), 14.06.2009; vrelska padina (e1.22), 14.06.2009; mountain home "đorđe živković" (e2.2), 07.07.2009; mouth of čestobrodica to grza (e2.2, e5.21), 12.07.2009; weekend estate "grza" (e2.2), 13.07.2009; barski vrh (e1.2), 15.07.2009; javoračke padine (e2.2), 15.07.2009; javorački vrh (f3.242c), 15.07.2009; mouth of čestobrodica to grza (e2.2, e5.21), 02.09.2009. 52. brenthis daphne (denis & schiffermüller, 1775) published data: grza (380-420 m, utm: ep 46), 15.07.2005. van swaay et al. (2007). new data: mouth of čestobrodica to grza (e2.2), 13.06.2009; weekend estate "grza" (e2.2), 13.06.2009; mountain home "đorđe živković" (e2.2), 14.06.2009; vrelska padina (e1.22), 14.06.2009; mountain home "đorđe živković" (e2.2), 07.07.2009; vrelo (g1.691), 08.07.2009; pištolje (e1.22), 09.07.2009; mouth of čestobrodica to grza (e2.2, e3.4, e5.21), 12.07.2009; weekend estate "grza" (e2.2), 13.07.2009; barski vrh (e1.2), 15.07.2009; vrelska padina (e1.22), 15.07.2009; javoračke padine (e2.2), 15.07.2009; javorački vrh (f3.242c), 15.07.2009. 53. brenthis hecate (denis & schiffermüller, 1775) published data: grza (380-420 m, utm: ep 46), 15.07.2005. van swaay et al. (2007). new data: mouth of čestobrodica to grza (e2.2), 13.06.2009; weekend estate "grza" (e2.2), 13.06.2009; mountain home "đorđe živković" (e2.2), 07.07.2009. 54. boloria dia (linnaeus, 1767) new data: mouth of čestobrodica to grza (e2.2), 02.09.2009. 55. vanessa atalanta (linnaeus, 1758) published data: grza (380-420 m, utm: ep 46), 15.07.2005. van swaay et al. (2007). new data: mountain home "đorđe živković" (e2.2), 28.07.2008; mountain home "đorđe živković" (e1.22), 29.07.2008; javoračke padine (e2.2), 31.07.2008; mouth of čestobrodica to grza (e2.2), 04.08.2008; mouth of čestobrodica to grza (e5.21), 13.06.2009; weekend estate "grza" (e2.2), 13.06.2009; mountain home "đorđe živković" (e2.2, e5.22), 07.07.2009; vrelo (g1.691), 08.07.2009; pištolje (e1.22, e5.22), 09.07.2009; mouth of čestobrodica to grza (e2.2, e5.21), 12.07.2009; weekend estate "grza" (e2.2), biologica nyssana 2 (2) december 2011: 107-117 nahirnić a. supplements of butterfly fauna… 113 13.07.2009; javoračke padine (e2.2), 15.07.2009; mouth of čestobrodica to grza (e2.2), 02.09.2009. 56. vanessa cardui (linnaeus, 1758) new data: mouth of čestobrodica to grza (e5.21), 13.06.2009; weekend estate "grza" (e2.2), 13.06.2009; mountain home "đorđe živković" (e2.2), 14.06.2009; pištolje (e1.22, e5.22), 09.07.2009; weekend estate "grza" (e2.2), 13.07.2009; mouth of čestobrodica to grza (e2.2), 12.07.2009; mouth of čestobrodica to grza (e2.2, e5.21), 02.09.2009. 57. aglais io (linnaeus, 1758) published data: grza (380-420 m, utm: ep 46), 15.07.2005. van swaay et al. (2007). new data: javoračke padine (e2.2), 31.07.2008; mountain home "đorđe živković" (e1.22, e2.2), 07.07.2009; weekend estate "grza" (e2.2), 13.07.2009; mouth of čestobrodica to grza (e2.2), 12.07.2009; javoračke padine (e2.2), 15.07.2009; mouth of čestobrodica to grza (e2.2), 02.09.2009. 58. aglais urticae (linnaeus, 1758) published data: grza (380-420 m, utm: ep 46), 15.07.2005. van swaay et al. (2007). new data: mouth of čestobrodica to grza (e2.2), 13.06.2009; mountain home "đorđe živković" (e2.2), 14.06.2009. 59. polygonia c-album (linnaeus, 1758) published data: grza (380-420 m, utm: ep 46), 15.07.2005. van swaay et al. (2007). new data: javoračke padine (e5.22), 31.07.2008; weekend estate "grza" (e2.2), 13.06.2009; mountain home "đorđe živković" (e5.22), 14.06.2009; vrelo (g1.691), 08.07.2009; mountain home "đorđe živković" (e5.22), 07.07.2009; mouth of čestobrodica to grza (e5.21), 12.07.2009; weekend estate "grza" (e2.2), 13.07.2009; javoračke padine (e5.22), 15.07.2009; mouth of čestobrodica to grza (e5.21), 02.09.2009. 60. araschnia levana (linnaeus, 1758) published data: grza (380-420 m, utm: ep 46), 15.07.2005. van swaay et al. (2007). new data: weekend estate "grza" (e2.2), 20.04.2009; mountain home "đorđe živković" (e5.22, g1.691), 07.07.2009; mouth of čestobrodica to grza (e2.2), 02.09.2009; mouth of čestobrodica to grza (e2.2), 02.09.2009. 61. nymphalis antiopa (linnaeus, 1758) new data: mouth of čestobrodica to grza (e5.22), 10.05.2009. 62. nymphalis polychloros (linnaeus, 1758) new data: mountain home "đorđe živković" (e5.22), 07.07.2009. 63. melitaea cinxia (linnaeus, 1758) new data: vrelska padina (e1.22), 14.06.2009. 64. melitaea arduinna (esper, 1783) published data: ep 56, grza river, 380 – 420 m, 14.06.2009. (termophile meadows in vicinity of oak forest) jakšić (2011). 65. melitaea trivia (denis & schiffermüller, 1775) new data: mouth of čestobrodica to grza (e5.21), 10.05.200; pištolje (e1.22), 09.07.2009. 66. melitaea didyma (esper, 1778) new data: mouth of čestobrodica to grza (e2.2, e3.4, e5.21), 13.06.2009; mountain home "đorđe živković" (e2.2), 14.06.2009; vrelska padina (e1.22), 14.06.2009; mountain home "đorđe živković" (e1.22, e2.2), 07.07.2009; mouth of čestobrodica to grza (e2.2), 12.07.2009; vrelska padina (e1.22), 15.07.2009; mouth of čestobrodica to grza (e2.2, e5.21), 02.09.2009. 67. melitaea aurelia (nickerl, 1850) published data: grza (380-420 m, utm: ep 46), 15.07.2005. van swaay et al. (2007). 68. melitaea athalia (rottemburg, 1775) published data: grza (380-420 m, utm: ep 46), 15.07.2005. van swaay et al. (2007). new data: mouth of čestobrodica to grza (e2.2, e3.4), 13.06.2009; weekend estate "grza" (e2.2), 13.06.2009; vrelska padina (e1.22), 14.06.2009. 69. limenitis camilla (linnaeus, 1764) new data: mountain home "đorđe živković" (e5.22), 28.07.2008; javoračke padine (e5.22), 15.07.2009. 70. neptis sappho (pallas, 1771) new data: mountain home "đorđe živković" (e5.22), 28.07.2008; vrelo (g1.691), 01.08.2008; mountain home "đorđe živković" (g1.761), 03.08.2008; mouth of čestobrodica to grza (e3.4), 10.05.2009; weekend estate "grza" (g1.691), 13.07.2009; mouth of čestobrodica to grza (e5.21, e5.22, g1.a46), 02.09.2009. biologica nyssana 2 (2) december 2011: 107-117 nahirnić a. supplements of butterfly fauna… 114 71. apatura ilia (denis & schiffermüller, 1775) published data: grza (380-420 m, utm: ep 46), 15.07.2005. van swaay et al. (2007). new data: mouth of čestobrodica to grza (e5.21), 13.06.2009; mouth of čestobrodica to grza (g1.a46), 12.07.2009. 72. apatura iris (linnaeus, 1758) new data: mountain home "đorđe živković" (g1.761), 07.07.2009. 73. kirinia roxelana (cramer, 1777) new data: barski vrh (g1.696), 15.07.2009, 1$m. 74. pararge aegeria (linnaeus, 1758) new data: vrelski krš (f3.242c), 30.07.2008; vrelo (g1.691), 01.08.2008; mouth of čestobrodica to grza (e3.4, e5.21, g1.a46), 10.05.2009; mountain home "đorđe živković" (g1.691), 14.06.2009; vrelska padina (g1.761), 14.06.2009; vrelo (g1.691), 08.07.2009; mountain home "đorđe živković" (g1.691), 07.07.2009; mouth of čestobrodica to grza (e3.4), 12.07.2009; weekend estate "grza" (g1.691), 13.07.2009; barski vrh (g1.696), 15.07.2009; javorački vrh (f3.242c), 15.07.2009. 75. lasiommata megera (linnaeus, 1767) new data: mouth of čestobrodica to grza (e5.21), 12.07.2009; mouth of čestobrodica to grza (e5.21), 02.09.2009. 76. coenonympha arcania (linnaeus, 1761) published data: grza (380-420 m, utm: ep 46), 15.07.2005. van swaay et al. (2007). new data: mouth of čestobrodica to grza (e2.2, e3.4, e5.21), 13.06.2009; mountain home "đorđe živković" (e2.2), 14.06.2009; vrelska padina (e1.22), 14.06.2009; pištolje (e1.22, e5.22), 09.07.2009; mouth of čestobrodica to grza (e3.4, e5.21, g1.a46), 12.07.2009; weekend estate "grza" (e2.2), 13.07.2009; barski vrh (e1.2), 15.07.2009; vrelska padina (e1.22), 15.07.2009; javoračke padine (e2.2), 15.07.2009. 77. coenonympha pamphilus (linnaeus, 1758) new data: mountain home "đorđe živković" (e2.2), 28.07.2008; vrelska padina (e1.22), 31.07.2008; barski vrh (e1.2), 31.07.2008; javoračke padine (e2.2), 31.07.2008; vrelska padina (e1.22), 02.08.2008; veliki kozji rog (e1.74), 02.08.2008; mountain home "đorđe živković" (e2.2), 03.08.2008; mouth of čestobrodica to grza (e2.2), 04.08.2008; mouth of čestobrodica to grza (e2.2), 10.05.2009; mouth of čestobrodica to grza (e2.2, e5.21), 13.06.2009; weekend estate "grza" (e2.2), 13.06.2009; mountain home "đorđe živković" (e2.2), 14.06.2009; vrelska padina (e1.22), 14.06.2009; mountain home "đorđe živković" (e2.2), 07.07.2009; pištolje (e1.22), 09.07.2009; weekend estate "grza" (e2.2), 13.07.2009; vrelska padina (e1.22), 15.07.2009; javoračke padine (e2.2), 15.07.2009; mouth of čestobrodica to grza (e2.2, e5.21), 02.09.2009. 78. aphantopus hyperantus (linnaeus, 1758) published data: grza (380-420 m, utm: ep 46), 15.07.2005. van swaay et al. (2007). new data: mountain home "đorđe živković" (e2.2), 28.07.2008; javoračke padine (e2.2), 31.07.2008; vrelo (g1.691), 08.07.2009; mountain home "đorđe živković" (e2.2), 07.07.2009; pištolje (e1.22, e5.22), 09.07.2009; barski vrh (e1.2), 15.07.2009; vrelska padina (e1.22), 15.07.2009; javoračke padine (e2.2), 15.07.2009. 79. maniola jurtina (linnaeus, 1758) published data: grza (380-420 m, utm: ep 46), 15.07.2005. van swaay et al. (2007). new data: mountain home "đorđe živković" (e2.2), 28.07.2008; vrelski krš (f3.242c), 30.07.2008; barski vrh (e1.2), 31.07.2008; javoračke padine (e2.2), 31.07.2008; vrelska padina (e1.22), 02.08.2008; veliki kozji rog (e1.74), 02.08.2008; mountain home "đorđe živković" (e2.2, g1.691), 03.08.2008; mountain home "đorđe živković" (e1.22), 04.08.2008; mouth of čestobrodica to grza (e2.2), 04.08.2008; mouth of čestobrodica to grza (e2.2, e3.4, e5.21, g1.a46), 13.06.2009; weekend estate "grza" (e2.2), 13.06.2009; mountain home "đorđe živković" (e2.2), 14.06.2009; vrelska padina (e1.22), 14.06.2009; mountain home "đorđe živković" (e1.22, e2.2, e5.22), 07.07.2009; vrelo (g1.691), 08.07.2009; pištolje (e1.22, e5.22), 09.07.2009; mouth of čestobrodica to grza (e2.2, e3.4, e5.21), 12.07.2009; weekend estate "grza" (e2.2), 13.07.2009; barski vrh (e1.2), 15.07.2009; vrelska padina (e1.22), 15.07.2009; javoračke padine (e2.2), 15.07.2009; javorački vrh (f3.242c), 15.07.2009; mouth of čestobrodica to grza (e2.2, e5.21), 02.09.2009. 80. erebia medusa (denis & schiffermüller, 1775) new data: vrelska padina (e1.22), 14.06.2009. biologica nyssana 2 (2) december 2011: 107-117 nahirnić a. supplements of butterfly fauna… 115 81. melanargia galathea (linnaeus, 1758) published data: grza (380-420 m, utm: ep 46), 15.07.2005. van swaay et al. (2007). new data: vrelska padina (e1.22), 31.07.2008; barski vrh (e1.2), 31.07.2008; javoračke padine (e2.2), 31.07.2008; vrelska padina (e1.22), 02.08.2008; weekend estate "grza" (e2.2), 13.06.2009; mountain home "đorđe živković" (e2.2), 14.06.2009; mountain home "đorđe živković" (e2.2, e5.22), 07.07.2009; vrelo (g1.691), 08.07.2009; pištolje (e1.22, e5.22), 09.07.2009; mouth of čestobrodica to grza (e2.2, e3.4, e5.21), 12.07.2009; weekend estate "grza" (e2.2), 13.07.2009; barski vrh (e1.2), 15.07.2009; vrelska padina (e1.22), 15.07.2009; javoračke padine (e2.2), 15.07.2009; javorački vrh (f3.242c), 15.07.2009. 82. minois dryas (scopoli, 1763) new data: mountain home "đorđe živković" (e2.2), 28.07.2008; vrelski krš (f3.242c), 30.07.2008; vrelska padina (e1.22), 31.07.2008; barski vrh (e1.2), 31.07.2008; vrelska padina (e1.22), 31.07.2008; veliki kozji rog (e1.74), 02.08.2008; mountain home "đorđe živković" (e2.2, g1.691, g1.761), 03.08.2008; vrelska padina (e1.22, g1.761), 15.07.2009; mouth of čestobrodica to grza (e2.2), 02.09.2009. 83. arethusana arethusa (denis & schiffermüller, 1775) new data: mountain home "đorđe živković" (e5.22), 03.08.2008. 84. brintesia circe (fabricius, 1775) published data: grza (380-420 m, utm: ep 46), 15.07.2005. van swaay et al. (2007). new data: javoračke padine (e2.2), 31.07.2008; veliki kozji rog (e1.74), 02.08.2008; mountain home "đorđe živković" (e1.22), 04.08.2008; mountain home "đorđe živković" (e2.2), 07.07.2009; pištolje (e1.22, e5.22), 09.07.2009; mouth of čestobrodica to grza (e2.2, e5.21), 12.07.2009; weekend estate "grza" (e2.2), 13.07.2009; barski vrh (e1.2), 15.07.2009; vrelska padina (e1.22), 15.07.2009; mouth of čestobrodica to grza (e2.2, e5.21), 02.09.2009. table 1. comparative review of specific elements of butterfly fauna of three valleys specific elements of butterfly fauna lazarev canyon grza river gorge ibar river gorge zerinthia cerisyi (godart, 1824) + + melitaea arduinna (esper, 1783) + + pyrgus sidae (esper, 1784) + iolana iolas (ochsenheimer, 1816) + maculinea alcon (d. & s., 1775) + aricia artaxerxes (fabricius, 1793) + melitaea aurelia (nickerl, 1850) + colias erate (esper, 1805) + pseudophilotes vicrama (moore, 1865) + euphidryas aurinia (rottemburg, 1775) + melanargia larissa (geyer, 1828) + disscussion and conclusion the total number of recorded species is 84. quantitative composition of established butterfly species is determined as moderate abundance, following the scale which determines the selection of prime butterfly areas in serbia (jakšić (ed.), 2008). there has been recorded 54 species of butterflies new to grza river gorge territory. compared with van swaay et al., (2007) data, all species were rerecorded, except argynnis adippe and melitaea аurelia, since they are rare in serbia. the data for melitaea arduinna is done on the basis of jakšić (2011). all species are inside the boundaries of their known area of distribution. we consider that level of fauna knowledge is satisfactory. zoogeographical division of established species is done according to jakšić (1998). in zoogeographical point of view middle-european species are dominant (30 species, 36%) followed by pontic-mediterranean group with 16 species (19%). sub-mediterranean species (12 species, 14%) are also significant members of grza river gorge fauna. biologica nyssana 2 (2) december 2011: 107-117 nahirnić a. supplements of butterfly fauna… 116 specific elements of butterfly fauna studied in this paper are compared to specific elements of butterfly fauna of lazarev canyon and the ibar river gorge on the basis of previous study (jakšić (ed.), 2008). lazarev canyon and the grza river gorge belong to moesian floristic province. ibar river gorge is under influence of illyric province. this comparision is presented in table 1. after analysing recorded species we can conclude that butterfly fauna of ibar gorge is clearly different because of the presence of species p. vicrama, e. aurinia and m. larissa and also because of the absence of z. cerisyi and m. arduinna. these elements make the main difference between illyric and moesian province. on the other side, species registered in the grza river gorge, but not in lazarev canyon: p. sidae, c. erate, i. iolas, m. alcon, a. artaxerxes and m. aurelia are species from open sunny worm habitats which are not present in covered plot of lazarev canyon. m. arduinna is the best represent of moesian province. habitat analysis is made consisting 81 species recorded by author, excluding a. adippe m. arduinna and m. aurelia. аnalysis showed that 96,3% of 81 species are recorded at e grasslands and lands dominated by forbs, mosses or lichens, 12,3% at f heathland, scrub and tundra and 30,9% at g woodland, forest and other wooded land. high percentage of butterfly species inhabit forests occurs due to dominance of forest habitats in the grza river gorge territory especially g1.69 moesian fagus forests (g1.691 and/or g1.696) where 19,7% species were recorded. e2.2 low and medium hay meadows are the most suitable habitat types for butterflies having in mind that 71,6% of species were recorded here. in e1 dry grasslands (e1.2, e1.22 and/or e1.74) were found 56,8% of species, while also high percentage 54,3% were registered in e5 woodland fringes and clearings and tall forb stands (e5.21 and/or e5.22). in e3.4 moist or wet eutrophic and mesotrophic grassland 19,7% species were found, although there is only one meadow of that type in investigated territory. g1.761 a2.111 moesian hungarian oak [quercus frainetto] and turkey oak [quercus cerris] woods and g1.a46 southeastern european ravine forests inhabits 7,4% and 11,1%, respectively. natural habitats in grza river gorge are still in good condition, but the intensive touristic development, as well as moderate traffic influence is potential risk for habitat destruction. what makes the special and much stronger reason for providing habitat protection in the grza river gorge is occurrence of 9 target species listed in prime butterfly areas in serbia (jakšić (ed.), 2008) (no. from list of species: 7, 9, 11, 25, 35, 37, 64, 67, 80) and 11 strictly protected species by low of republic serbia (anonymous, 2010) (no. from list of species: 3, 9, 11, 13, 25, 36, 37, 61, 67, 71, 72). acknowledgements. i would like to give special thanks to predrag jakšić (faculty of sciences and mathematics, university of niš) for helping me in the field examination and zoran nikolić (faculty of biology, university of belgrade) for helpful comments on an earlier version of the manuscript. the part of this study was financially supported by bid „josif pančić“ faculty of biology, university of belgrade and eko fond paraćin. i want to thank olivera košanin, teacher of english, for editing the english text. references anonymous, 2010. pravilnik o proglašenju i zaštiti strogo zaštićenih biljnih vrsta biljaka, životinja i gljiva. prilog 2. strogo zaštićene vrste. službeni glasnik rs br 5/10. davies, c.e. & moss, d. 2002: eunis habitat classification european habitats classification system, european environment agency & european topic centre on nature protection and biodiversity. hill, m.o., moss, d. & davies, c.e., 2004. revision of habitat descriptions originating from devillers et al (2001). european topic centre on nature protection and biodiversity, paris. jakšić, p., 1998. altitudinal and biogeographical division of the butterflies of balkan peninsula (lepidoptera: hesperioidea & papilionoidea). the university thought, 5(2): 77-88. jakšić, p. (ed.), 2008. prime butterfly areas in serbia. habiprot, beograd, 223 pp. jakšić, p., 2011. melitaea arduinna (lepidoptera: nymphalidae): a new species for serbia. phegea 39(1): 8-11. karsholt, o. & razowsky, j., 1996. the lepidoptera of europe, a distribution checklist, apollo books, stenstrup, 380 pp. lakušić, d., 2005a. ključ za identifikaciju staništa srbije. in: lakušić, d. (ed.), staništa srbije, rezultati projekta “harmonizacija nacionalne nomenklature u klasifikaciji staništa sa standardima međunarodne zajednice”, institut za botaniku i botanička bašta “jevremovac”, biološki fakultet, univerzitet u beogradu, ministarstvo za nauku i zaštitu životne sredine republike srbije, http://habitat.bio.bg.ac.rs/ lakušić, d., 2005b. veze između međunarodnih klasifikacija staništa i klasifikacija staništa srbije. in: lakušić, d. (ed.), staništa srbije, rezultati projekta “harmonizacija nacionalne nomenklature u klasifikaciji staništa sa standardima međunarodne zajednice”, institut za biologica nyssana 2 (2) december 2011: 107-117 nahirnić a. supplements of butterfly fauna… 117 botaniku i botanička bašta “jevremovac”, biološki fakultet, univerzitet u beogradu, ministarstvo za nauku i zaštitu životne sredine republike srbije, http://habitat.bio.bg.ac.rs/ lakušić, d., blaženčić, j., ranđelović, v., butorac, b., vukojičić, s., zlatković b., jovanović, s., šinđar-sekulić, j., žukovec, d., čalić, i., pavićević, d. 2005. staništa srbije – priručnik sa opisima i osnovnim podacima. in: lakušić, d. (ed.), staništa srbije, rezultati projekta “harmonizacija nacionalne nomenklature u klasifikaciji staništa sa standardima međunarodne zajednice”, institut za botaniku i botanička bašta “jevremovac”, biološki fakultet, univerzitet u beogradu, ministarstvo za nauku i zaštitu životne sredine republike srbije, 684 pp. http://habitat.bio.bg.ac.rs/ lakušić, d., medarević, m., 2010. tipovi staništa zastupjeni na teritoriji republike srbije. annex 1 – the rulebook on habitat types, the criteria for the selection of habitat types, on sensitive, endangered, rare and priority for protection habitat types and on the protection measures for their conservation. official gazette of rs no. 35/2010. mišić, v., 1981. the forest vegetation of gorges and canyons in eastern serbia. institut za biološka istrazivanja “siniša stanković”, beograd, 327 pp. [in serbian]. swaay, c., van, cuttelod, a., collins, s., maes, d., lópez munguira, m., šašić, m., settele, j., verovnik, r., verstrael, t., waren, m., wiemers, m. and wynhof, i. 2010. european red list of butterfies luxembourg: publications office of the european union, 47 pp. swaay, c., van, jakšić, p. & đurić, m., 2007. the notes on some summer butterflies (lepidoptera: hesperioidea & papilionoidea) of eastern serbia. acta entomologica serbica, 12(1): 1-10. beograd. zajić, v., lakušić, d., 2004. contribution to the flora of the grza river gorge (e. serbia). proceedings of the 2nd congress of ecologists of macedonia with international participation, 2529.10.2003, ohrid. special issues of macedonian ecological society 6, skopje. anticancer compounds from medicinal plants biologica nyssana 5 (2)  december 2014: 91-102 zlatković, i. et al.  taxonomical, phztogeographical and ecological … 91 original article received: 17 jul 2014 revised: 21 september 2014 accepted: 10 november 2014 taxonomical, phytogeographical and ecological analysis of the salt marsh flora of central and southern serbia ivana zlatković1*, bojan zlatković2, vladimir ranđelović2, dragana jenačković2, lidija amidžić3 1 college of agriculture and food technology, ćirila and metodija 1, 18400 prokuplje, serbia 2university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia 3 university singidunum, faculty of applied ecology, belgrade, serbia *corresponding autor: gajevicivana@yahoo.com abstract: zlatković, i., zlatković, b., ranđelović, v., jenačković, d., amidžić, l.: taxonomical, phytogeographical and ecological analysis of the salt marsh flora of central and southern serbia. biologica nyssana, 5 (2), december 2014: 91-102. the floristic studies of salt marshes of central and southern serbia in period 2000-2014 have shown presence of 333 taxa within 176 genera and 46 families. the phytogeographical structure is dominated by taxa with wide distribution (291 or 87.39%). the best represented chorological types are: eurasian, holarctic, mediterranean-submediterranean and cosmopolitan. the analysis of representation of life forms has shown that salt marsh flora in this part of serbia has therophytic character, with a significant participation of hemicryptophytes. presence of a high number of threatened taxa, including some listed in “red book of flora of serbia, 1” as critically endangered (cr) plant taxa, indicates pronounced importance of these habitats for biodiversity conservation. key words: flora, chorological type, halophyte, life form, salt marsh area apstrakt: zlatković, i., zlatković, b., ranđelović, v., jenačković, d., amidžić, l.: taksonomska, fitogeografska i ekološka naliza flore slatina centralne i južne srbije. biologica nyssana, 5 (2), decembar 2014: 91-102. florističkim istraživanjima slatina centralne i južne srbije u periodu od 2000-2014. godine, utvrđeno je prisustvo 333 taksona svrstanih u 176 rodova i 46 familija. u fitogeografskom smislu dominiraju taksoni širokog rasprostranjenja (291, 87,39%). najzastupljeniji horološki tipovi su: evroazijski, holarktički, mediteransko-submediteranski i kosmopolitski. analiza zastupljenosti pojedinih životnih formi pokazala je da flora slatina ovog dela srbije ima terofitski karakter, sa značajnim učešćem hemikriptofita. prisustvo velikog broja ugroženih taksona, od kojih su neki našli svoje mesto i u „crvenoj knjizi flore srbije, 1“ kao krajnje ugroženi (cr) biljni taksoni, ukazuje na veliki značaj ovih staništa u smislu očuvanja biodiverziteta. key words: flora, horološki tip, halofite, životna forma, slatinska područja 5 (2) • december 2014: 91-102 biologica nyssana 5 (2)  december 2014: 91-102 zlatković, i. et al.  taxonomical, phytogeographical and ecological analysis … 92 introduction the continental salt marshes belong to fragile, highly threatened habitats. they occupy large areas in pannonian and walachian plains and neighboring lowland regions. however, existence of halomorphic soil and salt marsh habitats is extremely rare in serbia south of vojvodina. in the area from the southern edge of pannonian basin to ovče polje in macedonia salt marshes include only a few small localities in river valleys of južna morava and toplica. in this area the salt marshes represent the relict habitats, where characteristic flora and vegetation have developed under the conditions of isolation and high anthropogenous pressure. vegetation of continental salt marshes is welldeveloped in the pannonian and walachian plains and neighboring lowland regions. the salt marsh areas of central and southern serbia are different from salt marshes of pannonian and walachian basins in the phytogeographical sense, as shown by studies of their flora. salt marsh flora in the study area was a subject of interest for many botanists, starting with p a n č i ć (1884). there are some scarce and non-systematized literature data on salt marsh flora recorded in vicinity of bujanovac, oslare and levosoje (s l a v n i ć , 1940), aleksandrovac (s l a v n i ć , 1940; z l a t k o v i ć et al., 2003), lalinac and oblačina salt marshes (p a n č i ć , 1884; n i k e t i ć , 1993; m i l o s a v l j e v i ć et al., 2002). halophyte vegetation of vicinity of prokuplje was also studied (r a n đ e l o v i ć et al., 2000). the goal of this paper is to present in a systematic manner all available literature and herbarium data, as well as data gathered in our own research and to perform a floristic analysis of these interesting habitats. investigated area the study area includes localities in southern and central serbia (fig. 1, tab. 1), divided into three groups: aleksandrovac salt marsh area, lalinac salt marsh area and bresničić salt marsh area. fig 1. geographical position of the investigated localities biologica nyssana 5 (2)  december 2014: 91-102 zlatković, i. et al.  taxonomical, phytogeographical and ecological analysis … 93 tabela 1. list of sites with geographical and climatic data (a – altitude, t mean annual temperature, m – mean annual precipitation) (temperature and precipitation data taken from diva-gis, 2002) locality n latitude e longitude a (m) t (co) m (mm) 1. levosoje 42°25'50'' 21°44'50'' 400 11,2 553 2. oslare 42°25'39'' 21°43'21'' 405 11,2 553 3. bujanovac 42°27' 21°45' 395 11,2 550 4. aleksandrovac 42°29'20'' 21°54'12'' 401 11,2 540 5. neradovac 42°31'17'' 21°52'56'' 403 11,0 550 6. lalinac 43°20'42'' 21°44'45'' 200 11,2 633 7. oblačina 43°18'26'' 21°40'54'' 285 11,1 636 8. lepaja 43°17'35'' 21°39'50'' 285 11,1 636 9. suva česma 43°13'58'' 21°30'43'' 257 10,9 655 10. bresničić 43°14'50'' 21°27'10'' 293 10,8 670 the aleksandrovac salt marsh area includes several smaller localities (aleksandrovac, oslare, levosoje, bujanovac and neradovac) with developed halomorphic soil and very interesting flora and vegetation. the greatest surface area of salt marshes in this area is in vicinity of aleksandrovac. the lalinac salt marsh area is situated at the foothills of mt. mali jastrebac. this area includes lalinačka, oblačinska and lepajska salt marshes. in the valley of river toplica, in the area between prokuplje and kuršumlija (bresničić salt marsh area), there are several salt marsh fragments. this paper includes data on the better-preserved salt marsh fragments suva česma and salt marshes near bresničić. material and methods the floristic list was composed according to literature data (s l a v n i ć , 1940; n i k e t i ć , 1995; m i l o s a v l j e v i ć et al., 2002; z l a t k o v i ć et al., 2003), herbarium collections (hmn) and our own studies in period 2000-2014. the list of localities is included in tab. 1. the collected material was herbarized and herbarium specimens stored in the herbarium collection “herbarium moesiacum” at the faculty of science and mathematics in niš (hmn). identification of plant material was performed by using keys in “flora europaea” (t u t i n et al., 1964-1980), “flora of serbia” (j o s i f o v i ć (ed.), 1970-1978; s a r i ć (ed.), 1986, 1992) and “flora of nr bulgaria” (j o r d a n o v (ed.) 1963-1979; v e l č e v 1979-1995). the nomenclature was correlated with med-checklist (g r e u t e r et al., 1984-1989), flora europaea (t u t i n et al., 19641980), and euro+med plantbase (euro+med 2006). the chorological types of plant taxa were defined by m e u s e l et al. (1965, 1978), m e u s e l and j ä g e r (1992) and s t e v a n o v i ć (1992). the life forms were presented according to raunkier’s principles, further developed by ellenberg and mueller-dombois (e l l e n b e r g & m u e l l e r -d o m b o i s , 1967 in m u e l l e r d o m b o i s & e l l e n b e r g , 1974) and modified according to s t e v a n o v i ć (1992) in “flora of serbia”. the abbreviations used for life forms follow the chart in “flora of serbia” (s t e v a n o v i ć , 1992b). the list of taxa follows this scheme: name of taxon – life form – chorological type (number of locality according to literature data) author (year); (number of locality in our study) results list of vascular plant taxa magnoliophyta magnoliopsida boraginaceae asperugo procumbens l. t rept ea (6) buglossoides arvensis (l.) i. m. johnston t scap ea (6, 10) heliotropium europaeum l. t scap – e(w)a (6) h. supinum l. t scap – e(w)a (2, 3, 4, 5) slavnić (1940) – (7) myosotis arvensis (l.) hill t scap-ros ea (6, 9) m. stricta link ex roemer & schultes t scap-ros ea (4) nonea pallens petrović t scap – pont/balk (6) campanulaceae legousia speculum-veneris (l.) chaix t scap – cemed-pont (6) caryophyllaceae cerastium brachypetalum pers. t scap – e(w)a (6) c. dubium (bast.) o. schwartz t caesp ce (4, 6, 9) c. glomeratum thuill. t caesp cosm (4, 6) c. pumilum curtis t scap-ros – ce-med-pont (4, 5, 6) c. semidecandrum l. t scap – e(w)a (4, 6) dianthus monadelphus vent. subsp. pallens (sibth. & sm.) w. greuter & burdet ch caesp – medsmed/balk (6) niketić (1995); biologica nyssana 5 (2)  december 2014: 91-102 zlatković, i. et al.  taxonomical, phytogeographical and ecological analysis … 94 d. pinifolius sibth. & sm. ch caesp – med-smed/balk (6) niketić (1995); d. viscidus bory & chaub t scap – med-smed/balk (2, 3, 4, 5) slavnić (1940); (4) gypsophila muralis l. t caesp ea (4, 7) holosteum umbellatum l. t scap – ea (4) minuartia viscosa (schreber) schinz & thell. t caesp – e(w)a (4) moenchia graeca boiss. & heldr. t caesp – medsmed/balk (4) m. mantica (l.) bartl. t scap – e(w)a (4, 6) petrorhagia prolifera (l.) p. w. ball & heywood t scap – e(w)a (6, 7, 10) scleranthus annuus l. t caesp ce-med (6) s. polycarpos l. t caesp ce-med (4) spergula arvensis l. t scap cosm (4) spergularia rubra (l.) j. & c. presl t caesp cosm (2, 3, 4, 5) slavnić (1940); (1, 4) vaccaria pyramidata medicus t scap cosm (6) chenopodiaceae atriplex prostrata boucher ex dc. t scap hol (5) slavinić (1940); (6, 9) a. rosea l. t scap – ea (2) slavinić (1940) a. tatarica l. t scap ea (2) slavinić (1940); (4, 10) camphorosma monspeliaca l. ch suffr caesp medpont (6) niketić (1995); (6) milosavljević & al. (2002); (6) chenopodium glaucum l. t scap ea (2, 3, 4, 5) slavnić (1940); (7, 9) c. hybridum l. t scap hol (4) c. rubrum l. t scap hol (1, 4, 10) c. urbicum l. t scap еа (10) polycnemum arvense l. t rept еа (6) salsola kali l. t scap еа (6) compositae achillea collina j. becker ex reichenb. h scap eа (6) a. crithmifolia waldst. & kit. h scap – med-pont (9) aster oleifolius (lam.) wagenitz h scap pont (6) niketić (1995); (6) a. sedifolius l. subsp. canus (waldst.& kit.) merxm. h scap – med-pont/balk (6) niketić (1995); (6) bellis perennis l. h ros ce-med (2, 3, 4, 5) slavinić (1940); (4) bidens tripartita l. t scap ea (4,5,2,3) slavinić (1940) carduus crispus l. t/h scap bienn ea (6) carthamus lanatus l. h scap ce-med-pont (6) centaurea pannonica (heuffel) simonkai h scap ce (6) niketić (1995); (4) c. salonitana vis. h scap pont (6) c. solsticialis l. t scap med-pont (6, 10) chamomilla recutita (l.) rauschert t scap ea (4, 6, 9) chondrilla juncea l. h scap – e(w)a (2, 3, 4, 5) slavnić (1940) cichorium intybus l. h scap cosm (4) cirsium arvense (l.) scop. h scap ea (6) crepis setosa haller fil. t scap med-smed (9) filaginella uliginosa (l.) opiz t scap ea (4,5,2,3) slavnić (1940); hypochoeris radicata l. h ros – e(w)a (4) inula britannica l. h scap ea (1, 6, 7) i. oculus-christi l. h scap-semiros – med-pont (6) lactuca saligna l. t/h scap bienn ce-med-pont (4) leucanthemum vulgare lam. h scap ea (6) onopordum acanthium l. t scap – e(w)a (6) picris echioides l. h scap med-smed (6) niketić (1995); (6,10) p. hieracioides l. t/h scap bienn e(w)a (6) pulicaria dysenterica (l.) bernh. h scap e(w)a (4) scorzonera cana (c. a. meyer) o. hoffm. h scap – ce-med-pont (6) niketić (1995); (6) milosavljević & al (2002); (4, 2, 1, 6, 9, 10) senecio aquaticus hill subsp. barbareifolius (wimmern & grab) h scap ce-med (1, 6) s. erucifolius l.h scap ce-med (6) s. vernalis waldst. & kit. t scap ce-med (6, 10) s. vulgaris l. t scap ea (6) sonchus asper (l.) hill t/h scap bienn ea (8) s. oleraceus l. t/h scap bienn ea (10) taraxacum erythrospermum andraz. ex besser h ros med-pont (4) t. palustre (lyons) symons h ros ce (4, 5) t. serotinum (waldst. & kit. ) poiret h ros med-pont (4) tragopogon pratensis l. h scap ce-med-pont (4) xanthium strumarium l. t scap antrop (2, 3, 4, 5) slavnić (1940) xeranthemum annuum l. t scap med-smed (6) x. cylindraceum sibth. & sm. t scap med-pont (6) crassulaceae sedum caespitosum (cav.) dc. t succ ce-med (4, 6, 10) cruciferae berteroa incana (l.) dc. t/h scap bienn ea (2, 3, 4, 5) slavnić (1940) bunias erucago l. t scap med-pont (6, 9) calepina irregularis (asso) thell. h scap med-pont (9) camelina sativa (l.) crantz t scap ce-med-pont (6) capsella bursa-pastoris (l.) medicus t scap-ros cosm (6) cardamine hirsuta l. t scap-ros ea (4, 6) cardaria draba (l.) desv. h scap ce-med (9) coronopus didymus (l.) sm. h scap ce-med (6) erophila verna (l.) chevall. t ros e(w)a (4, 6) erysimum repandum l. t scap e(w)a (6) lepidium perfoliatum l. t scap-ros ce-med-pont (6) milosavljević & al (2002); (4) zlatković & al (2003); (4, 6, 9, 10) l. ruderale l. t scap-ros e(w)a (1, 4, 6 ,7, 8, 10) myagrum perfoliatum l. t scap med-pont (6) neslia paniculata (l.) desv. subsp. thracica (velen.) bornm. t scap ea (6) rorippa sylvestris (l.) besser h scap-semiros ea (9) teesdalia coronopifolia (j. p. berg.) thell. t scap-ros ce-med-pont (4) thlaspi arvense l. t scap ea (10) dipsacaceae dipsacus fullonum l. t/h scap bienn e(w)a (6) knautia arvensis (l.) coulter t/h scap bienn e(w)a (6) euphorbiaceae euphorbia chamaesyce l. t scap – ea (6) biologica nyssana 5 (2)  december 2014: 91-102 zlatković, i. et al.  taxonomical, phztogeographical and ecological … 95 e. falcata l. t scap – ea (6) e. helioscopia l. t scap – ea (4, 6) e. salicifolia host h scap ce (10) gentianaceae centaurium erythraea rafin. t scap – med-pont (6, 7) c. littorale (d. turner) gilmour subsp. uliginosum (waldst. & kit.) rothm. t scap -ce-pont (6, 8) c. pulchellum (swartz) druce t scap ce-pont (4, 8) geraniaceae erodium cicutarium (l) l'her. t scap-ros cosm (4, 6) geranium dissectum l. t scap e(w)a (10) g. molle l. t scap e(w)a (10) guttiferae hypericum elegans stephan ex willd. h scap ea (6) haloragaceae myriophyllum spicatum l. hydt rad hol (4) labiatae lamium amplexicaule l. t scap – e(w)a (6) mentha aquatica l. h scap -cosm – (4) m. pulegium l. h scap ea (4, 6) m. spicata l. h scap antrop (6) phlomis tuberosa l. g tub/h scap – med-pont (6) niketić (1995); (6) salvia aethiopis l. t/h scap bienn ea (6, 7, 10) s. pratensis l. h scap – ce (6, 9, 10) stachys cassia (boiss.) boiss. h scap – med-smed (6) niketić (1995); s. germanica l. h scap – ce-med-pont (6) s. milanii petrović t scap – med-smed/balk (6) niketić (1995); (6) milosavljević & al (2002.); (6) teucrium chamaedrys l. ch suffr caesp e(w)a (6) t. polium l. ch suffr caesp – med-pont (6) t. scordium l. subsp. scordioides (schreber) maire & petitmengin h scap – med-smed (4, 6, 7,8) thymus pannonicus all. ch suffr rept ce (10) galega officinalis l. h scap e(w)a (6) lathyrus aphaca l t scap hol (6) l. hirsutus l. t scap ce-med (4, 6, 10) l. tuberosus l. g tub rept ea (6) lotus angustissimus l. t scap – med-pont (4, 6, 10) l. corniculatus l. h scap ce-med (6) l. tenuis waldst. & kit. ex willd. h scap – ce-med (2, 3, 4, 5) slavnić (1940) medicago arabica (l.) hudson t/h scap – med-pont (9) m. lupulina l. t scap ea (6) m. minima (l.) bartal. t scap ea (4, 6) m. rigidula (l.) all. t scap ce-med-pont (4, 6) m. officinalis (l.) pallas t/h scap bienn ea (6) ononis arvensis l. ch suffr caesp ea (4) o. spinosa l. ch suffr caesp ea (6) pisum sativum l. subsp. arvense (l.) asch. & graebn. t scap cemed-pont (6) trifolium dubium sibth. t scap ce-med-pont (10) t. echinatum bieb. h scap med-pont (6, 7, 8, 10) t. fragiferum l. h rept ea (4) t. incarnatum l. t scap med-smed (9) t. lappaceum l. t scap med-smed (6, 7, 10) t. nigrescens viv. t scap ce-med (4, 10) t. patens schreber t scap ce-med (6) t. pratense l. h scap ea (6) t. repens l. h rept hol (6) t. resupinatum l. h scap med-pont (4, 6, 9, 10) t. striatum l. h scap ce-med-pon (4) t. strictum l. h scap ea (6) t. subterraneum l. t rept med-pont (4) vicia grandiflora scop. t scap ce-med-pont (9) v. hirsuta (l.) s. f. gray t scap ce-med (9) v. narborensis l. t scap ce-med-pont (6) v. serratifolia jacq. t scap ce-med-pont (6, 10) linaceae linum corymbulosum reichenb. t scap med-smed (6) lythraceae lythrum hyssopifolia l. t scap cosm (1, 4) l. salicaria l. h scap ea (6) malvaceae althaea hirsuta l. t scap med-pont (1, 6) a. officinalis l. h scap ea (1, 6) onagraceae epilobium tetragonum l. h scap hol (10) papaveraceae fumaria officinalis l. t scap ea (6, 9) f. parviflora lam. t scap – phol-ptrop (6) f. vaillantii loisel. t scap ea (9) papaver dubium l. t scap – med-pont (2, 3, 4, 5) slavnić (1940) p. rhoeas l. t scap – med-pont (2, 3, 4, 5) slavnić (1940) plantaginaceae plantago coronopus l. t ros ce-med (2, 3, 4, 5) slavnić (1940); (4) zlatković & al (2003); (4) p. lanceolata l. h ros ea (4, 6, 9, 10) p. major l. subsp. intermedia (dc.) arcangeli h ros hol (3) slavnić (1940); (4) p. media l. h ros ea (8, 9) plumbaginaceae limonium gmelinii (willd.) o. kuntze h scap-ros/ch herb succ ea (6) niketić (1995); (6) milosavljević & al (2002.); (6, 7, 10) polygonaceae polygonum amphibium l. hydg rhiz hol (3) slavnić (1940) p. lapathifolium l. t scap hol (4) slavnić (1940) p. patulum bieb. t scap hol (2, 3, 4, 5) slavnić (1940); (4) rumex conglomeratus murray h scap ea (4, 6, 7) r. palustris sm. t scap ea (1, 7, 9) r. sanguineus l. h scap – med-pont (10) r. stenophyllus ledeb. h scap ea (7) portulacaceae montia fontana l. subsp. chondrosperma (fenzl) walters t rept cosm (4) ranunculaceae adonis aestivalis l t scap med-pont (9) a. flammea jacq. t scap med-pont (6) biologica nyssana 5 (2)  december 2014: 91-102 zlatković, i. et al.  taxonomical, phytogeographical and ecological analysis … 96 a. vernalis l. g rhiz caesp med-pont (6) niketić (1995); (10) consolida orientalis (gay) schodinger t scap e-medsmed-pon (2, 3, 4, 5) slavnić (1940); (6) c regalis s. f. gray t scap med-pont (2, 3, 4, 5) slavnić (1940); (6) myosurus minimus l. t ros med-pont – (4) zlatković & al (2003); (4, 6, 7) ranunculus acris l. h scap-semiros hol (4, 6) r. circinatus sibth. – hydt ea (4) r. ficaria l. subsp. calthifolius (reichenb.) arcangeli g tub rept ea (4) r. fluitans lam. – hydt ea (4) slavnić (1940); r. marginatus d'urv. t scap – ce-med-pont (4) r. ophioglossifolius vill. t/h scap bienn – med-smed (4) slavnić (1940) r. pedatus waldst. & kit. g tub/h scap ea (4, 6, 7, 9, 10) r. repens l. h rept ea (4, 6) r. sardous crantz t scap med-pon (9, 10) r. sceleratus l. t scap ea (7, 10) r. serbicus vis. h /g rhiz scap – med-smed/apembalk (6) r. trichophyllus chaix hydt cosm (4) slavnić (1940); (6) r. velutinus ten. h scap med-smed (4) thalictrum lucidum l. h scap ce (6) t. minus l. h scap ea (6) rosaceae potentilla reptans l. h rept ea (6) rubiaceae asperula purpurea (l.) ehrend. ch suffr caesp medsmed (6) galium parisiense l. t scap ce-med (4) g. tenuissimum bieb. t scap med-pont (6, 10) g. tricornutum dandy t scap ea (6) g. verum l. h scap ea (6, 10) sherardia arvensis l. t rept ea (4, 6) santalaceae comandra elegans (rochel ex reichenb.) reichenb. fil. ch suffr caesp – pont/balk (6) niketić (1995) scrophulariaceae kickxia elatine (l.) dumort. t rept e(w)a (2, 3, 4, 5) slavnić (1940); (4, 7, 10) k. spuria (l.) dumort. t rept e(w)a (2, 3, 4, 5) slavnić (1940); (4, 10) melampyrum arvense l. t scap ce-med (6) parentucellia latifolia (l.) caruel t scap ce-medpont (4) verbascum blattaria l. t/h scap bienn ea (6, 10) veronica acinifolia l. t scap ea (4, 6) v. anagallis-aquatica l. h scap ea (1, 7, 8) v. arvensis l. t scap ea (6) v. beccabunga l. h rept ea (4) v. hederifolia l. t caesp med-smed (6) v. polita fries t caesp ea (6) v. verna l. t caesp ea (10) umbelliferae anthriscus sylvestris (l.) hoffm. h scap ce (9) bifora radians bieb. t scap ce-med-pont (6) bupleurum rotundifolium l. t scap ea (6) b. tenuissimum l. t scap ce-med-pont (2, 3, 4, 5) slavnić (1940); (4, 6, 7, 10) oenanthe fistulosa l. h scap ce-med (1) o. silaifolia bieb h scap ce-med (4, 6, 10) pastinaca sativa l. t/h scap bienn ea (6) torilis nodosa (l.) gaertner -t scap ce-med-pont (7) turgenia latifolia (l.) hoffm. t scap ce-med-pont (6) valerianaceae valerianella carinata loisel. t scap ce-med (9) v. dentata (l.) pollich t scap ce-med (6) v. locusta (l.) laterrade t scap ce-med (4, 6) violaceae viola kitaibeliana schultes t caesp ce-med (6) monocotyledones alismataceae alisma plantago-aquatica l. hydg rhiz hol (4) slavnić (1940); (4, 8) amaryllidaceae leucojum aestivum l. g bulb – e(w)a (9) araceae arum orientale bieb. g rhiz caesp e(w)a (6) butomaceae butomus umbellatus l. hydg rhiz ea (1, 4) cyperaceae carex distans l. h caesp ea (1, 4, 5, 6, 9, 10) c. divisa hudson g rhiz caesp e(w)a (4, 3) slavnić (1940); (1, 4, 6, 7, 8, 9, 10) c. elata all. h caesp ce (4) slavinić (1940) c. hirta l. g rhiz caesp hol (9) c. hordeistichos vill. g rhiz caesp hol (7) c. otrubae podp. h/g rhiz caesp – e(w)a (4, 6, 9) c. tomentosa l. h/g rhiz caesp e(w)a (4, 6) c. vulpina l. h/g rhiz caesp e(w)a (1, 4, 6, 9, 10) cyperus flavescens l. t caesp cosm (2, 3, 4, 5) slavnić (1940); (8) c. fuscus l. t caesp hol (2, 3, 4, 5) slavnić (1940); (4, 7, 8, 9) c. longus l. g rhiz caesp ea (4) slavinić (1940); (1, 2) c. pannonicus jacq. t caesp ce-pont (4, 3) slavinić (1940); (4, 6, 9, 10) eleocharis palustris (l.) roemer & schultes hydg rhyz cosm (2, 3, 4, 5) slavnić (1940); (4, 6, 7) fimbristylis bisumbellata (forskål) bubani t caesp cosm (4) slavnić (1940); (4) zlatković & al (2003) scirpoides holoschoenus (l.) soják – h caesp/g rhiz ea (1) scirpus lacustris l. subsp. lacustris g rhiz caesp ea (4) slavnić (1940); s. lacustris l. subsp. tabenaemontani (c. c. gmelin) syme ea (4, 2, 6, 7, 8, 10) bolboschoenus maritimus (l.) palla g rhiz ceasp cosm (2, 3, 4, 5) slavnić (1940); (6) niketić (1995); (4) zlatković (2003); (1, 4, 6, 7, 9, 10) b. glaucus (lam.) s.g. sm. g rhiz ceasp cosm (6). biologica nyssana 5 (2)  december 2014: 91-102 zlatković, i. et al.  taxonomical, phztogeographical and ecological … 97 gramineae agropyron cristatum (l.) gaertner g rhiz caesp cemed-pont (6) niketić (1995); (6) agrostis capillaris l. h caesp hol (4) a. stolonifera l. h caesp hol (2, 3, 4, 5) slavnić (1940); alopecurus aequalis sobol. t rept ea (6) a. myosuroides hudson t rept e(w)a (6) a. pratensis l. h caesp ea (10) a. rendlei eig. t rept hol (4, 6, 9) a. odoratum l. h caesp ea (4) arrhenatherum elatius (l.) beauv. ex j. & c. presl h caesp – e(w)a (9) beckmannia eruciformis (l.) host h caesp hol (1) bromus arvensis l. t scap ea (4) b. commutatus schrader t scap ce-med (4) b. hordeaceus l. t scap hol (4, 9, 10) b. racemosus l. t scap ce (4) b. scoparius l. t scap med-pont-s sib (6) b. squarrosus l. t/h caesp bienn med-smed (6) b. tectorum l. t scap ce (9) calamagrostis arundinacea (l.) roth g rhiz caesp ce-pont (1) catabrosa aquatica (l.) beauv. g rhiz caesp hol (6, 8) crypsis aculeata (l.) aiton t caesp med-pont (2, 3) slavinić (1940); (4) zlatković & al (2003); (6, 10) c. alopecuroides (piller & mitt.) schrader t caesp ce-med-pont (4, 2, 3) slavinić (1940); (4, 8) c. schoenoides (l.) lam. t caesp med-pont (2, 3, 4, 5) slavinić (1940); (4, 6, 10) cynodon dactylon (l.) pers. g rhiz rept cosm (4, 6, 10) echinochloa crus-galli (l.) beauv. t caesp cosm (4, 3) slavinić (1940); eragrostis cilianensis (all.) f. t. hubbard t caesp ea (6) e. minor host t caesp hol (4) e. pilosa (l.) beauv. t caesp cosm (4, 10) festuca valesiaca schleicher ex gaudin h caesp cemed (7, 10) hordeum hystrix roth h caesp ce-med-pont (2, 3, 4, 5) slavinić (1940); (6) milosavljević & al (2002.); (4, 6, 8, 9, 10) h. murinum l. h caesp ce-med-pont (9) h. secalinum schreber h caesp hol (6, 7, 8, 10) lolium perenne l. h caesp ea (9) l. rigidum gaudin t caesp – med-smed (4, 10) melica transsilvanica schu h caesp ce (6) molineriella minuta (l.) rouy t scap medsmed/balk (4) phleum pratense l. subsp. bertolonii (dc) bornm h caesp ce-med (4) pholiurus pannonicus (host) trin. t caesp ce-medpont (4) zlatković & al (2003); (4, 6, 7, 8, 10) phragmites australis (cav.) trin. ex steudel hydg rhiz cosm (4) slavnić (1940); (4, 6, 8, 9) poa annua l. t caesp cosm (4, 6) p. bulbosa l. h caesp ea (4, 9) p. palustris l. h caesp hol (9) puccinellia distans (l.) parl. h caesp hol (2, 3, 4, 5) slavinić (1940); (6) niketić (1995); (6) milosavljević & al (2002.); (4, 6, 7, 8, 9, 10) p. festuciformis (host) parl. subsp. convoluta (hoernm.) w.e. hughes h caesp med-pont (4) zlatković & al (2003); (2, 1) sclerochloa dura (l.) beauv. t caesp ea(9) taeniatherum caput-medusae (l.) nevski t scap med-pont (10) tragus racemosus (l.) all. t caesp cosm (4) vulpia bromoides (l.) s. f. gray t scap med-pont (4) v. myuros (l.) c. c. gmelin t scap ce-med-pont (4, 6, 10) iridaceae crocus chrysanthus (herbert) herbert g bulb – medsmed/balk-anat (6) niketić (1995); juncaceae juncus articulatus l. g rhiz caesp hol (4, 3) slavinić (1940); (1, 4) j. bufonius l. t caesp hol (2, 3, 4, 5) slavinić (1940); (1, 4) j. compressus jacq. g rhiz caesp ea (6, 7, 9, 10) j. effusus l. g rhiz caesp cosm (6) j. gerardii loisel. g rhiz caesp hol (3, 4) slavnić (1940); (4, 6, 8, 10) j. inflexus l. g rhiz caesp hol (4) slavnić (1940); (6) j. ranarius song. & perr. t caesp ce-med-pont (2, 3, 4, 5) slavinić (1940); (7, 8) j. tenageia l. fil. t caesp e(w)a (1) lemnaceae lemna gibba l. hyd t cosm (7, 10) l. minor l. hyd t cosm (4) slavnić (1940); liliaceae allium cupani rafin. g bulb – med-smed/apen-balk (6) niketić (1995); a. guttatum steven subsp. dalmaticum (a. kerner ex janchen) g bulb med-smed/balk (6) niketić (1995); (4, 6, 10) a. scorodoprasum l. g bulb ce-med-pont (6) niketić (1995); (6) a. spaerocephalon l. g bulb ce-med-pont (6) a. vineale l. g bulb hol (6) muscari tenuiflorum tausch g bulb med-pont (6) ornithogalum divergens boreau g bulb ce-med-pont (5) o. pyramidale l. g bulb med-pont (6, 10) o. refractum kit. ex schlecht. g bulb med-pont (6) scilla autumnalis l. g bulb med-pont (4, 6) sternbergia colchiciflora waldst. & kit. g bulb cemed-pont (6) niketić (1995); orchidaceae orchis morio l. g tub scap ce-med-pont (4) spiranthes spiralis (l.) chevall. g tub scap ce-medpont (4) potamogetonaceae potamogeton pectinatus l. hydt cosm (4) slavnić (1940); p. polygonifolius pourret hydt cosm (4) slavnić (1940); biologica nyssana 5 (2)  december 2014: 91-102 zlatković, i. et al.  taxonomical, phytogeographical and ecological analysis … 98 sparganiaceae sparganium erectum l. hydg rhiz ea (4) slavnić (1940); typhaceae typha angustifolia l. hydg rhiz cosm (4) slavnić (1940); t. latifolia l. hydg rhiz cosm (4) slavnić (1940); (7, 10) zannichelliaceae zannichellia palustris l. hydt cosm (10) discussion the results of original studies performed in salt marsh fragments of central and southern serbia have shown presence of 334 taxa at species and subspecies level, belonging to 177 genera and 46 families. all taxa belong to division magnoliophyta. class magnoliopsida includes 232 species or 69.46% of total flora, while class liliopsida includes 102 species or 30.54% of flora in the study area. detailed taxonomic analysis was used to determine number of species and subspecies, genera and families in each salt marsh area (tab. 2). as expected, most species were recorded at lalinac salt marsh area (207 species) which includes the bestpreserved and so far best-studied fragments of salt marsh areas in serbia south of the pannonian basin. table 2. total taxa (nt), genera (ng) and families (nf) in salt marsh areas of central and southern serbia salt marsh area nt ng nf aleksandrovac 168 102 34 lalinac 207 126 39 bresničić 114 76 30 the analysis of representation of genera (tab. 3) and species in certain families (fig. 2) has shown that family gramineae has the highest number of taxa at aleksandrovac (19 genera, 27 species) and bresničić salt marsh areas (16 genera, 23 species), while family compositae had the highest number of taxa at lalinac salt marsh area (15 genera, 25 species). the pronounced diversity of family gramineae is attributed to strong steppe influence on formation of salt marsh flora. the high number of taxa from family compositae was expected as this family is richest in taxa both in balkan peninsula and in europe (t u r i l l 1929). analysis of representation of species in certain genera (fig. 3) has shown that the largest genera were ranunculus, trifolium, carex and juncus. table 3. comparative overview of families with the highest number of species in a salt marsh areas of central and southern serbia (a – aleksandrovac, l – lalinac, b – bresničić) a l b gramineae 30 21 23 compositae 18 24 9 cyperaceae 16 13 9 caryophyllaceae 13 12 3 ranunculaceae 13 13 5 legumonosae 11 21 12 cruciferae 6 11 7 liliaceae 3 10 5 labiatae 3 12 3 one important characteristic of flora in all three salt marsh areas is complete absence of highly halophilic genera such as suaeda and salicornia, in contrast to salt marshes of macedonia and pannonian plain. the phytogeographical analysis (tab. 4) has shown that floras of all three salt marsh areas are dominated by species with wide distribution, as a consequence of floristic uniformity of this habitat type as well as the pronounced anthropogenous influences on the broader regions. the dominance of taxa with eurasian range types indicates continental character of these areas. although they are few in number, the species of mediterranean-submediterranean distribution type play an important role in structure of the flora of aleksandrovac salt marsh area. this group also includes two endemic (allium guttatum subsp. dalmaticum and dianthus viscidus) and one subendemic taxon (moenchia graeca). dianthus viscidus and moenchia graeca are differential species in comparison to salt marshes of jastrebac foothills (bresničić and lalinac salt marsh area). group of differential species also includes the mediterraneanmacedonianthracian species molineriella minuta, which was first recorded in serbia during the studies of salt marshes of aleksandrovac area (z l a t k o v i ć et al., 2005). species puccinellia festuciformis also has a significant differential role, appearing as a dominant and characteristic species of association montiopuccinellietum festuciformis at the salt marshes in vicinity of vranje and bujanovac. flora of lalinac salt marsh area is predominantly characterized by species of pontiansouth siberian range type, with a differential character in comparison to aleksandrovac and bresničić salt marsh area. analysis of flora of lalinac salt marsh area has shown presence of the greatest number of endemics (stachys milanii and biologica nyssana 5 (2)  december 2014: 91-102 zlatković, i. et al.  taxonomical, phztogeographical and ecological … 99 allium guttatum subsp. dalmaticum) and subendemics (dianthus pinifolius, allium cupanii, ranunculus serbicus and achillea crithmifolia) in comparison to other salt marshes of central and southern serbia. this group may also include the species with ranges including part of balkan peninsula, pannonian plain and the western part of pontian-south siberian floristic region (comandra elegans, dianthus monadelphus, aster sedifolius, aster oleifolius, centaurea salonitana and nonea pallens). the greatest phytogeographical significance is certainly that of the endemic species stachys milanii. in addition, the lalinac salt marsh area also hosts another critically endangered species of serbian flora, camphorosma monspeliaca, creating characteristic chamaephyte associations. the bresničić salt marsh area was characterized by absence of any taxa of pontiansouth siberian range types, although the steppe influence is reflected in presence of 31 taxa with ranges mostly occupying the pontian-south siberian region. at the salt marshes of this area there was 30 18 16 13 13 11 21 24 13 13 12 21 23 9 9 5 3 12 0 5 10 15 20 25 30 35 gr am in ea e co m po sit ae cy pe ra ce ae ra nu nc ul ac ea e ca ry op hy lla ce ae le gu m in os ae aleksandrovac area lalinac area bresničić area fig. 2. comparative overview of families with the highest number of species in a salt marsh areas of central and southern serbia 10 6 6 5 4 4 3 1 6 6 5 7 2 5 5 5 3 5 2 6 2 1 1 1 0 2 4 6 8 10 12 ra nu nc ul us ca re x ju nc us tr ifo liu m br om us ce ra st iu m ve ro ni ca al liu m aleksandrovac area lalinac area bresničić area fig. 3. comparative overview of genera with the highest number of species in a salt marsh areas of central and southern serbia biologica nyssana 5 (2)  december 2014: 91-102 zlatković, i. et al.  taxonomical, phytogeographical and ecological analysis … 100 only one endemic (allium guttatum subsp. dalmaticum) and one subendemic taxon (achillea crithmifolia). flora of salt marshes of central and southern serbia includes 6 main (fig. 4) and two transitional life forms of plants. characteristic feature is significant participation of therophytes (about 50% of total flora in all three salt marsh areas) and complete absence of phanerophytes. the therophyte character of flora in these habitats is explained by presence of relatively arid and unsuitable habitats for plant life. additional factor favoring presence of therophytes and reducing the other ecological groups is substrate salinity. high levels of presence of therophytes within the spectrum of life forms of salt marsh flora are also contributed to by strong influence of the anthropogenous factor. in all salt marsh areas the hemicryptophytes table 4. comparative overview of presence of chorological types in the flora of salt marshes of central and southern serbia (n – number of species) s a l t m a r c h a r e a aleksandrovac lalinac bresničić chorological type n % n % n % cosmopolitan (cosm) 24 14.29 15 7.25 10 8.77 antropohoric (antrop) 1 0.60 1 0.48 0 0.00 holarctic (hol) 22 13.10 14 6.76 11 9.65 paleoholarctic-paleothropic (ph-pt) 0 0.00 1 0.48 0 0.00 eurasian (ea) 41 24.40 59 28.50 30 26.32 euro-west asian (e(w)a) 16 9.52 22 10.63 11 9.65 central european-mediterranean-pontic (ce-med-pont) 19 11.31 25 12.08 13 11.40 central european-pontic (ce-pont) 2 1.19 3 1.45 1 0.88 mediterranean-submediterranean-pontic (med-pont) 17 10.12 27 13.04 17 14.91 pontic-south siberian (pont) 0 0.00 4 1.93 0 0.00 central european (ce) 5 2.98 5 2.42 6 5.26 central european-mediterranean (ce-med) 13 7.74 15 7.25 9 7.89 mediterranean-submediterranean (med-smed) 8 4.76 16 7.73 6 5.26 fig. 4. comparative overview of the percentage representation of life forms in the flora of the investigated salt march areas biologica nyssana 5 (2)  december 2014: 91-102 zlatković, i. et al.  taxonomical, phztogeographical and ecological … 101 occupy the second place by number of species. the third place is occupied by the life form of geophytes. the pronounced presence of geophytes in salt marsh habitats is explained by their adaptation to unfavorable habitat and climate conditions, primarily during the arid summer period. presence of deeper water level in spring period and deeper temporary and permanent ponds enables appearance of hydrotherophytes and hydrogeophytes. the real hydrophytes were best represented at the aleksandrovac salt marsh area, due to proximity of accumulation lake. the water from the accumulation floods the coastal part of salt marsh in spring months, remaining for several months and enabling development of marsh vegetation of saline terrain. salt marshes are characterized by specific water regime. during the vegetation period they are flooded for a certain period of time. these conditions are not suitable for development of most chamaephytes. the percentage of chamaephytes is somewhat greater at lalinac salt marsh area than in other habitats, as a consequence of presence of welldeveloped steppe habitats. furthermore, the salt marshes of lalinac area are the only site where exclusively chamaephyte phytocoenoses of halophyte camhorosma mospelliaca were recorded. regarding the taxonomic diversity and endemism of flora, the salt marsh areas of central and southern serbia may be considered to be one of the more important centers of diversity for salt marsh flora and vegetation in the region south of the pannonian plain. as all three salt marsh areas are under strong anthropogenous influence, it is necessary to implement appropriate measure for conservation and protection of their flora and vegetation. acknowledgements. the ministry of education, science and technological development of the republic of serbia (grant 173030) supported this research. one part of the results was collected during the research activities in period 2003-2005, within the project “flora and vegetation of salt marshes of central and southern serbia and their valorization from the aspect of conservation”, financially supported by the institute for nature conservation of serbia. references euro+med, 2006-: euro+med plantbase the information resource for euro-mediterranean plant diversity. published on the internet http://ww2.bgbm.org/europlusmed/ greuter, w., burdet, h.m., long, g. (ed.), 19841989: med-checklist, 1, 3, 4. gèneve. йорданов, д., (ед.), 1963-1986: флора на нр българия, i-viii. издателство на бан. софия. josifović, m. (ed.), 1970-1977: flora sr srbije, iix. sanu. beograd. meussel, h., jager, e., weinert, e., 1965: vergleinchende chorologie der zentraleuropaischen flora. veb. gustav fischer verlag, 1. jena. meussel, h., jager, e., raischert, s., weinert, e., 1978: vergleinchende chorologie der zentraleuropaischen flora. veb. gustav fischer verlag, 2. jena. meusel, h., jager, e., 1992: vergleichende chorologie der zentraleuropaischen flora, karten, literatur, register. gustav fischer, jena, stuttgart, new york. milosavljević, v., ranđelović, v., zlatković, b., 2002: vegetacija lalinačke slatine kod niša. 7. simpozijum o flori srbije i susednih područja. zbornik rezimea, 47. dimitrovgrad. mueller-dombois, d., ellenberg, h., 1974: aims and methods of vegetation ecology. john wiley and sons. new york. niketić, m., 1995: pregled flore šireg područja lalinačke slatine kod niša. ii simpozijum o flori srbije (iv simpozijum o flori jugoistočne srbije). – zbornik rezimea, 34, vranje. pančić, j., 1884: dodatak flori kneževine srbije. kraljevska srpska državna štamparija. beograd. ranđelović, v., amidžić, l., ilić, n., 2000: halofitska vegetacija okoline prokuplja. 6. simpozijum o flori jugoistočne srbije i susednih područja. zbornik rezimea, 39. sokobanja. raunkiaer, c. 1934: the life forms of plants and statistical plant geography; being the collected papers of c. raunkiaer, translated into english by h. g. carter, a. g. transley and miss fausboll. london. sarić, m. (ed.), 1986: flora sr srbije, x. sanu. beograd. sarić, m. (ed.), 1992: flora srbije, i (2 izd.). sanu. beograd. slavnić, ž., 1940: prilog halofitskoj flori i vegetaciji jugoistočne srbije. glasnik skopskog naučnog društva, 22: 65-77, skoplje. stevanović, v. 1992: floristička podela teritorije srbije sa pregledom viših horiona i odgovarajućih flornih elemenata. in: sarić, m.r. (ed.), flora srbije, 1, 47-56. sanu, beograd. turrill, w.b., 1929: the plant-life of the balkan peninsula. a phytogeographical study. clarendon, oxford. 490 p. tutin, t. g., heywood, v. h., burges, n. a., valentine, d. h., walters, s. m. & webb, d. a. (eds.), 1964: flora europaea, 1. cambridge. biologica nyssana 5 (2)  december 2014: 91-102 zlatković, i. et al.  taxonomical, phytogeographical and ecological analysis … 102 велчев, в. (ед.), 1989: флора на нр българия, ix. издателство на бан. софия. zlatković, b, randjelović, v., amidzić, l., 2003: flora, vegetation and conservation of aleksandrovac's salth marsh. third international balkan botanical congress. abstracts, 134, sarajevo. zlatković, b, randjelović, v., amidzić, l., 2005: novi podaci o flori slatina centralne i južne srbije. 8. simpozijum o flori jugoistočne srbije i susednih područja. microsoft word bn-oa-0201-09 zivkovic & jovanovic biologica nyssana 2 (1) september 2011: 51-61 živković, d., jovanović, b. spatial morphometric plasticitz of spirlin… 67 original article ! spatial morphometric plasticity of spirlin alburnoides bipunctatus (bloch, 1782) phenotype from the nišava river, serbia, danube basin dušan živković1, boris jovanović*2 1 universityof niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, niš, serbia 2 interdepartmental toxicology program and fisheries biology program, departments of biomedical sciences and natural resource ecology and management, iowa state university, ames, ia, usa * e-mail: prcko@iastate.edu abstract: živković, d., jovanović, b.: spatial morphometric plasticity of spirlin alburnoides bipunctatus (bloch, 1782) phenotype from the nišava river, serbia, danube basin. biologica nyssana, 2 (1), september 2011: 67-77. variation of 22 morphometric and 4 meristic characters of the species alburnoides bipuuctatus from nišava river were analyzed. specimens were collected from four sampling stations along the putative mesohabitat gradient of river influence and were grouped into the categories with similar total length. morphometric characters varied immensely between different mesohabitats outlining morphometric plasticity. among several morphometry features that were influenced by different mesohabitats the most observable differences were noted for: postocular distance, anal fin height, minimal body height and preanal length. regarding the meristic features, number of soft rays in the anal fin did varied significantly among sampling stations while fourth hard ray in the anal fin for this species was reported for the first time ever. pharyngeal teeth formula of spirlin also showed variability across different mesohabitats. in conclusion, it appears that spirlin express a big morphological plasticity in relation to spatial and mesohabitat distribution and there is a possibility that spirlin from nišava river is a complex of neospecies in formation/morphotypes/ecotypes rather than a single population. key words: alburnoides bipunctatus, cyprinidae, mesohabitat, phenotype plasticity introduction ! spirlin alburnoides bipunctatus (bloch, 1782) – cyprinidae is a freshwater species reaching a maximal length of 13-14 cm (l e l e k , 1987). it is a sub-montane species typically present in upper and middle rithronic communities. it is widely distributed in europe and in north and west asia including the turkey and iran (b o g u t s k a y a , 1997, l e l e k , 1987). due to spirlin population abundance in majority of its areal, it is an important prey item of many predatory fish species, while its main food items are diatoms and green algae (t r e e r et al., 2006). spirlin is listed in iucn as a species of least concern, but locally threatened, and its status in serbia is lr (lc) (s i m o n o v i ć , 2001). several subspecies have been described for european region: a. bipunctatus rossicus (berg) from volga basin; a. bipunctatus strymonicus (chichkoff) from marica drainage; a. bipunctatus ohridanus (karaman) and a. bipunctatus fasciatus (nordmann) from ohrid lake and drim basin and a.bipunctatus prespensis (karaman) from lake prespa (l e l e k , 1987). at least six subspecies are present in asia (l e l e k , 1987). nominate subspecies inhabit both europe and asia. its areal is 2 (1) • september 2011: 67-77 biologica nyssana 2 (1) september 2011: 51-61 živković, d., jovanović, b. spatial morphometric plasticitz of spirlin… 68 from loire drainage in france eastward, in nearly all rivers draining to southern baltic, north, black and azov seas; caspian basin, in upper volga and from kura drainage southward to iranian tributaries of caspian (f r o e s e & p a u l y , 2011). nominate subspecies is also the one inhabiting nišava river (s i m o n o v i ć , 2001). recently, a relationship between morphometry and microhabitat utilization by a. bipunctatus has been established (copp et al., 2010; k o v a č et al., 2006). the differences observed were mainly due to different preference for water velocity, depth and substratum, within the same sampling station, as the fish get older (c o p p et al., 2010). such, segregation is likely connected with very specific abrupt isometric growth of a. bipunctatus two periods of isometric growth with different proportional values interrupted with short interval of allometric growth (k o v a č et al., 2006). such adaptation is increasing swimming performance of spirlin and provides better microhabitat utilization. while microhabitat analyses are based on identifying the segregation and preferences toward specific ecological factors within a habitat, mesohabitat modelling is based on a coarser scale covering longer sections of the river with mixture of ecological factors and adds the spatial component. the degree of spatial influence can be immense when analysing species with high morphometric plasticity potential. spatial component as a function of mesohabitat was not evaluated in the past regarding variation in morphometry for this species. therefore the aim of this study was to investigate whether the phenotypes of the spirilin will be significantly different along the same river. materials and methods stream and sampling site description the nišava river with its tributary ginska is a 202 km long with a watershed area of 4068 km2. it is situated in southeast serbia and bulgaria (upper one fourth of the river) and it is the largest tributary of the južna morava river, part of the southern danube river watershed (g a v r i l o v i ć & d u k i ć , 2002). the nišava has two distinct parts: upstream of the town of bela palanka it is a narrow, rapid mountain stream ranging from 6 to 76 m in width and 0.6 to 2 m in depth with velocity from 1 to 2.5 m s-1 and discharge of 0.3-270 m3 s-1 of water. after bela palanka and all the way to južna morava river, the nišava becomes a meandering stream with increased width and depth, and flow that carries up to 700 m3 s-1 of water (b r a n k o v i ć , 1997). sampling of spirlin was performed on four sites (fig. 1). the nišava river at ivko’s watermill, 1 km upstream from the city of dimitrovgrad was selected as site l1. this is the first section of the river as it enters serbia and there are no larger settlements upstream to this sampling site. in this section the river has a width of 3-6 m, and a depth of no more than 1 m. the bottom is composed of gravel, and the banks are covered with thick vegetation. the section is composed of light riffles intersperse with glide sections. the majority of the benthofauna is composed of ephemeroptera and trichoptera taxons, while in the ichthyofauna the most dominant species are: spirlin, brook barbel barbus balcanicus (kotlik, tsigenopoulos, rab & b e r r e b i 2002) cyprinidae and chub leuciscus cephalus (l. 1758) cyprinidae. figure 1. geographic region of nišava river. the four sampling stations (l1-l4) are indicated with dotted arrows biologica nyssana 2 (1) september 2011: 51-61 živković, d., jovanović, b. spatial morphometric plasticitz of spirlin… 69 site l2 – tributary of temska stream to nišava river. temska stream is the typical salmonid stream and the biggest right tributary of nišava river. the tributary section is enveloped in thick jungle-like vegetation, and river bottom is rocky with cobble size stones. the average water discharge is 10.5 m3 s-1 (ž i v i ć et al., 2005). mouth of the tributary is about 4 m wide with depth of 0.5 m and the whole section is with light riffles. benthofauna is almost exclusively composed of over dominant order trichoptera with occasional representatives of nematomorpha, hirudinea, odonata and megaloptera (ž i v i ć et al., 2005). ichthyofauna is similar to site l1 with addition of bleak alburnus alburnus (l. 1758) cyprinidae, barbel barbus barbus (l. 1758) – cyprinidae and nase chondrostoma nasus (l. 1758) cyprinidae. site l3 nišava river after town of bela palanka and before sićevo gorge. this section of river has biggest width, and it is essentially one long glide with mixture of sandy and muddy bottom with dept often over 1.5 m. as site l4, the nišava river before its entry of the major city of niš was selected (suburbia donja vrežina). the selected site is located on the last section of river, about 12-15 km before its mouth. in this section the nišava river is between 0.2 and 1.2 m deep and is a mixture of small riffle areas and stretches of slow moving water. the bottom is composed of gravel with copious cladophora sp. algae clinging on the bottom. benthofauna is composed predominantly of classes gastropoda, hirudinea, clitellata and malacostraca (gammaridae). icthyofauna is rich, with the predominant species such as: spirlin, barbel, brook barbel, chub, bleak, nase, perch perca fluviatilis (l. 1758) – percidae, bitterling rhodeus sericeus (p a l l a s , 1776) cyprinidae, carassius gibelio (b l o c h , 1783) cyprinidae, carp cyprinus carpio (l. 1758) cyprinidae, roach rutilus rutilus (l. 1758) cyprinidae, gudgeon gobio spp., as well as other typical lowland cyprinid species. morphometry several hundreds of spirlin specimens were sampled at each site ignoring the differences in microhabitats and sampling along the whole profile section. after the sampling, 40 specimens > 60 mm were chosen at random from each of the sampling sites and 22 morphometric and 4 meristic characters were measured or counted. list of measured morphometric characteristics is provided in fig. 2. all measurements were taken with 0.1 mm precision using vernier. list of meristic features counted is outlined in fig. 3. in adition mass of specimens were recorded using digital scale (kern 440’33, readability d = 0.01 g). figure 2. morphometry of a. bipunctatus a-b2: total length (longitudo totalis) a-b1: fork length (longitudo caudalis) a-v: standard length (longitudo corporis) c-g: proposal length (longitudo praedorsalis) g-d: base length of dorsal fin (longitudo dorsalis pinnae) hd: dorsal fin height (altitudo dorsalis pinnae) dc: postdorsal length (longitudo corporis postdorsalis pinnae) i-l: head length (longitudo capitis) i-j: preocular distance (spatium preorbitale) j-e: horisontal diameter of eye (diameter oculi horizonialis) k-l: postocular distance (spatium postorbitale) h-i-l: maximal head height (altitudo capitis) m-n: prepectoral length (longitudo praepectoralis) o-p: preventral length (longitudo praeventralis) o-a: preanal length (longitudo praeanalis) g-r: base length of anal fin (longitudo analis pinnae) hp: pectoral fin height (altitudo pectoralis pinnae) hv: ventral fin height (altitudo ventralis pinnae) ha: anal fin height (altitudo analis pinnae) t-v: caudal trunk length (longitudo pedunculi caudalis) h: maximal body height (altitudo corporis maxima) h: minimal body height (altitudo corporis minima) biologica nyssana 2 (1) september 2011: 51-61 živković, d., jovanović, b. spatial morphometric plasticitz of spirlin… 70 statistics for morphometry data sample from each sampling site was divided into two size categories based on the total length of the individuals: category a 60 80 mm; category b 80 100 mm. similar approach was previously used with success to assess difference in morphometry characteristics in mixed age sample for spirlin (k o v a č et al., 2006; c o p p et al., 2010). there were only five specimens in total longer than 100 mm (the longest was 122 mm) and these specimens were discarded from any further analysis. analysis of variance (anova) was used to depict any differences in characters between different sampling sites for each category separately followed by tukey – hsd post hoc comparison of means test. only values with p < 0.05 were considered statistically significant. results morphometrics morphometric results for each category are presented in tab. 1. total length of specimens in category a did not varied significantly between sampling sites (anova; df total = 63; f = 2.50; p > 0.05); however there was a significant difference in morphometry of 9 out of 22 characters (tab. 2). standard length (a-v) of specimens from site l1 was significantly smaller than the ones from l2 and l4 indicating a longer tail fin. specimens from l1 had shorter base of the dorsal fin (g-d) comparing to l4 specimens; l1 specimens also had shorter postdorsal length (dc) comparing to the l2 and l4 specimens. there was a small difference in preventral length (o-p) and preanal length (o-a) with o-p being shorter in l1 compared to l2 specimens and o-a being shorter in l1 as opposed to l4. caudal trunk length (t-v) was also shorter in l1 vs. l2 specimens. the most observable differences were for postocular distance (k-l); anal fin height (ha); and minimal body height (h) (fig. 4). postocular distance from l4 is significantly smaller comparing to specimens from any other sampling site. anal fin height was considerably bigger in l2 specimens comparing to any other sampling location while minimal body height of l1 was significantly smaller comparing to other samples. mass of the specimens varied slightly between localities for category a (anova; df total = 63; f = 4.59; p < 0.01). average mass with standard deviation of the specimens from l1-l4 were: 3.31 + 0.74; 4.64 + 0.53; 4.24 + 1.26; and 3.95 + 1.02 g respectively. category b significantly varied in total length between sampling locations (anova; f = 13.04; df total = 82; p << 0.001) (fig. 5). specimens from l1 and l4 sites had the same mean total length and were significantly different from l2 and l3 specimens. sample l2 and l3 had the approximately the same mean total length. since there was obvious difference in total length the rest of 21 morphometric features were compared only between the samples with the same mean. l1 vs. l4 and l2 vs. l3. results are presented in fig. 6. mass of the specimens varied significantly between localities for category b (anova; df total = 82; f = 16.55; p < 0.001). average mass with standard deviation of the specimens from l1-l4 were 6.34 + 1.23; 8.19 + 1.65; 9.32 + 1.46; and 6.42 + 1.20 g respectively. meristics number of scales in lateral line did not varied significantly between sampling sites (anova; df total = 159; f=0.41; p >> 0.05) and the average scale number formula for the whole river profile is 43-50 8-10/3-5. there was no difference in average number of hard or soft rays in the dorsal fin between sampling sites (anova; df total = 159; figure 3. meristic characters of a. bipunctatus. d: number of rays in dorsal fin separately for hard and soft (numerus radiorum pinnae dorsalis) a: number of rays in anal fin separately for hard and soft (numerus radiorum pinnae analis) ll: number of sacles in the lateral line (squamae linea lateralis) shown in red; number of scales above and below the lateral line (squamae linea trasversalis superior et squamae linea trasversalis inferior) shown in ocher. df: number of pharyngeal teeth (dentes pharyngeales) biologica nyssana 2 (1) september 2011: 67-77 živković, d., jovanović, b. spatial morphometric plasticitz of spirlin… 71 l1 l2 l3 l4 78 80 82 84 86 88 90 92 94 96 98 ab2 l1 l2 l3 l4 5.0 5.2 5.4 5.6 5.8 6.0 6.2 6.4 6.6 6.8 7.0 7.2 7.4 7.6 7.8 8.0 kl l1 l2 l3 l4 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 12.0 ha l1 l2 l3 l4 4.8 5.0 5.2 5.4 5.6 5.8 6.0 6.2 6.4 6.6 6.8 7.0 h figure 4. difference in a. bipunctatus category a morphometry characteristics between four sampling locations. y axis is in mm corresponding to the proper measurement (figure 1). l1 – l4 are different sampling locations. boxes refer to standard error and whiskers to standard deviation figure 5. distribution of a. bipunctatus category a total length morphometry across four sampling locations. y axis is in mm of total length. l1 – l4 are different sampling location. boxes refer to standard error and whiskers to standard deviation biologica nyssana 2 (1) september 2011: 51-61 živković, d., jovanović, b. spatial morphometric plasticitz of spirlin… 72 table 1. morpfometric analysis of a. bipunctatus. all data except column % are in mm. % refers to the percent of given character compared to total length of the specimen. std refers to standard deviation. category a category b mean % std min max mean % std min max a-b2 70.9 100.0 5.3 60.7 79.0 89.1 100.0 5.3 80.0 100.0 a-b1 64.5 91.0 4.9 54.5 72.4 81.4 91.4 5.1 72.1 96.0 a-v 58.0 81.8 4.5 50.0 65.7 73.3 82.3 4.6 64.2 83.0 c-g 30.6 43.2 2.7 25.5 37.3 38.6 43.3 2.8 33.0 44.5 g-d 7.29 10.3 0.9 5.2 9.7 9.4 10.5 0.8 7.7 11.5 hd 11.7 16.5 1.2 8.2 14.2 15.2 17.1 1.4 12.0 19.0 d-c 19.7 27.8 1.8 16.0 23.6 24.6 27.6 1.9 20.0 29.0 i-l 14.5 20.5 1.2 12.0 17.0 18.0 20.2 1.2 14.0 20.6 i-j 3.9 5.5 0.6 2.6 5.2 4.9 5.5 0.4 4.0 6.0 j-e 4.2 5.9 0.5 3.3 6.5 5.0 5.6 0.3 4.0 5.7 k-l 6.3 8.9 0.7 4.9 8.0 8.0 9.0 0.9 4.9 10.1 h-i-l 11.1 15.7 0.9 9.5 13.0 13.9 15.6 1.0 12.2 16.7 m-n 14.5 20.5 1.3 11.8 17.4 18.1 20.3 1.4 14.0 21.3 o-p 27.1 38.2 2.8 20.0 32.6 34.3 38.5 2.3 28.6 40.0 o-a 37.2 52.5 3.0 30.5 43.0 47.5 53.3 3.6 40.0 57.2 g-r 9.6 13.5 1.2 7.0 12.0 12.6 14.1 1.3 10.0 17.0 hp 11.2 15.8 1.1 8.6 13.2 14.2 15.9 1.2 12.0 17.4 hv 8.8 12.4 1.0 7.0 11.4 11.0 12.3 1.1 9.0 13.0 ha 9.5 13.4 1.3 6.0 12.0 11.8 13.2 1.3 9.0 15.2 t-v 11.9 16.8 1.3 9.7 15.2 15.1 16.9 1.3 12.7 18.0 h 15.7 22.1 1.6 12.6 20.0 21.0 23.6 2.1 16.4 25.0 h 5.9 8.3 0.67 4.4 7.0 7.5 8.4 0.7 5.8 9.0 table 2. analysis of variance and post hoc comparison of means of a. bipunctatus category a morphometry. l1 – l4 refers to the sampling site. +; ++; +++ means that the effect is statistically significant with p value being < 0.05; < 0.01; and < 0.001 respectively. ns means that the effect is not statistically significant. a-v g-d dc k-l o-p o-a ha t-v h p value + + + +++ + + ++ + ++ anova p – value tukey hsd l1 vs. l2 + ns + ns + ns ++ + + l1 vs. l3 ns ns ns ns ns ns ns ns + l1 vs. l4 + + + +++ ns + ns ns + l2 vs. l3 ns ns ns ns ns ns + ns ns l2 vs. l4 ns ns ns + ns ns ++ ns ns l3 vs. l4 ns ns ns +++ ns ns ns ns ns biologica nyssana 2 (1) september 2011: 51-61 živković, d., jovanović, b. spatial morphometric plasticitz of spirlin… 73 table 3. distribution of hard and soft rays in the anal fin of a. bipunctatus from nišava river. l1 – l4 refers to sampling locations. roman numerals refer to hard rays while arabic refers to soft rays. data are presented as % of sample. table 4. distribution of pharyngeal teeth of a. bipunctatus from nišava river. l1 – l4 refers to sampling locations. data are presented as % of the sample. f = 0.06; p >> 0.05). the overall number of rays in dorsal fin was d, ii-iii + (7-8) 9 (10). number of hard rays in anal fin did not varied statistically among the samples (anova; df total = 159; f=0.84; p >> 0.05), however for the first time in science a fourth hard ray in anal fin was detected for this species which has never been documented elsewhere. the fourth hard ray was present only in the specimens from sampling station l2 and l3. on the other hand, number of soft rays in the anal fin did varied significantly among sampling stations (anova; df total = 159; f = 5.61; p < 0.01). there was significantly higher number of soft rays in the specimens from l2. distribution of hard and soft rays from anal fin across sampling stations is presented in tab. 3. formula of anal fin rays is: a, (ii) iii (iv) + (10) (12) 13-15 (16). pharyngeal teeth of spirlin are in two rows. first row have two teeth, while the number of teeth in second raw is variable. specimens from l3 had more pharyngeal teeth on the right branchial arch than specimens from any other sample (anova; df total = 159; f = 9.08; p < 0.001). commonly these specimens had 7 teeth on the right branchial arch while specimens from other samples had 5 or 6 (tab. 4). discussion it is important to note that for the overall sample (category a + b) individuals from l2 and l3 on average have the biggest total length and mass. also, the biggest total length of any individual was 120 mm for l2 and 104.6 for l3 while the same parameter was only 90 mm for l1 and 91 mm for l4. one of the possible reasons for this might be better feeding conditions on sites l2 and l3. the studies performed in the fair vicinities of these localities demonstrated abundance of diatoms (t r a j k o v i c et al., 2008) which are spirlin’s primary food item (t r e e r et al., 2006) as well as caddis flies (ž i v i ć et al., 2005) which are secondary food item (t r e e r et al., 2006). better feeding can lead to faster growth rate which for this species is particularly characterized with two periods of isometric growth with different proportional values interrupted with short interval of allometric growth (k o v a č et al., 2006). thus, a difference in a rate of allometric growth can result in drastic changes of morphometry. in general, l2 and l3 spirlin had biggest anal fin height, as well as the biggest maximal body height and maximal observed total length, indicating better growth than spirlin ii iii iv 10 11 12 13 14 15 16 l1 12.5 87.5 0 0 0 5 45 37.5 12.5 0 l2 12.5 82.5 5 0 0 2.5 12.5 42.5 37.5 5 l3 5 92.5 2.5 0 0 10 30 50 10 0 l4 5 95 0 2.5 0 15 17.5 50 12.5 2.5 average 8.75 89.38 1.875 0.625 0 8.13 26.3 45 18.125 1.875 left branchial arch right branchial arch 3 4 5 6 7 3 4 5 6 7 l1 12.5 22.5 30 27.5 7.5 5 7.5 20 17.5 50 l2 2.5 17.5 35 35 10 5 12.5 20 25 37.5 l3 5 10 35 45 5 0 2.5 5 12.5 80 l4 2.5 17.5 37.5 37.5 5 5 5 35 37.5 17.5 average 5.625 16.875 34.375 36.25 6.875 3.75 6.875 20 23.125 46.25 biologica nyssana 2 (1) september 2011: 67-77 živković, d., jovanović, b. spatial morphometric plasticitz of spirlin… 74 l1 l4 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 kl c l1 l4 15.0 15.5 16.0 16.5 17.0 17.5 18.0 18.5 19.0 i-l a l1 l4 15.0 15.5 16.0 16.5 17.0 17.5 18.0 18.5 19.0 m -n a l2 l3 12.5 13.0 13.5 14.0 14.5 15.0 15.5 16.0 hi-l b l2 l3 32 33 34 35 36 37 38 op b l2 l3 42 44 46 48 50 52 54 oa c l2 l3 13.5 14.0 14.5 15.0 15.5 16.0 16.5 17.0 17.5 t-v b l2 l3 18 19 20 21 22 23 24 25 h c l2 l3 6.5 7.0 7.5 8.0 8.5 h b figure 6. difference in a. bipunctatus category b morphometry characteristics. y axis is in mm corresponding to the proper measurement (figure 1). l1 – l4 are different sampling locations. boxes refer to standard error and whiskers to standard deviation from other localities. spirlin of both categories from the l1 locality compared to spirlin from other samples had shorter base of the dorsal fin, shorter postdorsal, preventral, preanal, caudal trunk and minimal body length while having longer head, post ocular and prepoctaral length and theoretically longer tail fin. essentially this gives them a streamlined, torpedo-like shape with powerful head to pectoral region and slender, narrow streamline body for propelling themselves through the faster water current. fish with more streamline body have less drag (by minimizing viscous force) in a faster current, spending less energy by utilizing continuous, rather than burst-and-coast swimming mode (m ü l l e r et al., 2000; p e t r e l l & j o n e s , 2000). results of this investigation suggest that morphometry of spirlin can vary significantly for some characters, while others are not affected. comparison of this study with previous investigation of nominate subspecies features inhabiting different mesohabitat type (river gradac) identified head length (i-l); maximal head height (hi-l); base length of dorsal fin (g-d); caudal trunk length (t-v); maximal body height; and minimal body height (h) as characters with biggest variability (s t a n i s a v l j e v i ć et al., 1999). in conclusion, it appears that spirlin express a big morphological plasticity in relation to spatial and mesohabitat distribution. whether the plasticity was based on the influence of environmental factors, or it resulted from the expression of an organism's genes as well as the interactions between the two so far is not known. however, it is known that the karyotype of spirlin varies significantly on a spatial scale (k i l i c -d e m i r o k & ü n l ü , 2004). biologica nyssana 2 (1) september 2011: 51-61 živković, d., jovanović, b. spatial morphometric plasticitz of spirlin… 75 coupled with the number of known subspecies (l e l e k , 1987); the number of newly discovered sister species which were until recently classified as nominate subspecies (b o g u t s k a y a et al., 2010; c o a d & b o g u t s k a y a , 2009); and the new meristic characteristics presented in this manuscript it is reasonable to conclude that taxon a. bipunctatus bipunctatus is most likely complex of cryptic species/subspecies/neospecies in formation/morphotypes/ecotypes rather than a single nominate subspecies. it is possible (especially for species in formation) that species complex has the same genetic material (genome) which can be accounted for successful reproduction among different members, but completely different gene expression (transcriptome). it is hard to carry out such experiments for the species for which genome is not yet sequenced and most of the time pieces of information can be obtained through expressed sequence tag (est) sequencing coupled with genomics which is a laborious process based on pure luck of proper est extraction (j o v a n o v i ć et al., in press). however, the recent advances in "next generation" sequencing platforms and technologies, for quantitative comparative transcriptomics allowed for successful differentiation in the hepatic transcriptomes between two lake trout ecotypes, and demonstrated that this approach could be used to simultaneously discover and quantify differentially expressed genes in a nonmodel organism lacking a sequenced genome (g o e t z et al., 2010). this would be the right approach to discern whether spirlin from nišava river is a complex of different transcriptomic populations/ecotypes or not, and determine the level of crypticism within a complex if any. acknowledgements. we are thankful to dr stevan maletin for the useful comments regarding morphometry techniques. references bogutskaya, n.g., (1997). contribution to the knowledge of leuciscine fishes of asia minor. part 2. an annotated check-list of leuciscine fishes (leuciscinae, cyprinidae) of turkey with descriptions of a new species and two new subspecies. mitteilungen hamburgisches zoologisches museum und institut 94, 161-186. bogutskaya, n.g., zupančič, p. & naseka, a.m. (2010). two new species of freshwater fishes of the genus alburnoides, a. fangfangae and a. devolli (actinopterygii: cyprinidae), from the adriatic sea basin in albania. proceedings of the zoological institute ras, 314 (4): 448-468. branković, s. 1997. biosistematske i hematološke karakteristike riba iz gornjeg i donjeg toka reke nišave. bsc thesis. prirodno-matematički fakultet. priština. coad, b.w. & bogutskaya, n. g. (2009). alburnoides qanati, a new species of cyprinid fish from southern iran (actinopterygii, cyprinidae). zookeys, 13, 67-77. copp, g.h., kováč, v. & siryová, s. (2010). microhabitat use by stream-dwelling spirlin alburnoides bipunctatus and accompanying species: implications for conservation. folia zoologica, 59 (3): 240–256. froese, r. & pauly, d. editors. (2011). fishbase. world wide web electronic publication. www.fishbase.org, version (02/2011). gavrilović, lj. & dukić, d. (2002). reke srbije. zavod za izdavanje udžbenika, beograd. goetz, f., rosauer, d., sitar, s., goetz, g., roberts, s., johnson, r., murphy, c., charles, b. & mackenzie, s. (2010). a genetic basis for the phenotypic differentiation between siscowet and lean lake trout (salvelinus namaycush). molecular ecology, 19, 176-196. jovanović, b., goetz, f.w., goetz, g.w. & palić, d. (in press). different immunological stimuli change expression of genes in fathead minnow (pimephales promelas rafinesque, 1820). journal of fish biology. kilic-demirok, n & erhan ünlü, e. (2004). karyotype of the cyprinid fish alburnoides bipunctatus (cyprinidae) from the tigris river. folia biologica (kraków), 52 (1-2): 57-59. kováč, v., katina, s., copp, g.h. & siryová, s. (2006). ontogenetic variability in external morphology and microhabitat use of spirlin alburnoides bipunctatus from the river rudava (danube catchment). journal of fish biology, 68, 1257–1270. lelek, a. (1987). the freshwater fishes of europe. vol. 9. threatened fishes of europe, balogh scientific books. 343 p. müller, u.k., stamhuis, e.j. & videler, j.j. (2000). hydrodynamics of unsteady fish swimming and the effects of body size: comparing the flow fields of fish larvae and adults. the journal of experimental biology, 203, 193–206. petrelland, r.j. & jones, r.e. (2000). power requirement of swimming in chinook salmon and atlantic salmon and implications for food conversion and growth performance. aquacultural engineering, 22 (3): 225-239. simonović, p. (2001). ribe srbije. nnk international, zavod za zaštitu prirode srbije i biološki fakultet. beograd. 247 p. biologica nyssana 2 (1) september 2011: 51-61 živković, d., jovanović, b. spatial morphometric plasticitz of spirlin… 76 stanisavljević, p., janković, a., ranisavljević, n. & đukić, g. (1999). taksonomska i ekološka svojstva pliske alburnoides bipunctatus (bloch 1782) iz reke gradac (sliv kolubare), oš “miša dudić”. valjevo. trajkovic, s., brankovic, s. & gocic, m. (2008). analysis of biological water quality parameters of the river nisava upstream of water treatment plant "mediana". balwois 2008 – ohrid, republic of macedonia, 27-31 may. http://www.balwois.com/balwois/administration/ full_paper/ffp-946.pdf treer, t., piria, m., aničić, i., safner, r. & tomljanović, t. (2006). diet and growth of spirlin, alburnoides bipunctatus in the barbel zone of the sava river. folia zoologica, 55(1): 97–106. živić, i., marković, z. & ilić, j. (2005). composition, structure, and seasonal dynamics of macrozoobenthos in the temska and visočica rivers (serbia). archive of biological sciences, 57 (2): 107-118. microsoft word bn-oa-0201-03 karalija, paric 29 original article ! the effect of ba and iba on the secondary metabolite production by shoot culture of thymus vulgaris l. erna karalija, adisa parić* faculty of science, university of sarajevo, zmaja od bosne 33-35, 71 000 sarajevo, bosnia and herzegovina * e-mail: adisacausevic@hotmail.com abstract: karalija, e., parić, a.: the effect of ba and iba on the secondary metabolite production by shoot culture of thymus vulgaris l.. biologica nyssana, 2 (1), september 2011: 29-35. in vitro shoots of the common thyme (thymus vulgaris l.) were established, and the effects of different concentrations of the cytokinin ba and the auxin iba on secondary metabolite production were investigated. the highest number of shoots was obtained through cultivation on the ms medium containing 2 mg/l ba and 0.1 mg/l iba and 4 mg/l ba and 0.1 mg/l iba (14.3 and 13.3 respectively). quantitative changes in chlorophyll a, chlorophyll b and carotenoid content were recorded in response to the effect of varying concentrations of growth regulators in the medium. furthermore, after addition of plant growth regulators (0.5 mg/l ba + 0.1 mg/l iba) to the medium, elicitation of phenolic compounds was recorded in plantlets. the concentration range of ba from 2 to 4 mg/l improved the production of flavonoids (0.61 and 0.64 mg/g fw). in contrast, plantlets cultivated on the same treatment, showed a decrease in monomeric anthocyanins . key words: common thyme, in vitro, micropropagation, secondary metabolite production introduction ! the common thyme or thymus vulgaris l., a member of the lamiaceae family, is an aromatic herb growing up to 30 cm in height and flowering from july to september (g r u e n w a l d et al., 2004). it is native to southern europe, where it is often cultivated as a culinary herb. thyme is used as spice and medicinal herb almost everywhere in the world (m o r a l e s , 2002). many pharmacological in vitro experiments carried out during the last decade revealed well defined pharmacological properties of both, the thyme essential oil and the plant extracts. the non-medicinal use of thyme is worthy of attention, because thyme is used in the food and aroma industries and it is widely used as a culinary ingredient and it serves as a preservative for foods especially due to its antioxidant effect (z a r z u e l o & c r e s p o , 2002). the active substances in the thyme essential oil are thymol, carvacrol, p-cymene, β pinene, γterpinene, β-caryophyllene, 1-borneol, 1, 8-cineole, etc (j u v e n et al., 1994). recent studies showed that thyme essential oils have strong antibacterial, antifungal, and antioxidative properties (c r u z et al., 1989; p i c c a g l i a et al., 1993; c o n s e n t i n o et al., 1999; k a r a m a n et al., 2001; v a r d a r et al., 2003, p r a b u s e e n i v a s a n et al., 2006; b u d k a & k h a n , 2010). essential oils of different species of genus thymus inhibit a broad spectrum of bacteria, with gram-positive bacteria generally being more susceptible than gram-negative bacteria (b l a k e w a y , 1986; f a r a g et al., 1986; d e a n s & r i t c h i e , 1987; k n o b l o c h et al., 1988). recently, the antibacterial property of thyme oil against some important food-borne pathogens, like salmonella enteritidis, escherichia coli, staphylococcus aureus, listeria monocytogenes, and 2 (1) • september 2011: 29-35 biologica nyssana 2 (1) september 2011: 29-35 karalija e., parić a. the effect of ba and iba on the secondary metabolite... 30 campylobacter jejuni, has been tested (s m i t h p a l m e r et al., 1998). several in vitro and in vivo screenings have shown that volatile oils may be used against fungal diseases, especially those from plants of the genus thymus (b l a k e w a y , 1986; f a r a g et al., 1986; d e a n s & r i t c h i e , 1987). a few studies demonstrate the antiviral effects of thyme extracts (h e r r m a n n & k u c e r a , 1967). micropropagation techniques play an important role in conservation and genetic improvement of pharmaceutical and medicinal plants. today, there is a tendency to develop in vitro systems of production as an alternative to field production of plants used as a source of active compounds. such system is not limited by sources of nutrients or climate changes and can thus be better controlled. for effective induction of secondary metabolite production, plant growth regulators (pgrs) are required (z h o n g et al., 1996; a l -s a n e ' et al., 2005; shilpashree & ravishankar, 2009), since they have significant effects on the metabolism of secondary metabolites. the quality, as well as the quantity, of plant growth regulators, plays a major role in the production capability of a given in vitro culture. pgrs affect cell multiplication and division, which increases production of secondary metabolites (k u r z & c o n s t a b e l , 1979; s t a b a , 1980). the effects of different pgrs on secondary metabolite production have previously been studied and confirmed in vrious plants (f u r u y a et al., 1971; i k u t a et al., 1975; i k e d a et al., 1976; i k u t a & i t o k a w a , 1982; n a i r et al., 1992; w e a t h e r s et al., 2005; k h a n et al., 2008). the aim of this study was to develop an efficient protocol for mass multiplication of t. vulgaris, through manipulation of different concentrations of plant growth regulators, and to examine the relationship between pgrs and the production and accumulation of antioxidants, polyphenols and chlorophyll. materials and methods plant material and treatments commercially purchased seeds of t. vulgaris l. (sjemenarna ljubljana d.d., slovenija) were surface-sterilised with 20% commercial bleach for 15 min followed by 3x5 min rinsing in sterile distilled water and were then germinated on a ms basal medium (m u r a s h i g e & s k o o g , 1962). shoot multiplication was investigated on the ms basal medium (bh; control) or on media supplemented with 0.5, 2, 4 mg/l ba and 0.1 mg/l iba (bi1; bi2 and bi4). the ph of all media was adjusted to 5.8 with 1 m koh or 1 m hcl before autoclaving at 121°c for 20 minutes. all cultures were incubated at 24±1°c under cool white fluorescent lights (3000 lux) and with the 16 h light photoperiod. the aerial parts of t. vulgaris were collected for analysis. photosynthetic pigments – extraction was undertaken in accordance to porra et al. (1989). quantification was done by spectrophotometric determination of absorbance on 663 nm (chlorophyll a), 646 nm (chlorophyll b) and 440 nm (carotenoids). the concentrations of photosynthetic pigments were expressed as mg of pigments per g of fresh weight (mg/g fw). secondary metabolite production – samples used for the analysis of concentrations of phenols, flavonoids and anthocyanins were frozen in liquid nitrogen and stored at -20°c. extraction of phenols and flavonoids was performed in 100% methanol. total phenol content was determined according to w o l f e et al. (2003), with folin-ciocalteu reagent through spectrophotometric readings of absorbance at 765 nm. total phenol content was expressed as mg of catechin per g of fw. total flavonoid content was analysed according to o r d o n et al. (2006) by absorbance readings at 415 nm. total flavonoid content was expressed as mg of ruthin per g of fw. monomeric anthocyanins were determined according to m a n c i n e l l i (1984). extraction was performed in acidified methanol (1% hcl in methanol), and the concentration was expressed as mg of cyanidin 3-glucoside per l of extract. observations and statistical analysis each treatment had 5 replicates containing 10 explants and all experiments were repeated three times. all analyses were done after 30 days of cultivation with no subcultivation. the data was analysed using spss 15.0 by employing parametric (pearson correlation, one tailed) and non-parametric (kruskal-walis analysis) tests. results and discussion shoot multiplication – the induction of shoot formation started after a week of culture cultivation with initiation of shoot meristemswhich was followed by growth of shoot buds. no multiplication was recorded on the ms medium without plant growth regulators (control) (fig. 1a), while a high rate of root induction was observed on the same medium. shoot apices grown in the presence of 0.5 mg/l ba and 0.1 mg/l iba, produced on average 8.2 axillary buds at the base of the first pair of leaves (tab. 1, fig. 1b). treatments with higher concentrations of ba (2 mg/l) increased the number of shoots, but shoots were much shorter and biologica nyssana 2 (1) september 2011: 29-35 karalija e., parić a. the effect of ba and iba on the secondary metabolite... 31 table 1. the induction of adventive shoots formation in thymus vulgaris l. treatment number of shoots per explant rate of regeneration (%) induction of root formation (%) bh 2.9 (±0,71) 100 81.25 bi1 8.2 (±0,56)* 100 0 bi2 14.3 (±0,82)* 100 0 bi4 13.3 (±0,84)* 100 0 the results represent mean values of three replications (±standard deviation) after 30 days on the ms basal medium with no plant growth regulators and with addition of different concentrations of ba and iba. *statistically important differences at 0.01 level with respect to control. bh 0 m g/l ba + 0 mg/l iba; bi1 0.5 mg/l ba + 0.1 mg/l iba; bi2 2 mg/l ba + 0.1 mg/l iba; bi4 4 mg/l ba + 0.1 mg/l iba; table 2. the effect of ba and iba on photosynthetic pigments in shoot cultures of t. vulgaris treatment chlorophyll a (mg/g) chlorophyll b (mg/g) total chlorophylls (mg/g) carotenoids (mg/g) bh 0.29 (±0.02) 0.16 (±0.01) 0.45 (±0.02) 0.51 (±0.03) bi1 0.27*(±0.01) 0.40* (±0.02) 1.25* (±0.03) 0.20* (±0.03) bi2 0.24*(±0.24) 0.39* (±0.01) 1.14* (±0.01) 0.22* (±0.01) bi4 0.10*(±0.10) 0.17* (±0.02) 0.48* (±0.01) 0.65* (±0.02) the results represent mean values of three replications (±standard deviation) after 30 days on the ms basal medium with no plant growth regulators and with addition of different concentrations of ba and iba. *statistically important differences at 0.01 level with respect to control. bh 0 m g/l ba + 0 mg/l iba; bi1 0.5 mg/l ba + 0.1 mg/l iba; bi2 2 mg/l ba + 0.1 mg/l iba; bi4 4 mg/l ba + 0.1 mg/l iba; table 3. the effect of ba and iba on secondary metabolite production in shoot cultures of t. vulgaris treatment phenols (mg/g) flavonoidis (mg/g) monomeric anthocynins (mg/l) bh 2.09 (±0.14) 0.50 (±0.01) 6.62 (±0.32) bi1 3.05* (±0.11) 0.55 (±0.02) 3.38*(±0.05) bi2 1.70* (±0.01) 0.61*(±0.01) 2.95*(±0.01) bi4 2.32 (±0.04) 0.64*(±0.01) 2.13* (±0.04) the results represent mean values of three replications (±standard deviation) after 30 days on the ms basal medium with no plant growth regulators and with addition of different concentrations of ba and iba. *statistically important differences at 0.01 level with respect to control. bh 0 m g/l ba + 0 mg/l iba; bi1 0.5 mg/l ba + 0.1 mg/l iba; bi2 2 mg/l ba + 0.1 mg/l iba; bi4 4 mg/l ba + 0.1 mg/l iba; had developed necrosis (fig. 1c). on the medium with 4 mg/l ba and 0.1 mg/l iba, an average of 13.3 shoots was obtained directly from the apical or the axillary buds (tab. 1, fig. 1d). similar results were observed in previous studies on the common thyme (b e h e s h t i & m o r t e z a , 2005). a high capability of explant multiplication was observed on the medium with the highest cytokinin/auxin ratio (2 and 4 / 0.1 mg/l) and leads us to consider these combinations as efficient promoters of t. vulgaris multiplication. photosynthetic pigments – the content of the total chlorophylls ranged from 0.45 mg/g fw for control to 1.25 mg/g fw for bi1 treatment (tab. 2). the increase in the total chlorophyll content was a result of a significant increase in chlorophyll b content, while chlorophyll a content decreased significantly when compared to the control. according to our results an increase in the cytokinin concentration in the medium (bi1 and bi2) significantly increases the amount of chlorophyll b produced, except for bi4 medium. on the other hand it shows reverse correlation with the chlorophyll a content. it was not possible to establish a direct link between concentrations of pgrs used and the content of photosynthetic pigments in all cases (s t e t l e r & l a e t s c h , 1965; e d e l m a n & h a n s o n , 1971). cytokinins as well as auxins can improve concentration of photosynthetic pigments (v e r m a & s e n , 2008; p a r s a e i m e h r et al., 2010; v a m i l et al., 2010). furthermore, it was shown that the biologica nyssana 2 (1) september 2011: 29-35 karalija e., parić a. the effect of ba and iba on the secondary metabolite... 32 biosynthesis pathway of chlorophyll is highly insensitive to light and cytokinins (y a r o n s k a y a et al., 2006). changes in the content of chlorophylls, caused by pgrs, could, most likely, be related to growth rate and primary and secondary metabolic activities (l i c h t e n t h a l e r , 1987). secondary metabolite production – the total phenol content was significantly higher on bi1 treatment, while total flavonoid content was higher on all treatments when compared with the control (tab. 3). it was previously reported that the maturation of cells always corresponds to an increase in secondary metabolites (s h e t t y & l a b b e , 1998; b i o n d i et al., 2002; p a r s a e i m e h r et al., 2010). on the other hand, the quality and the quantity of applied plant growth regulators have a significant effect on the metabolism of secondary metabolites (k h a n et al., 2008). in plant cell cultures the production of secondary metabolites supports cell multiplication and division, hence plant growth regulators are bound to have a major role in determining the production of potentiality of a given culture. during our research presence of higher concentrations of ba promoted shoot proliferation and flavonoid production. accumulation of flavonoids is usually correlated to morphogenesis during plant development (p a s q u a et al., 2003). however, no such positive influence of higher ba concentrations was observed for phenol content. only the ba concentration that tested the lowest (0.5 mg/l) induced phenol accumulation. the concentration of monomeric anthocyanins was significantly lower on all treatments in comparison to the control (tab. 3). anthocyanins exhibited positive correlation for chlorophyll a and carotenoids, but a negative correlation for flavonoids, with the significance at the 0.01 level. the reverse relation between the in vitro shoot proliferation and the production of anthocyanins production, which was found in this study, is a general complication found in in vitro secondary metabolites production. several studies dealing with this problem observed that 2.4-d or naa increase anthocyanin production and decrease growth rate. on the other hand some of the studies observed that ba or kin enhance production of anthocyanins, while inhibiting cell growth (m e y e r & v a n s t a d e n , 1995). the difficulties expressed through the reverse relationship between the production of anthocyanins and cell growth can cause problems in all secondary products induction media and additional research is needed to establish specific requirements of each plant species. a b c d figure 1. induction of adventive shoots formation on ms medium with ba and iba a – bh 0 m g/l ba + 0 mg/l iba; b – bi1 0.5 mg/l ba + 0.1 mg/l iba; c – bi2 2 mg/l ba + 0.1 mg/l iba; d – bi4 4 mg/l ba + 0.1 mg/l iba; conclusion our research shows that the shoot multiplication of common thyme is depended upon the treatment that is used. the highest number of adventive shoots was obtained on the ms media supplemented with 2 or 4 mg/l ba and 0.1 mg/l iba, which employed the highest concentration of cytokinins. an increase in the concentration of cytokinins above 2 mg/l was followed by a decrease in photosynthetic pigments content, and total monomeric anthocyanins content. cytokinins (0.5 mg/l) in combination with low auxin levels (0.1 mg/l) can enhance production of phenolic compounds, and higher concentrations of cytokinins can induce an increase in total biologica nyssana 2 (1) september 2011: 29-35 karalija e., parić a. the effect of ba and iba on the secondary metabolite... 33 flavonoid content. this study successfully established the optimal conditions for the in vitro growth of common thyme, with a potential for secondary metabolite production. further investigations are necessary in order to increase the production of secondary metabolites by in vitro cultures. references al-sane', k.o., shibli, r.a., freihat, n.m., hammouri, m.k. 2005: cell suspension culture and secondary metabolites production in african violet (saintpaulia ionantha wendl.). jordan journal of agricultural sciences, 1 (1): 84-92. beheshti, h.b., morteza, k. 2005: effects of media and growth regulators on garden thyme (thymus vulgaris l.) micropropagation. iranian journal of horticultural science and technology, 6 (2): 61-68. biondi, s., scaramagli, s., oksman-caldentey, k., poli, f. 2002: secondary metabolism in root and callus cultures of hyoscyamus muticus l.: the relationship between morphological organization and response to methyl jasmonate. plant science, 163 (2): 563−569. blakeway, j. 1986: the antimicrobial properties of essential oils. soap, perfumery & cosmetics, 59 (4): 201-203. budka, d., khan, n.a. 2010: the effect of ocimum basilicum, thymus vulgaris, origanum vulgare essential oils on bacillus cereus in rice-based foods. european journal of biological sciences, 2 (1): 17-20. consentino, s., tuberoso, c.i.g., pisano, b., satta, m., arzedi, e., palmas, f. 1999: in vitro antimicrobial activity and chemical composition of sardinian thymus essential oils. letters in applied microbiology, 29 (2): 130–135. cruz, t., cabo, m.p., cabo, m.m., jiménez, j., cabo j., ruiz, c. 1989: in vitro antibacterial effect of the essential oil of thymus longiflorus boiss. microbios, 60 (242): 59–61. deans, g.g., ritchie, g. 1987: antibacterial properties of plant essential oils. international journal of food microbiology, 5 (2): 165-180. edelman, j., hanson, a.d. 1971: sucrose suppression of chlorophyll synthesis in carrot callus culture. planta, 98: 150-156. farag, r.s., salem, h., badei, a.z.m.a., hassanein, d.e. 1986: biochemical studies on the essential oils of some medicinal plants. fette, seifen, anstrichmittel, 88 (2): 69-72. furuya, i., kojima, h., syono, k. 1971: regulation of nicotine biosynthesis by auxins in tobacco callus tissue. phytochemistry, 10: 1529-1532. gruenwald, j., bendler, t., jaenicke, c. 2004: pdr for herbal medicines. medical economics company. montvale (nj). herrmann, e.c., kucera, l.s. 1967: antiviral substances in plants of the mint family (labiatae) 111. peppermint (mentha piperita) and other mint plants. proceedings of the society for experimental biology and medicine, 124: 874-878. ikeda, t., matsumoto, t., noguchi, m. 1976: formation of ubiquinone by tobacco plant cells in suspension culture. phytochemistry, 15: 954959. ikuta, a., itokawa, h. 1982: berberine and other protoberberine alkaloids callus tissue of thalictrum minus. phytochemistry, 21: 14191421. ikuta, a., syono, k., furuya, t. 1975: alkaloids in plants regenerated from coptis callus cultures. phytochemistry, 14: 1209-1210. juven, b.j., kanner, j., schveol, f., weisslowicz, h. 1994: factors that interact with the antibacterial action of thyme essential oils and its active constituents. the journal of applied bacteriology, 76 (6): 626-631. karaman, s., digrak, m., ravid, v., iclim, a. 2001: antibacterial and antifungal activity of the essential oils of thymus revolutus celak from turkey. journal of ethnopharmacology, 76 (2): 183–186. khan, t., krupadanam, d., anwar, s.y. 2008: the role of phytohormone on the production of berberine in the calli cultures of an endangered medicinal plant, turmeric (coscinium fenestratum l.). african journal of biotechnology, 7 (18): 244-3246. knobloch, k., pauli, a., iberl, b., weis, n. weigand, h. 1988: mode of action of essential oil components on whole cell of bacteria and fungi in plate tests. in: schreier, p. (ed.), bioflavour 87: 287-299, de gruyter, berlin, new york. kurz, w.e.w., constabel, f. 1979: plant cell cultures, a potential source of pharmaceuticals. advances in applied microbiology, 25: 209-240. lichtenthaler, h. k., 1987: chlorophylls and carotenoids, the pigments of photosynthetic biomembranes. in: douce, r., packer, l. (ed.), methods in enzymology 350–382, academic press inc., new york. mancinelli, a.l. 1984: photoregulation of anthocyanin synthesis. plant physiology, 75: 447-453. biologica nyssana 2 (1) september 2011: 29-35 karalija e., parić a. the effect of ba and iba on the secondary metabolite... 34 meyer, h.j., van staden, j. 1995: the in vitro production of anthocyanin from callus cultures of oxalis linearis. plant cell, tissue and organ culture 40: 55-58. morales, r. 2002: the history, botany and taxonomy of the genus. in: stahl-biskup, e., sáez, f. (ed.), medicinal and aromatic plants – industrial profiles 17: 1-43, taylor & francis, london. murashige, t., skoog, f. 1962: a revised medium for rapid growth and bioassays with tobacco tissue culture, physiologia plantarum, 15: 473497. nair, a.j., sudhakaran, p.r., madhusudana, j.r., ramakrishna, s.v. 1992: berberine synthesis by callus and cell suspension cultures of coscinium fenestratum. plant cell, tissue and organ culture, 29: 7-10. ordon, a.a.l., gomez, j.d., vattuone, m.a., isla, m.i. 2006: antioxidant activities of sechium edule (jacq.) swart extracts. food chemistry, 97: 452–458. parsaeimehr, a., sargsyan, e., javidnia, k. 2010: a comparative study of the antibacterial, antifungal and antioxidant activity and total content of phenolic compounds of cell cultures and wild plants of three endemic species of ephedra. molecules, 15 (3): 1668-1678. pasqua, g., avato, p., monacelli, b., santamaria, a.r., argentieri, m.p. 2003: metabolites in cell suspension cultures, calli, and in vitro regenerated organs of hypericum perforatum cv. topas. plant science, 165: 977–982. piccaglia, r., marotti, m., giovanelli, e., deans, s.g., eaglesham, e. 1993: antibacterial and antioxidant properties of mediterranean aromatic plants. industrial crops production, 2 (1): 7–50. porra, r.j., thompson, w.a., kriedemann, p.e. 1989: determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectrometry. biochimica et biophysica acta, 975: 384–394. prabuseenivasan, s., jayakumar, m., ignacimuthu, s. 2006: in vitro antibacterial activity of some plant essential oils. bmc complementary and alternative medicine, 6 (1): 39. shetty, k., labbe, r.g. 1998: food-borne pathogens, health and role of dietary phytochemicals. asia pacific journal of clinical nutrition, 7 (3/4): 270-276. shilpashree, h.p., ravishankar, r. 2009: in vitro plant regeneration and accumulation of flavonoids in hypericum mysorense. international journal of integrative biology, 8: 43-49. smith-palmer, a., stewart, j., fyfe, l. 1998: antimicrobial properties of plant essential oils and essences against five important food-borne pathogens. letters in applied microbiology, 26 (2): 118-122. staba, e.j. 1980: plant tissue culture as a source of biochemicals.crc press, boca raton, florida. stetler, d.a., laetsch, w.m. 1965: kinetin-induced chloroplast maturation in cultures of tobacco tissue. science, 149: 1387-1388. vamil, r., aniat-ul-haq, agnihotri, r.k. 2010: plant growth regulators as effective tool for germination and seedling growth for bambusa arundinaceae. research journal of agricultural sciences, 1 (3): 233-236. vardar, u.g., candan, f., sokmen, a., daferera, d., polissiou, m., sokmen, m., donmez, e., tepe, b. 2003: antibacterial and antioxidant activity of the essential oil and methanol extracts of thymus pectinatus fisch. et mey var. pectinatus (lamiaceae). journal of agricultural and food chemistry, 51 (14): 63–67. verma, p., sen, n.l. 2008: the impact of plant growth regulators on growth and biochemical constituents of coriander (coriandrum sativum l.). journal of herbs, spices & medicinal plants, 14 (3/4): 144 – 153. weathers, p.j., bunk, g., mccoy, m.c. 2005: the effect of phytohormones on growth and artemisinin production in artemisia annua hairy roots. in vitro cellular & developmental biology, plant, 41 (1): 47-53. wolfe, k., wu, x., liu, r.h. 2003: antioxidant activity of apple peels. journal of agricultural and food chemistry, 51 (22): 609–614. yaronskaya, e., vershilovskaya i., poers, y., alawady, a.e., averina, n., grimm, b. 2006: cytokinin effects on tetrapyrrole biosynthesis and photosynthetic activity in barley seedlings. planta, 224 (3): 700–70. zarzuelo, a., crespo, e. 2002: medicinal and aromatic plants – industrial profiles. in: stahlbiskup, e., sáez, f. (ed.), thyme 24: 278, taylor & francis, united kingdom. zhong, j.j., bai, y., wang, s.j. 1996: effects of plant growth regulators on cell growth and ginsenoside saponin production by suspension cultures of panax quinquefolium. journal of biotechnology, 45 (3): 227-234. microsoft word bn020201a jotic et al biologica nyssana 2 (2) december 2011: 00-00 nahirnić a. supplements of butterfly fauna… 91 original article ! the vascular flora of the vučje hill near pirot city (eastern serbia) branko jotić1, marija marković1, bojana petrović2, ivana fusijanović1, dragana pavlović3, vladimir ranđelović1 1university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia 2 institute for nature conservation of serbia, dr. ivana ribara 91, 11070 novi beograd, serbia 3university of kragujevac, faculty of sciences and mathematics, institute of biology and ecology, radoja domanovića 14, 34000 kragujevac, serbia * e-mail: branko_jotic@live.com abstract: jotić, b., marković, m., petrović, b., fusijanović, i., pavlović, d., ranđelović, v.: the vascular flora of the vučje hill near pirot city (eastern serbia). biologica nyssana, 2 (2), december 2011: 91-106. as a result of the floristic investigations of the vučje hill carried out betwenn 2008 and 2010, 534 plant taxa belonging to 277 genera and 67 plant families were recorded. phytogeographical analysis showed that the species of eurasian area type are the most abundant ones. there are 10 endemic taxa in flora of investigated area. the hemicriptophytic character of flora with significant contribution of therophytes and geophytes, is established by analyzing the presence of plant life forms. 31 species are listed as protected or strictly protected in national legislature, while some of them are of international importance for conservation. key words: endemic, flora, pirot city, vučje hill introduction ! the vučje hill (1188 m) is situated in eastern serbia, 8 km se of pirot city, above the krupac village (fig. 1). this is the part of mt. vidlič, westernmost part of the mountain system stara planina. the hill is elongated in the southeasternnorthwestern direction. in contrast to other regions of mountain system stara planina, mt. vidlič are represented by unique geological structure and climate conditions. the geological structure of the most part of mt. vidlič is represented by limestone, while other serbian parts of stara planina are built of silicate (v i d a n o v i ć , ed., 1960). the pedological substrate is consisted of reddish soil tiller and mountain soil. the climate of this area is transitional humid continental, between dry climate of pirot region and wet climate on mt. stara planina (v i d a n o v i ć , ed., 1960). although the flora of this part of mt. stara planina was investigated by p a n č i ć (1884) and a d a m o v i ć (1911), it has not been well known. the vučje hill flora is not investigated up to now. materials and methods the flora of vučje hill were studied in the period between 2008 and 2010. herbarium specimens are deposited at the herbarium moesiacum (hmn). determination of plant material was carried out using the keys from the regional flora reference (j o s i f o v i ć , ed. 1970-1977; v e l č e v , ed. 19821989). the nomenclature follows med-checklist (greuter et al., 1984-1989) and flora europaea (t u t i n et al., eds., 1964-1980). 2 (2) • december 2011: 91-106 biologica nyssana 2 (2) december 2011: 91-106 jotić b. et al. the vascular flora of the vučje hill… 92 for the area types classification m e u s e l et al. (1965, 1978), m e u s e l and j ä g e r (1992) and s t e v a n o v i ć (1992) were used. life forms were determined according to m u e l l e r d o m b o i s and e l l e n b e r g (1974) and s t e v a n o v i ć (1992a). results as a result of the floristic investigations of vučje hill in eastern serbia, 534 plant taxa belonging to 277 genera and 67 families were recorded (tab. 1). figure 1. geographical position of investigated area table 1. list of vascular flora with life forms and area-types taxa life form area type polypodiopsida aspleniaceae asplenium ceterach l. semp ch herb caesp me asplenium ruta-muraria l semp ch herb caesp hol asplenium trichomanes l. semp ch herb semiros csm dryopteridaceae dryopteris filix-mas (l.) schoot a meg g rhiz hol hypolepidaceae pteridium aquilinum (l.) kuhn a meg-alt g rhiz csm polypodiaceae polypodium vulgare l. mac fo semp g rhiz hol woodsiaceae cystopteris fragilis (l.) bernh. fo dec ch herb semiros csm biologica nyssana 2 (2) december 2011: 91-106 jotić b. et al. the vascular flora of the vučje hill… 93 taxa life form area type pinopsida cupressaceae juniperus communis l. a semp np scap hol magnoliopsida acanthaceae acanthus balcanicus heywood & i.b.k.richardson a meg-alt h scap semiros eas aceraceae acer campestre l. fo dec mes p scap eas acer hyrcanum fischer & c. a. meyer subsp. intermedium (pančić) bornm. fo dec mes p scap me acer monspessulanum l. fo dec mi-mes p scap med-smed apiaceae aegopodium podagraria l. v-a mes-meg h scap/g rhiz eas angelica sylvestris l. a mac-alt h scap bor bupleurum flavicans boiss. & heldr. a mi-mes t scap med-smed bupleurum praealtum l. v-a mi-mes t scap med-smed bupleurum veronense turra a mes t scap eas caucalis platycarpos l. v-a mes t scap eas chaerophyllum aureum l. a mes-alt h scap eas daucus carota l. a meg t/h scap hol eryngium campestre l. v-a mes-meg h semiros eas eryngium palmatum pančić et vis. a mes-meg h scap semiros med-smed freyera cynapoides (guss.) griseb. v-a mes-mac g bulb eamt orlaya grandiflora (l.) hoffm. a mes t scap eas scandix pectin-veneris l. v-a mes t scap med-smed smyrnium perfoliatum l. a mes-meg h scap med-smed torilis arvensis (hudson) link a meg t scap csm torilis heterophylla guss. a mes t scap med-smed torilis leptophylla (l.) reichenb. a mes t scap eas apocynaceae vinca herbacea waldst. et kit. v-a mi-mes ch suff rept p araliaceae hedera helix l. aut fo semp alt s lig me aristolochiaceae aristolochia clematitis l. v-a mes-meg g rad eas asarum europaeum l. fo semp ch herb rept eas asclepiadaceae vincetoxicum hirudinaria medicus a mes-alt h scap eas compositae (asteraceae) achillea clypeolata sibth. & sm. a mes-meg h scap med-smed achillea crithmifolia waldst. et kit. a mes-meg h scap msm achillea millefolium l. a meg h scap hol achillea pannonica scheele a meg h scap hol ambrosia artemisiifolia l. a-aut mes t scap adv anthemis arvensis l. a mes-mac t scap med-smed anthemis ruthenica bieb. a mes-meg t scap medsmed artemisia alba turra aut meg fo dec ch suffr caesp eamt artemisia scoparia waldst. et kit. a-aut meg t scap bienn eas artemisia vulgaris l. aut meg-alt h scap hol bellis perennis l. v-a mi-mes h ros eas biologica nyssana 2 (2) december 2011: 91-106 jotić b. et al. the vascular flora of the vučje hill… 94 taxa life form area type carduus acanthoides l. a mac h scap me carduus candicans waldst. et kit. subsp. globifer (velen.) kazmi a mes-meg h scap msm carduus nutans l. a mes-meg h scap me carlina vulgaris l. a mes-meg t/h scap bienn eas centaurea biebersteinii dc. a mes-meg h scap eas centaurea calcitrapa l. a mes-meg h caesp eas centaurea cyanus l. v-a mes-mac t scap csm centaurea scabiosa l. a meg h scap eas centaurea stenolepis a. kerner. a mes-meg h scap med-smed centaurea triumfetti all. a mes-meg h scap eamt chamomilla recutita (l.) rauschert a mes-meg t scap csm chondrilla juncea l. a mes-meg t scap bienn med-smed cichorium intybus l. a mac h scap csm cirsium acaule scop. a mes h ros eas cirsium vulgare (savi) ten. a mes-alt t/h scap bienn eas conyza canadensis (l.) cronquist a-aut mes-alt t scap adv crepis biennis l. v-a mac h scap bienn me crepis foetida l. a mes-meg t ros eas crepis sancta (l.) babcock v mi-mes t scap eas crepis setosa haller a mes-alt t scap med-smed crupina vulgaris cass v-a mes t scap eas doronicum columnae ten. v mes-meg h scap/g rhiz eamt erigeron acer l. a mes-meg h scap bienn hol hieracium cymosum l. a mes-meg h scap ros eamt hieracium pilosella l. a mi-mes h ros eas hieracium praealtum vill. ex gochnat subsp. bauhinii (besser) petunnikov a meg-alt h scap me inula britannica l. a mes-meg h scap eas inula conyza dc. a mes-meg h scap eas inula oculus-christi l. a mes-meg h scap p lactuca saligna l. a meg-alt t/h scap bienn eas lactuca serriola l. a-aut mes-mac t scap bienn eas lactuca viminea (l.) presl. a mes-mac h scap bienn eas lapsana communis l. a meg-alt t scap eas leontodon crispus l. a mi-mes h ros med-smed leontodon hispidus l. a mi-mac h ros eas leucanthemum vulgare lam. a mes-meg h scap eas logfia minima (sm.) dumort. a mi-mes t scap eas matricaria perforata mérat a-aut mes-meg t/h scap bienn eas mycelis muralis (l.) dumort. a mes-alt h scap eas onopordum acanthium l. a meg-alt h scap bienn eas picris hieracioides l. a-aut meg-alt h scap bienn eas ptilostemon afer (jacq.) w. greuter a mac-meg h scap bienn med-smed scorzonera cana (c. a. meyer) o.hoffm. a mes-mac h scap eas scorzonera hispanica l. a mac-meg h scap p senecio vernalis waldst. et kit. v mes t scap eas sonchus asper (l.) hill a-aut meg-alt t scap bienn eas sonchus oleraceus l. a meg-alt t/h scap bienn csm taraxacum officinale weber v-aut mes h ros eas tragopogon balcanus velen. a mes-meg h scap bienn med-smed tragopogon pratensis l. a mes-meg h scap eas xanthium spinosum l. a mes-meg t scap adv xeranthemum annuum l. a mes-meg t scap med-smed biologica nyssana 2 (2) december 2011: 91-106 jotić b. et al. the vascular flora of the vučje hill… 95 taxa life form area type boraginaceae anchusa barrelieri (all.) vitman a mac-alt h scap bienn eas anchusa officinalis l. a alt h scap bienn eas asperugo procumbens l. v mi-mes t scap eas buglossoides arvensis (l.) i. m. johnston v-a mes t scap eas buglossoides purpurocaerulea (l.) i. m. johnston v-a mac h scap eas echium italicum l. v-a mac-meg h scap bienn med-smed echium vulgare l. a mac h scap eas myosotis arvensis (l.) hill v-a mi-mes t scap hol myosotis caespitosa f.c.schultz. a mes h caesp bienn hol myosotis sylvatica hoffm. v mes t/h scap ros eas nonea pulla (l.) dc. v-a mes-mac h scap bien eas pulmonaria officinalis l. v mes-mac h scap me symphytum tuberosum l. v mes-mac h scap/g tub msm criciferae (brassicaceae) aethionema saxatile (l.) r. br. a mi-mes t scap med-smed alliaria petiolata (bieb.) cav. & grande v-a mes-meg t/h scap eas alyssum alyssoides (l.) l. v mi-mes t scap eas alyssum montanum l. v mi-mes fo dec ch suffr rept eas alyssum petraeum ard. a mes h scap ros bienn med-smed alyssum repens baumg. v mi-mes fo dec ch suffr p arabidopsis thaliana (l.) heuff. v mi-mes t scap ros eas arabis glabra (l.) bernh. a meg-alt h scap bienn hol arabis hirsuta (l.) scop. a mes-meg h ros bienn csm arabis recta vill. v meg h scap bienn eas arabis sagittata (bertol.) dc. a mes-meg h ros bienn eas arabis turrita l. v-a meg h scap semiros eas berteroa incana (l.) dc. a mes-mac t/h scap eas camelina rumelica velen. a mes t scap-ros med-smed capsella bursa – pastoris (l.) medicus v-aut mi-meg t/h ros bienn csm cardamine bulbifera (l.) crantz v-a mes-mac g rhiz eas cardamine hirsuta l. a mi-mes t scap ros eas cardaria draba (l.) desv. v-a mes-meg h scap eas draba muralis l. v mi-meg t scap csm erophila verna (l.) besser v mi-mes t ros eas erysimum cuspidatum (bieb.) dc. a mac t/h ros bienn p erysimum diffusum ehrh. a mes-meg t/h scap eas erysimum hieracifolium l. a mes h scap bienn me erysimum odoratum ehrh. a meg t scap bienn me lepidium campestre (l.) r. br. a meg t/h scap bienn eas sinapis arvensis l. v-a mes-mac t scap eas sysimbrium orientale l. a meg t scap bienn eas thlaspi arvense l. v mes-mac t scap eas thlaspi kovatsii heuff. a mes-alt h scap p thlaspi perfoliatum l. v mi-mes t scap semiros eas campanulaceae asyneuma canescens (w. et k.) gris. et sch. a mes-meg h scap eas campanula bononiensis l. a mes-meg h scap eas campanula glomerata l. a mes-mac h scap eas campanula persicifolia l. a mes-meg h scap me campanula sparsa friv. a mes-mac t scap me caprifoliaceae lonicera xylosteum l. fo dec mi p caesp eas biologica nyssana 2 (2) december 2011: 91-106 jotić b. et al. the vascular flora of the vučje hill… 96 taxa life form area type sambucus ebulus l. a alt g rad/h scap eas sambucus nigra l. fo dec mi p scap eas viburnum lantana l. fo dec n-mi p caesp eas caryophyllaceae agrostemma githago l. a meg t scap eas arenaria serpyllifolia l. v-a mi-mes t scap eas cerastium brachypetalum desp. a mi-mes t scap eas cerastium glomeratum thuill. v-a mi-mes t scap csm cerastium pumilum curt. a mes h scap eas cerastium semidecandrum l. v mi-mes t scap eas dianthus carthusianorum l. a mes-mac h scap caesp eas dianthus cruentus griseb. a mes-meg h scap caesp eamt herniaria hirsuta l. a mi-mes t/h rept eas herniaria incana lam. a mi h caesp rept eas holosteum umbellatum l. a mi t scap eas lychnis coronaria (l.) desr. a mes-meg h scap eas minuartia verna (l.) hiern. a mi-mes h caesp hol petrorhagia illyrica p.w.ball et heywood a-aut mes h/ch herb caesp med-smed petrorhagia prolifera (l.) p. w. ball & heywood a mes t scap eas petrorhagia saxifraga (l.) link a mes-mac ch caesp eas scleranthus neglectus rochel ex baumg. a mes t caesp eas scleranthus perennis l. subsp. dichotomus schur a mes-mac h scap rept eas silene bupleuroides chater et walters a mes-mac h scap eas silene otites (l.) wiebl a mes-meg h scap eas silene vulgaris (moench.) garcke a mes-meg h scap/g rad eas stellaria holostea l. a mes-mac h scap eas stellaria media (l.) vill. a mes-meg h scap rept csm celastraceae evonymus europaeus l. fo dec n-mi p caesp eas evonymus latifolius (l.) miller fo dec n-mi p caesp eas evonymus verrucosus scop. fo dec n-mi p caesp eas cistaceae fumana procumbens (dunal) gren. & gordon v-a mi-mes ch frut eas helianthemum ledifolium (l.) miller a mi t scap med-smed helianthemum nummularium (l.) miller a mes-meg fo dec ch suffr caesp eas helianthemum salicifolium (l.) miller v mi-mes t scap med-smed convolvulaceae convolvulus arvensis l. a mes-alt s herb eas convolvulus canthabrica l. a mes-meg h scap eas cuscuta approximata bab. t/s par hol cuscuta epithymum (l.) l. t/s par hol cuscuta europaea l. t/s par eas cornaceae cornus mas l. fo dec mi p caesp eas cornus sanguinea l. fo dec mi-mes p caesp me corylaceae carpinus betulus l. fo dec meg-alt p caesp scap eas carpinus orientalis miller fo dec mi-mes p caesp med-smed corylus avellana l. fo dec meg-alt p caesp eas corylus colurna l. fo dec mes p scap med-smed crassulaceae jovibarba heuffelii a. et d. löve a mes fo semp ch herb ros succ p biologica nyssana 2 (2) december 2011: 91-106 jotić b. et al. the vascular flora of the vučje hill… 97 taxa life form area type sedum acre l. a mi-mes ch herb scap succ me sedum album l. a mi-mes ch herb scap succ med-smed sedum annuum l. a n-mi ch herb scap succ aa sedum hispanicum l. a mi t/h succ eas sedum ochroleucum chaix. a mes ch herb succ med-smed sedum sexangulare l. a mi-mes ch herb succ me sedum telephium l. subsp. maximum (l.) krock. a mac h scap succ eas dipsacaceae cephalaria laevigata (waldst. & kit.) schrader a meg-alt ch suffrut med-smed knautia arvensis (l.) coult. v-a mes-alt h scap eas knautia integrifolia (l.) bertol a mes-meg t scap med-smed scabiosa argentea l. a mes-meg h scap p euphorbiaceae euphorbia amygdaloides l. a mes-mac t scap eas euphorbia cyparissias l. v-a mes-mac h scap eas euphorbia esula l. a meg-alt h scap me euphorbia falcata l. a-aut mi-mes t scap eas euphorbia helioscopia l. a mi-meg t scap eas euphorbia nicaeensis all. subsp. glareosa (pallas ex bieb.) a. r. sm. v-a mes h scap eas euphorbia taurinensis all. v-a mes t scap med-smed euphorbia seguierana necker subsp. niciciana (borbás ex novák) rech. fil. a mes-meg h scap eas euphorbia platyphyllos l. a-aut mes-mac t scap eas euphorbia salicifolia host. v-a mes h scap eas leguminosae (fabaceae) anthyllis vulneraria l. a mes-meg h scap eas astragalus glycyphyllos l. a mes-meg h scap rept eas astragalus onobrychis l. a mes fo ch suffrut rept eas chamaecytisus austriacus (l.) link v-a mes-mac fo dec ch suffrut caesp msm chamaecytisus ciliatus (wahlenb.) rothm. v-a mes-mac fo dec ch suffrut caesp msm chamaecytisus glaber (l. fil.) rothm. v-a mes-meg fo dec ch frut caesp eamt chamaecytisus rochelii (wierzb.) rothm. v-a mes-meg fo dec ch frut caesp msm chamaespartium sagittale (l.) p. gibbs a mes fo dec ch suffrut caesp eas coronilla coronata l. v-a mes fo dec ch suffrut caesp eas coronilla emerus l. a fo dec np caesp med-smed coronilla scorpioides (l.) koch v mi-mes t scap med-smed coronilla varia l. a mes-meg h scap eas cytisus procumbens (waldst. & kit. ex willd.) sprengel a mes fo dec ch suffrut caesp med-smed dorycnium pentaphyllum scop. subsp. germanicum (gremli) gams a mes fo dec ch suffrut caesp msm genista ovata waldst. et kit. a mes-mac fo dec ch suffrut caesp eas lathyrus aphaca l. a mes t/s herb eas lathyrus cicera l. a mes-mac t scap med-smed lathyrus inconspicuus l. a mes t scap med-smed lathyrus nissolia l. a mes-meg t scap eas lathyrus pallescens (bieb.) c. koch. a mes-mac t scap p lathyrus pratensis l. a mes-meg h scap eas lathyrus setifolius l. a mes-mac t rept med-smed lathyrus venetus (mill.) wohlf. a mes-meg h scap p lotus corniculatus l. a mes h scap hol medicago lupulina l. a mes t/h scap hol biologica nyssana 2 (2) december 2011: 91-106 jotić b. et al. the vascular flora of the vučje hill… 98 taxa life form area type medicago minima (l.) bartal v mi-mes t scap eas medicago rigidula (l.) all. v mi-mes t scap eas medicago sativa l. a mes-meg h scap adv medicago sativa l. subsp. falcata (l.) arcangeli a mes-meg h scap eas melilotus officinalis (l.) pallas a meg t/h scap bienn eas onobrychis alba (waldst. et kit.) desv. a mes-meg h scap med-smed onobrychis arenaria (kit.) dc. v-a mes-meg h scap eas onobrychis ocellata g. beck. a mes h scap med-smed onobrychis viciifolia scop. a mes-meg h scap med-smed onоnis pusilla l. a-aut mi-mes fo dec ch suffrut caesp eas ononis spinosa l. a mes-meg fo dec ch suffrut caesp eas oxytropis pilosa (l.) dc. a mes h scap eas trifolium alpestre l. a mes-mac h scap eas trifolium arvense l. a mes t/h scap bienn eas trifolium badium schreber a mes-meg h scap eamt trifolium campestre schreber a mes-mac t scap eas trifolium incarnatum l. a mes-meg t scap eas trifolium medium l. v-a mes-mac h scap eas trifolium montanum l. a mes h scap eas trifolium pratense l. a mes h scap eas trifolium repens l. a mi h rept hol trifolium scabrum l. a mi-mes t scap med-smed trifolium striatum l. a mes t scap eas vicia cracca l. a meg-alt h/s scap herb eas vicia hirsuta (l.) s. f. gray a mes-meg t/s scap herb eas vicia lathyroides l. v-a mes-meg t scap eas vicia pannonica crantz v-a mes t/s scap herb p vicia sativa l. subsp. nigra (l.) ehrh. v-a mes-meg t scap bienn eas vicia tenuifolia roth. a mac-alt h/s scap herb eas vicia tetrasperma (l.) schreber v-a mes t/s herb eas fagaceae fagus moesiaca (k. maly) czecz. fo dec meg-alt p scap me quercus cerris l. fo dec mes p scap med-smed quercus petraea (mattuschka) liebl. fo dec meg-alt p scap me quercus pubescens willd. fo dec mes p scap med-smed fumariaceae corydalis solida l. v mes g tub me fumaria officinalis l. a mes t scap eas fumaria vaillantii loisel. a mi-mes t scap eas gentianaceae gentiana cruciata l. a mes-meg g rad scap eas geraniaceae erodium ciconium (l.) l’her. v-a mi-mes t scap msm erodium cicutarium (l.) ľ herit. v-a mi-mes t ros csm geranium columbinum l. a mes-meg t scap eas geranium dissectum l. a mi-meg t scap eas geranium molle l. v mes t scap eas geranium rotundifolium l. a mi-meg t caesp eas geranium sanguineum l. v-a mes h semiros eas globulariaceae globularia punctata lapeyr. v-a mes h scap eas biologica nyssana 2 (2) december 2011: 91-106 jotić b. et al. the vascular flora of the vučje hill… 99 taxa life form area type grossulariaceae ribes alpinum l. fo dec mi p caesp scap eas hypericaceae hypericum barbatum jacq. a mes-mac h scap eas hypericum montanum l. a mes-meg h scap me hypericum perforatum l. a mes-meg h scap eas hypericum rumelicum boiss. a mi-mes h scap med-smed labiatae (lamiaceae) acinos alpinus (l.) moench a mes t scap/g rhiz eas acinos arvensis (lam.) dandy a mi-mes t scap/g rhiz eas ajuga chamaepytus (l.) shreber subsp. chia (schreber) arcangeli a mi-mes t scap med-smed ajuga genevensis l. v-a mes-mac h scap ros eas ajuga laxmannii (l.) bentham v-a mes h scap/g rhiz p ajuga reptans l. a mes h rept eas ballota nigra l. a meg h scap eas clinopodium vulgare l. a mes-meg h scap hol glechoma hirsuta waldst. et kit. a mes h rept eas hyssopus officinalis l. aut mes-meg ch frut eas lamiastrum galeobdolon (l.) ehrend. & polatschek v-a mes-mac h scap me lamium amplexicaule l. v mi-mes t scap hol lamium bifidum cyr. v-a mi-mes t scap med-smed lamium maculatum l. v mes-mac h scap eas lamium purpureum l. v mi-mes t scap eas marrubium peregrinum l. a mes-meg h scap eas marrubium vulgare l. a meg h scap eas mentha longifolia (l.) hudson a mes-meg h scap hol nepeta nuda l. a mac-meg h scap eas origanum vulgare l. a mes-meg h scap eas prunella laciniata (l.) l. a mes-mac h scap eas prunella vulgaris l. a mi-mes h scap semiros eas salvia aethiopis l. a mes-mac h scap p salvia austriaca jacq. a mes-mac h scap p salvia nemorosa l. a mes-meg h scap eas satureja montana l. subsp. kitaibelii (wierzb.) p.w. ball a mes ch suffrut caesp eamt sideritis montana l. a mi-mes t scap eas stachys germanica l. a meg h scap msm stachys recta l. a mes-meg h scap eas teucrium chamaedrys l. a mes ch suffrut caesp eas teucrium montanum l. a mes ch suffrut caesp eamt thymus glabrescens willd. a mi-mac ch herb rept eas thymus pannonicus all. a mi-mac ch rept me thymus praecox opiz subsp. jankae (čelak.) jalas a mi ch herb rept pulv med-smed thymus pulegioides l. a mi-mes ch herb rept me ziziphora capitata l. a mi-mes t scap med-smed linaceae linum hologynum reichenb. a mes-mac h caesp med-smed linum tenuifolium l. a mesmac h scap eas malvaceae althaea hirsuta l. a mes t scap eas althaea officinalis l. a meg h scap bienn eas biologica nyssana 2 (2) december 2011: 91-106 jotić b. et al. the vascular flora of the vučje hill… 100 taxa life form area type malva neglecta wallr. v-a mes-mac t/h scap eas malva sylvestris l. v-a mes-alt h scap eas oleaceae fraxinus ornus l. fo dec mi-mes p scap eas ligustrum vulgare l. fo dec mi p caesp eas syringa vulgaris l. fo dec mi p caesp med-smed orobanchaceae orobanche gracilis sm. a mes-mac ep par g eas orobanche nana (reuter) noë ex g. beck a mi-mes ep par g p papaveraceae papaver dubium l. a meg t scap eas papaver rhoeas l. a mes-meg t scap eas plantaginaceae plantago argentea chaix a mi-meg h ros eamt plantago lanceolata l. a mi-meg h ros eas plantago maior l. a mes-meg h ros csm plantago media l. a mes-meg h ros eas polygalaceae polygala comosa schkuhr a mi-mes h scap eas polygala vulgaris l. a mi-mes h scap eas polygonaceae fallopia convolvulus (l.) á. löve a-aut mes t/s rept-herb eas rumex patientia l. a mes-alt h scap semiros eas rumex sanguineus l. a meg h scap hol primulaceae anagallis arvensis l. v-a mi-mes t scap csm primula veris l. v mi-mes h ros eas ranunculaceae adonis flammea jacq. a mes t scap p adonis vernalis l. v mes-meg g rhiz p anemone nemorosa l. v mi-mes g rhiz hol anemone ranunculoides l. v mi-mes g rhiz me clematis vitalba l. a dec s lig eas consolida regalis s. f. gray a mes-meg t scap eas delphinium fissum waldst. et kit. a meg-alt g tub med-smed helleborus odorus waldts. & kit. v meg g rhiz me isopyrum thalictroides l. v mes g rhiz eas nigella arvensis l. a-aut mes t scap eas nigella damascena l. a mes t scap med-smed pulsatilla montana (hoppe) reichenb. subsp. bulgarica rummelspecher v mes h scap semiros eas ranunculus bulbosus l. a mes-meg h scap eas ranunculus ficaria l. v mi-mes g tub me ranunculus millefoliatus wahl. v-a mes h scap semiros med-smed ranunculus polyanthemos l. a meg h scap semiros eas ranunculus sardous crantz a mes-meg t scap eas thalictrum aquilegifolium l. a meg-alt h scap me resedaceae reseda lutea l. a mes-meg t/h scap bienn eas biologica nyssana 2 (2) december 2011: 91-106 jotić b. et al. the vascular flora of the vučje hill… 101 taxa life form area type rosaceae agrimonia eupatoria ledeb. a meg h scap eas aremonia agrimonioides (l.) neck. v mi-mes h scap ros eamt cotoneaster integerrimus medicus a mac ch frut rept me crataegus monogyna jacq. fo dec mi p caesp eas filipendula vulgaris moench a mes-meg h scap eas fragaria moschata duchesne a mes h rept ros eas fragaria vesca l. a mes h rept csm fragaria viridis duchesne a mes h rept eas geum urbanum l. a mes-meg h scap hol malus pumila miller fo dec mi-mes p scap p malus sylvestris miller fo dec mi-mes p scap eas potentilla argentea l. a mes-meg h scap me potentilla cinerea chaix ex vill. v mi-mes h caesp rept eas potentilla micrantha ramond ex d.c. v mi-mes h scap med-smed potentilla recta l. a mes-meg h scap eas prunus spinosa l. fo dec n p caesp eas prunus tenella batsch fo dec n p caesp eas pyrus amygdaliformis vill. fo dec n p caesp med-smed pyrus pyraster burgsd. fo dec mes p scap eas rosa canina l. fo dec n p caesp eas rosa micrantha borrer ex sm. fo dec n p caesp eas rosa pimpinellifolia l. fo dec n p caesp eas sanguisorba minor scop. a mes-meg h scap eas sorbus torminalis (l.) crantz fo dec mes p scap eas rubiaceae asperula aristata l. subsp. scabra (j. & c. presl) nyman a mes-meg h scap me asperula cynanchica l. v-a mes-mac h scap eas crucianella angustifolia l. a mi-mes t scap med-smed cruciata glabra (l.) ehrend. v-a mi-mes h scap eas cruciata laevipes opiz v-a mes-mac h scap eas cruciata pedemontana (bellardi) ehrend. v-a mes-meg h scap med-smed galium album miller a meg-alt h/s scap herb me galium aparine l. v-a mes-meg t/s herb csm galium verum l. a mes-meg h scap eas sherardia arvensis l. v-a mi-mes t scap med-smed rutaceae dictamnus albus l. v-a mes-alt ch suffrut caesp eas salicaceae salix purpurea l. fo dec mi p caesp eas santalaceae thesium alpinum l. a mes-mac h scap eamt thesium arvense horvatovszky a mi-mes h scap eas thesium divaricatum jan ex mert. & koch a mes h scap med-smed scrophullariaceae chaenorhinum minus (l.) lange a-aut mi-mes t scap eas digitalis lanata ehrh. a mac-alt h scap me euphrasia pectinata ten. a mi-mes t scap/ep semipar eas kickxia spuria (l.) dumort a-aut mes t rept eas lathraea squamarria l. a mes-meg g par eas linaria angustissima (lois.) borb. a mes h/g scap rhiz med-smed biologica nyssana 2 (2) december 2011: 91-106 jotić b. et al. the vascular flora of the vučje hill… 102 taxa life form area type linaria genistifolia (l.) mill. a mes-mac h scap eas linaria genistifolia (l.) miller subsp. dalmatica (l.) maire & petitmengin a meg-alt h scap eas linaria rubioides vis. & pančić subsp. nissana (petrović) niketić & tomović a mes h scap med-smed linaria vulgaris l. a mes-mac h scap eas rhinanthus rumelicus velen. a mi-mac t scap/ep semipar eas scrophularia canina l. a mes-mac h scap med-smed verbascum lychnitis l. a mac-alt t/h scap ros bienn eas verbascum phlomoides l. v-a mes-alt h ros eas verbascum phoeniceum l. v-a mac-meg h ros eas veronica austriaca l. a mes-mac h scap eas veronica chamaedrys l. v-a mi-mes h scap eas veronica hederifolia l. v mi-mes t scap eas veronica persica poiret v-aut mi-mes t scap adv veronica praecox all. v mi-mes t scap bienn eas veronica verna l. v-a mi-mes t scap bienn me solanaceae hyascyamus niger l. v-a mes-meg t scap bienn eas tiliaceae tilia platyphyllos scop. fo dec mes p scap me ulmaceae ulmus glabra hudson fo dec mes p scap me ulmus minor mill. fo dec mes p scap med-smed ulmus procera salisb. fo dec mes p scap me urticaceae urtica dioica l. a mes-meg t/h scap hol valerianaceae valeriana officinalis l. a meg-alt h scap eas valerianella coronata (l.) dc. v mi-mes t scap med-smed valerianella dentata (l.) pollich v mi-mes t scap eas valerianella locusta (l.) laterrade v mes t scap csm valerianella rimosa bast. v mes t scap eas verbenaceae verbena officinalis l. a mes-meg h scap eas violaceae viola alba besser v mi-mes h rept semiros eas viola arvensis murray v-aut mi-mac t scap csm viola canina l. v mi-mes h scap semiros hol viola hirta murray v mi-mes h scap me viola jordanii hanry v mes h scap ros p viola kitaibeliana schultes v n-mi t scap eas viola odorata l. v mi-mes h rept ros eas viola tricolor l. subsp. macedonica (boiss. & heldr.) a. schmidt v mi-mes t/h scap med-smed liliopsida araceae arum maculatum l. v mes-meg g rhiz me cyperaceae carex caryophyllea latourr. a mes-meg h caesp eas biologica nyssana 2 (2) december 2011: 91-106 jotić b. et al. the vascular flora of the vučje hill… 103 taxa life form area type carex divulsa stokes v-a mes-mac h caesp hol carex halleriana asso v mes-mac h caesp med-smed carex humilis leysser a mi-mes h caesp eas carex pilosa scop. v-a mes-meg h caesp eas dioscoreaceae tamus communis l. v alt s/g herb rhiz med-smed iridaceae crocus adami gay v mi g bulb msm liliaceae allium albidum fischer ex bieb. a mes g bulb p allium cupani rafin a mes g bulb med-smed allium flavum l. a mes-mac g bulb msm allium moschatum l. a mes g bulb eas allium scorodoprasum (l.) subsp. rotundum (l.) stearn a mes-meg g bulb eas allium sphaerocephalon l a mes-meg g bulb eas anthericum ramosum l. a mes-meg g rhiz eas asparagus officinalis l. a meg-alt g rhiz p asparagus tenuifolius lam. a meg-alt g rhiz eas colchicum autumnale l. aut mes g bulb eas erythronium dens – canis l. v mes g bulb eas gagea lutea (l.) ker-gawler v mes g bulb eas gagea arvensis (pers.) dumort. v mi-mes g bulb eas gagea minima (l.) ker-gawler v mi g bulb eas gagea pratensis (pers.) dumort. a mi-mes g bulb eas gagea pusilla (f. w. schmidt) schultes & schultes fil v mi g bulb eas hyacinthella leucophaea (c. koch) schur a mi-mes g bulb eas muscari comosum (l.) miller v mes g bulb eas muscari neglectum guss. ex ten. v mes g bulb eas muscari tenuiflorum tausch v mes g bulb p ornithogalum collinum guss. v-a mi-mes g bulb eas ornithogalum ortophyllum ten. subsp. kochii (parl.) zahar. v mi-mes g bulb eas ornithogalum pyramidale l. v mi-mes g bulb eas ornithogalum umbellatum l. v-a mes g bulb eas polygonatum odoratum (miller) druce a mes-meg g rhiz eas scilla bifolia l. v mes g bulb eas veratrum nigrum l. a meg-alt g rhiz eas orchidaceae listera ovata (l.) r. br. a mes g tub scap eas ophrys apifera hudson a mes g tub scap eas ophrys cornuta steven a mes g tub scap eas orchis morio l. v-a mes g tub scap eas orchis pallens l v mes g tub scap eas poaceae aegilops geniculata roth. a mi-mes t caesp med-smed agropyron cristatum (l.) gaertne a mes-meg g rhiz caesp eas agrostis capillaris l a mes-meg h caesp hol alopecurus pratensis l. a mes-meg h caesp eas antoxanthum odoratum l. a mes-meg h caesp eas avenula pubescens (hudson) dumort. a mes-alt h caesp eas biologica nyssana 2 (2) december 2011: 91-106 jotić b. et al. the vascular flora of the vučje hill… 104 taxa life form area type brachypodium pinnatum (l.) beauv. a mac-alt h caesp eas brachypodium sylvaticum (hudson) beauv. a mac-alt h caesp eas briza media l. a mes-meg h caesp me bromus commutatus schrader a meg t scap msm bromus hordeaceus l. a mes-meg t caesp eas bromus ramosus hudson a meg-alt h caesp eas bromus squarosus l. a mes-mac t caesp bienn med-smed chrysopogon gryllus (l.) trin. a alt h caesp eas cleistogenes serotina (l.) keng a mes-mac h caesp med-smed dactylis glomerata l. a meg h caesp eas dasypyrum villosumr. br. ex fresen. a mes-meg t caesp med-smed dichanthium ischaemum (l.)roberty a mes-meg h caesp eas elymus repens (l.) gould a mes-meg g rhiz caesp csm festuca pratensis huds. a mac-meg h caesp eas festuca rubra l. a meg h caesp hol festuca valesiaca schleicher ex gaudin a mes-mac h caesp eas holcus mollis l. a mes-mac h caesp eas hordeum murinum l. subsp. leporinum (link) arcangeli a mes t caesp med-smed koeleria eryostachya pančić a meg h caesp me koeleria glauca (schreder) dc. a mes-meg h caesp eas koeleria glaucovirens domin a mes-meg h caesp med-smed koeleria macrantha (ledeb.) schultes a mes-mac h caesp hol koeleria nitidula velen. a mes-meg h caesp med-smed lolium perenne l. a mes-mac h caesp eas melica ciliata l. a mes-meg h caesp eas melica transsilvanica schur a mes-meg h caesp p melica uniflora retz. a mes-mac h caesp me phleum pratense l. a mes-alt h caesp me poa annua l. a-aut mi-mes t/h caesp csm poa bulbosa l. a mes-meg h caesp eas poa compressa l a mes-mac h caesp csm poa nemoralis l. a mes-meg h caesp hol poa pratensis l. a mes-meg h scap eas sesleria latifolia (adamović) degen a mes-meg h caesp eamt sesleria rigida heuffel ex reichenb. a mes-meg h caesp eamt setaria viridis (l.) beauv. a mes-mac h caesp eas sorghum halepense (l.) pers. a-aut meg-alt g rhiz caesp adv stipa capillata l. a mac-meg h caesp eas stipa pulcherrima c. koch. a mac-alt h caesp eas tragus racemosus (l.) all. a mi-mes t caesp-rept csm vulpia myuros (l.) c. c. gmelin a mes-meg t caesp csm disscusion taxonomic analysis divisio pteridophyta was represented by single class polypodiopsida with only 7 species. pinophyta was represented with single class pinopsida and just one species. divisio magnoliophyta was represented by 526 species, from which 439 are classified as dicotyledons (magnoliopsida) and 87 as monocotyledons (liliopsida). in taxonomic spectrum of the flora of investigated area the most numerous in genera were compositae (36), gramineae (29), labiatae (20), criciferae (17) and leguminosae (17). families with the most number of species were compositae (63), leguminosae (55), gremineae (47) and labiatae (36). the analysis of the particular genera occurrence in flora of the vučje hill has shown biologica nyssana 2 (2) december 2011: 91-106 jotić b. et al. the vascular flora of the vučje hill… 105 domination of representatives of genera trifolium (11), euphorbia (10), lathyrus (8), viola (8), vicia (7) sedum (7), following by the genuses centaurea, veronica and allium with 6 species in each genus. phytogeographical analisys for the phytogeographical analysis, all plant taxa were classified into 10 area types (fig. 2) according to the classification by s t e v a n o v i ć (1992). eurasian area type (eas) was the most dominant in the flora of the vučje hill with 303 taxa (56.7%). the second one was mediterraneansubmediterranean area type (med) with 75 species (7.9%). me 8% med 14% p 4% hol 6% csm 5% adv 1% eam 3% eas 57% msm 2% ab 0% figure 2. area spectrum of the flora of vučje hill: csm – cosmopolitan, hol – holarctic, ab – arctoalpian + boreal, me – middle-european, eas – euroasian, eamt – euroasian mountain, p – pontic, msm – merridional-submerridional, med – mediterranean-submediterranean, adv adventitious there were 10 balcan endemic taxa in the flora of the vučje hill. these represents 3,48% of the total number of balkan endemics in serbian flora, which includes 287 taxa in rank of species and subspecies (s t e v a n o v i ć et al., 1999). small number of endemic floral elements are expected due to uniformity of habitats in the wider area of vučje. the recorded balkan endemics are: acanthus balcanicus, acer hyrcanum subsp. intermedium, bupleurum flavicans, eryngium palmatum, hypericum rumeliacum, thymus praecox subsp. jankae, pulsatilla montana subsp. bulgarica, linaria rubioides subsp. nissana, viola tricolor subsp. macedonica and sesleria latifolia. biological spectrum the analysis of plant life forms (fig. 3) showed that flora of investigated area was of hemicryptophyta type, as well as the flora of serbia (d i k l i ć , 1984) and balkan peninsula (turill, 1929). the hemicryptophytes were represented with 45%; theropytes followed with 26,9% and geophytes with 11,1%. the climatics characteristics of this area caused the high percentage of hemicryptophyta. h 45% g 11% t 27% s 3% p 7% ch 7% figure 3. biological spectrum of the flora of vučje hill: p – phanerophyta, ch – chamaephyta, h – hemycryptophyta, g – geophyta, t – terophyta, s scandetophyta threatened status even the flora of the vučje hill is diverse and rich, consisting of 526 plant taxa, 63 representatives are considered to be threatened in flora of serbia. in the flora of the vučje hill were registered 9 strictly protected species: acer hyrcanum subsp. intermedium, vinca herbacea, sideritis montana, adonis vernalis, pulsatilla montana subsp. bulgarica, gagea minima, ophrys apifera, orchis pallens and stipa pulcherrima (anonimous, 2010). also, in the flora of the vučje hill were registered 22 protected species, such as: achillea clypeolata, corylus colurna, allium albidum, lilium martagon, listera ovata, prunus tenella, ajuga laxmanii, salvia austriaca and other (anonimous, 2010). in the flora of the study area registered 33 species, that are covered by regulation on collecting, use and sale, such as: hedera helix, asarum europaeum, achillea millefolium, pulmonaria officinalis, primula veris, rosa canina, teucrium chamaedrys and other (anonimous, 2010a). references adamović, l., 1911: flora jugoistočne srbije. jazu. zagreb. anonimous, 2010: službeni glаsnik republike srbije br. 5/10, prаvilnik o proglаšenju i zаštiti biologica nyssana 2 (2) december 2011: 91-106 jotić b. et al. the vascular flora of the vučje hill… 106 strogo zаštićenih i zаštićenih divljih vrstа biljаkа, životinjа i gljivа anonimous, 2010a: službeni glаsnik republike srbije br. 31/05, 45/05-isprаvkа, 22/07, 38/08 i 9/10, uredbа o stаvljаnju pod kontrolu korišćenjа i prometа divlje flore i fаune ćirić, j., živković, v., 1974: krupac. geografija, istorija, društveni život, narodnooslobodilački rat. izdanje muzeja ponišavlja – pirot i balkanološkog instituta univerziteta – niš. diklić, n., 1984: životne forme biljnih vrsta i biološki spektar flore sr srbije. in: sarić, m. (ed.): vegetacija sr srbije, i: 291-316. sanu, posebna izdanja. beograd. greuter, w., burdet, h.m., long, g., eds., 19841989: med-checklist, 1, 3, 4. gèneve. javorka, s., csapody, v., 1991: iconographia florae partis austro-orientalis europe centralis. akademia kiado. budapest. josifović, m., ed., 1970-1976: flora sr srbije, i-ix. sanu. beograd. marinkov, j., 1999: zabrđe. prirodno – privredni potencijali. narodna biblioteka dimitrovgrad. dimitrovgrad. meussel, h., jäger, e., weinert, e., 1965: vergleinchende chorologie der zentraleuropaischen flora. veb. gustav fischer verlag, 1. jena. meussel, h., jäger, e., raischert, s., weinert, e., 1978: vergleinchende chorologie der zentraleuropaischen flora. veb. gustav fischer verlag, 2. jena. meusel, h., jäger, e., 1992: vergleinchende chorologie der zentraleuropaischen flora. veb. gustav fischer verlag, 3. jena. mueller – dombois, d., ellenberg, h., 1974: aims and methods of vegetation ecology. john wiley and sons. new york. pančić, j., 1884: dodatak flori kneževine srbije. kraljevska srpska državna štamparija, beograd. stevanović, v., ed., 1999: crvena knjiga flore srbije. 1, iščezli i krajnje ugroženi taksoni. ministarstvo za životnu sredinu republike srbije, zavod za zaštitu prirode srbije. beograd. stevanović, v., 1992: floristička podela teritorije srbije sa pregledom viših horiona i odgovarajućih flornih elemenata. in sarić, m. (ed.): flora srbije, i. (drugo izdanje). sanu. beograd. 49 – 65. stevanović, v., 1992a: klasifikacija životnih formi flore srbije. in sarić, m. (ed.): flora srbije i. (drugo izdanje). sanu. beograd. 39 – 46. turill, w.b., 1929: the plant life of the balkan peninsula. a phytogeographical study. oxford. tutin, t.g., heywood, v.h., burges, n.a., moore, d.m., valentine, d.h., walters, s.m., webb, d.a., eds., 1964-1980: flora europaea, i-v. cambridge university press. london. vidanović, g., 1960: vidlič – zabrđe, prilog poznavanju privrednog tipa, ekonomsko – geografska studija, srpska akademija nauka, posebna izdanja geografskog instituta, knjiga 15, beograd. microsoft word bn-aa-0201-00 mitrovic et al biologica nyssana 2 (1) june 2011: 1-12 dimitrijević d. et al. the nature of the variability of the morphological characteristics… 1 review article ! lichens as source of versatile bioactive compounds tatjana mitrović*, slavisa stamenković, vladimir cvetković, miloš nikolić, svetlana tošić, dragana stojičić department of biology and ecology, faculty of sciences and mathematics, university of niš * e-mail: tatjanamitrovic@hotmail.com abstract: mitrović, t., stamenković, s., cvetković, v., nikolić, m., tošić, s., stojičić, d.: lichens as source of versatile bioactive compounds, biologica nyssana, 2 (1), september 2011: 1-6. lichens represent unique symbiosis of fungi (mycobionts) and algae (photobionts). living in extreme conditions they developed various compounds to survive. many of these original compounds have proven biological activities (antibiotic, antimycotic, antiviral, antitumor, antioxidant, etc) . this paper is synthesis of currently known data about lichens extracts and their potential use in pharmaceutics and medicine. key words: lichens, bioactive compounds, phytomedicine introduction ! pharmaceutical industry is forced to continual chase and development of new pharmacological active molecules. similar to higher plants, lichens are considered as potential source of novel biologically active compounds. lichens are complex symbiotic associations between a fungus (mycobiont) and an alga (photobiont) with unique characteristics in plant kingdom. it is estimated that there are approximately 25,000 species of lichens (c h a p m a n , 2009). they are proven as the earliest colonizers of terrestrial habitats on the earth with a worldwide distribution from artic to tropical regions and from the plains to the highest mountains (taylor et al., 1995). specific, even extreme, conditions of their existence, slow growth and long duration (age of several thousand years) are result of numerous protective compounds against different physical and biological influences (d e n t o n & k a r l e n , 1973). these metabolites are acters of lichen extracts’ bioactivity of various importance for modern pharmaceutics and medicine. short review of utilization of lichens back in time and modern investigation of lichen extracts and components and their application are given below. a brief history of lichen utilization throughout the ages, lichens are used in nutrition of many animals and humans during famine (for example, during leningrad siege) (r i c h a r d s o n , 1988; k a r a g o z et al., 2009). the desert species lecanora esculenta is considered as “biblical manna” (t r e a s e & e v a n s , 1978). since egyptians, lichens were used as dyes, perfumes and remedies in folk medicine (v a r t i a , 1973). it is known that romans dyed their togas with orchil, a purple pigment from roccella sp. and crottal, brown pigment from parmelia, ochrolechia and evernia sp. (m u g g i a et al., 2009). lichensdyed textile reached considerable economic importance in 18th century in some parts of the world as in the canary islands (m u g g i a et al., 2009). litmus, a blue coloring matter from lichen fermentation, was used as dye for textile and beverages (b e e c k e n et al., 1961). also, paper strips impregnated with litmus, a water extracted 2 (1) • september 2011: 1-6 biologica nyssana 2 (1) september 2011: 1-6 mitrović t. et al. lichens as source of versatile bioactive compounds 2 dyes from roccella sp., are used as ph indicators in laboratories from ancient times till present. extracts of some species of lichens, like evernia prunastri, are contents of perfumes (t r e a s e & e v a n s , 1978). naturally, the most important and studied application of lichens is the one in traditional medicine for treatment of animals and humans. for instance, new zealand maori traditionally use long, pendulous species of usnea for nappies and sanitary pads (p e r r y et al., 1999). also, usnea species have been used in asia, africa and europe for pain relief and fever control (o k u y a m a at al., 1995). usnea densirostra, known as “barba de la piedra” served as a cure for various disorders in argentina’s folk medicine (c o r r e c h e at al, 1998). two lichen species, parmelia caperata and umbilicaria sp. are reported in study of chilean traditional medicine (m u n o z et al., 1981). ramalina thrausta is used in finland for treatment of wounds, athlete’s foot or other skin diseases and taken to relieve sore throat and toothache (v a r t i a , 1973). cetraria islandica is ancient cough remedy known as “tonicum amarum” accepted as a mucilage drug (m u l l e r , 2001). various species worldwide are used in traditional medicine and said that they are able to cure dyspepsia, bleeding piles, diabetes, bronchitis, pulmonary tuberculosis, spermatorrhoea and other diseases of the blood and heart (r i c h a r d s o n , 1988). lichen metabolites generally, lichens metabolites can be divided into two groups: primary and secondary. primary metabolites are proteins, lipids, carbohydrates and other organic compounds involved in lichen’s metabolism and structure. secondary metabolites, also known as lichens substances, are produced mainly by the fungus and secreted onto the surface of the lichen’s hyphae either in amorphous forms or as crystals. they are in focus of this review.hhh lichen substances secondary metabolites are complex, but predominantly small molecules, which comprise up to 20% of lichen’s dry weight (m u g g i a et al., 2009). structures of more then 1000 different lichen substances are determined to date and many of them are pharmaceutically relevant (m u g g i a et al., 2009). secondary metabolites are products of polyketide pathway, mainly monocyclic and/or bicyclic phenols joined by an ester bond (depsides), both ester and ether bonds (depsidones) or furan heterocycle (dibenzofurans and usnic acid), antraquinones, xanthones, chromones and secondary aliphatic acids and esters (s t o j a n o v i ć et al., 2011). some of them are produced by the fungus or the alga per se, while others are exclusively produced by synergistic action of both partners in lichens. whole spectrum of lichen metabolites evolved for protective purposes against various physical and biological environmental factors (d e n t o n & k a r l e n , 1973). large amounts of phenolic compounds fungal melanins are synthesized and accumulated in the tallus in order to absorb uvb light and shelter the photobiont from excessive radiation (g a u s l a a & s o l h a u g , 2001). these photoprotectors have great antioxidant capacity (h i d a l g o et al., 1994, f e r n a n d e z et al., 1996) and can be used as preservatives in cosmetic products (m u l l e r , 2001). certain phenolic compounds protect lichens from herbivores (l a w r e y , 1989). theirs toxicity, feeding deterrence to insect larvae and nematocidal activity has been demonstrated (a h a d et al. 1991, e m m e r i c h et al., 1993). other lichen metabolites have antibiotic properties which prevent microbial degradation of the thallus (e m m e r i c h et al., 1993). some of lichen metabolites are involved in maintaining of the symbiotic equilibrium (h u n e c k , 1999), while others dissolved rocks for better attachment of lichens (s e a w a r d ,1997). biological activity of lichens lichen metabolites exert manifold biological activity. first, antibiotic properties of lichen extracts are known for decades. first study by burkholder originated from 1944. later, v a r t i a (1973) reported antimicrobial activity of several lichen species. according to wide screening of antimicrobial activity of lichen extracts, it seems that bacterial inhibitions can vary within the lichen extract, solvent used for extraction and bacteria tested. r a n k o v i c et al. (2007a; 2007b) tested aqueous, acetone and methanol extracts of cladonia furcata, parmelia caperata, parmelia pertusa, hypogimnia physodes, umbilicaria polyphylla, lasallia pustulata, parmelia sulcata, umbilicaria crustulosa and umbilicaria cylindrica from serbia on six species of bacteria and ten species of fungi. the strongest activity was observed with methanol extracts of parmelia pertusa and parmelia sulcata and the weakest activity was manifested by parmelia caperata and umbilicaria cylindrical. aqueous extracts of all tested lichen species were inactive. bacillus mycoides was the most sensitive bacterial species tested, whereas candida albicans was the most sensitive fungal species examined. other studies monitored ramalina farinacea and 69 species of lichens from new zealand and showed biologica nyssana 2 (1) september 2011: 1-6 mitrović t. et al. lichens as source of versatile bioactive compounds 3 their inhibitory effect against a lot of bacteria such as bacillus, pseudomonas, e.coli, streptococcus, staphylococcus, enterococcus, mycobacterium (e s i m o n e & a d i k w n , 1999; p e r r y et al., 1999). b e h e r a et al. (2005) reported that acetone, methanol and light petroleum extracts of lichen usnea ghattensis were effective against bacillus licheniformis, b. megaterium, b. subtilis and s. aureus. also, k a r a g o z et al. (2009) evaluated aqueous and ethanol extracts of 11 different species from turkey and determined potent antibacterial activity of aqueous extract of peltigera polydactyla and ethanol extract of ramalina farinacea. chemical identification of anti-gram-positive activities revealed evernic acid, vulpinic acid and hirtusneanoside as main acters (l a w r e y , 1986; r e z a n k a & s i g l e r , 2007). recently, m i t r o v i ć et al. (2011) studied antibacterial activity of methanol extracts of five lichen species (flavoparmelia caperata, evernia prunastri, hypogymnia physodes and cladonia foliacea). two lichen species were tested for the first time (evernia prunastri and cladonia foliacea). the analysis of their antibacterial potential were performed on 15 strains of bacteria and revealed the strongest inhibitory effect, especially on gram (+) bacteria, of hypogymnia physodes and cladonia foliacea. second, antifungal activities of certain lichen substances are also revealed. m a n o j l o v i c et al. (2005) reported antifungal activity of the anthraquinone parietin isolated from caloplaca cerina. two years later, antifungal properties were observed in extracts of the andean lichens protousnea poeppigii and usnea rigida, which contain divaricatinic acid, isodivaricatinic acid, usnic acid and 5-resorcinol (schmeda-hirschmann et al., 2008). also, m i t r o v i ć et al. (2011) determined strong antifungal effect of evernia prunastri and hypogymnia physodes. while evernia prunastri exerted the best effect on yeasts, hypogymnia physodes were better on filamentous fungi. third, antiviral properties has been attributed to specific lichen secondary metabolites. for instance, p e r r y et al. (1999) showed antiviral activity of usnic acid against herpes simplex type 1 and polio type 1 viruses. parietin extracted from teloschistes chrysophthalmus proved as virucidal for junin and tacaribe arenaviruses (f a z i o et al., 2007). lichenan, widely distributed in lichens, demonstrated inhibition of tobacco mosaic virus (s t u b l e r & b u c h e n a u e r , 1996). fourth, antitumor activities of lichens are of major importance as source of potential drugs for lethal malignant diseases. usnic acid, the most extensively studied lichen metabolite since its first isolation in 1844, exhibited an antiproliferative effect on human leukemia cells (k562) and endometrial carcinoma (hec-50) cells (c a r d e r e l l i et al., 1997, i n g o l f s d o t t i r , 2002, k r i s m u n d s d o t t i r et al., 2002). another lichen compound depsidone pannarin inhibited cell growth and induces apoptosis in human prostate carcinoma du-145 and human melanoma m14 cells (r u s s o et al. 2006, 2008). lichen polysaccharide cfp-2 reduced the viability of hl-60 and k562 cells (l i n et al., 2003). protolichesterinic acid isolated from cetraria islandica showed inhibition of growth of brest cancer cell lines and mitogenstymulated lymphocytes (o g m u n d s d o t t i r et al., 1998). inhibition of enzyme 5-lipoxygenase involved in inflammation, non-specific binding to dna polymerase β and dna ligase i are possible mechanisms of antitumor activity of protolichesterinic acid (m u l l e r , 2001). b u c a r et al. (2004) revealed inhibitory activities on 12(s)hete inside antiproliferative effect of several lichen substances in human platelets. furthermore, antipyretic and analgesic effects of lichen components were demonstrated on animal studies. for instance, usnic acid from usnea diffracta inhibited acetic-acid-induced writhing in mice and raised the pain threshold in dosedependent manner (o k u y a m a et al., 1995). finally, antioxidant properties, already mentioned previously concerned with phenolic content of lichens. j a y a p r a k a s h a and r a o (2000) examined antioxidant capacities of methyl orsellinate, atranorin, osellinic acid and lecanoric acid. b h a t t a r a i et al. (2008) noticed stronger antioxidant activities in lichens from antarctic that the one in lichens from native to temperate or tropical regions. m i t r o v i ć et al. (2011) compared the chemical content of lichen extracts (flavoparmelia caperata, evernia prunastri, hypogymnia physodes and cladonia foliacea) and their free radical scavenging ability. they observed strong correlation according to previous conclusions of r a n k o v i ć et al. (2010). hypogymnia physodes with the highest phenolic content showed the strongest antioxidant effect. conclusions lichens represent powerful source of new bioactive molecules for various pharmaceutical purposes. structure of more then 1000 lichen substances are available, but even more remain to be characterized, which is delayed by their natural occurrence in low concentrations. lichens were frequently ignored by pharmaceutical industries because of their slow growth. difficulties biologica nyssana 2 (1) september 2011: 1-6 mitrović t. et al. lichens as source of versatile bioactive compounds 4 encountered with collecting substantial amounts of plant materials may be circumvented by establishing lichen tissue-cultures that may be promising sources of novel biologically active compounds. isolation into pure culture was attempted on 1,183 species of lichen-forming and lichenicolous fungi (c r i t t e n d e n et al., 1995). however, it should be noted that lichen metabolites produced in mycobiontic cultures are not always identical to those produced from lichens themselves (m u l l e r , 2001). as an alternative method, the transfer of genes responsible for the production of lichen metabolites are suggested (h u n e c k , 1999). lichen tissue culture and gene transfer techniques may considerable enlarge the access to lichen derived substances and their excessive pharmaceutical screening. eventual drug-promising molecules from lichens can be improved with increased stabilities and reduced toxicities by modern computer-aided drug design programs and combinatorial chemistry (m u l l e r , 2001). acknowledgements. research study mentioned in this review was supported by the ministry of science and education of the republic serbia (project iii41018). references ahad, a.m., goto, y., kiuchi, f., tsuda, y., kondo, k., sato, t. 1991: nematocidal principles in “oakmoss absolute” and nematocidal activity of 2,4-dihydroxybenzoates. chem. pharm. bull., 39: 1043-1046. beecken, h., gottschalk, e-m., v. gizycki, u., kramer, h., maassen, d., matthies, h-g., musso, h., rathjen, c., zahorsky, u.i. 1961: orcein and lackmus. angew chem. 73: 665-688. behera, b.c., verma, n., sonone, a., makhija, u. 2005: antioxidant and antibacterial activities of lichen usnea ghattensis in vitro. biotechnol. lett., 27: 991-995. bhattarai, h.d., paudel, b., hong, s.g., lee, h.k., yim, j.h. 2008: thin layer chromatography analysis of antioxidant constituents of lichens from antarctica. j. nat. med., 62: 481-484. bucar, f., schneider, i., ogmundsdottir, h., ingolfsdottir, k., 2004: anti-proliferative lichen compounds with inhibitory activity on 12(s)hete production in human platelets. phytomedicine, 11: 602-606. burkholder, p.r., evans, a.w., mcveigh, i., thorton, h.k. 1944: antibiotic activity of lichens. proc. natl. acad. sci. usa, 30: 250-255. cardarelli, m.a., serino, g., campanella, l., ercole, p., de cicco-nardone, f., alesiani, o., rossiello, f. 1997 : antimitotic effects of usnic acid on different biological systems. cell. mol. life. sci., 53: 667-672. chapman, a.d. 2009: numbers of living species in australia and the world. australian biological resources study(abrs). canberra. 80 p. correche, e. r., carrasco, m., escudero, m. e. 1998 : study of the cytotoxic and antimicrobial activities of usnic acid and derivates. fitoterapia, 69: 493-501. crittenden, p.d., david, j.c., hawksworth, d. l., campbell, f.s. 1995: attempted isolation and success in the culturing of a broad-spectrum of lichen-forming and lichenicolous fungi. new phytol. , 130: 267-297. denton, g.h., karlen, w. 1973: lichenometry: its application to holocene moraine studies in southern alaska and swedish lapland. artic alpine res., 5: 347-372. emmerich, r., giez, i., lange, o.l., proksch, p. 1993: toxicity and antifeedant activity of lichen compounds against the polyphagous herbivorous insect spodoptera littoralis. phytochemistry, 33: 1389-1394. esimone, c.o., adikwn, m.u. 1999: antimicrobial activity of the cytotoxicity of ramalina farinacea. fitoterapia, 7: 428-431. fazio a.t., adler, m.t., bertoni, m.d., sepulveda, c.s., damonte e.b., maier, m.s. 2007: lichen secondary metabolites from the cultured lichen mycobionts of teloschistes chrysophthalmus and ramalina celastri and their antiviral activities. z. naturforsch, 62: 543-549. fernandez, e., quilhot, w., gonsalez, i., hidalgo, m.e., molina, x., meneses, i. 1996 : lichen metabolites as uv-b filters. cosmetics and toiletries, 111: 69-74. gauslaa, y., solhaug, k.a. 2001: fungal melanins as sun screen for symbiotic green algae in the lichen lobaria pulmonaria. oecologia, 126: 462471. huneck, s. 1999: the significance of lichens and their metabolites. naturwissenschaften, 86: 559570. hidalgo, m.e., fernandez, e., quilhot, w., lissi, e.a. 1994 : antioxidant capacity of depsides and depsidones. phytochemistry, 37: 1585-1587. ingondolfsdottir, k. 2002: usnic acid. phytochemistry, 61: 729-736. jayaprakasha, g.k., rao, l.j. 2000: phenolic constituents from the lichen parmotrema stuppeum (nyl.) hale and their antioxidant activities. z. naturforsch., 55: 1018-1022. karagoz, a., dogruoz, n., zeybek, z., aslan, a. 2009: antibacterial activity of some lichen extracts. journal of medicinal plants research, 3: 1034-1039. biologica nyssana 2 (1) september 2011: 1-6 mitrović t. et al. lichens as source of versatile bioactive compounds 5 kristmundsdottir, t., aradottir, h.a.e., ingolfsdottir, k., ogsmundsdottir, h. m. 2002: solubilization of the lichen metabolite (+) –usnic acid for testing in tissue culture. j. pharm. pharmacol., 54: 1447-1452. lawrey, j.d. 1986: biological role of lichen substances. bryologist, 89: 11-122. lawrey, j. d. 1989: lichen secondary compounds: evidence for a correspondence between antiherbivore and antimicrobial function. bryologist, 92: 326-328. lin, x., cai, y.j., li, z. x., chen, q., liu, z. l., wang, r. 2003 : structure determination, apoptosis induction, and telomerase inhibition of cfp-2, a novel lichenin from cladonia furcata. biochem. biophys.acta, 1622: 99-108. manojlović, n.t., solujić, s., sukdolak, s., milošev, m. 2005: antifungal activity of rubia tinctorum, rhamnus frangula and caloplaca cerina. fitoterapia, 76: 244-246. mitrović, t., stamenković, s., cvetković, v., tošić, s., stanković, m., radojević, i., stefanović, o., čomić, lj., đačić, d., ćurčić, m., marković, m. 2011: antioxidant, antimicrobial and antiproliferative activities of five lichen species. int. j. mol. sci., 12: 5428-5448. muggia, l., schmitt, i., grube, m. 2009: lichens as treasure chests of natural products. sim news, 59: 85-97. muller, k. 2001: pharmaceutically relevant metabolites from lichens. appl. microbiol. biotechnol. 56: 9-16. munoz, m., barrera, e., meza, i. 1981 : el uso medicinal y alimenticio de plantas nativas y naturalizadas en chile. publicacion ocasional no. 33 museo national de historia natural : santiago de chile, 8. perry, n. b., benn, m.h., brennan, n. j., burgess, e. j., ellis, g., galloway, d. j., lorimer, s.d., tangney, r.s. 1999: antimicrobial, antiviral and cytotoxic activity of new zealand lichens. lichenologist, 31: 627-636. okuyama, e., umeyama, k., yamazaki, m., kinoshita, y., yamamoto, y. 1995: usnic acid and diffractaic acid as analgesic and antipyretic components of usnea diffracta. planta med, 61: 113-115. ogmundsdottir, h.m., zoega, g.m., gissurarson, s.r., ingolfsdottir, k. 1998: anti-proliferative effects of lichen-derived inhibitors of 5lipoxygenase on malignant cell lines and mytogen stimulated lymphocytes. j. pharm. pharmacol., 50: 107-115. ranković, b., mišić, m. sukdolak, s. 2007 a: antimicrobial activity of the lichens cladonia furcata, parmelia caperata, parmelia pertusa, hypogimnia physodes and umbilicaria polyphylla. british journal of biomedicinal science, 64: 143-148 ranković, b., mišić, m. sukdolak, s. 2007 b: evaluation of antimicrobial activity of the lichens lasallia pustulata, parmelia sulcata, umbilicaria crustulosa and umbilicaria cylindrica. microbiology, 76: 723-727. ranković, b., ranković, d., marić, d. 2010: antioxidant and antimicrobial activity of some lichen species. microbiology, 79: 809–815. rezanka, t., sigler, k. 2007: hirtusneanoside, an unsymmetrical dimeric tetrahydroxanthone from the lichen usnea hirta. j. nat. prod., 70: 14871491. richardson, d.h.s. 1988. medicinal and other economic aspects of lichens. in: galun, m. (ed.), crc handbook of lichenology 1: 93-108, crc press, boca raton, florida. russo, a., piovano, m., lombardo, l., vanella, l., cardile, v., garbarino, j. 2006: pannarin inhibits cell growth and induces cell death in human prostate carcinoma du-145 cells. anticancer drugs, 17:1163-1169. russo, a., piovano, m., lombardo, l., garbarino, j., cardile, v. 2008: lichen metabolites prevent uv light and nitric oxide-mediated plasmid dna damage and induce apoptosis in human melanoma cells. life. sci. , 83: 468-474. seaward, m.r.d. 1997: major impacts made by lichens in biodeterioration processes. int. biodeterior. biodegrad., 40: 269-273. schmeda-hirschmann, g., tapia, a., lima, b., pertino, m., sortino, m., zacchino, s., rojas de arias, a., and feresin, g.e. 2008: a new antifungal and antiprotozoal depside from the andean lichen protousnea poeppigii. phytother. res., 22: 349-355. stojanović, i. ž., radulović, n.s., mitrović, t. lj., stamenković, s.m., stojanović, g. s. 2011: volatile constituents of selected parmeliacea lichens. j. serb. chem. soc. 76: 987-994. stubler, d., buchenauer, h. 1996: antiviral activity of the glucan lichenan (poly-β{1 → 3, 1 →4} danhydroglucose) 1. biological activity in tobacco plants. j. phytopathol., 144: 37-43. taylor, t. n., hass, h., remy, w., kerp, h. 1995: the oldest fossil lichen. nature, 378: 244-244. trease, g.e., evans, w.c. 1978: pharmacognosy, 11th edition, balliere tindall, london, 75-76. vartia, k. o. 1973: antibiotics in lichens. in: ahmadjian, v., hale, m.e. (ed.), the lichens, 547-561, academic press, new york. microsoft word bn020203 gnjatovic zikic biologica nyssana 2 (2) december 2011: 119-122 gnjatović i., žikić v. new data on longhorn beetles… 119 original article ! new data on longhorn beetles for the territories of serbia and montenegro (coleptera, cerambycidae) with the detailed description of callimoxys gracilis (brullé 1832) ivan gnjatović, vladimir žikić university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia * e-mail: ivangnjatovic81@gmail.com abstract: gnjatović, i., žikić, v.: new data on longhorn beetles for the territories of serbia and montenegro (coleptera, cerambycidae) with the detailed description of callimoxys gracilis (brullé 1832), biologica nyssana, 2 (2), december 2011: 119-122. investigated sample of longhorn beetles discovers 24 species from 20 genera of 11 tribes from three subfamilies. the species vadonia moesiaca daniel 1891 apears to be a balkan endemic. also, very rare species callimoxys gracilis (brullé 1832) is recorded for the second time for the serbian fauna. the examined material was collected during the period of 2010-2011 on the territories of serbia and montenegro. key words: cerambycidae, callimoxys gracilis, sićevo gorge, torilis ucranica introduction ! the fauna of cerambycidae is more or less well investigated on the teritorry of serbia, starting from b o b i ć (1891) over the detailed monograph of the yugoslav cerambycids in three volumes by m i k š i ć at al. (1971, 1973, 1985) to the last publication of i l i ć (2005). the data from the recent studies shows that there are 259 species of longhorn beetles in serbia (p i l & s t o j a n o v i ć , 2009) and 142 species for the territory of montenegro (ć u r č i ć al., 2003). the genus callymoxys belongs to the subfamily: cerambycinae and the tribe: stenopterini comprising only two species: the blood-necked longhorn beetle, c. sanguinicollis (o l i v i e r , 1795) inhabiting north america and c. gracilis (b r u l l é , 1832) which occurs in europe. materials and methods all specimens were collected during 20102011 on the territories of serbia and montenegro. the sampling of longhorn beetles has been performed mostly by using an entomological net by sweeping over plants or picking the specimens from the ground or plants by hand. the total number of collected and examined specimens is 56. the specimens of the species callimoxys gracilis (b r u l l é , 1832), one female and three males, were found on the plant torilis ucranica spreng 1820 , apiales: apiaceae. this species had been recorded previously only once for the fauna of serbia. there has been no data for the territory of montenegro yet. identification of the sampled specimens was performed using adequate determination keys: m i k š i ć & g e o r g i j e v i ć (1973), and b e n s e (1995). additional confirmation was performed by checking the data of the fauna of longhorn beetles 2 (2) • december 2011: 119-122 biologica nyssana 2 (2) december 2011: 119-122 gnjatović i., žikić v. new data on longhorn beetles… 120 for the territory of serbia and montenegro, and also by comparing with the literature data of a l h t o f f & d a n i l e v s k y (1997), i l i ć (2005) and g n j a t o v i ć & ž i k i ć (2010). results the list of collected specimens: subfamily cerambycinae tribe clytini chlorophorus hungaricus seidlitz 1791 2♂, serbia: fruška gora mt., 01.06.2011, leg. p. jakšić. tribe stenopterini callimus angulatus (schrank 1789) 2♂, montenegro: bečići, 23.04.2011, leg. v. žikić. callimoxys gracilis (brullé, 1832) 1♀, 3♂, serbia, niš, sićevo gorge, 15.05.2011., leg. v. žikić. tribe purpuricenini purpuricenus budensis (goeze 1783) 1♀, serbia: niš, sićevačka gorge, 19.07.2011, leg. v. žikić. subfamily lamiinae tribe acanthocinini exocentrus punctipennis mulsant & guillebeau 1856, 1♂, serbia: niš, 15.07.2011, leg. v. žikić. tribe agapanthiini calamobius filum (rossi 1790) 2♀, 2♂, serbia: bovan lake, 13.06.2011, leg. v. žikić. agapanthia kirby (gyllenhal 1817) 1♀, serbia: miljkovačka gorge, 08.06.2011, leg. i. simonović; 2♂, montenegro: budva, 25.05.2011, leg. v. žikić. agapanthia intermedia ganglbauer 1884 1♂, montenegro: budva, 25.05.2011, leg. v. žikić. agapanthia dahli (richter 1821) 1♂, montenegro: bečići, 18.05.2011, leg. v. žikić. tribe lamiini leiopus nebulosus (linanneus 1758) 1♂, serbia: niš, popovac, 22.05.2011, leg. v. žikić. lamia textor (linanneus 1758) 1♀, serbia: boljevac, progor, 06.07.2011, leg. p. jakšić. tribe obereini oberea (amaurostoma) erythrocephala (schrank 1776) 1♀, montenegro: budva, 25.05.2011, leg. v. žikić. tribe phytoeciini musaria affinis (harrer 1784) 1♀, serbia: niš, 02.07.2011, leg. i. gnjatović. phytoecia caerulea (scopoli 1772) 1♀, montenegro: bečići, 25.05.2011, leg. v. žikić. tribe saperdini saperda scalaris (linanneus 1758) 1♀, 1♂, serbia: niš, sićevačka gorge, 31.05.2011, leg. v. žikić. subfamily lepturinae tribe lepturini alosterna tabacicolor (de geer 1775) 1♀, 1♂, serbia: niš, gornji matejevac, sv. jovan, 03.06.2010. leg. i. gnjatović. paracorymbia pallens (brulle 1832) 1♀, 3♂, serbia: niš, gornji matejevac, sv. jovan, 03.06.2010. leg. i. gnjatović. stictopleura scutellata (fabricius 1781) 1♀, serbia: kopaonik mt., 25.08.2010, leg. v. žikić. stenurella melanura (linanneus 1758) 2♀, 2♂, serbia: bosilegrad, dukat mt., 07.08.2010, leg. v. žikić. stenurella nigra (linanneus 1758) 5♀, 4♂, serbia: niš, gornji matejevac, sv. jovan, 03.06.2010. leg. i. gnjatović. stenurella septempunctata (fabricius 1792) 1♀, serbia: miljkovačka gorge, 08.06.2011. leg. i. simonović. vadonia moesiaca daniel 1891 1♀, serbia: niš, sićevačka gorge, 31.05.2010, leg. v. žikić. tribe rhagiini gaurotes (carilia) virginea (linanneus 1758) 3♀, 4♂, serbia: kopaonik mt., 27.08.2010. leg. v. žikić. pachyta quadrimaculata (linanneus 1758) 1♀, serbia: kopaonik mt., metođe, 20.07.2010, leg. s. stanković. disscusion identified taxa of the specimes which are listed here belong to the three subfamilies out of seven known from the fauna of serbia and montenegro, and out of eight from the fauna of europe: cerambycinae, lepturinae and lamiinae. comparing the taxonomical results for each noted subfamily, according to available data from the repesentative literature (m i k š i ć & g e o r g i j e v i ć , 1973; i l i ć , 2005), the biologica nyssana 2 (2) december 2011: 119-122 gnjatović i., žikić v. new data on longhorn beetles… 121 faunaeuropea database (a u d i s i o , 2005) and our results, we give here the total number of the recorded species: 65 from the subfamily cerambycinae, 55 from lepturinae and 77 from lamiinae. we have chosen the species callimoxys gracilis here to describe in details because of the suspicion on a new species. we were not able to find the original description of this species to compare with our specimens, therefore in this work we are not declaring this particular taxa as a new species for science. the first record of callimoxys gracilis (b r u l l é , 1832) for serbia was mentioned in the check-list of longhorn beetles by a l t h o f f & d a n i l e v s k y (1997) which encompass the whole territory of europe. there was no detailed information about habitat of this species or any other relevant data. in sićevo gorge, niš, serbia, we have found the adults of this particular longhorn species on the blossoms of torilis ucranica feeding on it. this piece of information is new about its biology. description: callimoxys gracilis (brullé, 1832) (fig. 1) original name: stenopterus gracilis brullé, 1832 material examined: 1♀, 3♂, niš, sićevo gorge, 15.05.2011. leg. i. gnjatović. the body is black, slightly greenish or bluish with metallic shine. length is about 7 (males) to 13 mm (female). head: semi-prognate. antennae: do not reach the elytra apex, quite longer in males. eyes: large, with small and fine omatidia (diurnal). pronotum is greenish to bluish in males, in female red, with black frontal and posterior margins (fig. 1). elytra are broad at the base, fine rasp punctuated, slightly narrow at the apex. hind wings: partly exposed reaching the apex of abdomen. lateral and ventral parts of both, thorax and abdomen, covered with tiny hairs. legs: all femora are slightly swollen. outer edges of hind tibia serrate (the most important taxonomic characteristic of the genus callimoxys). biology: adults apear from april to june. development is inadequately known. larvae have been found in dry branches of broadleaf trees: paliurus, prunus, quercus (š v a c h a & d a n i l e v s k y , 1987). locality: sićevo gorge, 15 km eastern of niš, serbia. territory under protection by the serbian government acts as a natural resource of great importance (t r a j k o v i ć & b r a n k o v i ć , 2007). substrate is limestone, important refuge of the arcto – tertiary flora during the last ice age. distribution: croatia (slavonia), northern bosnia and herzegovina, and montenegro (m i k š i ć & g e o r g i j e v i ć , 1973), as well in macedonia and romania, (i l i ć , 2005) and bulgaria, (g e o r g i e v at al., 2005; i l i ć , 2005). figure 1. callimoxys gracilis (brullé 1832), female (left) and male (right) biologica nyssana 2 (2) december 2011: 119-122 gnjatović i., žikić v. new data on longhorn beetles… 122 conclusion twenty four species of longhorn beetles, belonging to the three subfamilies were listed: cerambycinae (4), lepturinae (9) and lamiinae (11). the number of identified species in serbia is 18 and for the territory of montenegro it is six species. by analyzing the available literature, we deliver five species marked as rare: chlorophorus hungaricus seidlitz 1791, exocentrus punctipennis mulsant & guillebeau 1856, lamia textor (linanneus.1758), paracorymbia pallens (b r u l l e , 1832 (i l i ć , 2005) and callimoxys gracilis (b r u l l é , 1832) where we gave detailed description. the species vadonia moesiaca daniel 1891 belongs to the endemics of the balkan peninsula. acknowledgements. the authors wish to express their sincere thankfulness to saša stanković, ivan simonović and dr predrag jakšić for the loan of specimens. for the present work the authors were supported by the ministry of education and science of the republic of serbia (grant no. 43001). references audisio, p. 2005: fauna europaea: cerambycidae. u: sama g. (ur.) fauna europaea: coleoptera 2. fauna europaea, version 1.2, http://www.faunaeur.org althoff, j., danilevsky, m., 1997: a check-list of longicorn beetles (coleoptera, cerambycidae) of europe. slovensko entomološko društvo štefana michielija, ljubljana, 64 p. bense, u., 1995: longhorn beetles. illustrated key to the cerambycidae and vesperidae of europe. margraf verlag, weikersheim, 512 p. ćurčić, s. b., brajković, m. m., tomić, v. t., mihajlova, b., 2003: contribution to the knowledge of longicorn beetles (cerambycidae, coleoptera) from serbia, montenegro, the republic of macedonia and greece. arch. biol. sci., belgrade, 55(1-2): 33-38. georgiev, g., simov, n., stojadinova, a., doychev, d., 2005: new and interesting records of longhorn beetles (coleoptera, cerambycidae) in some bulgarian mountains. acta zoologica bulgarica, 57(2): 131-138. gnjatović, i., žikić, v., 2010: cerambycids of southeast serbia (coleoptera, cerambycidae). biologica nyssana, niš, 1(1-2): 111-115. ilić, n., 2005: strižibube srbije (coleoptera, cerambycidae) – faunistički pregled. autorsko izdanje, beograd, 179 p. mikšić, r., georgijević, e., 1973: cerambycidae jugoslavije, 2. deo. akademija nauka i umjetnosti bosne i hercegovine, sarajevo, 153 p. pil, n., stojanović, d., 2009: teophilea subcylindricollis hladil, 1988 a new longhorn beetle (coleoptera, cerambycidae) for serbian fauna, acta entomologica serbica, 14(1): 125128. švácha, p., danilevsky, m., 1987: cerambycoid larvae of europe and soviet union (coleoptera, cerambycoidea). part ii. acta universitatis carolinae – biologica 31, univerzita karlova, (3-4): 121-284. trajković, s., branković, s., 2007: sićevo and jelašnica gorges environment status monitoring. institute for nature conservation of serbia, faculty of civil engineering and architecture, niš, 191 p. anticancer compounds from medicinal plants biologica nyssana 5 (2)  december 2014: 83-90 dragović, r. et al.  accumulation of heavy metals in different parts … 83 original article received: 14 november 2014 revised: 8 december 2014 accepted: 20 december 2014 accumulation of heavy metals in different parts of russian thistle (salsola tragus, chenopodiaceae), a potential hyperaccumulator plant species ranko dragović1*, bojan zlatković1, snežana dragović2, jelena petrović3, ljiljana janković mandić2 1university of niš, faculty of sciences and mathematics, višegradska 33, 18000 niš, serbia 2university of belgrade, vinča institute of nuclear sciences, p.o. box 522, 11001 belgrade, serbia 3university of belgrade, institute for the application of nuclear energy, banatska 31b, 11080 belgrade * corresponding author: dragovicr@pmf.ni.ac.rs abstract: dragović, r., zlatković, b., dragović, s., petrović, j., janković mandić, lj.: accumulation of heavy metals in different parts of russian thistle (salsola tragus, chenopodiaceae), a potential hyperaccumulator plant species. biologica nyssana, 5 (2), december 2014: 83-90. distribution and accumulation of 10 heavy metals was observed in plant parts of species s. tragus, a native representative of serbian flora. according to high bioaccumulation values (baf), more than a half of the analyzed elements were bioaccumulated in roots, as well as in the aerial part of the plant. translocation factor (tf) value was calculated, showing the ratio between the concentration of metals in roots and aerial parts of the plant. the results indicate that s. tragus may accumulate significant amounts of cd, co, cr and pb. concentrations of some of the individual elements are shown to be statistically different depending on the sampled plant parts. potential capacity of s. tragus for hyperaccumulation of heavy metals and possible utilization of this plant for phytoremediation is discussed. key words: salsola tragus, heavy metals, plant parts, accumulation, serbia apstrakt: dragović, r., zlatković, b., dragović, s., petrović, j., janković mandić, lj.: akumulacija teških metala u različitim delovima sonjače (salsola tragus, chenopodiaceae), potencijalne hiperakumulatorske biljne vrste. biologica nyssana, 5 (2), decembar 2014: 83-90. istraživana je distribucija i akumulacija 10 teških metala u delovima biljke s. tragus, autohtonog predstavnika flore srbije. prema visokim vrednostima faktora bioakumulacije (baf), vise od polovine analiziranih elemenata je bioakumulirano u korenu, kao i u nadzemnim delovima biljke. izračunat je faktor translokacije (tf), koji predstavljaja odnos koncentracije datog elementa u korenu i one u nadzemnim delovima biljke. rezultati su pokazali da s. tragus može akumulirati značajne koncentracije cd, co, cr i pb. pokazano je da se koncentracije elemenata statistički razlikuju u zavisnosti od analiziranih delova biljke. diskutovan je potencijalni kapacitet hiperakumulacije teških metala ove biljke i mogućnost njene primene u fitoremedijaciji. ključne reči: salsola tragus, teški metali, delovi biljke, akumulacija, srbija 5 (2) • december 2014: 83-90 biologica nyssana 5 (2)  december 2014: 83-90 dragović, r. et al.  accumulation of heavy metals in different parts … 84 introduction anthropogenic activities have resulted in increased deposition and migration of potentially toxic heavy metals, posing a major risk for both human health and the environment. heavy metals may disrupt the physical, chemical and biological balance of the soil and therefore affect the growth of plants. however, many plant species have adapted to tolerate high concentrations of heavy metals without any detrimental effects on growth or survival (b r o o k s , 1998). a hyperaccumulator may be defined as a plant whose tissues and organs contain a metallic element at a concentration exceeding a specified threshold, when growing in nature (v a n d e r e n t et al., 2013). the threshold concentration should be two or three orders of magnitude higher than in leaves of most species on normal soils, and at least one order of magnitude greater than the usual range found in plants from metalliferous soils. based on these criteria, the proposed nominal threshold values (mg kg-1 of dry leaf tissue) are: 100 for cd, se and tl; 300 for co, cr and cu; 1000 for as, ni and pb; 3000 for zn and 10,000 for mn (v a n d e r e n t et al., 2013). salsola tragus l. (syn. s. kali subsp. ruthenica), commonly known as russian thistle or tumbleweed, is one of the most common species of tumbleweed belonging to s. kali aggregate. it is an annual, weedy plant species, which at maturation forms spherical bushes approximately 1 m in diameter, detaches from the root system and tumbles in the wind, spreading its seed (b a r g e r o n & s w e a r i n g e n , 2010). native distribution of the species overlaps large areas of europe and asia, but it is also naturalized in north, central and south america, africa and australia (m o s y a k i n , 2003). it has a comparatively wide distribution in serbia, especially in the northern and northeastern parts of the country (s l a v n i ć , 1972). it is generally distributed in sandy areas in coastal regions and semiarid and arid areas in lowlands, deserts, but it is also common at ruderal habitats and disturbed places in the settlements, across the roadsides, and cultivated fields. due to its wide distribution, adequate life form and other biological traits, this taxon is a good candidate for a biomonitoring species. it has been shown that tumbleweed is able to capture elements from the ambient air and that has capabilities for being used as biomonitor of environmental pollution by heavy metals (b e n i t e z , 2009). the tumbleweed is also able to grow in areas polluted by radionuclides (w a r r e n , 2001). the aim this study was to assess the hyperaccumulating capacity of aerial as well as underground parts of tumbleweed (s. tragus) collected in the urban and sub-urban area of the city of niš, southeast serbia. in this study we demonstrated distribution and accumulation of cadmium (cd), cobalt (co), chromium (cr), mercury (cu), iron (fe), manganese (mn), nickel (ni), lead (pb), zinc (zn) and vanadium (v) in the tissues of s. tragus. material and methods study area the study area covers the urban and suburban area of the city of niš, i.e. the peripheral parts of niš valley, a small part of the sićevo gorge and alluvial terraces of nišava river and the south morava river at their confluence. niš is a city in southern serbia, situated in the composite valley of the river nišava near its confluence with the south morava. the city of niš has an area of 597 km2 including niška banja and 68 suburban and rural settlements. with 393,357 inhabitants (census of 2011) it is the second largest city in serbia and one of the most important industrial centers in serbia (electronics, mechanical engineering, textile and tobacco industry). the nišava valley is located at the contact of young and accumulated layers of broken rocks belonging to the carpatho-balkanides and paleozoic formations belonging to rhodopian crystalline mass. in niš basin the following rock types are distributed: magmatites, metamorphites, carbonate rocks, clastites and non-consolidated or partly consolidated neogene sediments (k r s t i ć et al., 1980; r a k i ć et al., 1973). at the rims of niš basin, above these rocks the alluvial sediments are deposited. the alluvial soils dominated in the area. sićevo gorge is formed by vertical fluvial erosion in the pliocene limestones between the southern slopes of the svrljig mountains and the mountain suva planina. sampling locations in sićevo gorge are located next to the main road junction with high traffic frequency (v u j i s i ć et al., 1980). at the study sites, there are no significant deposits of heavy metals. sampling and sample preparation the samples of tumbleweed plants and underlying soils were collected in 2013 from 15 locations with different intensity and types of anthropogenic influence. the selection of sampling sites was influenced by spatial distribution of tumbleweed populations in the investigated area. underground (roots) and aerial parts (stems, leaves and inflorescences) of plants from the same location were separated and washed in a series of fresh and de-ionized water to remove any particles of soil. the biologica nyssana 5 (2)  december 2014: 83-90 dragović, r. et al.  accumulation of heavy metals in different parts … 85 material was chopped, dried at room temperature for two weeks and finally stored in paper bags, prior to chemical analysis. voucher specimens are deposited at the department of biology and ecology (hmn), faculty of science and mathematics, university of niš, under the acquisition numbers 7315-7324. the mineralization of soil samples was performed by slightly modified methods described by e d g e l l (1988). approximately 1-3 g (depending of expected total metal concentrations in soil) of air-dried, ground and sieved (< 0.2 mm) soil was weighed and transferred into cuvettes. each sample was weighed in two replications. the 10 ml of hno3 was added and the samples were heated on the heating plate gradually, until no brown fumes were emitted. the solution was allowed to evaporate approximately to 5 ml. after cooling, 2 ml of water and 3 ml of 30% h2o2 were added and heated until effervescence subsided and the solution cooled. additional h2o2 was added until effervescence ceased (but no more than 10 ml h2o2 was added). the heating was continuing, and the solution was allowed to evaporate to approximately 5 ml. dried and ground plant materials (root and aerial parts, separately) were weighed (approximately 0.5 g) and transferred into cuvettes for mineralization. each sample was weighed in two replications. the mineralization was performed by wet procedure, i.e. with the mixture of hno3 (65%) and h2so4 (min. 95%) (iso 6636/2: 1981). fig. 1. s. tragus at the collection site in suburban area of the city of niš both soil and plant extract were cooled, filtered through satorius stedim biotech filter paper (quantitative grade 391) and adjusted to the appropriate volume with hno3/h2o (3:1) solution. analytical methods soil and plant samples were analyzed by using an atomic absorption spectrometer shimadzu aa-7000. measurements was performed by flame technique with the detection limits (3σ) being approximately 0.1 ppm for v, 0.6 ppm for cr, 0.01 ppm for cu, zn and mn, 0.2 ppm for ni, 0.07 ppm for pb, 0.7 ppm for fe, 0.3 ppm for co and 0.03 for cd. the deuterium lamp (d2) method was used as background correction function. control software wizaard was used for shimadzu atomic absorption spectrophotometer. reference standard materials nist 1515 (apple leaves) and bipea quality control sample (soil 90-0115-0106) were used for the verification of the measurement. the bioaccumulation factor is calculated as follows (f e i j t e l et al., 1997; g o b a s & m o r r i s o n , 2000; m a c k a y & f r a s e r , 2000): bfi = ci/cs (1) where ci is the mean concentration of metal in roots (i=a) or aerial (i=b) parts of tumbleweed (mg kg-1) and cs is the mean metal concentration in underlying soil (mg kg-1). the plant could be considered as hyperaccumulator if the baf is higher than 1. translocation factor (tf) is calculated as ratio of the metal concentration in aerial parts of the plants to those in the roots (c u i et al., 2007; l i et al., 2007; s o u s a et al., 2008; m a l i k et al., 2010): tf = cb/ca (2) where cb is the mean metal concentration in aerial parts (mg kg-1) and ca is the mean metal concentration in roots of plant (mg kg-1). the tf˃1 indicates that the plant translocates metals effectively from the roots to its aerial parts (b a k e r & b r o o k s , 1989). statistical methods analysis of variance (anova): one-way anova was used in order to determine whether there are any differences in concentrations of heavy metals among different examined parts of the plant, and among the localities. the results of this analysis should also pinpoint the most important variables regarding concentrations of metal in tissues of two plant parts. correlation analysis: in order to affirm relationships between heavy metal concentrations in roots and aerial parts of tumbleweed, pearson’s correlation analysis was applied. statistical data processing was carried out by statistica 8 software. biologica nyssana 5 (2)  december 2014: 83-90 dragović, r. et al.  accumulation of heavy metals in different parts … 86 results and discussion the mean concentrations of heavy metals in different parts of tumbleweed and in underlying soils and values of bioaccumulation and transfer factors are presented in tab. 1. different plant species and genera grown on the same soil may display different metal uptake and movement in plant (m i l e s & p a r k e r , 1979). in addition to availability of metals in soil to plants, there are several factors that cause metal uptake and transport through the plant. the biological factors, including the capability to transport particular metals through transport systems, strongly affect the accumulation behavior of the plant. heavy metal absorption and accumulation varies according to plant taxa and presence of suitable metal forms for the plant uptake in the soil. the studies of de la r o s a et al. (2004) and g a r d e a -t o r r e s d a y et al. (2005) suggested that tumbleweeds could be considered as potential hyperaccumulating species for cadmium and chromium, respectively. one of the species, s. soda has also found to be hyperaccumulator for lead (l o r e s t a n i et al., 2011; 2013) and zinc (l o r e s t a n i et al., 2013) and therefore suitable candidate for phytoremediation of contaminated soils. the obtained baf values pointed out the tendency of s. tragus to accumulate cd, co, cr, ni, pb and v in both underground and aerial parts of the plant (tab. 1). the highest bioaccumulation was observed for cd, with baf values of 6.32 and 6.73 in underground and aerial parts of the plant, respectively. for most studied elements, the plants have shown higher bioaccumulation capacity in their upper parts, except for v, which was better represented in roots than in aerial parts. however, the number of analyzed cases with favorable transport of absorbed metals from root zone into the aerial parts of plant, shown as translocation factor (tf), was much lower. in this study, high values of translocation factor were recorded only for cd, co, cr and pb, in cases of realistic possibility of uniform accumulation of these elements in aerial parts of plant species s. tragus. while the translocation factor indicates the relative ratio of concentrations of a certain metal in root and aerial parts, the transport of metals through the plant is directly influenced by various ecological characteristics of the plant, primarily the type and level of development of transport tissues, xylem and phloem, or existence of special physiological mechanisms. the data collected on ten elements in both plant parts in all localities were used to determine the interaction among metal concentrations in different plant parts. in combination with the tf values this could provide additional information on possibility of their transport through plant tissues. the derived correlation coefficients, as a measure of linear strength of connection between a pair of observed factors, indicate strong connection between concentrations of pb, fe and ni in roots and in aerial parts of plant (table 2). there was also a strong and statistically highly significant (p < 0.001) positive correlation of concentrations of fe and pb in aerial and underground part of plant. at the same time other types of correlation between concentrations of other variables were also recorded. however, the values of the correlation coefficients were weak or medium-strength, indicating that in the species s. tragus there is a clear interaction between concentrations of adopted elements in aerial and underground parts of plant. existence of favorable transport through the tissues and successful overcoming of the barriers that might potentially disrupt transport of heavy metals through plants, speaks in favor of the possibility that s. tragus performs hyperaccumulation of pb, fe and ni in cases when these metals are present in the environment in greater concentrations. samples of roots and aerial parts of plant species s. tragus collected in the study area show variability in concentrations for all 10 analyzed elements. the original hypothesis assumed that, as table 1. mean concentrations of heavy metals in underground (ca) and aerial (cb) parts of tumbleweed and underlying soil cs (mg kg -1 d.w.) and mean values of bioaccumulation (baf) and transfer (tf) factors parameter cd co cr cu fe mn ni pb zn v ca 5.56 79.7 86.4 8.12 6120 19.9 75.2 185 14.9 140 cb 5.92 85.9 102 10.4 6470 43.5 72.2 188 28.7 87.7 cs 0.88 14.7 41.6 54.7 27310 320 41.7 101 63.0 43.4 bafa 6.32 5.42 2.08 0.15 0.22 0.06 1.80 1.82 0.24 3.18 bafb 6.73 5.85 2.46 0.19 0.24 0.14 1.73 1.85 0.46 2.02 tf 1.06 1.08 1.18 1.27 1.06 2.18 0.96 1.02 1.92 0.63 biologica nyssana 5 (2)  december 2014: 83-90 dragović, r. et al.  accumulation of heavy metals in different parts … 87 table 2. correlations between concentrations of heavy metals in roots and aerial parts of tumbleweed (high r values in bold) cdb cob crb cub feb mnb nib pbb znb vb cda 0.72 0.17 0.21 -0.08 -0.32 -0.09 0.05 -0.25 0.19 -0.11 coa 0.29 0.42 0.10 -0.32 -0.41 -0.15 -0.26 -0.48 0.25 0.03 cra -0.16 -0.10 0.49 0.52 0.17 0.20 0.21 0.11 -0.21 -0.37 cua -0.48 0.02 -0.06 0.30 0.40 0.32 0.01 0.43 -0.11 -0.47 fea -0.35 -0.15 -0.14 0.29 0.94 0.71 0.18 0.96 -0.03 -0.49 mna -0.43 0.05 0.03 0.33 0.67 0.65 0.24 0.70 0.03 -0.54 nia -0.05 -0.23 0.23 0.27 0.35 0.41 0.84 0.38 0.05 -0.06 pba -0.42 -0.25 -0.13 0.32 0.94 0.62 0.24 0.98 -0.02 -0.48 zna -0.15 -0.04 -0.00 0.10 -0.03 0.10 0.07 -0.04 0.38 -0.08 va -0.24 0.04 0.12 0.11 0.13 -0.21 -0.16 0.17 -0.56 0.10 a – roots, b-aerial parts different parts of plant differ in anatomicallymorphological and functional sense, the movement and potential accumulation of metals in tissues would also be different. therefore the differences in mean values of measured concentrations of metals in various parts of plants were tested, and characters with greatest impact in differentiation of samples from aerial and underground plant parts were determined. the results of unifactorial analysis have shown that there are statistically significant differences in concentrations of heavy metals depending on plant organ, but only for some metals (tab. 3). comparison of aerial and underground parts of plant s. tragus, at level of 15 collected samples, shows statistically highly significant concentrations of mn, zn and v. the possibility of differentiation is most influenced by mn, which is characterized by the highest f value among all statistically significant variables. the measured mean values of mn were greater (more than double) in tissues of aerial part than in the root of the analyzed plant species. the manganese is one of the most important elements, influencing the process of photosynthesis and some other metabolic processes, but it is believed to be easy to transport through transport tissues and accumulated primarily in green parts of plants (m a r s c h n e r , 1995). the manganese shows a general tendency of greater accumulation in shoots than in root of the same plant, as demonstrated in samples of a number of different plant species (m i l l a l e o et al., 2010). it was proven that mn is transported through xylem relatively easily, in contrast to transport in the opposite direction, through the phloem part of transport system toward the underground plant organs (p a g e & f e l l e r , 2005). the aerial parts of s. tragus have included concentration of zn almost double to that in the underground part. the zinc is similar to mn in being an important nutrient for plant organisms, playing a significant role in process of photosynthesis and shows tendency to accumulate in the aerial parts of plants. in experimental conditions it was proven that this metal accumulates in leaves, and in case of increased concentration in substrate there may be hyperaccumulation in leaves of a number of cultivated plant species (s e k a r a et al., 2005). table 3. analysis of variance of heavy metal concentrations between the root and the aerial parts of tumbleweed (significant values in bold) effect root/aerial part root/ locality aerial part/ locality variable f (df 1) f (df 10) f (df 10) cd 0.51 0.71 0.78 co 1.01 1.16 1.22 cr 0.30 2.14 1.04 cu 1.84 1.40 0.62 fe 0.11 3.22 3.20 mn 44.92 1.82 0.77 ni 0.09 3.22 6.95 pb 0.02 4.39 4.18 zn 22.13 0.69 1.32 v 12.68 0.49 1.14 athe significance levels: p˂0.05;  p˂0.01; p˂0.001 distribution of v in parts of plant body is different than distribution of other elements. depending on sampling locality, values of this element in roots of s. tragus were greater on average than the values for aerial parts (tab. 1). the mean values of all analyzed samples show concentrations of v in roots to be more than 1.5 times greater than in rest of the plant. in contrast to mn and zn which play important roles in various physiological processes in plants, the phenomenon of accumulation and transport of v in plants is still biologica nyssana 5 (2)  december 2014: 83-90 dragović, r. et al.  accumulation of heavy metals in different parts … 88 largely understudied. most plants absorb v in very small amounts in comparison to its availability in soil. results of several studies, including our research, indicate that this element is accumulated in the root and poorly transported toward the aerial organs of the plant (s z i k l a i et al., 1987; n o w a k o w s k y , 1993). possibility of stronger accumulation of v in the root than in stems, fruits and green leaves, was also proven experimentally (g i l et al., 1995; v a c h i r a p a t a m a et al., 2001). increased concentration of this element in environment and in plant and animal organisms may have a toxic effect on consumers. increase of level of v in water and soil is attributed to burning of coal and oil derivatives, as well as to use of phosphate fertilizers (r i n g e l b a n d & h e h l , 2000). according to literature data, increased level of v was primarily recorded in plants growing on alluvium soil (g i l et al., 1995). it must be noted that almost all samples of species s. tragus analyzed in this study were collected at agricultural land or recently abandoned cultivated land, formed on alluvial deposits of river valleys. although s. tragus is able to accumulate v, according to tf values for this element (0.63) it is similar to most other plants in not being able to hyperaccumulate it and transport it to aerial parts in favorable amounts. results of analyzing the concentrations of heavy metals in a single part of plant among 11 groups of localities defined according to the distance from the potential source of pollution (highway) paint a slightly different picture (tab. 3). it is assumed that determination of important variables is crucially affected by contamination of soil with heavy metals, unlike the previous case where the important factor was affinity for accumulation in plant organs. if roots and aerial parts of plants are compared according to spatial distribution of sampling sites, concentrations of fe, ni and pb become more important in differentiation of sample groups. comparison of mean values shows that approximately identical concentrations of ni and pb were recorded in roots and aerial parts of plants, while fe is characterized by a significantly higher concentration in the aerial part. the f value was most prominent for concentration of pb, which was shown to be more variable and statistically more significant than concentration of other metals regarding the concentration of metals in the root. the lead belongs to metals most commonly mentioned as primary contaminants in the environment. in this case, the role of this metal in differentiation of samples probably accentuates the level of contamination in sampling sites, which were situated in vicinity of traffic routes as contaminant sources. the significance of species s. tragus as an indicator organism for lead contamination of environment, as concentration of lead in plants is dependent on distance from traffic routes where lead gasses are emitted, was stressed by s i n e g a n i (2007). considering the aerial parts of plant, ni is more significant and represents a more significant variable than either pb or fe. the nickel does not have any great role as a plant nutrient, and increase of its concentration in plants is mostly caused by emission from industrial sources or by prolonged use of mineral fertilizers and pesticides containing that element (s e r e g i n & k o z h e v n i k o v a , 2006). conclusion according to its ability to accumulate several heavy metals, s. tragus may represent an interesting model of phytoremediation or biomonitoring. this plant can uptake cd, co, cr and pb from the soil and make significant deposits in its aerial parts. distribution and accumulation of heavy metals is not equal in all parts of a plant. differences in concentrations of heavy metals between the underground and the aerial parts of a plant are mostly determined by concentrations of mn, zn and v. the levels of mn and zn were higher in the green parts of the plant, while concentration of v was higher in the roots. possibility of differentiation in statistical sense was particularly high for concentration of mn. the positive correlation was determined between the concentrations of pb, fe and ni in the roots and their concentrations in upper parts of the plant. acknowledgements. this study was supported by the ministry of education, science and technological development of the republic of serbia (projects iii43009 and 173030). references baker a.j.m., brooks r.r., 1989: terrestrial higher plants which hyperaccumulate metallic elementsa review of their distribution, ecology and phytochemistry. biorecovery, 1 (2): 81–126. bargeron, c., swearingen, j., 2010: invasive plant atlas of the united states. university of georgia center for invasive species and ecosystem health. http://www.invasiveplantatlas.org. benitez, t., 2009: salsola kali (tumbleweed): a possible biomonitoring device for the detection of airborne heavy metals. university of texas at el paso. usa. biologica nyssana 5 (2)  december 2014: 83-90 dragović, r. et al.  accumulation of heavy metals in different parts … 89 brooks, r.r., 1998: plants that hyperaccumulate heavy metals. cab international. wallingford, uk. cui s., zhou q., chao l., 2007: potential hyperaccumulation of pb, zn, cu and cd in endurant plants distributed in an old smeltery, northeast china. environmental geology, 51 (6): 1043–1048. de la rosa, g., peralta-videa, j.r., montes, m., parsons, j.g., cano-aguilera, i., gardeatorresday, j.l., 2004: cadmium uptake and translocation in tumbleweed (salsola kali), a potential cd-hyperaccumulator desert plant species: icp/oes and xas studies. chemosphere, 55 (9): 1159-1168. edgell, k., 1988: usepa method study 37 sw846 method 3050 acid digestion of sediments, sludges, and soils, epa contract no. 68-033254. feijtel, t., kloepper-sams, p., den haan, k., van egmond, r., comber, m., heusel, r., wierich, p., ten berge, w., gard, a., wolf, w., niessen, h., 1997: integration of bioaccumulation in an environmental risk assessment. chemosphere, 34 (11): 2337-2350. gardea-torresday, j.l., de la rosa, g., peraltavidea, j.r., montes, m., cruz-jimenez, g., cano-aguilera, i., 2005: differential uptake and transport of trivalent and hexavalent chromium by tumbleweed (salsola kali). archives of environmental contamination and toxicology, 48 (2): 225-232. gil, j., alvarez, c.e., martinez, m.c., perez, n., 1995: effect of vanadium on lettuce growth, cationic nutrition, and yield. journal of environmental science and health, 30 (1): 7387. gobas, f.a.p.c., morrison, h.a., 2000: bioconcentration and biomagnification and food chain accumulation in the aquatic environment. in: boethling, r.s., mackay, d. (eds.), handbook of property estimation methods for chemicals: 189-231, crc press, boca raton, fl. iso 6636/2, 1981: fruits, vegetables and derived products determination of zinc content part 2: atomic absorption spectrometric method, international standard organization, geneva, switzerland. krstić, b., veselinović, m., divljan, m., rakić, m., 1980: guide of the geological map of socialist federal republic of yugoslavia, 1:100000, sheet 'aleksinac', federal geological institute, belgrade (in serbian). li m.s., luo y.p., su, z.y., 2007: heavy metal concentration in soils and plant accumulation in a restored manganese mineland in guangxi, south china. environmental pollution, 147 (1): 168–175. lorestani, b., cheraghi, m., yousefi, n., 2011: accumulation of pb, mn, cu and zn in plants and choice of hyperaccumulator plant in the industrial town of vian, iran. archive of biological sciences, 63 (3): 739-745. lorestani, b., yousefi, n., cheraghi, m., farmany, a., 2013: phytoextraction and phytostabilization potential of plants grown in the vicinity of heavy-metal contaminated soils: a case study at an industrial town site. environmental monitoring and assessment, 185 (12): 1021710223. mackay, d., fraser, a., 2000: bioaccumulation of persistent organic chemicals: mechanisms and models. environmental pollution, 110 (3): 375391. malik, r.n., husain, s.z., nazir i., 2010: heavy metal contamination and accumulation in soil and wild plant species from industrial area of islamabad, pakistan. pakistan journal of botany, 42 (1): 291-301. marschner, h., 1995: mineral nutrition of higher plants. academic press, san diego, 889. millaleo, r., reyes-diaz, m., ivanov, a.g., mora, m.l., alberdi, m., 2010: manganese as essential and toxic element for plants: transport, accumulation and resistance mechanisms. journal of soil science and plant nutrition, 10 (4): 470 – 481. miles, l.j., parker, g.r., 1979: heavy metal interaction for andropogon scoparius and rudbeckia hirta grown on soil from urban and rural sites with heavy metal additions. journal of environmental quality, 8 (4): 443-449. mosyakin, s. l., 2003: salsola. in: flora of north america editorial committee (eds.), flora of north america north of mexico, 4. oxford university press, new york, 356-357. nowakowsky, w., 1993: vanadium bioaccumulation in pisum sativum l. seedlings. biologia plantarum, 35 (3): 461-465. page, v., feller, u., 2005: selective transport of zinc, manganese, nickel, cobalt and cadmium in the root system and transfer to the leaves in young wheat plants. annals of botany 96 (3): 425-434. rakić, m., dimitrijević, m.d., terzin, v., cvetković, d., petrović, v., 1973: guide of the geological map of socialist federal republic of yugoslavia, 1:100000, sheet 'niš', federal geological institute, belgrade (in serbian). ringelband, u., hehl, o., 2000: kinetics of vanadium bioaccumulation by the brackish water biologica nyssana 5 (2)  december 2014: 83-90 dragović, r. et al.  accumulation of heavy metals in different parts … 90 hydroid cordylophora caspia (pallas). bulletin of environmental contamination and toxicology, 65 (4): 486-493. sekara, a., poniedziaek, m., ciura, j., jedrszczyk, e., 2005: zinc and copper accumulation and distribution in the tissues of nine crops: implications for phytoremediation. polish journal of environmental studies, 14 (6): 829835. seregin, i.v., kozhevnikova, a.d., 2006: physiological role of nickel and its toxic effects on higher plants. russian journal of plant physiology, 53 (2): 285-308. sinegani, a.a.s., 2007: temporal and spatial variability of lead levels in salsola kali near razan-hamadan highway. journal of applied science and environmental management, 11 (3): 143-146. slavnić, ž., 1972: salsola l. in: josifović, m. (ed.), flora sr srbije 3: 49-52, srpska akademija nauka i umetnosti, beograd. sousa, a.i., caçador, i., lillebø, a.i., pardal, m.a., 2008: heavy metal accumulation in halimione portulacoides: intraand extra-cellular metal binding sites. chemosphere, 70 (5): 850-857. sziklai, i.l., ordogh, m., molnar, e., szabo, e., 1987: distribution of trace and minor elements in hungarian spice paprika plants. journal of radioanalytical and nuclear chemistry, 122 (2): 233-238. van der ent a., baker, a.j.m., reeves, r.d., pollard, a.j., schat, h., 2013: hyperaccumulators of metal and metalloid trace elements: facts and fiction. plant and soil, 362 (1-2): 319–334. vujisić, t.j., navala, m., kalenić, m., krstić, b., maslarević, lj., marković, b., buković, j., 1980. guide of the geological map of socialist federal republic of yugoslavia, 1:100000, sheet 'bela palanka', federal geological institute, belgrade (in serbian). vachirapatama, n., jirakiattikul, y., dicinoski, g., townsend, a.t, haddad, p.r., 2001: effect of vanadium on plant growth and its accumulation in plant tissues. songklanakarin journal of science and technology, 33 (3): 255-261. warren, r.w., 2001: sorption and transport of radionuclides by tumbleweed from two plasticlined radioactive waste ponds. journal of environmental radioactivity, 54 (3): 361-376. anticancer compounds from medicinal plants biologica nyssana 5 (2)  december 2014: 123-129 zlatković, b., bogosavljević, s.  report for the new floristic data… 123 original article received: 23 november 2014 revised: 11 december 2014 accepted: 20 december 2014 report on the new floristic data from serbia bojan zlatković1*, stefan bogosavljević2 1 university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia 2 "filly farm pharmacy", health institution, dušanova 31, 18000 niš, serbia *corresponding autor: bojanzlat@yahoo.com abstract: zlatković, b., bogosavljević, s.: report on the new floristic data from serbia. biologica nyssana, 5 (2), december 2014: 123-129. new chorological data for 16 taxa of vascular flora of serbia (records no. 1-16) are given in the article. the taxa represented belong to the following families: asteraceae (1-8), euphorbiaceae (9), geraniaceae (10), juncaceae (13), lamiaceae (11), salicaceae (12), poaceae (14, 15) and typhaceae (16). artemisia alba var. biasolettiana is reported for the first time within the territory of serbia. key words: new chorological data, serbia, vascular flora apstrakt: zlatković, b., bogosavljević, s.: izveštaj o novim florističkim podacima iz srbije. biologica nyssana, 5 (2), decembar 2014: 123-129. prikazani su novi horološki podaci za 16 taksona vaskularne flore srbije (podaci br. 1-16). taksoni pripadaju sledećim familijama: asteraceae (1-8), euphorbiaceae (9), geraniaceae (10), juncaceae (13), lamiaceae (11), salicaceae (12), poaceae (14, 15) i typhaceae (16). prisustvo artemisia alba var. biasolettiana do sada nije bila poznato u flori srbije. ključne reči: novi horološki podaci, srbija, vaskularna flora introduction the goal of this paper is presentation of new chorological data for 16 taxa in vascular flora of serbia. according to our survey of literature, one infraspecies taxon was reported in this paper for the first time within the territory of serbia. the new records may be used as additional data for distribution of taxa presented in the edition series “the flora of sr serbia (i-x)” (j o s i f o v i ć , ed., 1970-1977; s a r i ć & d i k l i ć , 1986; d i k l i ć , 1977; n i k o l i ć et al., 1986), as well as the taxa that were reported from serbia after the monograph was published but will be included within the new edition “the flora of serbia” (s t e v a n o v i ć , 1992, 2012) and for completing the datasets on general distribution of recorded taxa. 5 (2) • december 2014: 123-129 biologica nyssana 5 (2)  december 2014: 123-129 zlatković, b., bogosavljević, s.  report for the new floristic data… 124 material and methods the collected plant material was stored at the herbarium of the institute of botany and botanical garden “jevremovac”, university of belgrade (beou), herbarium of the faculty of science and mathematics, university of niš (hmn) and herbarium of the faculty of sciences and mathematics, university of novi sad (buns). acquisition numbers are given in the brackets. distribution of the studied taxa within the territory of serbia was determined and mapped in utm grid system (10 x 10 sq. km., utm zone 34t). the nomenclature and classification of taxa was matched with the euro+med (2006). we used a geographic regionalization of serbia according to m a r k o v i ć (1970), modified by s t e v a n o v i ć (1999). results and discussion asteraceae 1. erigeron sumatrensis retz. (syn. conyza sumatrensis (retz.) e. walker, c. albida spreng.) (central): the town of niš, batušinac village, ruderal, 186 m, artemisietea vulgaris, en69, 15.09.2014, coll. b. zlatković (hmn 9245). (east): the town of gadžin han, grkinja village, ruderal, 350 m, artemisietea vulgaris, en88, 27.09.2014, coll. m. ranđelović & s. bogosavljević (hmn 9244). (east): the town of niš, gorica hill, ruderal, 358 m, artemisietea vulgaris, en79, 02.10.2014, coll. b. zlatković & s. bogosavljević (hmn 9246). (south): the town of leskovac, ruderal, 250 m, artemisietea vulgaris, en76, 21.09.2014, coll. m. ranđelović & s. bogosavljević (hmn 9243). presence of invasive species e. sumatrensis in serbia was first recorded in 2002 in vegetation of urban area of belgrade (n i k e t i ć & j o v a n o v i ć , 2002). this species originated in tropical and subtropical regions, and its presence in european countries, where it has invasive character, was first recorded in 1875 (c a s a s a y a s , 1989). spread of species e. sumatrensis in territory of serbia matches the invasive potential shown by this species in other countries of southeastern europe and balkan peninsula (a n a s t a s i u & m e m e d e m i n , 2010; v l a d i m i r o v , 2009). 2. dittrichia graveolens (l.) greuter (syn. inula graveolens (l.) desf.) (east): sićevo gorge, gradište village, limestone, ruderal places, roadside, 400 m, artemisietea vulgaris, en99, 27.09.2011, coll. b. zlatković (beou 16839). (east): the town of dimitrovgrad, srećkovac village, limestone, ruderal places, roadside, 470 m, artemisietea vulgaris, fn36, 26.10.2013. coll. b. zlatković, m. miljković & m. marković (beou 16840). dittrichia graveolens is a therophyte species with mediterranean-submediterranean range type, with additional parts of range in middle east and the atlantic region of europe (m e u s e l & j ä g e r , 1992). recently this species was introduced to north america, australia and south africa, showing a strong invasive character (b r o w n s e y et al., 2013). its range increase in central europe started in mid-20th century and continues to gain intensity (k i r á l y et al., 2014). it is assumed that spread of this species is greatly influenced by its anthropochorous type of dispersal, particularly facilitated by construction of traffic routes and formation of ruderal-type habitats. the new localities are situated along the high-frequency international traffic routes, so it is assumed that the anthropogenous factor is greatly beneficial to its range increase in serbia. it was previously recorded only in vicinity of suva reka in kosovo and metohija (g a j i ć , 1975a). 3. achillea chrysocoma friv. (southeast): vlasina plateau, vlasina rid, schists, in vegetation of mountain pastures (nardion strictae) close to the lake, 1200 m, fn03, 16.06.2013, coll m. ranđelović & s. bogosavljević (beou 16833). endemic species characteristic for vegetation of high-mountain pastures and stony ground in the central and southern part of balkan peninsula. in the territory of serbia it has been reported from high mountains of kosovo and metohija (šar-planina, koritnik), as well as besna kobila mt. (d i k l i ć , 1977; n i k o l i ć et al., 1986) and crni vrh close to vranje (a d a m o v i ć , 1908). t o m o v i ć (2007) considers the distribution data for this species in region of vlasina (gorge of masurićka reka) to be doubtful due to incomplete herbarium material in collection of beou. the new record in distribution of achillea chrysocoma in serbia supports the claim that range of this species may realistically include the area of vlasina plateau. biologica nyssana 5 (2)  december 2014: 123-129 zlatković, b., bogosavljević, s.  report for the new floristic data… 125 4. artemisia alba turra var. biasolettiana (vis.) ch. gussev (east): valley of visočica, village slavinja, carbonates, marls, 734 m, fn47, 13.08.2010, coll b. zlatković & d. jović (hmn 9247). artemisia alba is a relatively common species with pontian-mediterranean type of distribution, and within its wide range it shows a pronounced polymorphism of morphological characters. in serbia it appears in several types of thermophilous habitats, primarily on stony ground on limestone and serpentinite. besides the type variety (var. alba), this study has revealed the first record of taxon a. alba var. biasolettiana in serbia. it is characterized by presence of very thick white indumentum that completely covers the leaves, stem and inflorescences of this plant (fig. 1c, 1d). the closest known locality where this taxon was recorded is situated in western part of bulgaria (g u s s e v , 2012). 5. artemisia campestris l. (east): the town of knjaževac, rgošte village, alluvium, ruderal places near railroad, 230 m, artemisietea vulgaris, ep92, 13.10.2013, coll. b. zlatković & s. bogosavljević (beou 16832). according to g a j i ć (1975), artemisia campestris is a species distributed almost throughout serbia. however, our review has shown that this species is rare in southern and eastern parts of serbia where its distribution is insufficiently known. 6. carlina graeca heldr. & sartori (south): mt rujan, slavujevac village, silicate, dry pastures, 800 m, festuco-brometea, em68, 31.07.2008, coll. b. zlatković & n. smiljković (beou 16835). distribution of this species in serbia is still insufficiently studied and important, as it form the northern edge of its area from mediterranean region toward inner part of balkan peninsula. it was reported from the few localities from southern and southeastern region of serbia (n i č i ć , 1902; z l a t k o v i ć & r a n đ e l o v i ć , 2004; z l a t k o v i ć , 2006). 7. jurinea polycephala formánek (syn. j. bipinnata adamović) (south): mt rujan, orljak, silicate, 742 m, festuco-brometea, em67, 26.06.2009, coll. b. zlatković & n. smiljković (beou 16849). (southeast): the town of bosilegrad, izvor village, limestone, dry pastures and rocky grounds, 995 m, festuco-brometea, fc20, 21.06.2014, coll. b. zlatković & g. tomović (hmn 9372). this species inhabits steppe-like grassy habitats and thermophilous rocky ground on silicate and limestone geological substrate. records of j. arachnoidea in vicinity of sukovo (a d a m o v i ć , 1899), prizren (k o š a n i n , 1939), and bosilegrad (r a n đ e l o v i č et al., 1988), probably refers to j. polycephala. besides the new data on distribution this study has also proven the presence of this species in vicinity of bosilegrad, at the slopes of mt. rudina in serbia. 8. centaurea melanocephala pančić (fig. 1a, 1b) (central): studena planina, peak cvetalica, slopes toward brezanska reka river, serpentinite, mountain pastures and glades in oak forest, exp. nw, 1055 m, festucobrometea, dp72, 11.09.2002, coll. b. zlatković (hmn 9248). until now, this endemic species was reported from very few localities in the ibar valley and slopes of mt. stolovi in central serbia (p a n č i ć , 1874, a d a m o v i ć , 1907) and surroundings of kosovska mitrovica and đakovica in kosovo and metohija (w r a b e r , 1993). the new data offers first confirmation for existence of this extremely rare species close to its “locus classicus” (“mt. stol”= mt. stolovi) after it was described by p a n č i ć (1874). euphorbiaceae 9. euphorbia velenovskyi bornm. (east): the town of babušnica, bežište village, alluvium, 600 m, phragmitetea, fn07, 28.05.2008, coll. b. zlatković (beou 16848). (south): the town of vranje, buštranje village, silicate, wet meadows, 450 m, molinioarrhenatheretea, em79, 17.05.2008, coll. b. zlatković & n. smiljković (beou 16857). (south): the town of vranje, ribnice village, alluvium, wet meadows, 370 m, phragmitetea, en70, 17.05.2008, coll. b. zlatković & n. smiljković (beou 16834). euphorbia velenovskyi is an insufficiently studied species of serbian flora, and the only data on its distribution in serbia are from several sites in vicinity of bosilegrad in southeast serbia (u r u m o f f , 1935). certain authors consider this taxon to be a synonym for species e. palustris, but biologica nyssana 5 (2)  december 2014: 123-129 zlatković, b., bogosavljević, s.  report for the new floristic data… 126 figure 1. a, b centaurea melanocephala; c, d artemisia alba var. biasolettiana; e distribution of trachynia distachya in serbia. e. velenovskyi clearly differs by pointed tips and sharply serrated margins of leaf blades (k u z m a n o v , 1979). we believe that e. velenovskyi may have much wider distribution in serbia, but it is necessary to perform a review of data on distribution of e. palustris in vicinity of bujanovac (j a n k o v i ć & n i k o l i ć , 1972), vranje, pirot and sukovo (a d a m o v i ć , 1898). geraniaceae 10. geranium pratense l. (east): the town of dimitrovgrad, mazgoš village, alluvium, 661 m, salicetum albofragilis, fn56, 22.06.2013, coll. b. zlatković & n. stanković (hmn 9250). (east): the town of dimitrovgrad, protopopinci village, alluvium, 666 m, wet meadows, biologica nyssana 5 (2)  december 2014: 123-129 zlatković, b., bogosavljević, s.  report for the new floristic data… 127 molinio-arrhenatheretea, fn56, 22.06.2013, coll. b. zlatković & n. stanković (hmn 9249). in serbia, it was reported only for šumadija region (v e l j o v i ć , 1967). lamiaceae 11. salvia verbenaca l. (east): the town of dimitrovgrad, kozarica mt. slopes, limestone, ruderal places, roadside, 474 m, festuco-brometea, fn46, 30.04.2007, coll. b. zlatković (beou 16855). (east): the town of pirot, sarlak hill, limestone, ruderal places, roadside, 450 m, festucobrometea, fn28, 30.04.2007, coll. b. zlatković (beou 16854). (southeast): the municipality of trgovište, crnovce village, molasses, steppe-like pastures in silicate, astragalo-stipetum capillatae, 800 m, festuco-brometea, em89, 17.05.2008, coll. b. zlatković (beou 16838). presence of this species was recently recorded in several localities in eastern and southwestern parts of serbia (z l a t k o v i ć et al., 2005). monitoring of its distribution has shown spread pattern along the traffic routes as well as in ruderal habitats in human settlement, mostly in southern warmer parts of serbia. salicaceae 12. salix elaeagnos scop. (southeast): vlasina plateau, bratanov del, silicate, near stream, 1238 m, querco-fagetea, fn02, 31.07.2013, coll. b. zlatković (beou 16850). this species is distributed along the river courses of western and southwestern serbia and kosovo and metohija (j o v a n o v i ć & t u c o v i ć , 1972; j o v a n o v i ć , 1997), however the amount of substantiated data on its distribution in other regions of serbia is insufficient. juncacee 13. juncus capitatus weigel (south): rujan planina, borovac village, eroded areas along the road, 422 m, em69, 31.07.2013, coll. b. zlatković (hmn 9251). this is the second confirmed set of data on existence in serbian flora of this threatened taxon (cr b2ab(i,iii,iv)), which used to be known from a single locality at the extreme southeast of serbia (t o m o v i ć et al., 2009). at the new locality, this species appears in a localized, sparse population, within the mostly therophytic vegetation, at sandy areas around streams and springs. poaceae 14. rostraria cristata (l.) tzvelev (syn. koeleria phleoides (vill.) pers.) (east): sićevo gorge, sićevo village, limestone, 420 m, festuco-brometea, en89, 11.07.2010, coll. b. zlatković & s. bogosavljević (hmn 9254). this mediterranean grass is casual in serbia. it was reported from a few localities in eastern and southeastern parts of the country (š m a r d a , 1968; z l a t k o v i ć & s t e v a n o v i ć , 2007). 15. trachynia distachya (l.) link (syn. brachypodium distachyon (l.) p. beauv. ) (east): sićevo gorge, gradište village, arid grassy locations, en99, 05.07.1999, coll. b. zlatković (buns). (south): mt rujan, orljak, silicates, 742 m, em67, 26.06.2009, coll. b. zlatković & n. smiljković (beou 16841). (south): mt rujan, slavujevac village, arid pastures, 800 m, em68, 31.07.2008, coll. b. zlatković & n. smiljković (beou 16845). (south): preševska povija, mamince village, limestone, dry pastures, 450 m, therobrachypodietea, em58, 23.07.1998, coll. b. zlatković, n. ranđelović & v. ranđelović (beou 16843). (south): the town of bujanovac, srpska kuća village, marls, clays and sandstones, arid pastures, 421 m, en60, 31.07.2008, coll. b. zlatković & n. smiljković (beou 16846). (south): the town of vranje, buštranje village, limestone, dry pastures, 516 m, therobrachypodietea, em79, 17.05.2008, coll. b. zlatković & n. smiljković (beou 16836). (southeast): pčinja river gorge, brnjare village, marls, shales and sandstones, ephemeral pastures of dry sand area, ornithopodituberarietum guttatae, 510 m, em79, 28.06.2004, coll. b. zlatković & m. jušković (beou 16847). (metohija): the town of prizren, našec village, limestone, thermophilous pastures on shallow substrate, 390 m, thero-brachypodietea, dm77, 30.06.1996, coll. b. zlatković (beou 16837). (metohija): the town of prizren, thermophilous pastures on shallow substrate, limestone, 371 m, dm77, 30.06.1996, coll. b. zlatković (beou 16844). biologica nyssana 5 (2)  december 2014: 123-129 zlatković, b., bogosavljević, s.  report for the new floristic data… 128 mediterranean, therophyte species recorded in serbia for the first time near sredska (k r i v o š e j et al., 1993-1994), and later also in gorge of prizrenska bistrica (z l a t k o v i ć et al., 2014) at kosovo and metohija. supported by influence of warm climate from the adriatic and aegean mediterranean areas, following the valleys of larger rivers, this species has spread its range significantly in metohija, southern, southeastern and eastern serbia (fig. 1e). this species is a constituent of vegetation of thermophilous pastures and stony ground, appearing at lower altitudes. typhaceae 16. typha laxmannii lepech. (northeast): the town of zaječar, rgotina village, rgotsko lake, 185 m, fp07, 03.08.2014, coll. s. bogosavljević (hmn 9255). the first record of this species in serbia was from vojvodina (budak, 1975, 1986), while recently it was recorded at several localities in eastern and southeastern serbia (zlatković et al., 2007). at the locality in northeastern serbia typha laxmannii forms a relatively abundant population inhabiting shallow pools and ponds that dry out in summer months. the habitats are anthropogenous in origin, situated in immediate vicinity of stone quarries or gravel excavation pits. in some cases it may also appear along the stream that flows into the accumulation of artificial lake near the settlement rgotina. acknowledgements. the ministry of education, science and technological development of the republic of serbia (grant 173030) supported this research. references adamović, l., 1898: die vegetationsformationen ostserbiens. englers botanische jahrbuch, 26 (2): 124-218. adamović, l., 1899: neue beiträge zur flora von serbien. botanisches centralblatt, 78: 1-8. adamović, l., 1907: sitniji prilozi flori srbije. – nastavnik, 18 (3-4): 95-98. adamović, l., 1908: neue glieder der serbischen flora. allgemeine botanische zeitschrift, 14 (6): 85-87. anastasiu, p., memedemin, d., 2010: conyza sumatrensis: a new alien plant in romania. botanica serbica, 36 (1): 37-40. brownsey, r., kyser, g. b., ditomaso, j. m., 2013: stinkwort is rapidly expanding its range in california. california agriculture, 67 (2): 110– 115. budak, v., 1975: typha laxmannii lepech. in: sarić, m. & diklić, n. (eds), flore de la republique socialiste de serbie. vol. 10, pp. 255-266. acad. serbe sci. & arts, belgrade (in serbo-croatian). budak, v., 1986: typha laxmannii lepech. in: sarić mr & diklić n (eds.), flora sr srbije 10, pp. 255-256, srpska akademija nauka i umetnosti, beograd (in serbo-croatian). casasayas, t., 1989: la flora al-lòctona de catalunya / exotic flora of catalunya. tesis doctoral. facultat de biologia. universidad de barcelona / phd thesis. faculty of biology. barcelona university (in spanish). diklić, n., 1977: dopuna flori sr srbije novim podacima o rasprostranjenju biljnih vrsta in: josifović, m. (ed.). flora sr srbije. 9: 205-210. srpska akademija nauka i umetnosti. beograd. gajić, m., 1975: artemisia l. in: josifović, m. (ed.). flora sr srbije. 7: 121-129. srpska akademija nauka i umetnosti. beograd. gajić, m., 1975a: inula l. in: josifović, m. (ed.). flora sr srbije. 7: 46-59. srpska akademija nauka i umetnosti. beograd. gusev, č., 2012: artemisia l. in: peev, d. r. (ed.): flora na narodna republika b'lgaria 11: 388411. b'lgarskata akademija na naukite, institut po bioraznoobrazie i ekosistemni izsledvania, sofija (in bulgarian). janković, m., nikolić, v., 1972: euphorbia palustris l.: in josifović, m. (eds.). flora sr srbije. 3:544. srpska akademija nauka i umetnosti. beograd (in serbo-croatian). josifović, m. (ed.), 1970-1977: flora sr srbije 1-9. srpska akademija nauka i umetnosti, beograd. jovanović, b., tucović, a., 1972: salix l. in: josifović, m. (ed.). flora sr srbije. 3: 431-457. srpska akademija nauka i umetnosti. beograd. jovanović, b., 1997: krajrečna aluvijalna vegetacija. in: sarić, m. (ed.). vegetacija srbije. 2(1): 107-158. srpska akademija nauka i umetnosti. beograd. király, g., eliáš, p. jun., dítě, d. 2014: two thermophilic alien species new to the flora of slovakia, thaiszia, 24 (2): 125-134. košanin, n., 1939: über die vegetation von nordalbanien. spomenik srpske kraljevske akademije, lxxxix. prvi razred. 20: 73-107. krivošej, z., tatić, b., atanacković, b., vasić, p., 1993-1994: nova nalazišta nekih značajnih biljnih vrsta na teritoriji kosova i metohije (ii). zaštita prirode, 46-47: 151-155. kuzmanov, b., 1979: euprhorbiaceae juss. in: jordanov, d. (ed.), flora na narodna republika b’lgarija. 7: 110-177. b’lgarskata akademija na nauki. sofia. biologica nyssana 5 (2)  december 2014: 123-129 zlatković, b., bogosavljević, s.  report for the new floristic data… 129 marković, j., 1970: geografske oblasti socijalističke federativne republike jugoslavije. zavod za izdavanje udžbenika i nastavna sredstva srbije, beograd. meusel, h., jäger, e., 1992: vergleichende chorologie der zentraleuropäischen flora 3. karten, literatur, register. gustav fischer, jena, stuttgart, new york. niketić, m., jovanović, s., 2002: conyza sumatrensis nova adventivna vrsta u flori srbije vii simpozijum o flori jugoistočne srbije i susednih područja sa međunarodnim učešćem, zbornik rezimea, dimitrovgrad, 23. nikolić, v., sigunov, a., diklić, n., 1986: dopuna flori sr srbije novim podacima o rasprostranjenju biljnih vrsta. in: sarić, m., diklić, n. (eds.), flora sr srbije. 10: 259-336. srpska akademija nauka i umetnosti. beograd. ničić, đ. i., 1902: još nekoliko biljka iz vranjske okoline koje nisu navedene u građi za floru u okolini vranja. izveštaj gimnazija nemanjina u vranju. štamparija d. dimitrijevića. beograd. pančić, j., 1874: flora kneževine srbije. državna štamparija. beograd xxxiv + 802 pp. randželovič, n., stamenkovič, v., sotirov, s., randželovič, v., 1988: florata v okolnostta na bosilegrad iii. most, 112: 47-53. sarić, m., diklić, n., (eds.), 1986: flora sr srbije 10. srpska akademija nauka i umetnosti, beograd. stevanović, v., (ed.), 1992: flora sr srbije 1 (second edition). srpska akademija nauka i umetnosti, beograd. stevanović, v., (ed.), 1999: crvena knjiga flore srbije 1, iščezli i krajnje ugroženi taksoni. ministarstvo za životnu sredinu republike srbije, biološki fakultet univerziteta u beogradu, zavod za zaštitu prirode republike srbije, beograd. stevanović, v., (ed.), 2012: flora sr srbije 2 (second edition). srpska akademija nauka i umetnosti, beograd. šmarda, i., 1968: výsledsky biogeografických cest do jugoslávie v letech 1964-1967 ii. československá akademie vĕd geografický ústav v brnĕ. brnĕ, 128. the euro+med plantbase the information resource for euro-mediterranean plant diversity. 2014. http://ww2.bgbm.org/europlusmed/ tomović, g., 2007: fitogeografska pripadnost, distribucija i centri diverziteta balkanske endemične flore u srbiji. doktorska disertacija. prirodno-matematički fakultet, univerzitet u beogradu. beograd (manuscr.). 232 pp. tomović, g., zlatković, b., niketić, m., perić, r., lazarević, p., duraki, š., stanković, m., lakušić, d., anačkov, g., knežević, j., szabados, k., krivošej, z., prodanović, d., vukojičić, s., stojanović, v., lazarević, m., stevanović, v., 2009: threat status revision of some taxa from “the red data book of flora of serbia 1”. botanica serbica, 33 (1): 33-43. urumoff, i. k., 1935: florata na kjustendilskij okržja (flora des kustendiler kreises). sbornik na b’lgarskata akademija na nauki, 30: 1-235. veljović, v., 1967: vegetacija okoline kragujevca, glasnik prirodnjačkog muzeja, 22 (b): 1-109, beograd. vladimirov, v., 2009: erigeron sumatrensis (asteraceae): a recently recognized alien species in the bulgarian flora. phytologia balcanica, 15: 361-365. wraber, t. (1993): rastline na serpentinitu. proteus, 56: 22-30. zlatković, b., 2006: carlina corymbosa l. in: greuter, w. & raab-straube, e. (eds.). euro+med notulae, 2. willdenowia, 36 (2): 709. zlatković, b., ranđelović, v., jušković, m., marković, m., 2005. novi podaci o rasprostranjenju biljnih vrsta u srbiji. in: ranđelović, n. (ed.). viii simpozijum o flori jugoistočne srbije i susednih regiona. zbornik rezimea. 35-36. prirodno-matematički fakultet u nišu. niš zlatković, b., stevanovic, v. 2007: reports 139140. in: vladimirov, v., dane, f., stevanović, v., tan, k. (eds). new floristic records in the balkans: 6. phytologia balcanica, 13 (3): 433455. zlatković, b., ranđelović, v., jušković, m., 2007: reports 136-138. in: vladimirov, v., dane, f., stevanović, v., tan, k. (eds). new floristic records in the balkans: 6. phytologia balcanica, 13(3): 433-455. zlatković, b., bogosavljević, s., smiljković, n., ranđelović, v. 2014: report on the new and insufficiently studied taxa in the flora of serbia. biologica nyssana, 5 (1): 63-69. anticancer compounds from medicinal plants biologica nyssana 5 (2)  december 2014: 103-112 papović, o. et al.  phytogeographical analysis and endemism of the flora … 103 original article received: 21 september 2014 revised: 06 december 2014 accepted: 20 december 2014 phytogeographical characteristics and endemism of the flora of rogozna mt. (sw serbia) olivera papović1, milica miljković2, novica ranđelović2, vladimir ranđelović2 1university of pristine, faculty of sciences and mathematics, department of biology, ive lole ribara 29, 38220 kosovska mitrovica, serbia 2university of niš, faculty of sciences and mathematics, department of biology and ecology, višegradska 33, 18000 niš, serbia * corresponding autor: olja.bio@open.telekom.rs abstract: papović, o., miljković, m., ranđelović, n., ranđelović, v.: phytogeographical characteristics and endemism of the flora of rogozna mt. (sw serbia). biologica nyssana, 5 (2), december 2014: 103-112. based on presence of area types and area groups, the phytogeographical analysis show that rogozna mt. is area with eurasian-submediterranean-centraleuropean-pontic characteristics. eurasian area type is dominant in the flora of rogozna mt. detailed analysis of the eurasian area type showed a numerous presence of the species with central european-mediterranean (73) and central european-mediterranean-pontic (73) types of distribution. the influence of the mediterranean region is especially pronounced. there are 117 species with mediterranean-submediterranean type of distribution, many of which are endemics or subendemics. typical pontic elements of flora are present in very low percent (1.27%), but taxa with mediterranean-pontic (14.7%) and central-european-mediterranean-pontic (9.38%) are numerous. the presence of an imposing number of endemic taxa (51) has a great significance from the aspect of biodiversity and conservation of the area. key words: area-types and groups, endemites, flora apstrakt: papović, o., miljković, m., ranđelović, n., ranđelović, v.: fitogeografske karakteristike i endemizam flore planine rogozne (jz srbija). biologica nyssana, 5 (2), decembar 2014: 103-112. na osnovu zastupljenosti areal-tipova i areal-grupa, fitogeografskom analizom je utvrđeno da planina rogozna predstavlja područje sa evroazijsko-submediteransko-srednjeevropsko-pontskim karakteristikama. evroazijski areal-tip je dominantan u flori rogozne. detaljna analiza evroazijskog areal-tipa pokazuje brojno učešće taksona srednjeevropsko-mediteranskog (73) i srednjeevropsko-mediteransko-pontskog (73 taksona) tipa distribucije. uticaj mediteranskog regiona je naročito izražen. prisutno je 117 taksona mediteranskosubmediteranskog tipa distribucije, od kojih su mnoge endemiti i subendemiti. tipični pontski elementi flore je prisutan sa malim procentom (1.27%), ali su taksoni sa mediteransko-pontskim (14.7%) i srednjeevropskomediteransko-pontskim (9.38%) brojni. prisustvo impozantnog broja endemičnih taksona (51) ima veliki značaj sa aspekta biodiverziteta i očuvanje područja. ključne reči: areal-tipovi i grupe, endemiti, flora 5 (2) • december 2014: 103-112 biologica nyssana 5 (2)  december 2014: 103-112 papović, o. et al.  phytogeographical analysis and endemism of the flora … 104 introduction rogozna is the mountain located in southwestern serbia (p a p o v i ć et al., 2014). according to phytogeographical division of the southeastern europe (h o r v a t et al., 1974) the rogozna mt. is situated near the border of the illyrian, west mesian and scardo-pindhian provinces of the balkan floristic subregions (fig. 1). such position of the mountain reflects to a large extent on the genesis of its flora. in addition, geological composition of the terrain has a significant influence on the phytogeographical and ecological characteristics of the flora. it is represented by upper cretaceous flysch of kosovska mitrovica which seals the immediate contact between the ophiolite mélange of the vardar zone and drina–ivanjica metamorphic unit in the east and west, respectively (b o r o j e v i ć š o š t a r i ć et al., 2012). in the central part of mountain, serpentine rocks (ophiolite) are brought to the surface. east and west of them widespread dacite-andesite and quartz latite (m i l o v a n o v i ć & k a r a m a t a , 1960; u r o š e v i ć et al., 1966, 1970). on the surface of serpentine rocks, very fragile and fine-grained, poor soil is developed, whose water and mineral regime are unfavourable (p r o d a n o v i ć et al., 2010). areas of the soil-rock systems, which may be grouped together as serpentines occur in many parts of the world and, wherever they occur, are known by their remarkable plant life (w h i t t a k e r , 1954). the plants which develop on serpentines are called serpentinophytes (p a v l o v i ć , 1962; v a s i ć & d i k l i ć , 2001). according to preference for the serpentine supstrate, serpentinophyte can be obligate (plants which are mainly growing on serpentine) and facultative (plants which often grow on serpentine, but also inhabit other kinds of supstrate) (s t e v a n o v i ć et al., 2003). the aims of this study are to determine phytogeographical characteristics of the flora of rogozna mt. based on the presence of area types and groups of plant taxa. special attention was paid to the obligate serpentinophytes and endemics. material and methods the analysis is based on the floristic list shown in the article “analysis of the flora of rogozna mountain in southwestern serbia“ (p a p o v i ć et al., 2014) supplemented (see tab. 1.) by data from the literature (r e x h e p i , 1979; k r i v o š e j et al., 2013; p r o d a n o v i ć et al., 2013) and herbarium collection (hmn). fig 1. position of rogozna mt. on phytogeographical map of the southeastern europe based on h o r v a t et al. (1974) biologica nyssana 5 (2)  december 2014: 103-112 papović, o. et al.  phytogeographical analysis and endemism of the flora … 105 nomenclature was correlated with medchecklist (g r e u t e r et al., 1984-1989), flora europaea (t u t i n et al., 1964-1980), and euro+med plantbase (euro+med, 2006). area types, groups and floristic elements of the plant species were defined by m e u s e l et al. (1965, 1978), m e u s e l and j ä g e r (1992) and s t e v a n o v i ć (1992). results revision of the flora of rogozna mt. list of the species shown in previous article (p a p o v i ć et al., 2014) is supplemented with 17 species (tab. 1). after herbarium revision, it was established that misidentification occurred in the case of 6 species, where data related to other taxa (tab. 2). some of the species do not grow on the rogozna mt., such as andromeda polyfolia l., potentilla leucopolitana p.j. mueller, angelica verticilaris l., hacquetia epipactis (scop.) dc., tordilium apulum l., cerastium transsilvanicum schur., dianthus ciliatus guss., dianthus serotinus waldst. & kit. and melampyrum fimbriatum vandas. phytogeographical analysis for the phytogeographical analysis, all plant taxa were classified into 12 area types and 20 area groups (tab. 3). as expected, the largest number of the species has eurasian type of distribution (360 taxa), but also numerous taxa that have a mediterranean (118) and meridional-submeridional (121) type of distribution. the other area types and groups contribute with small percentages to the chorological composition of the rogozna mt. flora. table 1. the list of supplemented plant species according to the literature and herbarium data plant species data asparagus tenuifolius lam., asparagaceae rexhepi, 1979 bupleurum tenuissimum l., apiaceae prodanović et al., 2013 laburnum alpinum (mill.) bercht. et j. presl., fabaceae prodanović et al., 2013 ophioglossum vulgatum l., ophioglossaceae krivošej et al., 2013 hedera helix l., araliaceae hmn sedum album l., crassulaceae hmn carex caryophyllea latour., cyperaceae hmn cheilanthes persica (bory) kuhn, adiantaceae hmn euphorbia amygdaloides l., euphorbiaceae hmn robinia pseudoacacia l., fabaceae hmn quercus frainetto ten., fagaceae hmn erodium cicutarium (l.) l'herit, geraniaceae hmn scilla bifolia l., liliaceae hmn corydalis solida (l.) clairv., plantaginaceae hmn plantago lanceolata l., plantaginaceae hmn p. major l., plantaginaceae hmn anthoxanthum odoratum l., poaceae hmn table 2. revision of the list of flora of rogozna mt. from p a p o v i ć et al. (2014) incorrectly listed taxa correct name alnus viridis (chaix) lam. et d.c. a. glutinosa (l.) gaertn. cerastium grandiflorum waldst. & kit. c. decalvans schlosser & vuk. cerastium sylvaticum waldst. & kit. c. fontanum baumg. dianthus pinifolius sibth. & sm. ssp. pinifolius d. pinifolius sibth. & sm. ssp. serbicus wettst. silene bupleuroides l. ssp. bupleuroides s. bupleuroides l. ssp. staticifolia (sibth. & sm.) chowd. sempervivum heuffelii schott jovibarba heuffelii (schott) á. löve & d. löve tulipa scardica bornm. t. serbica tatić et krivošej biologica nyssana 5 (2)  december 2014: 103-112 papović, o. et al.  phytogeographical analysis and endemism of the flora … 106 table 3. phytogeographical spectrum of the rogozna mt. (n – number of taxa, % percent of the total flora) area type/area group/area subgroup n % cosmopolitan (cosm) 23 2.92 holarctic (hol) 38 4.82 palaeoholarctic-palaeotropic (ph-pt) 6 0.76 boreal (bor) 7 0.89 arctic-alpine (a-a) 2 0.25 antropohoric (ant) 7 0.89 eurasian (ea) 358 45.43 eurasian (ea) 213 27.03 eurasian (ea) 117 14.83 west eurasian (eaw) 96 12.17 central european-mediterranean-pontic (ce-m-p) 73 9.26 c. european-mediterranean-pontic-oriental-turanian (ce-med-pont-or-tur) 3 0.38 c. european-mediterranean-pontic-oriental (ce-med-pont-or) 22 2.79 c. european-mediterranean-pontic (ce-med-pont) 45 5.70 c. european-pontic (ce-pont) 3 0.38 central european-mediterranean (ce-med) 73 9.26 central european-mediterranean (ce-med) 66 8.37 central european-submediterranean (ce-smed) 7 0.89 eurasian mountain (eam) 50 6.34 eurasian mountain (eam) 6 0.76 eurasian mountain (eam) 2 0.25 west eurasian mountain (eamw) 4 0.51 south european mountain (sem) 18 2.28 s. european mountain (sem) 12 1.39 apenninic-balkanic-anatolian (ap-balk-an) 1 0.13 balkanic (balk) 5 0.63 central s. european mountain (csem) 26 3.30 csem-caucasian (csem-cau) 1 0.13 central s. european mountain (csem) 18 2.28 (illyrian)-balkanic ((illyr)-balk) 5 0.64 balkanic-anatolian (balk-an) 1 0.13 alpine-balkanic-carpathian (alp-balk-carp) 1 0.13 central european (ce) 51 6.46 centraleuropean (ce) 41 5.20 alpine-apenninic-balkanic (alp-apen-balk) 1 0.13 alpine-balkanic-carpathian (alp-balk-carp) 1 0.13 balkanic-carpathian (balk-carp) 3 0.38 pannonian-balkanic (pan-balk) 1 0.13 balkanic (balk) 4 0.51 pontic-south siberian (pont-ss) 10 1.27 balkanic-pontic (balk-pont) 1 0.13 pontic (pont) 9 1.14 meridional-submeridional (msm) 118 14.96 mediterranean-pontic-oriental (med-pont-or) 4 0.51 mediterranean-submediterranean-oriental-turanian (med-smed-or-turan) 1 0.13 mediterranean-submediterranean-oriental (med-smed-or) 3 0.38 medeterranean-pontic (med-pont) 114 14.45 atlantic-medeterranean-pontic (atl-med-pont) 2 0.25 medeterranean-pontic (med-pont) 35 4.44 submediterranean-pontic (smed-pont) 77 9.76 mediterranean-submediterranean (med-smed) 118 14.95 mediterranean-submediterranean (med-smed) 51 6.46 biologica nyssana 5 (2)  december 2014: 103-112 papović, o. et al.  phytogeographical analysis and endemism of the flora … 107 area type/area group/area subgroup n % mediterranean-submediterranean (med-smed) 28 3.55 mediterranean-e. submediterranean (med-esmed) 3 0.38 submediterranean (smed) 20 2.53 e. mediterranean-e. submediterranean (emed-esmed) 67 8.19 e. mediterranean 12 1.52 alpine-(illyrian)-balkanic (alp-(illyr)-balk) 5 0.64 alpine-balcanic-carpathian (alp-balk-carp) 1 0.13 apenninic-(dacian-illyrian)-balkanic (apen-(dac-illyr)-balk) 4 0.51 apenninic-balkanic-anatolian (apen-balk-anatol) 1 0.13 (illyrian)-balkanic-anatolian-(crimean) ((illyr)-balk-anat-(crim)) 5 0.64 balkanic-dacian-(danubian) (balk-dac-(danub) 2 0.25 illyrian-balkanic-anatolian-caucasian (illyr-balk-anat-cau) 1 0.13 balkanic-caucasian (balk-cau) 2 0.25 illyrian-balkanic-crimean (illyr-balk-crim) 1 0.13 (illyrian)-balkanic-carpathian (balk-carp) 4 0.51 (illyrian)-balkanic ((illyr)-balk) 29 3.66 total 789 100.00 table 4. list of the obligate serpentinophytes distributed on rogozna mt. plant taxa area type floristic element stachys recta l. subsp. baldaccii (k. malý) haye sem dinaric-scardo-pindhian bromus pannonicus kummer & sendtner msm balkan-pontic-pannonian asplenium cuneifolium viv. ce central european silene paradoxa l. med-smed mediterranean-submediterranean paragymnopteris marantae (l.) k. h. shing med-smed mediterranean-submediterranean hypochaeris illyrica k. malý med-smed e illyrian-moesian halacsya sendtneri (boiss.) dörfler med-smed moesian-scardo-pindhian scrophularia tristis (k. malý) šilić med-smed illyrian-moesian alyssum bertolonii desv. med-smed moesian-scardo-pindhian alyssum markgrafii o. e. schulz ex markgraf med-smed moesian-scardo-pindhian fumana bonapartei maire & petitmengin med-smed illyrian-scardo-pindhian sedum serpentini janchen med-smed moesian-scardo-pindhian scabiosa fumarioides vis. & pančić med-smed moesian-scardo-pindhian thymus lykae degen med-smed w moesian-scardo-pindhian tulipa serbica tatić & krivošej med-smed w moesian orobanche nowackiana markgraf med-smed dinaric-scardo-pindhian helleborus serbicus adamović med-smed dinaric-w moesian galium rubrum l. med-smed c mediterranean-submediterranean haplophyllum boissieranum vis. & pančić med-smed moesian-scardo-pindhian obligate serpentinophytes and its phytogeographical characteristics on the rogozna mt. 19 plant taxa from the group of obligate serpentinophytes based on s t e v a n o v i ć et al. (2003) were recorded (tab. 4). one of these tulipa serbica, is the local endemic for rogozna mt. (t a t i ć & k r i v o š e j , 1997). most of the other obligate serpentinophytes are the balkan endemics (12 taxa) or subendemics (2 taxa), while 4 taxa have a wider distribution – central european or mediterraneansubmediterranean. endemism of the flora the endemic plants are very important plant group for understanding the florogenetic tendencies and phytogeographic characteristics of each region (r a n đ e l o v i ć et al., 2008). based on the complete list of plant taxa inhabiting the rogozna mt., a list of 51 balkan endemic taxa was prepared (tab. 5). the largest number of the endemic taxa has a mediteranean-submediteranean type of distribution (fig. 2). biologica nyssana 5 (2)  december 2014: 103-112 papović, o. et al.  phytogeographical analysis and endemism of the flora … 108 table 5. list of the balkan endemics distributed on rogozna mt. plant taxa area type floristic element campanula sparsa friv. subsp. sphaerothrix (griseb.) hayek ce adriatic-egean-macedonic-thracianmoesian-dacian trifolium medium l. subsp. balcanicum velen. ce dinaric-moesian-scardo-pindhian acer hyrcanum fischer & c. a. meyer subsp. intermedium (pančić) bornm. ce dinaric-moesian-scardo-pindhianpeloponnesian acer heldreichii orph. ex boiss. subsp. visianii k. malý ce moesian-scardo-pindhian trifolium velenovskyi vandas ce moesian-scardo-pindhian knautia dinarica (murb.) borbás subsp. dinarica eam/csem dinaric-moesian stachys alpina l. subsp. dinarica murb. eam/csem dinaric-scardo-pindhian viola aetolica boiss. & heldr. subsp. kopaonikensis pančić ex tomović & niketić, ined. eam/csem dinaric-w moesian pastinaca hirsuta pančić eam/csem e moesian tephroseris crassifolia (schultes) griseb. & schenk eam/csem illyrian-moesian silene sendtneri boiss. subsp. sendtneri eam/csem illyrian-moesian-scardo-pindhian campanula moesiaca velen. eam/csem moesian-scardo-pindhian melampyrum scardicum wettst. eam/csem moesian-scardo-pindhian trifolium trichopterum pančić eam/csem scardo-pindhian-macedonic-thracian cerastium decalvans schlosser & vuk. subsp. leontopodium (stoj. et stef.) niketic eam/sem dinaric-moesian-scardo-pindhianhellenic-peloponnesian dianthus cruentus griseb. subsp. cruentus eam/sem dinaric-moesian-scardo-pindhianpeloponnesian stachys scardica (griseb.) hayek eam/sem dinaric-moesian-scardo-pindhianthessaly acinos alpinus (l.) moench subsp. albanicus (kümmerle & jáv.) niketić eam/sem dinaric-scardo-pindhian stachys recta l. subsp. baldaccii (k. malý) haye eam/sem dinaric-scardo-pindhian pimpinella serbica (vis.) bentham & hooker fil. ex drude eam/sem moesian-scardo-pindhian erysimum kuemmerlei jáv. eam/sem moesian-scardo-pindhianmacedonic-thracian dianthus pinifolius sibth. & sm. subsp. serbicus wettst. med-smed dacian-moesian-scardo-pindhian linum hologynum reichenb. med-smed dacian-moesian-scardo-pindhian galatella albanica degen med-smed dinaric-adriatic-w moesian-scardopindhian eryngium palmatum pančić & vis. med-smed dinaric-moesian-scardo-pindhian trifolium pignantii fauché & chaub. med-smed dinaric-moesian-scardo-pindhianthessaly alyssum markgrafii o. e. schulz ex markgraf med-smed dinaric-scardo-pindhian orobanche nowackiana markgraf med-smed dinaric-scardo-pindhian melampyrum heracleoticum boiss. & orph. med-smed dinaric-scardo-pindhian-thessaly helleborus serbicus adamović med-smed dinaric-w moesian linaria rubioides vis. & pančić subsp. rubioides med-smed dinaric-w moesian melampyrum hoermannianum k. malý med-smed e illyrian-dinaric-moesian hypochaeris illyrica k. malý med-smed illyrian-moesian scrophularia tristis (k. malý) šilić med-smed illyrian onosma echioides l. dalmatica (scheele) peruzzi & n. g. passal. med-smed illyrian-adratic-dinaric hieracium tommasinianum k. malý med-smed illyrian-moesian-scardo-pindhian scabiosa fumarioides vis. & pančić med-smed illyrian-moesian-scardo-pindhian acanthus hungaricus (borbás) baenitz med-smed illyrian-moesian-scardo-pindhianmacedonic-thracian thymus praecox opiz subsp. jankae (čelak.) jalas med-smed illyrian-moesian-scardo-pindhianmacedonic-thracian fumana bonapartei maire & petitmengin med-smed illyrian-scardo-pindhian biologica nyssana 5 (2)  december 2014: 103-112 papović, o. et al.  phytogeographical analysis and endemism of the flora … 109 plant taxa area type floristic element halacsya sendtneri (boiss.) dörfler med-smed illyrian-w moesian-scardo-pindhian sedum serpentini janchen med-smed illyrian-w moesian-scardo-pindhian potentilla visianii pančić med-smed illyrian-w moesian-scardo-pindhian haplophyllum boissieranum vis. & pančić med-smed illyrian-w moesian-scardo-pindhian eryngium serbicum pančić med-smed moesian tragopogon pterodes pančić ex petrović med-smed moesian-macedonic-thracian alyssum montanum l. subsp. serbicum novák med-smed moesian-scardo-pindhian bupleurum apiculatum friv. med-smed moesian-thessaly-macedonicthracian-aegean tulipa serbica tatić & krivošej med-smed w moesian linum tauricum willd. subsp. serbicum (podp.) petrova med-smed w moesian thymus lykae degen med-smed w moesian-scardo-pindhian fig 2. representation of area types in the endemic flora of the rogozna mt. discussion based on presence of area types and area groups, the phytogeographical analysis show that rogozna mt. is area with eurasian-submediterraneancentraleuropeanpontic characteristics. eurasian area type is dominant in the flora of rogozna mt. the dominance of eurasian floristic elements is decisive for the genesis of the flora on this mountain. however, detailed analysis of the eurasian area type showed a numerous presence of the species with central european-mediterranean (73) and central european-mediterranean-pontic (73) types of distribution, which indicates a significant influence of these floristic chorions on the genesis of the flora. however, in comparison to the ibar river valley, where the pontic flora and related area groups (central europeanmediterranean-pontic and mediterranean-pontic) are numerous (p r o d a n o v i ć , 2007; p r o d a n o v i ć et al. 2012), pontian floristic region made less influence on the genesis of the rogozna mt. flora (fig. 3). the influence of the mediterranean region is especially pronounced. there are 117 species with mediterranean-submediterranean type of distribution, many of which are endemics or subendemics. out of these, 51 species (6.46%) are widespread in the mediterranean. that is more than the percent of these floristic elements in the flora of kosovo (5.23%) (r e x h e p i , 1997). the mediterranean exhibits influence through the valley of the river ibar. as confirmation of this, there is more pronounced presence of mediterranean species in this river valley (72 taxa, 8.79%) (fig. 3) (based on the floristic list from p r o d a n o v i ć , 2007). most of the obligate serpentinophytes has a mediterranean type of distribution (tab. 4). serpentine massifs are characterized by significant presence of the pontic taxa. according to s t e v a n o v i ć et al. (2003) the spread of pontic flora throughout the central and east balkans was especially pronounced during ice age. serpentine habitats were very suitable for these plants (j a k o v l j e v i ć et al., 2011). on the rogozna mt. typical pontic elements of flora are present in very low percent (10 taxa or 1.27%), but taxa with mediterranean-pontic (116 or 14.7%) and centraleuropean-mediterranean-pontic (74 or 9.38%) are numerous. small presence of the plant taxa with pontic type of distribution is a consequence of large distance from the pontic region. additionally, the high massif of kopaonik mt. is a significant barrier which prevents the spread of pontic elements of flora from the east. floristic endemism of the serpentine areas in the balkans is very pronounced (s t e v a n o v i ć et al., 2003; m i l l a k u et al. 2008; p a v l o v a , 2007; t o m o v i ć , 2007; t o m o v i ć et al., 2014). the presence of an imposing number of endemic taxa has biologica nyssana 5 (2)  december 2014: 103-112 papović, o. et al.  phytogeographical analysis and endemism of the flora … 110 fig 3. comparative spectrum of the basic area groups in the flora of the rogozna mt. (tab. 3) and the ibar river valley (according to the data of p r o d a n o v i ć , 2007) fig 4. spectrum of the floristic elements corresponding to the floristic provinces (total) and area type (different colour) of the endemic flora of rogozna mt. biologica nyssana 5 (2)  december 2014: 103-112 papović, o. et al.  phytogeographical analysis and endemism of the flora … 111 a great significance from the aspect of biodiversity and conservation of the area, emphasizing the fact that the rogozna mt. represents an important center of the endemic balkan flora in serbia. the total list of the endemic flora of the rogozna mt. can be divided into four area-groups (fig. 2). the strong influence of the mediterraneansubmediterranean floristic chorion, which advances through the ibar valley, is observable in the areaspectrum of the endemic flora. the largest number of endemics (30 taxa) belongs to the mediterraneansubmediterranean area group. half of them belong to a group of obligate serpentinophytes (tab. 4). they are distributed into 20 floristic elements (tab. 5), where illyrian-w moesian-scardo-pindhian element is represented by 4 taxa, while others are represented by one or two taxa. the geomorphological characteristics of the area, primarily altitude (1479 m) (p a p o v i ć et al., 2014), have enabled a strong influence of the eurasian mountainous chorion on florogenesis. the consequence of this influence is the presence of much endemics belonging to the south european mountainous (7 taxa) and central south european mountainous (9 taxa) area group (tab. 5). except for the prevailing illyrian-moesianscardo-pindhian endemic taxa (including illyrian-w moesian-scardo-pindhian, illyrian-moesian and illyrian-scardo-pindhian) from different area groups (10 taxa), it should be noted that the moesianscardo-pindhian endemics (including w moesianscardo-pindhian) (7 taxa), regardless of the area group, represent one of the dominant group in the endemic flora of the rogozna mt. additionally, all other endemics at least partially by its own areal spread into one of these floristic provinces (tab. 5, fig 4). this is confirmation of the fact that this region is located at the border of illyrian, west moesian and scardo-pinhian provinces of the balkan floristic subregion. acknowledgements. the ministry of education, science and technological development of the republic of serbia (grant 173030) supported this research. references borojević-šoštarić, s., cvetković, v., neubauer, f., palinkaš, l. a., bernroider, m., & genser, j., 2012: oligocene shoshonitic rocks of the rogozna mts. (central balkan peninsula): evidence of petrogenetic links to the formation of pb–zn–ag ore deposits. lithos, 148: 176-195. euro+med, 2006-: euro+med plantbase the information resource for euro-mediterranean plant diversity. published on the internet http://ww2.bgbm.org/europlusmed/ greuter, w., burdet, h.m., long, g. (ed.), 19841989: med-checklist, 1, 3, 4. gèneve. horvat, i., glavač, v., ellenberg, h., 1974: vegetation sudosteuropas. geobotanica selecta, band 4. gustav fischer verlag. stuttgart. 768 p. jakovljević, k., lakušić, d., vukojičić, s., tomović, g., šinžar-sekulić, j., stevanović, v., 2011: richness and diversity of pontic flora on serpentine of serbia. central european journal of biology, 6 (2): 260-274. krivosej, z., prodanović, d., lazarević, p., vasić, p., 2013: ophioglossum vulgatum l. (ophioglosaceae) – in the flora of kosovo and metohija (serbia). natura montenegrina, 12 (2): 395-404. marin, p., tatić, b., 2001: serpentine soil and plant diversity, with emphasis balkan peninsula. bocconea, 13: 145-150. meussel, h., jager, e., weinert, e., 1965: vergleinchende chorologie der zentraleuropaischen flora. veb. gustav fischer verlag, 1. jena. meussel, h., jager, e., raischert, s., weinert, e., 1978: vergleinchende chorologie der zentraleuropaischen flora. veb. gustav fischer verlag, 2. jena. meusel, h., jager, e., 1992: vergleichende chorologie der zentraleuropaischen flora, karten, literatur, register. gustav fischer, jena, stuttgart, new york. millaku, f., heiselmayer, p., rexhepi, f., krasniqi, e., eichberger, c., haziri, a., 2008: endemic, stenoendemic and relict plants in serpentines of kosova. sauteria, 16: 149-162. milovanović, b., karamata, s., 1960: über den diapirsrismus serpentinischer massen. international geological congress, xxi session, part xviii, copenhagen. papović, o., miljković, m., ranđelović, n., ranđelović, v., 2014: analysis of the flora of rogozna mountain in southwestern serbia. biologica nyssana, 5 (1): 17-30. pavlova, d., 2007: endemics and rare plants growing on serpentines in the rodopes mountains (bulgaria). collection of papers devoted to academician kiril micevski, 158170. pavlović, z., 1962: karakteristieni elementi serpentinske flore srbije. glasnik prirodnjačkog muzeja srpske zemlje, serija b, 18: 3-20. prodanović, d., 2007: serpentinska flora kosovskog dela ibarske doline, doktorska disertacija. univerzitet u prištini, prirodno matematički fakultet, kosovska mitrovica. biologica nyssana 5 (2)  december 2014: 103-112 papović, o. et al.  phytogeographical analysis and endemism of the flora … 112 prodanović, d., krivošej, z., amidžić, l., 2012: ecological features on steppe flora on the ibar valley serpentine, northern kosovo. natura montenegrina, 11 (3): 405-424. prodanović, d., krivošej, z., amidžić, l., bartula, m., 2013: floristic and chorological news from northern kosovo, in the ibar river valley. natura montenegrina, 12 (2): 257-269. prodanović, d., krivošej, z., lazarević, p., amidžić, l., 2010: contribution to the knowledge of serpentine flora in ibar waley. botanica serbica, 34 (2): 81-86. ranđelović, v.n., zlatković, b.k., milosavljević, v.n., ranđelović, n.v., 2008: the endemic flora of bosilegrad surroundings (krajište region) in se serbia. phytologia balcanica, 14 (3): 367375. rexhepi, f., 1979: prilog poznavanju flore na serpentinima kosova. biotehnika, 7 (1-2): 53-70. rexhepi, 1997 mediterranean, submediterranean and illyric floristic elements in the kosovo flora. bocconea, 5, 451-456. stevanović, v., 1992: floristička podela teritorije srbije sa pregledom viših horiona i odgovarajućih flornih elemenata. in: sarić, m.r. (ed.), flora srbije, 1, 47-56. sanu, beograd. stevanović, v., tan, k., iatrou, g., 2003: distribution of the endemic balkan flora on serpentine. plant systematic and evolution, 242: 149-170. tatić, b., krivošej, z., 1997: tulipa serbica (liliaceae), a new species from serbia. bocconea, 5: 733-736. tomović, g., 2007: phytogeographycal reference, distribution and diversity centres of the balkan endemic flora in serbia. phd thesis, university of belgrade. (in serbian, with english abstract). tomović, g., niketić, m., lakušić, d., ranđelović, v., stevanović, v., 2014: balkan endemic plants in central serbia and kosovo regions: distribution patterns, ecological characteristics, and centres of diversity. botanical journal of the linnean society, 176 (2): 173-202. tutin, t.g., heywood, v.h., burges, n.a., moore, d.m., valentine, d.h., walters, s.m., webb, d.a. (ed.), 1964-1980: flora europaea, i-v. cambridge, university press. london. urošević, m., pavlović, z, klisić, m., karamata, s., malešević, m., stefanović, m., marković, o., trifunović, s., 1966: tumač za list ogk novi pazar, 77. savezni geološki zavod, beograd. urošević, m., pavlović, z, klisić, m., brković, t., malešević, m., trifunović, s., 1970: osnovna geološka karta 1:100000, list novi pazar, k 3430. savezni geološki zavod, beograd. vasić, o., diklić, n., 2001: the flora and vegetation on serpentinites in serbia a review. bocconea, 13: 151-164. whittaker, r.h., 1954: the ecology of serpentine soils. ecology, 35 (2): 258-288.